About ISEV
The International Society for Extracellular Vesicles is the leading professional society
for researchers and scientists involved in the study of microvesicles and exosomes.
With
nearly 1,000 members, ISEV continues to be the leader in advancing the study of
extracellular vesicles. Founded in 2012 in Sweden, ISEV has since moved its Headquarters
to
the United States. Through its programs and services, ISEV provides essential training
and
research opportunities for those involved in exosome and microvesicle research.
Mission Statement
Advancing extracellular vesicle research globally.
Vision
Our vision is to be the leading advocate and guide of extracellular vesicle research
and to
advance the understanding of extracellular vesicle biology.
ISEV2020 Annual Meeting
The International Society for Extracellular Vesicles is the is the premier international
conference of extracellular vesicle research, covering the latest in exosomes, microvesicles
and more. With an anticipated 1,000 attendees, ISEV2020 will feature presentations
from the
top researchers in the field, as well as providing opportunities for talks from students
and
early career researchers.
ISEV2020 International Organizing Committee
IOC Chairs: Alissa Weaver (USA), Lucia Languino (USA), Cherie Blenkiron (New
Zealand), Amy Buck (United Kingdom), Dolores Di Vizio (USA), Uta Erdbrugger (USA),
Andrew
Hoffman (USA), Michael Pfaffl (Germany), Kenneth Witwer (USA), Hang Yin (China).
Journal of Extracellular Vesicles: Editors in Chief
Jan Lotvall (Sweden)
Oral Presentations
Featured Abstracts
FA01
Ral GTPases promote metastasis by controlling biogenesis and
organotropism of extracellular vesicles
Shima Ghoroghi
a, Benjamin
Marya, Annabel LARNICOLa, François Delalandeb, Christine
Carapitoc, Nicodème Paula, Raphael Carapitoa, Olivier
Lefebvrea, Jacky Goetzd and Vincent Hyennee
aINSERM UMR_S1109, Tumor Biomechanics, Université de
Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg,
France, Strasbourg, France; bLSMBO, Institut Pluridisciplinaire Hubert Curien,
Strasbourg, France, Strasbourg, France; cLSMBO, Institut Pluridisciplinaire
Hubert Curien, Strasbourg, France, Strasbourg, France; dINSERM UMR_S1109, Tumor
Biomechanics, Université de Strasbourg, Fédération de Médecine Translationnelle de
Strasbourg (FMTS), Strasbourg, France, Strasbourg, France; eTumor Biomechanics
INSERM UMR_S 1109 Institut d’hématologie et d’immunologie, CNRS SNC5055, Strasbourg,
France,
Strasbourg, France
Introduction: Primary tumours secrete large amounts of extracellular vesicles
(EVs), which play critical roles in preparing distant sites for a pre-metastatic niche
formation, thereby promoting metastasis and even determining metastatic organotropism.
Whether biogenesis, secretion rates and organotropism of EVs are linked remains unknown.
We
have recently shown that Ral GTPases control EVs secretion in nematodes as well as
in mouse
mammary tumour cells (Hyenne et al. JCB 2015). Since both RalA and RalB are overexpressed
or
over-activated in various human cancers, we aimed to investigate the mechanisms by
which
these two GTPases control EVs secretion and to determine how this affects metastatic
progression, with a focus on breast cancer.
Methods: We used 4T1 mouse mammary carcinoma cells knocked down for either
RalA or RalB and determined their ability to induce orthotopic tumours and metastasis
in a
syngeneic mouse model. In vitro, we investigated EV secretion mechanisms using confocal
and
electron microscopy (EM). EVs were isolated either by UC or SEC and characterized
by NTA,
EM, RNA sequencing and mass spectrometry. The function of EVs was assessed using a
transwell
assay. Finally, we tracked the organotropism of fluorescently labelled EVs and their
capacity to induce pre-metastatic niches in mice.
Results: We show that RalA and RalB promote lung metastasis of breast cancer
cells in mice without affecting their invasive behaviours. We found that RalA and
RalB
control the biogenesis of exosomes, by acting on the formation of multi-vesicular
bodies
though the phospholipase PLD1. As a consequence, knock down of RalA or RalB reduces
the
levels of secreted EVs and modifies their RNA and protein contents. These differences
alter
the pro-tumoural function of EVs, as demonstrated with an in vitro permeability test.
Importantly, we show in vivo that EVs from RalA or RalB depleted cells have a decreased
lung
organotropism and, as a consequence, are less efficient in priming lung metastasis.
Finally,
we show that high expression of RalA or RalB is associated with a bad prognosis in
human
breast cancer patients.
Summary/Conclusion: Altogether, our study identifies Ral GTPases as central
molecules linking the mechanisms of EVs secretion, their dissemination and their capacity
to
promote metastasis.
FA02
Nuclear proteins are recruited into tumour-derived extracellular
vesicles upon expression of tetraspanin Tspan8
Elena Grueso Navarro
a, Andrea
Grossb, Amal Mohamedb, Patrick Thenc, Frank
Garwec, Domitille Schvartzd, Jean-Charles Sanchezd,
Andreas Kellere, Joaquin Jurado Maquedaf, Carla Oliveiraf
and Irina Nazarenkog
aInstitute for Infection Prevention and Hospital
Epidemiology; Medical Center-University of Freiburg, University of Freiburg, Freiburg,
Germany, Freiburg, Germany; bInstitute for Infection Prevention and Hospital
Epidemiology; Medical Center-University of Freiburg, University of Freiburg, Freiburg,
Germany., Freiburg, Germany; cLeibniz Institute of Photonic Technology, Jena,
Germany, Jena, Germany; dDepartment of Human Protein Sciences, Centre Médical
Universitaire, Geneva, Switzerland, Geneva, Switzerland; eClinical
Bioinformatics, University Hospital Saarland University, Saarbrücken, Germany, Saarbrücken,
Germany; fBioinf2Bio, Porto, Portugal. i3S-Instituto de Investigação e Inovação
em Saúde Universidade do Porto, Porto, Portugal. Ipatimup-Institute of Molecular Pathology
and Immunology University of Porto, Porto, Portugal., Porto, Portugal; gInstitute
for Infection Prevention and Hospital Epidemiology; Medical Center – University of
Freiburg,
Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany, Freiburg, Germany
Introduction: Tetraspanin Tspan8 is a transmembrane protein that exhibits a
unique expression pattern, being overexpressed in many cancer types, but undetectable
in
most healthy tissues. Although there is increasing evidence of an effect of Tspan8
in
invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular
mechanisms of Tspan8 are yet not fully understood.
Methods: To study the function of Tspan8, we have established a fibrosarcoma
model consisting of the parental cell line (HT1080) and its derivatives expressing
Tspan8
(HT1080-Tspan8) either fused with different fluorescent tags or tag-free. Life imaging,
STED
and STORM microscopy were used to determine the intracellular localization of Tspan8.
Co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect
interacting partners of Tspan8. Small EVs were purified from cell-conditioned media
using
SEC and subjected to mass spectroscopy and NGS for a comprehensive comparative analysis
of
the proteome and transcriptome of the EVs.
Results: The results of the proteome analysis showed a strong effect in the
protein cargo of EVs upon Tspan8 expression. Remarkably, among 20 of the most regulated
targets, several histones and ribosomal proteins were enriched in the EVs derived
from
HT1080-Tspan8 cells. In line with this finding, life imaging and super-resolution
microscopy
revealed that, while a majority of the intracellular Tspan8 is located on the cell
membrane
or intracellular membranes, -as it is known for other tetraspanins-, a portion of
Tspan8 is
located on the nuclear envelope. In fact, several histones co-immunoprecipitated with
Tspan8, indicating their interaction.
Summary/Conclusion: Our data show that the expression of Tspan8 in the tumour
cells greatly impacts EV cargo. Moreover, localization of Tspan8 on the nuclear envelope,
together with the enhanced recruitment of nuclear and ribosomal proteins to the EVs,
suggests a new mechanism of action of Tspan8.
Funding: European Union’s Horizon 2020 research and innovation program under
the Marie Sklodowska-Curie grant agreement No 722148.
FA03
Genetically encoded probes provide insight into extracellular
vesicle cargo release in cells
Bhagyashree Joshi
a; Marit de
Beerb; Ben N. G. Giepmansc; Inge S. Zuhornc
aUniversity Medical Center Groningen, Groningen, Netherlands;
bPostdoctoral researcher, Nijmegen, Netherlands; cAssociate
Professor, Groningen, Netherlands
Introduction: Extracellular vesicles (EVs) modulate tissue development,
regeneration and disease through the transfer of proteins, nucleic acids and lipids
between
cells. Currently, the mechanism of cytosolic delivery of EV cargo is largely unknown.
Here,
we unravel how EVs release their cargo in recipient cells.
Methods: EVs were isolated from GFP-CD63 and CD63-RFP expressing HEK293 T
cells by ultracentrifugation. GFP-CD63 and CD63-RFP EVs were added to HEK293 T cells
stably
expressing anti-GFP fluobody and fluorescently tagged galectin-3, respectively. CLEM
and
fluorescence microscopy were employed to visualize fluorescent markers in recipient
cells.
Bafilomycin A1 and U18666A were used to inhibit endosomal acidification and cholesterol
export from lysosomes, respectively.
Results: Fluorescent galectin-3 which binds to beta-galactosides present at
the luminal side of endosomes was used to detect endosomal permeabilization. The absence
of
galectin-3 recruitment to endosomes in presence of CD63-RFP EVs showed that endosome
permeabilization is not the mechanism behind EV cargo release. GFP-CD63 EV addition
to cells
expressing anti-GFP fluobodies resulted in the formation of fluobody punctae, reflecting
cytosolic exposure of EV cargo. Subsequent CLEM of the fluobody punctae revealed endosomes
as the underlying cellular compartments from where cargo release takes place. Neutralization
of endosomal pH and accumulation of endosomal cholesterol blocked cargo release, showing
that EV cargo release is dependent on endosomal pH and cholesterol level.
Summary/Conclusion: We show that genetically encoded cytosolic probes and CLEM
offer an excellent approach to study both the mechanism and efficiency of EV cargo
release
in cells. We provide experimental evidence that EV cargo release occurs from endosomes.
Funding: The research was supported by Dutch technology foundation TTW and
Netherlands organization for scientific research NWO, de Cock-Hadders Stichting, and
Erasmus
Mundus NAMASTE scholarship.
FA04
Towards reference intervals of extracellular vesicles in human
plasma by flow cytometry
Bo Li
a, Edwin van der
Polb, Lei Zhengc and Rienk Nieuwlandd
aDepartment of Laboratory Medicine, Nanfang Hospital,
Southern Medical University, Guangzhou, China (People’s Republic); bDepartment of
Clinical Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands;
Vesicle Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the
Netherlands; Department of Biomedical Engineering and Physics, Amsterdam UMC, University
of
Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands; cDepartment of
Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515,
China, guangzhou, China (People’s Republic); dDepartment of Clinical Chemistry,
Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Vesicle Observation
Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam,
Netherlands
Introduction: Flow cytometers with submicrometer sensitivity can characterize
single extracellular vesicles (EVs) in clinical samples. Hitherto, there is no consensus
about the concentrations of EVs in plasma from healthy humans. To determine cut-off
values
for diagnoses, reference intervals of EVs in plasma are needed. To establish such
reference
intervals, (1) a significant number of healthy donors should be included, (2) the
presence
of non-EV particles, residual platelets, lipoproteins, and haemolysis should be quantified,
(3) flow cytometry signals should be in SI units. The long-term aim of this study
is to
determine reference intervals of EV concentrations in human plasma within known dynamic
ranges of the detectors.
Methods: (1) To establish a clinical reference, we collected blood from 224
healthy volunteers and prepared platelet-free plasma. (2) We performed quality control
measurements including residual platelet count, serum index, and lipid spectrum. (3)
We
measured all samples by flow cytometry (Apogee A60-Micro) and used custom software
(MATLAB
R2018b) to automate calibration of all signals and data processing. Scatter signals
were
calibrated in comparable units of scattering cross-section (nm2) and diameter (nm).
Fluorescence signals were calibrated in units of molecules of equivalent soluble
fluorophores (MESF).
Results: The quality controls showed that most residual platelet
concentrations ranged from 10^5 to 10^6 per mL except for one outlier, while the serum
index
and lipid spectrum were normally distributed. Preliminary results of the first 21
donors
analysed, show that within the EV size range of 162–1,000 nm, the median concentration
of
CD61+ EVs is 3.9∙10^8 per mL (APC>150 MESF), CD62p+ EVs is 1.1∙10^7 per mL (PE>83
MESF), CD235a+ EVs is 5.2∙10^7 per mL (PE>123 MESF), and CD45+ EVs is 1.8∙10^7 per
mL
(APC>91 MESF).
Summary/Conclusion: We have developed reliable procedures for establishing
reference intervals of EV concentrations, within a well-defined size and fluorescence
intensity range, in human plasma by flow cytometry. We are currently applying these
procedures to 224 samples to obtain, for the first time, EV reference intervals for
human
plasma.
Funding: Pol, E. van der is supported by the Netherlands Organisation for
Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research
programmes
VENI 15924.
OT01
Symposium Session 01: Advances in Separation and Concentration I
Chair:
Juan Manuel Falcon-Perez – Exosomes laboratory and Metabolomics Platform, CIC bioGUNE,
CIBERehd, Bizkaia, Spain. / IKERBASQUE, Basque Foundation for Science, Bizkaia,
Spain.
Chair: Navneet Dogra – Icahn Shool of Medicine at Mount
Sinai
OT01.1
Beyond size-exclusion: dual-mode chromatography improves purity
of extracellular vesicles from plasma
Jan Van Deun, Ala Jo, Huiyan Li, Hsing-Ying
Lin, Ralph Weissleder, Hyungsoon Im and Hakho Lee
Center for Systems Biology, Massachusetts General Hospital, Harvard
Medical School, Boston, USA
Introduction: Purifying extracellular vesicles (EVs) from complex biological
fluids is a critical step for reliable EV analysis. Plasma lipoprotein particles (LPPs)
are
a significant confounding factor as they outnumber EVs >10,000-fold. Given their size
overlap, LPPs cannot be completely removed using standard size-exclusion chromatography
(SEC). We noticed the contrast in surface charge properties between EVs (-) and
ApoB100-containing LPPs (+). Exploiting these charge discrepancies, we combined ion
exchange
in tandem with size-exclusion chromatography to obtain an LPP-depleted EV population.
Methods: The dual-mode chromatography (DMC) column was constructed as a
layered combination of size-exclusion and ion exchange resins. DMC performance for
EV
enrichment from plasma samples was compared to standard SEC by Western blot (CD63
and
ApoA1), ELISA (ApoB100), nanoparticle tracking analysis and transmission electron
microscopy
(TEM). Additional analytical methods included single vesicle imaging and integrated
magnetoelectrochemical exosome (iMEX) assay.
Results: The DMC strategy removed the majority of plasma LPPs: >97% of
high-density lipoprotein/HDL (similar to SEC) and >99% of (very) low-density
lipoproteins/(V)LDL (>60-fold more efficient than SEC). Additionally, applying DMC
resulted in a relatively high EV isolation yield. TEM provided qualitative confirmation
of
LPP removal after DMC operation. Furthermore, DMC-prepared samples led to better analytical
outcomes in single vesicle imaging and iMEX. DMC operation was simple, fast (15 min/sample)
and equipment-free (i.e. gravity-driven).
Summary/Conclusion: DMC is a novel, single-step chromatography approach for EV
enrichment. It produced enriched EV populations and improved outcome of EV immunoassays
by
lowering biological background. We envision further investigations with different
biofluids
and analytical modalities (e.g., nucleic acid detection) to broaden its applicability.
Such
efforts would further confirm DMC as a powerful EV preparation strategy that can seamlessly
replace the current SEC-based EV isolation.
Funding: This work was supported in part by U.S. NIH Grants P01CA069246 (R.W.,
H.L.), R01CA229777 (H.L.), 1R01CA204019 (R.W.), U01CA233360 (H.L.), T32CA 79443 (H.-Y.L.),
W81XWH1910199 (H.L.), DOD-W81XWH1910194 (H.L.); R00CA201248 (H.I.), R21CA217662 (H.I.),
P30AG062421 (H.I.); Belgian American Educational Foundation fellowship (J.V.D.); MGH
Scholar
Fund (H.L.), MGH Fund for Medical Discovery Fellowship (H.-Y.L.); the Institute for
Basic
Science IBS-R026-D1 (H.L.), South Korea. Huiyan Li thanks a postdoctoral fellowship
from the
Canadian Institutes of Health Research.
OT01.2
Tangential flow for analyte capture of extracellular
vesicles
Mehdi Dehghani
a, Kilean
Lucasb, Shayan Gholizadehc, Munther Alsudaisc, Jonathan
Flaxb, James McGrathb and Thomas Gaborskic
aRochester Institute of Technology, Medford, USA;
bUniversity of Rochester, Rochester, USA; cRochester Institute of
Technology, Rochester, USA
Introduction: The use of extracellular vesicles for diagnostic and therapeutic
applications has seen a major interest increase in recent years because of their capacity
to
exchange components such as nucleic acids, lipids and proteins between cells. Isolation
of a
pure population of EVs is the first step in studying their physiological functions
since
contamination of EV preparations with non-EV proteins can lead to incorrect conclusions
about their biological activities. We have developed a new method termed tangential
flow for
analyte capture (TFAC) using ultrathin nanomembranes to purify extracellular vesicles
from
pure, highly complex biological fluids such as blood plasma, resulting in a new method
for
extracellular vesicle purification.
Methods: The TFF microfluidic devices are assembled through a layer stack
process using patterned Polydimethylsiloxane (PDMS) sheets with the membranes sandwiched
between top and bottom channels. Undiluted plasma was tested in both normal flow filtration
(NFF) and tangential flow filtration (TFF) modes on ultrathin nanomembranes. We have
utilized a pore patterning technique called nanosphere lithography (NSL) that uses
close-packing of nanoscale beads to pattern pores in an ultrathin membrane.
Results: NFF of undiluted plasma resulted in a protein cake of ~8 μm on the
membrane, which prevented further transport across the membrane and EVs were buried
in the
formed cake that were impossible to identify. However, TFAC as a modified version
of TFF,
led to capturing CD63 positive EVs on the pores of the membrane with little evidence
of
protein fouling.
NSL allows us to fabricate nanopockets (bowls with a single pore at the base) with
various
diameter, depth and pore diameter. Using NSL, we further utilize nanopocket membranes
to
purify EV samples in TFAC devices. This nanomanufacturing technology will allow us
to
pattern nanopockets with various diameter, depth and pore diameter which increases
the
efficiency of capturing of EVs. Furthermore, nanopockets can be modified and coated
by
specific EV markers to capture different subpopulation of EVs based on size and affinity
and
further allows identifying the phenotypic subsets of EVs by combining both size and
affinity-based techniques.
Summary/Conclusion: We have developed a method for the capture and release of
nanoparticles such as EVs called TFAC using ultrathin nanomembranes. NSL technology
can be
applied to fabricate nanopockets with different physical and biochemical properties.
Utilizing nanopocket membranes in TFAC system will allow us to separate different
subpopulations of EVs based on size and affinity.
Funding: This project was supported in part by the National Science Foundation
(IIP 1660177) to J.L.M and T.R.G., Department of Defence (CA170373) to J.L.M., and
the
National Institutes of Health (R35GM119623) to T.R.G.
OT01.3
The addition of a size exclusion chromatography step to various
urinary extracellular vesicle concentrating methods reveals differences in the small
RNA
profile
Jenni Karttunen
a, Sarah E.
Stewartb, Andrew Grantc, Lajos Kalmarc, Fiona E. Karet
Frankld and Tim Williamsc
aDepartment of Veterinary Medicine, University of Cambridge,
Cambridge, UK; bInstitute of Metabolic Science, University of Cambridge,
Cambridge, UK, Cambridge, UK; cDepartment of Veterinary Medicine, University of
Cambridge, Cambridge, UK, Cambridge, UK; dDepartment of Medical Genetics,
University of Cambridge, Cambridge, UK, Cambridge, UK
Introduction: Urinary extracellular vesicles (EVs) and their RNA cargo are a
novel source of biomarkers for various diseases, however non-vesicular RNA (e.g. associated
with proteins) is also present within urine. This study aimed to identify the optimal
method
for isolating and enriching EVs from human urine prior to small RNA analysis.
Methods: Three EV concentration methods, ultracentrifugation (UC); a
precipitation-based kit (PK); and ultrafiltration (UF), were compared using 50 mL
aliquots
of pooled healthy volunteer urine. EVs were then separated from protein contaminants
by
size-exclusion chromatography (SEC). Presence of EVs was confirmed by transmission
electron
microscopy and Western blotting, and EVs were quantified using nanoparticle tracking
analysis (NTA). Small RNA content of concentrated urine and fractions obtained by
SEC (EVs
and proteins) were evaluated with the Agilent Bioanalyzer small RNA chip.
Results: EV recovery following SEC of concentrated samples was 35–78%, however
particle: protein ratio (indicating EV purity) was approximately 10x greater after
SEC,
regardless of the concentrating method used. UF+SEC yielded the highest number of
EVs (per
mL of urine) compared with PK+SEC and UC+SEC. Small RNA analysis from UF-concentrated
urine
(prior to SEC treatment) identified peaks at 20 nucleotides (nt) and 60 nt. Following
SEC,
RNA analysis indicated that EV fractions contained mostly small RNA of ~60 nt, whereas
the
protein factions contained small RNA of ~20 nt in size (consistent with miRNAs).
Summary/Conclusion: UF+SEC provided the best balance between EV recovery (per
mL urine) and particle: protein ratio. These data indicate that most of the 20 nt
sized
RNAs, presumably miRNAs, are not within EVs in urine. EV preparations obtained after
UC, PK
and SEC (regardless of concentrating method) contain predominantly ~60 nt sized small
RNA.
These data outline the importance of removing non-vesicular proteins and RNA from
urine EV
preparations prior to small RNA analysis.
Funding: This research has been funded by PetPlan Charitable Trust.
OT01.4
The use of rEV for the optimization of EV separation and
characterization by AF4
Lien Lippens
a, Edward
Geeurickxb, Robin Boiyb, Sarah Devillec, Bruno De
Geestd, Olivier de Wevere and An Hendrixe
aLaboratory of Experimental Cancer Research, Department of
Human Structure and Repair, Ghent University, Ghent, Belgium, Ghent, Belgium;
bLaboratory of Experimental Cancer Research, Department of Human Structure and
Repair, Ghent University, Ghent, Belgium, 9000, Belgium; cFlemish Institute for
Technological Research (VITO), Health Unit, Mol, Belgium; dBiopharmaceutical
Technology Unit, Department of Pharmaceutics, Ghent University, Ghent, Belgium, 9000,
Belgium; eLaboratory of Experimental Cancer Research, Department of Human
Structure and Repair, Ghent University, Belgium; Cancer Research Institute Ghent,
Belgium,
Ghent, Belgium
Introduction: The reproducibility of extracellular vesicle (EV) research has
been hampered by the infinite number of separation and measurement techniques and
the lack
of appropriate reference materials (Van Deun et al., Nat Methods, 2017). Recombinant
extracellular vesicles (rEV) were developed as a biological reference material to
overcome
these limitations (Geeurickx et al. Nat Comm 2019). Since rEV have EV-like physical
an
biochemical characteristics and as they are trackable and distinguishable from sample
EV
they can be used as a spike-in material for data normalization and method development,
and
as a quality control. We used rEV to optimize EV separation by asymmetrical flow field-flow
fractionation (AF4).
Methods: An AF4 long channel column with a frit inlet driven by the eclipse
system (Wyatt) was coupled to a UV detector (Shimadzu), MALS Dawn Helios-II (Wyatt)
and
fluorescent detector (Agilent). A spacer of 350 µm and a regenerated cellulose membrane
of
10 kDa were used. PBS supplemented with 0.02% NaN3 was used as a running buffer. Light
scatter profiles and UV profiles were analysed as well as the fluorescent emission
spectrum
as the rEV are GFP positive. Fractions were collected and analysed by nanoparticle
tracking
analysis (NTA) and western blot. We also estimated the repeatability and reproducibility
of
the AF4 technique by light scatter and fluorescence profiles as well as the recovery
efficiency by NTA.
Results: In a first step 4*10^10 rEV isolated from conditioned medium by a
velocity gradient were injected in the AF4 system to optimize the EV characterization
protocol. Later concentrated conditioned medium was spiked with 1*10^11 rEV and injected
in
the AF4 column to optimize EV separation from non-EV contaminants. The most optimal
separation protocol was obtained by varying detector and cross-flow settings. This
protocol
shows elution of monodisperse particles at each time point and size distribution estimations
by AF4 correspond to size determination by NTA and electron microscopy.
Summary/Conclusion: We were able to optimize the AF4 protocol for
characterization of EV by AF4 as well as for separation of EV from crude conditioned
medium
samples by using rEV. We demonstrate that rEV are suitable for method development
and that
AF4 has high potential as an EV separation technique.
OT01.5
Comparative evaluation of EV isolation methods for EV
subpopulation analysis in human urine, plasma and cell culture media
Liang Dong, Richard Zieren, Kengo Horie, Sarah
Amend and Kenneth Pienta
The Brady Urological Institute, Johns Hopkins University School of
Medicine, Baltimore, USA
Introduction: Extracellular vesicles (EVs) are membrane-enclosed particles of
variable sizes that are released by any cell types to the extracellular space and
are
identified in all body fluids. A shortcoming in EV research is the lack of standardized
isolation protocol for various sample types, resulting in heterogeneous outcomes in
downstream analyses. In this study, we compared the EV isolation purity and efficiency
among
ultracentrifugation (UC), precipitation, size-exclusion chromatography (SEC) and a
microfluidic tangential flow filtration device (Exodisc) in human plasma, urine and
cell
culture media (CCM).
Methods: All EVs were isolated by different isolation methods and
characterized per MISEV2018 guidelines. Single-particle interferometric reflectance
imaging
senor (SP-IRIS) with optional fluorescence and nano-flow (nFCM) were used for single
particle analysis.
Results: In CCM, total particle yield of Exodisc was about 5 times higher than
those of the rest three methods. Size distribution differed per sample, but the ranges
were
comparable between the different isolation methods. The total protein amount of SEC,
precipitation and Exodisc were similar which were 6–10 times higher than that of UC.
UC had
the highest particle-to-protein ratio followed by Exodisc. Precipitation and SEC had
low
ratios. When loading 9ug of total protein for Western blot, CD9, CD81, CD63 and Flot1
could
only be detected in UC and Exodisc samples, but not precipitation or SEC. SP-IRIS
and nFCM
demonstrated consistent purity findings. In urine, total particle yields of Exodisc
and SEC
were about 4 times higher than those of the rest two methods. The total protein amount
of
precipitation was 3 times higher than Exodisc and SEC, 10 times higher than UC. SEC
had the
highest particle-to-protein ratio followed by UC and Exodisc. Precipitation had low
ratios.
In plasma, total particle yields of Exodisc and precipitation were 100 times higher
than
those of the rest two methods. And so were the total protein amount. SEC had the highest
particle-to-protein ratio followed by UC. Exodisc and precipitation had low ratios.
Western
blot, SP-IRIS and nFCM demonstrated consistent purity findings in urine and plasma.
To
evaluate particle capture efficiency, we spiked a known number of density-gradient
UC
purified EVs to each method and the recovery rate of UC, precipitation, Exodisc and
SEC was
8.6%, 31%, 50.1% and 42%, respectively.
Summary/Conclusion: The order of EV isolation purity in CCM is UC, Exodisc,
SEC and precipitation. In urine it’s SEC, Exodisc, UC and precipitation. And in plasma,
this
order is SEC, UC, Exodisc and precipitation. Exodisc and SEC have similar high isolation
efficiency followed by precipitation. UC has low efficiency for EV capture.
OT01.6
A capillary-channelled polymer (C-CP) fibre spin-down tip
approach for the isolation and biomarker characterization of extracellular vesicles
of
ovarian cancer origin
Kaylan D. Kelsey, Rhonda R. Powell, Terri F.
Bruce and R. Kenneth Marcus
Clemson University, Clemson, USA
Introduction: Extracellular vesicle (EVs) profiling has shown promise for
disease detection through less invasive sampling (liquid biopsies). Current diagnostic
tools
for ovarian cancer are invasive or only semi-informative. Thus, use of EVs could prove
useful in early disease detection. Demonstrated is a hydrophobic interaction chromatography
(HIC)-based capillary-channelled polymer (C-CP) fibre tip spin-down process for the
isolation of ovarian cancer EVs for use in diagnostics.
Methods: Polyester C-CP fibre micropipette tips are employed in the isolation
of EVs from biological matrices including cell culture media, urine, and blood plasma
in a
spin-down solid-phase extraction (SPE) approach. EVs were isolated from standards
of healthy
urine origin and from SKOV3 cells (human ovary adenocarcinoma). The C-CP fibre isolation
method (taking less than 5 mins and 10 μL sample volumes) preserves the morphology
and
functionality of EVs as confirmed by SEM, TEM, and confocal fluorescence microscopy.
Results: The dynamic binding capacity of EV standards on a 1 cm PET C-CP fibre
tip was found to be ~7E11 particles (50%). The release of EVs was confirmed using
dot blot
analysis for CD9, CD81, and CD63 tetraspanin proteins. Immobilized EVs were subjected
to
immunolabeling to allow the positive identification of a profile of ovarian cancer
biomarker
proteins (HER2, CD24, EGFR, EPCAM, CA125).
Summary/Conclusion: This new EV isolation method introduces a simple capture
mode, allowing for direct immuno-characterization and imaging on the fibre surface.
This
offers a unique and cost-effective opportunity for clinical analyses related to early
detection and diagnosis of ovarian cancers (and others). The long-term goal is the
creation
of a rapid EV isolation and biomarker detection platform.
Funding: Support from the National Science Foundation, Eppley Foundation for
Scientific Research, Gibson Foundation, Prisma Health System and ITOR Biorepository
are
gratefully acknowledged.
OT01.7
Development and optimization of purification method of exosomes
by tangential flow filtration and ion-exchange chromatography approach
Tek Lamichhane, Ali Navaei, Sandeep Choudhary,
Yonatan Levinson and Senthil Ramaswamy
Cell & Gene Therapy R&D, Lonza Inc, Rockville, USA
Introduction: Extracellular vesicles (EVs) such as exosomes have significant
therapeutic potential, however, translation of EV-based therapies has been slowed
down
because of the biomanufacturing challenges. The isolation of EVs, especially exosomes,
is
inherently challenging due to their small size, and heterogeneity in the mixture.
The
current isolation methods either have low recovery rate, aggregation, damaging the
structure, time consuming or co-precipitation of contaminants. Specially, it is difficult
to
process larger sized samples by centrifugation-based or immunoaffinity based methods
because
of the time and cost associated with these methods.
Methods: To overcome these roadblocks, we developed and optimized alternative
purification techniques to isolate EVs with higher purity and yield by using tangential
flow
filtration (TFF) coupled with ion-exchange chromatography. We used bioreactor platform
to
produce EVs from serum-free medium using BM-MSC and HEK293 s cells. BM-MSCs were cultured
on
stirred tank bioreactors using microcarriers which provide a high surface area to
volume
ratio for the optimal cell growth and EVs production. Impellers were used to enhance
mixing
and maintain homogeneous culture conditions that can be easily monitored and controlled.
Results: Depth filtration was applied for clarification of conditioned medium.
We screened different types of filters during depth filtration for the best recovery
of EVs.
TFF membranes with different pore sizes were used to optimize the purity and yield
of EVs.
Because of the negatively charged nature of EVs, anion exchange chromatography was
chosen to
capture and separate TFF purified vesicles by their surface charge characteristics.
We
compared monolith based and membrane-based anion exchange columns to remove contaminants
and
purify exosomal fractions. The purity, size and presence of exosomal markers in isolated
EVs
at each step of purification was evaluated by F-NTA, nano-FCM and tetraspanins based
ELISA
kits.
Summary/Conclusion: In summary, our optimized methods improved the speed of
isolation and purity of EVs to the clinical grade. The production and isolation methods
of
exosomes that we developed here will be easily expandable to support large-scale and
cGMP
compatible bio-manufacturing in the future.
OT01.8
Use of an alternating current electrokinetic microelectrode chip
to positively identify oncology, neurology, and infectious disease samples through
plasma
extracellular vesicle analysis
Juan Pablo Hinestrosa, Jean Lewis, David
Searson, Orlando Perrera, Alfred Kinana, Heath Balcer and Rajaram Krishnan
Biological Dynamics, Inc., San Diego, USA
Introduction: Cancer, neurological, and infectious diseases are leading causes
of death, with early detection needed to improve outcomes. Extracellular vesicles
(EVs) in
the blood contain disease biomarkers, but current methods do not allow rapid analysis,
and
are often limited to one biomarker type.
Methods: We developed methods using alternating current electrokinetics (ACE)
to isolate EVs from blood-based samples and analyse the EVs in situ with downstream
assays
for protein and nucleic acid biomarkers. We investigated if we could identify tuberculosis
(TB) donor samples, protein and nucleic acid biomarkers in EVs derived from cancer
cell
lines, and Alzheimer’s disease (AD) protein biomarker levels.
Results: EV isolation was confirmed by positive identification of the proteins
CD9, CD63, and CD81 and measurement of EV mRNAs using a direct RT-ddPCR assay. Different
disease models were analysed following method development.
TB was used as a model for infectious disease, with 20 TB positive and 20 TB negative
samples isolated on ACE chips and analysed for levels of lipoarabinomannan and Ag85.
Using a
cut-off above the negatives, the AUC of ROC curves were 0.9975 and 1.0, respectively.
For oncology, cancer cell lines were cultured and EVs isolated from supernatants were
spiked into human plasma for analysis. Levels of PD-L1 or Glypican-1 on EVs were able
to be
measured following ACE capture. Additionally, DNA and RNA mutations known to be present
in
the cell lines were able to be detected using NGS and qRT-PCR, respectively.
Using AD samples as a neurological disease model, Tau and phospho-tau T181 (p-tau
T181) in
human donor plasma were detected. In 8 AD and 5 healthy donor samples, p-tau T181
signal
increased 134% in diseased versus healthy donors.
Summary/Conclusion: ACE chips are an innovative EV isolation and analysis
platform that allow rapid disease sample detection in a wide range of studies with
high
sensitivity and specificity.
OT02
Symposium Session 02: Cancer Progression
Chair: Hector Peinado – Microenvironment and Metastasis Group, Molecular Oncology
Program, Spanish National Cancer Research Center (CNIO)
Chair: Hidetoshi Tahara – Department of Cellular and Molecular Biology, Graduate
School of Biomedical & Health Sciences, Hiroshima University
OT02.1
Identification of critical modifying factors in EV-based
communication between colorectal cancer and stromal cells using the organoid
model
Zsuzsanna Szvicseka, Adam Oszvaldb, Anikó
Zeölda, Andrea Kelemena, Tamás Tölgyesc, Kristóf
Dedec, Attila Bursicsc, Edit Buzásd and Zoltán
Wiener
a
aSemmelweis University, Department of Genetics, Cell and
Immunobiology, Molecular Cancer Research Group, Budapest, Hungary, Budapest, Hungary;
bSemmelweis University, Department of Genetics, Cell and Immunobiology,
Budapest, Hungary; cUzsoki Hospital, Budapest, Hungary, Budapest, Hungary;
dSemmelweis University, Department of Genetics, Cell and Immunobiology, MTA-SE
Immune-Proteogenomics Extracellular Vesicle Research Group, Budapest, Hungary and
HCEMM_SE
Extracellular Vesicle Research Group, Budapest, Hungary
Introduction: Colorectal cancer (CRC) is one of the most frequent causes of
cancer-related death. In the majority of CRC patients, mutation in the Apc gene is
among the
first genetic events. It leads to uncontrolled activation of the Wnt pathway, and
thus, to
adenoma formation. Some of these adenomas may then further progress to CRC with the
accumulation of other mutations. The 3D organoids maintain the cellular and genetic
heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model
of
human cancers. Here we analysed the EV-based communication between cancer cells and
fibroblasts by i) identifying factors that substantially increase EV release from
intestinal
cancer cells and ii) by determining cargo components of EVs that enhance tumour cell
proliferation.
Methods: We used commercially available and patient-derived fibroblasts and
CRC organoids. The Medical Research Council of Hungary approved all experiments with
human
samples and informed consent was obtained from patients. EVs were studied by using
antibody-coated beads, TRPS, NTA, TEM and Western-blotting. We introduced Apc mutation
into
wild type murine small intestinal organoids by CRISPR-Cas9.
Results: We found that in CRC patient-derived organoid cultures, small EVs
were preferentially secreted. We observed that Apc mutation and the accumulation of
the
extracellular matrix component collagen critically enhanced EV secretion in intestinal
organoids. Furthermore, we showed that amphiregulin, present on fibroblast-derived
EV,
contributed to the maintenance of the intestinal stem cell pool and to cell proliferation
in
epidermal growth factor-dependent CRC organoids.
Summary/Conclusion: By proving the key role of mutations, collagen deposition
and EV-bound amphiregulin in the release intensity and functions of the EVs, we identified
novel mechanisms in the progression if CRC.
Funding: This work was funded by OTKA-NN 118018, by the National
Competitiveness and Excellence program NVKP_16-0007 (National Research, Development
and
Innovation Office, Hungary) and by the National Excellence Program in Higher Education
(Ministry of Human Resources, Hungary).
OT02.2
Prostate cancer-derived EVs induce a pro-inflammatory phenotype
in the stroma
Blandine F. Victor
a, Dolores Di
Viziob, Andrew Chinc, Tatyana Vagnerb, Javier
Mariscalb, Mandana Zandiana, Catherine Grassoa, Roberta
Gottlieba and Helen Goodridgea
aCedars-Sinai Medical Center, Los Angeles, USA;
bCedars-Sinai Medical Center, West Hollywood, USA; cCedars-Sinai, Los
Angeles, USA
Introduction: Since 90% of patients with metastatic prostate cancer (PC)
develop bone metastasis, identifying the mechanism that drives this process is essential.
Most EV research has been focused on the role of exosomes in mediating the pre-metastatic
niche formation. However, most of these studies do not separate exosomes from large
EVs. Our
preliminary studies have demonstrated that a subclass of EVs known as Large Oncosomes
(LO)
can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis
and
enhancing the migration and invasion of PC cells in vitro and tumour growth in vivo.
The
bone marrow is the initial site of entry into the bone microenvironment for disseminating
tumour cells (DTCs) and is a rich source of nutrients that houses various cells types
including bone marrow derived mesenchymal stem cells (BM-MSC) and immune cells such
as
neutrophils, which have been implicated in metastasis. Here we investigate the role
of LO in
reprogramming BM-MSCs and driving bone metastasis in PC.
Methods: Differential centrifugation, density gradient centrifugation, TRPS,
RNA sequencing, qPCR, migration assay, invasion assay, chemotaxis assay.
Results: We report that PC-derived EVs induce distinct gene expressions
changes in BM-MSCs. RNA-Seq analysis identified inflammatory and immune regulating
cytokines
as top differentially expressed genes (DEG) in BM–MSC. Moreover, LO induced a more
potent
response in BM-MSC in comparison to Exo and to non-treated controls. The genes enriched
in
LO treated BM-MSC were associated with tumour cell motility. In agreement with the
gene
expression data, LO-treated BM-MSC attracted migration and invasion of significantly
more PC
cells than Exo -treated BM-MSCs. In addition, the top DEG expressed in EV treated
BM-MSC
were identified as potent neutrophil chemoattractant proteins. In line with the RNAseq
findings, the LO-treated BM-MSC demonstrated enhanced chemotaxis of neutrophils towards
them
in comparison with Exo or vehicle-treated BM-MSC. Finally, we show that the observed
differences in BM-MSC’s response to LO and Exo may be mediated by distinct molecular
pathways.
Summary/Conclusion: The results from this study provide novel insight into how
tumour derived EVs alter the bone marrow microenvironment and how they may drive bone
metastasis in prostate cancer.
OT02.3
The αvβ6 integrin in cancer cell-derived small extracellular
vesicles enhances angiogenesis
Shiv Ram Krishn
a, Israa
Salemb, Fabio Quagliab, Nicole M. Naranjoc, Ekta
Agarwald, Peter A. McCueb, Paul H. Weinrebe, Shelia M.
Violettef, Dario C. Altierig and Lucia R. Languinob
aThomas Jefferson University, Philadelphia, USA,
Philadelphia, USA; bThomas Jefferson University, Philadelphia, USA;
cThomas Jefferson University, Philadelphia PA, USA; dThe Wistar
Institute, Philadelphia, USA; eBiogen Inc., Cambridge, USA; fBiogen,
Cambridge, USA; gImmunology, Microenvironment and Metastasis Program, The Wistar
Institute, Philadelphia, PA 19104 USA, Philadelphia, USA
Introduction: Prostate cancer (PrCa) cells crosstalk with the tumour
microenvironment by releasing small extracellular vesicles (sEVs). sEVs isolated from
PrCa
cell media, express the epithelial-specific αvβ6 integrin, a surface receptor for
fibronectin and vitronectin. The αvβ6 integrin is not detectable in healthy prostate
tissues
but is highly expressed in PrCa. In this study, we hypothesized that αvβ6 in cancer
sEVs
plays a crucial role in angiogenesis.
Methods: The sEVs isolated from PrCa cell media were characterized by
nanoparticle tracking analysis, iodixanol density gradients and expression of sEV
markers.
The αvβ6-negative endothelial cells (HMEC1) were incubated with αvβ6-positive sEVs
from PrCa
cells to evaluate the transfer of αvβ6 by immunoblotting (IB) and FACS. The effect
of
αvβ6-positive sEVs on motility, tube formation and angiogenic signalling were assessed
by
Boyden chamber, angiogenesis assays and IB in HMEC1.
Results: We demonstrate for the first time that the αvβ6 is de novo expressed
on endothelial cell surface by sEV-mediated protein transfer. PrCa cell-derived
αvβ6-positive sEVs, significantly promote the motility and the formation of nodes,
junctions
and tubules by HMEC1. Mechanistically, we demonstrate that HMEC1 treatment with sEVs
from
PC3 cells that endogenously express αvβ6, decreases pSTAT1(Y701), a negative regulator
of
angiogenesis, while upregulating survivin, an inducer of angiogenesis. HMEC1 treatment
with
sEVs isolated from PC3 cells harbouring CRISPR/Cas9-mediated downregulation of β6,
or
shRNA-mediated downregulation of β6, results in increased levels of pSTAT1(Y701).
This sEV
treatment also results in a decrease of survivin in sEVs and HMEC1.
Summary/Conclusion: Overall, our findings show that αvβ6 in prostate cancer
sEVs regulates a novel pro-angiogenic signalling pathway.
Funding: This study was supported by NCI R01-224769 (LRL); P01-140043 (LRL and
DCA).
OT02.4
Prostate cancer exosomes promote bone metastasis in a
cholesterol-dependent manner
Stephen E. Henrich
a, Kaylin
McMahona, Michael Plebaneka, Fabio Tavorab, Andre De
Souzac, Anthony Megac, Benedito Carneirob and Colby
Thaxtond
aDepartment of Urology, Feinberg School of Medicine,
Northwestern University, Chicago, IL, 60611, Chicago, USA; bBrown University,
Providence, RI, 02912, Providence, USA; cLifespan Cancer Institute, Providence,
RI, 02903, Providence, USA; dDepartment of Urology, Feinberg School of Medicine,
Northwestern University, Chicago, IL, 60611; Simpson Querrey Institute for
BioNanotechnology, Northwestern University, Chicago, IL, 60611; Robert H. Lurie
Comprehensive Cancer Center Northwestern University, Chicago IL, 60611, Chicago, USA
Introduction: Advanced prostate cancer (PCa) is associated with elevated
cholesterol levels; however, the mechanisms underlying this association are not well
understood. Further, the development of bone metastases is one of the most lethal
processes
in PCa, and is also associated with elevated cholesterol. Interestingly, recent work
has
demonstrated that exosome-mediated intercellular communication is dependent upon cholesterol
levels in the target cell population. Here, we investigated whether PCa exosomes promote
pre-metastatic niche formation and bone metastasis via intercellular communication
with bone
marrow-resident myeloid cells in a cholesterol-dependent fashion.
Methods: Exosomes from enzalutamide resistant (EnzR) PCa cells (CWR-R1) and
normal prostate epithelial cells (PNT2) were isolated via ultracentrifugation, while
exosomes from patient serum samples were isolated using ExoQuick Ultra (System Biosciences).
Intracardiac injection of luciferase-expressing EnzR CWR-R1 cells into C.B.-17 SCID
mice was
used as a bone metastatic PCa mouse model, and tumour burden was monitored by BLI.
Alterations in extracellular matrix (ECM) composition were determined via
immunohistochemistry and RNA sequencing. Sequencing of small RNAs was performed on
exosomal
RNA samples obtained from conditioned media and patient serum. A nanoparticle mimic
of
high-density lipoprotein was used for targeted reduction of myeloid cell cholesterol.
Results: Data revealed that educating mice with PCa exosomes altered the
composition of the bone marrow ECM in a pro-tumorigenic manner. Specifically, EnzR
CWR-R1
exosomes reduced thrombospondin-1 (TSP1) expression and enhanced versican (VCAN) expression.
Both changes have been reported to occur in advanced PCa. Furthermore, educating mice
with
EnzR CWR-R1 exosomes enhanced metastatic tumour burden in a PCa mouse model.
Exosome-mediated pre-metastatic niche formation and enhanced tumour burden were both
significantly diminished by targeted reduction of myeloid cellular cholesterol. Moreover,
we
found that exosome-induced downregulation of TSP1 was specifically mediated by exosomal
miR-4443. This miRNA was also over-represented in the serum of late stage PCa patients
compared to patients with early stage disease and healthy controls. Finally, reduced
TSP1
and increased VCAN expression were also found in bone tissues obtained from patients
with
bone metastatic PCa.
Summary/Conclusion: These results offer insights into mechanisms of PCa bone
metastasis, and specifically suggest an exosome-mediated mechanism for the link between
elevated cholesterol and advanced, bone metastatic PCa. Finally, these results suggest
that
exosomal miR-4443 may be a viable diagnostic and therapeutic target for PCa.
Funding: This work was funded in part by a grant from the Prostate Cancer
Foundation.
OT02.5
Low aggressive HNSCC-derived EVs regulates metastatic potential
of highly metastasized HNSCC.
Shinya Sato
a, Michael
Korrerb, Sohini Royb, Young Kimb and Alissa
Weaverb
aDivision of Molecular Pathology and Genetics, Kanagawa
Cancer Center Research Institute, Yokohama, Japan; bVanderbilt University,
Nashville, USA
Introduction: Extracellular vesicles (EVs) are secreted from cells, and carry
bioactive proteins and RNA cargoes. Increasing numbers of studies have identified
key roles
for exosomes in driving aggressive tumour behaviours, including metastasis. However,
the
detailed mechanisms and responsible factors in the EV cargo are still unclear. Recently,
immune system has been considered as an important factor in establishing and maintaining
metastasis. Our goal is to identify the role of head and neck squamous cell carcinoma
(HNSCC) derived small EVs (SEVs) in tumour metastasis from the study analysing the
effects
of SEVs on metastasis and tumour immunity.
Methods: SEVs were collected from the conditioned media of HNSCCs and purified
through cushioned density gradient ultracentrifugation. An orthotopic mouse model
was used
for the assessment of tumour angiogenesis and metastasis. MOC1 (inflammation-inducing
rarely
metastasizing murine HNSCC line) and MOC2 (highly metastasizing murine HNSCC line)
were used
for this study. MOC1 and MOC2 cells were transplanted into mice tongues orthotopically,
and
MOC1/MOC2 derived SEVs or PBS were injected into the tumour twice in a week. Two weeks
after
tumour transplantation, mice were sacrificed and tumours were sectioned for pathological
analysis and FACS analysis. In FACS analysis, the number and species of tumour-infiltrated
immune cells were measured.
Results: Injection of SEVs from MOC1 into MOC2 tumours suppressed frequency of
lymph node metastasis. On the other hand, injection of SEVs from MOC2 into MOC1 tumours
didn’t promote metastasis. CD4 positive T-cell distribution in MOC2 tumour was significantly
changed by MOC1 SEV injection. T-cell deprivation treatment using anti-CD3 antibody
increased the frequency of metastasis in MOC1-SEV treated MOC2 tumours. From the result
of
proteomics analysis on MOC1 and MOC2 SEVs, immune-regulated proteins and
metastasis-suppressing proteins were observed in MOC1 SEVs.
Summary/Conclusion: We find that low aggressive HNSCC SEVs affect metastasis
of highly metastasized HNSCC, and also find that changing immune cell distribution
may be
related to the result. This mechanism and finding contributes to understanding the
possible
role of HNSCC SEVs on metastasis as well as on the tumour immune microenvironment.
Funding: This work was supported by the NIH under award numbers R01CA163592
and R01CA206458 to AW.
OT02.6
Desmoglein 2 enhances squamous cell carcinoma tumour development
through extracellular vesicles in an IL-8/miR-146a-dependent mechanism
Joseph P. Flemming
a, Mohammed
Haqueb, Brianna L. Hillc, James Wahld, Kenneth
Tsaie, Peter Wermuthf, Andrew Overmillerg and My
Mahoneyh
aThomas Jefferson University, Haddonfield, USA;
bThomas Jefferson University, Lansdale, USA; cThomas Jefferson
University, Mohrsville, USA; dUNMC, Omaha, USA; eMoffitt Cancer
Center, Tampa, USA; fTJU, Philadelphia, USA; gThomas Jefferson
University, Bethesda, USA; hThomas Jefferson University, Philadelphia, USA
Introduction: The cadherin Dsg2 is a stem cell marker that is upregulated in
many different cancers, including SCCs, and its expression correlates with poor prognosis.
Dsg2 activates mitogenic signalling and plays a key role in cell proliferation, migration,
and survival. We recently showed that Dsg2 enhances EV release, but the mechanism
by which
these EVs modulate tumorigenesis is not fully understood.
Methods: We established SCC cell lines stably expressing wildtype Dsg2 or a
palmitoylation deficient mutant, Dsg2cacs. EVs were isolated by sequential
ultracentrifugation, iodixanol gradient separation, or qEV Izon column, and analysed
by NTA
and BCA. Tumour xenografts were established by subcutaneous injection of 106 cells
in SCID
mice and monitored up to 4 weeks. Cytokine profiling was determined by antibody array.
miRNA
expression was analysed by RNAseq and confirmed by qPCR.
Results: Dsg2 enhanced EV release by 40% and promoted a ~ fivefold increase in
tumour size in xenograft models. Tumour growth was increased when control cells were
treated
with a single 20 µg dose of EVs. Loss of palmitoylation, which altered membrane trafficking
of Dsg2, reduced EV release (~55%) as well as tumour development. Plasma EVs from
xenograft
mice reflected in vitro particle counts from SCC cell lines. A cytokine array analysis
was
performed revealing that Dsg2-EVs were enriched with pro-inflammatory cytokines including
IL-8, a potent chemotactic and angiogenic factor. Most importantly, IL-8 was surface-bound
on EVs. Furthermore, RNAseq revealed miR-146a, a negative regulator of IL-8, to be
significantly downregulated in response to Dsg2. Treatment with miR-146a mimic or
miR-146a
inhibitor decreased or increased, IL-8 expression in SCC cells, respectively.
Summary/Conclusion: In summary, Dsg2 plays a key role in SCC tumour
development by increasing EV biogenesis and downregulating miR-146a, which in turn
upregulates IL-8 synthesis and release which can promote invasion, angiogenesis and
metastasis.
Funding: NIH R01
OT03
Symposium Session 03: Cardiovascular Disease
Chair: Elena Aikawa, MD PhD –
Center for Interdisciplinary Cardiovascular Sciences, Division of Cardiovascular Medicine,
Brigham and Women’s Hospital, Harvard Medical School
Chair: Edit Buzás – Semmelweis University, Department of Genetics, Cell and
Immunobiology, MTA-SE Immune-Proteogenomics Extracellular Vesicle Research Group,
Budapest, Hungary and HCEMM_SE Extracellular Vesicle Research Group
OT03.1
Towards identification of protein components mediating cardiac
repair by cardiac progenitor cell-derived extracellular vesicles
Marieke Roefs
a, Pieter
Vaderb and Joost Sluijtera
aDepartment of Experimental Cardiology, University Medical
Center Utrecht, Utrecht University, The Netherlands, Utrecht, Netherlands;
bLaboratory of Clinical Chemistry and Hematology, UMC Utrecht; Department of
Experimental Cardiology, UMC Utrecht, The Netherlands, Utrecht, Netherlands
Introduction: Stem- and progenitor cell transplantation therapy holds great
promise for regenerating damaged heart tissue. Several lines of evidence suggest that
its
efficacy is mainly caused by secreted extracellular vesicles (EVs). Indeed, cardiac
progenitor cell (CPC)-derived EVs have been shown to protect the myocardium against
ischaemia/reperfusion injury in several preclinical models. However, the underlying
mechanisms for CPC-EV-mediated cardioprotection remain elusive. Here, we utilized
the
proteomic composition of CPC-EVs released during different culture conditions, to
unravel
protein-mediated effects of CPC-EVs on the endothelium.
Methods: CPCs were stimulated with calcium ionophore (ca ion-EVs) or vehicle
(control-EVs) for 24 hours and EVs were isolated from serum-free conditioned medium
using
size exclusion chromatography. EV concentration and size was assessed using NTA. EVs
were
functionally characterized based on endothelial cell activation by western blotting
and an
endothelial cell scratch assay. The proteomic composition of both EV conditions was
profiled
using mass spectrometry. CPC-EV knockouts for specific proteins were generated using
CRISPR/Cas9 technology.
Results: We found enhanced phosphorylation of ERK1/2 and AKT in endothelial
cells and increased wound closure after stimulation with control-EVs, but not after
stimulation with ca ion-EVs. Proteomic analysis identified a total of 1411 EV-associated
proteins, with 79 proteins uniquely expressed in control-EVs. Another 68 proteins
were
revealed as candidate proteins, based on their relative enrichment in control-EVs
compared
with ca ion-EVs. GO analysis demonstrated that differentially expressed proteins were
involved in vascular endothelial growth factor signalling, extracellular matrix organization
and angiogenesis. To investigate the involvement of the individual candidate proteins
on
endothelial cell activation, knockout EVs of multiple proteins were generated and
functionally characterized.
Summary/Conclusion: A specific set of EV proteins is identified that may be
functionally responsible for the activation of endothelial cells upon exposure to
CPC-EVs.
Generating knockout EVs for each of these proteins will help to investigate their
individual
roles. This may lead to a better mechanistic understanding of the use of CPC-EVs as
therapeutics for cardiac repair.
Funding: ERC-2016-COG-725229 EVICARE grant.
OT03.2
Hypoxia enhances the therapeutic potential of human CD34+ stem
cell exosomes in ischaemic hindlimb repair
Shweta Lodha
a, Morad
Asadia, Susmita Sahooa, Marta Adamiaka, Cherrie
Shermana, Douglas Losardob, Rajesh Guptac, Luca
Musanted, Uta Erdbrueggere, Michael Davisf and David
Kimb
aCardiovascular Research Center, Icahn School of Medicine,
Mount Sinai, New York, USA; bFeinberg Cardiovascular Research Institute, Feinberg
School of Medicine, Northwestern University, Chicago, IL, Evanston, USA;
cUniversity of Toledo, Toledo, USA; dUniversity of Virginia School of
Medicine, Charlottesville, USA; eUniversity of Virginia School of Medicine,
Charlotesville, USA; fEmory University, Atlanta, USA
Introduction: Human CD34+ stem cell therapy has emerged as a promising
approach for the treatment of ischaemic cardiovascular disease. We have previously
shown
that human CD34+ cell-derived exosomes (CD34Exo) improve perfusion and function of
the
ischaemic tissues. Hypoxia is shown to modulate the secretion and content of exosomes
in
both cardiovascular and cancer research. Therefore, we hypothesized that hypoxia can
modulate the content and regenerative efficacy of human CD34Exo.
Methods: CD34Exo were isolated from primary human CD34+ stem cells cultured
under hypoxia (1.5% O2, H-CD34Exo) or normoxia (20% O2, N-CD34Exo) using density gradient
ultracentrifugation. CD34Exo size was measured using TRPS, NTA, and DLS and surface
protein
expression was determined using imaging flow cytometry. Function of CD34Exo was assessed
using cell viability, migration and Matrigel tube formation assays in vitro and a
mouse hind
limb ischaemia model (HLI) in vivo. Protein content of hypoxic or normoxic CD34Exo
was
evaluated via LC-MS/MS and 2-D -DIGE followed by LC-MS/MS.
Results: We did not observe any significant differences in size or in quantity
of exosomes secreted from H- or N-CD34 cells. Both H- and N-CD34Exo expressed CD9,
CD81 and
CD63 surface markers. Interestingly, H-CD34Exo significantly improved cell viability,
migration and tube formation of HUVECs in vitro compared to N-CD34Exo. In the same
line,
H-CD34Exo also significantly improved perfusion (ratio: 0.93 ± 0.05 v 0.77 ± 0.02)
and
prevented ischaemic limb amputation (0% v 37.5%) as compared to N-Exo (p < 0.05; n = 7–8)
in a murine (BalbC nude) model of hind limb ischaemia. Flow cytometry and confocal
microscopy indicated that H-Exo was uptaken by endothelial cells in the ischaemic
limb.
Remarkably, we detected several proteins (including a fragment of hemopexin) and miRNAs
(mir-210) that could be responsible for the proangiogenic and beneficial function
of
H-CD34Exo. We have also demonstrated that removal of surface proteins diminished the
pro-angiogenic function of CD34Exo.
Summary/Conclusion: Hypoxia enhanced the proangiogenic and regenerative
potential of CD34Exo, and thus, may represent a more efficient clinical strategy for
CD34Exo
therapy. Our research is clinically important to improve therapeutic angiogenesis
in
diabetic and cardiovascular patients with compromised stem cell populations.
OT03.3
MiRNA-192 and −432 depleted c-kit+ progenitor cell exosomes
promotes the proliferation, migration of mesenchymal stem cells and regulates
inflammation
Hyun-Ji Park
a, Jessica R.
Hoffmanb and Michael Davisb
aEmory University, Decatur, USA; bEmory
University, Atlanta, USA
Introduction: Exosomes, a subset of membrane nanovesicles, transfer cellular
information by passing proteins and nucleic acids between cells. Exosomes have been
implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome
synthesis
abrogates the effects of cell therapy following cardiac injury. More importantly,
increasing
evidence indicates that miRNAs (miRs) within exosomes serve as important signalling
molecules to regulate inflammation, recruit stem cells, and repair diseased tissue.
Among
exosomal miRs, miR-192 and −432 are known to decrease angiogenesis, cell migration,
and
increase inflammation in various types of cells. Here, we investigated the inhibition
of
these negative miRs as a means to improve the reparative capacity of c-kit+ progenitor
cell
(CPCs) exosomes.
Methods: CPCs were isolated from three paediatric patients using magnetic-bead
sorting. 2ʹ-O-methylated RNA duplexes inhibited miR-192 and −432 expressions in CPCs.
Exosomes (inhExos) were isolated from miR-inhibited CPC conditioned medium. MiR expression
in exosomes and CPC was quantified by qRT-PCR. Migration and proliferation of mesenchymal
stem cells (MSCs) were assessed two days post-exosome treatment. For inflammation
analysis,
THP1 cells with/without TNFα exposure were treated with exosomes and the expression
of IL-6,
−8, and −10 was quantified by qRT-PCR. Finally, the angiogenic potential of inhExos
was
tested by tube formation of cardiac endothelial cells.
Results: Inhibitor treatment of CPCs decreased exosomal miR-192 and −432
expression. Treatment with inhExos enhanced MSC migration and proliferation compared
with
normal CPC exosome (norExo). Moreover, inhExos showed promising results for immune
regulation, as TNFα-induced inflammation was decreased in THP1 exposed to inhExos
for 4 h.
However, tube formation capacity is slightly decreased (~20%) by inhExo compared to
norExo.
Summary/Conclusion: Exosomes from miR-192 and −432-depleted CPCs may be a
promising strategy for the treatment of various cardiac diseases, as they enhanced
stem cell
recruitment and proliferation, and regulated inflammation and angiogenesis. While
other
studies focus on boosting the reparative potential of exosomes by increasing positive
miR
and mRNA cargo, the inhibition of negative miR in exosomes could be an overlooked
strategy
for the treatment of cardiac disease.
OT04
Symposium Session 04: Cellular Uptake and
Fusion
Chair: David R F. Carter – Oxford Brookes
University
Chair: Neta Regev-rudzi – Weizmann Institute of
Science
OT04.1
Endo-lysosomes as an alternative intracellular location for EV
cargo delivery with disease relevance
Giona Pedrioli
a, Marialuisa
Barberisa, Diego Moroneb and Paolo Paganettia
aLaboratory for Biomedical Neurosciences, LBN-EOC,
Taverne-Torricella, Switzerland; bIstituto di Ricerca in Biomedicina, Bellinzona,
Switzerland
Introduction: Extracellular vesicles (EV) are lipid-bilayer nanovesicles that
carry macromolecules and act as paracrine vectors for cell-to-cell communication.
The
processes regulating EV biogenesis are largely known, whereas how EV cargo is delivered
to
recipient cells remains poorly understood. A simple mechanism proposed is direct EV
fusion
with the cell membrane that liberate cargo into the cytosol. In this study, we observed
that
cargo release occurs also at an alternative intracellular location and that this acquires
a
disease relevance.
Methods: EV were isolated by serial centrifugation and characterized. For
uptake studies, EV were traced by labelling donor cells with a lipophilic dye or by
overexpressing GFP-CD63. Uptake was assessed by cytofluorimetry or by live confocal
imaging.
Co-localization studies were performed with ectopic marker expression or by immune
staining.
Protein-protein interaction was analysed by bi-molecular fluorescence complementation
(BiFC). Prion-like transmission was studied using a pro-fibrillogenic Tau fragment
in donor
cells and full-length Tau in recipient cells. For quantification of subcellular
localization, an automated algorithm based on machine learning was developed. Lysosomal
stress was monitored by nuclear translocation of TFE3 and lysotracker staining. Antibodies
directed against pathogenic epitopes of Tau were employed to assess prion-like
transmission.
Results: EV were taken up by recipient cells through an endocytic process and
accumulated in endo-lysosomes (EL). When cells were exposed to EV carrying a
pro-fibrillogenic Tau, recipient cells accumulated Tau within EL by an autophagic
process.
Direct interaction of EV-Tau and cellular Tau in EL favoured the appearance of pathological
epitopes. Cells displaying this condition showed an increased EL stress and
cytotoxicity.
Summary/Conclusion: In this study, for the first time we report that EL
represent a critical subcellular location where transcellular prion-like transmission
mediated by EV of a neurodegeneration-associated protein occurs. Thus, the degradative
pathway most likely involved in the recycle of EV and endogenous proteins is highjacked
in
disease. These findings represent a novel mechanism for EV acting as vector for
transcellular propagation of Tau, which opens up new therapeutic interventions trying
to
halt the disease.
Funding: Supported by Gelu foundation.
OT04.2
Anti-human Fab fragment of CD9 antibody prevents the endocytosis
of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer
of their cargos
Aurelio Loricoa
, Mark
Santosb, Jana Karbanovác, Kevin Huanga, Tony
Huynha, Gyunghwi Wooa, Cheryl Vanierd, Chikao
Morimotoe, Germana Rappaa and Denis Corbeilf
aTouro University Nevada College of Medicine, Henderson, USA;
bTouro University College of Medicine, Hendersom, USA; cTechnische
Universität Dresden, Dresden, Germany; dTouro University Nevada, Henderson, USA;
eJuntendo University, Tokyo, Japan; fTissue Engineering
Laboratories, Technische Universität Dresden, Dresden, Germany
Introduction: Interfering with the mechanisms regulating intercellular
communication mediated by extracellular membrane vesicles (EVs) may find relevance
especially in oncology where cancer cell-derived EVs have an implication in the malignant
transformation of tumour microenvironment. Our laboratories recently demonstrated
a novel
intracellular pathway in which a fraction of endocytosed EV-associated proteins is
transported into the nucleoplasm of the host cell via a subpopulation of Rab7+ late
endosomes entering into the nucleoplasmic reticulum. Here, we have investigated the
effect
of a monovalent Fab antibody against the tetraspanin CD9 (referred hereafter as CD9
Fab), on
the internalization of EVs and nuclear transfer of their cargo proteins.
Methods: To monitor the intracellular transport of EV-associated proteins, we
used bioengineered fluorescent EVs containing CD9-GFP fusion protein from FEMX-I melanoma,
SW480 colorectal cancer and bone marrow-derived mesenchymal stromal cells (MSC) as
donors
and the same cell types as recipients. EVs were enriched by differential centrifugation
from
72 h serum-free conditioned media and characterized by ZetaView nanoparticle tracking
analysis, zeta-potential and immunoblotting. CD9 Fab was prepared from 5H9 hybridoma
cells
using the Pierce Fab purification kit.
Results: We previously demonstrated that silencing CD9 both in EVs and
recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear
transfer
of their cargos. Here we show that CD9 Fab significantly reduced the cellular uptake
of
CD9-GFP+ EVs and the nuclear transfer of their proteins in melanoma, colorectal cancer
and
MSC used as receptor cells in a dose-dependent manner. The effect on the nuclear transfer
is
probably a direct consequence of the endocytosis inhibition of EVs. In contrast, the
divalent, intact CD9 antibody stimulated both events.
Summary/Conclusion: The effect of CD9 Fab appears independent of the used
EV-donor cell types or receptor cells, probably due to the widespread expression of
CD9 both
at plasma membrane and EV surface. In conclusion, by impeding intercellular communication
in
the tumour microenvironment, CD9 Fab-mediated inhibition of EV uptake, combined with
direct
targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic
strategies.
OT04.3
A bright, versatile reporter for multivesicular body trafficking
and exosome secretion and uptake
Bong Hwan Sung, Ariana von Lersner, Jorje
Guerrero, Evan Krystofiak, David Inmann, Roxanne Pelletier, Andries Zijlstra, Suzanne
Ponik
and Alissa Weaver
Vanderbilt University, Nashville, USA
Introduction: Live imaging of exosomes is one of the required tools to
understand the function of exosomes. Our previous live-cell reporter, pHluorin-CD63
allows
dynamic subcellular monitoring of exosome secretion in migrating and spreading cells.
However, there were some caveats to its use, including dim fluorescence and the inability
to
make cell lines that stably express the protein.
Methods: A stabilizing mutation, M153 R is incorporated in the pHluorin moiety
and now exhibits stable expression in cells and superior monitoring of exosome secretion.
A
dual-tag reporter was created by incorporating a further pH-insensitive red fluorescent
protein, mScarlet to the C-terminus of pHluo_M153 R-CD63. Cancer cells stably expressing
the
constructs were imaged using a variety of microscopy techniques in vitro as well as
in vivo.
Purified small EVs labelled with pHuo_M153 R-CD63 were imaged using immunogold transmission
electron microscopy (TEM) and quantitated for the half-life in the blood circulation
using
flow cytometry.
Results: pHluo_M153 R-CD63 and pHluo_M153 R-CD63-mScarlet are exclusively
detected in exosome-enrich small EV preparations. Immunogold TEM visualizes the pHluo_M153 R
tag is located on the surface of small EVs. Live cell imaging reveals
pHluo_M153 R-CD63–positive puncta left behind migrating cells suggesting the deposition
consists of exosomes. Those puncta and trails are not only positive for exosome markers
such
as CD63, Alix, and TSG101 but also correspond to small EVs observed by a scanning
electron
microscopy. The dual-tag reporter allows visualization of the exosome lifecycle, including
multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome
acidification.
Summary/Conclusion: Using pHluo_M153 R-CD63 construct, we demonstrate superior
visualization of exosome secretion in multiple contexts and a role of exosomes in
promoting
leader-follower behaviour in collective migration by observing that exosomes are secreted
at
the front of migrating cells and left behind in exosome trails. The dual-tag reporter
allows
visualization of the entire exosome lifecycle. We anticipate that these reporters
will be
broadly useful to investigate regulation and functions of exosome secretion and uptake
in
diverse physiological conditions.
Funding: R01GM117916, R01CA206458, 3U19CA179514-05S1, R01CA216248.
OT04.4
Uncovering novel genes regulating EV-mediated functional RNA
transfer using a CRISPR/Cas9-based reporter system
Olivier G. de Jong
a, Daniel E.
Murphyb, Imre Mägerc, Eduard Willmsd, Sander A.A.
Kooijmansa, Jacco van Rheenene, Raymond M. Schiffelersa,
Samir El-Andaloussif, Matthew J.A. Woodc and Pieter
Vaderg
aLaboratory of Clinical Chemistry and Hematology, UMC
Utrecht, The Netherlands, Utrecht, Netherlands; bLaboratory of Clinical Chemistry
and Hematology, UMC Utrecht, Utrecht, Netherlands; cDepartment of Paediatrics,
University of Oxford, Oxford, UK; dLa Trobe University, Oxford, UK;
eDepartment of Molecular Pathology, Oncode Institute, Netherlands Cancer
Institute, Amsterdam, Netherlands; fDepartment of Laboratory Medicine, Clinical
Research Center, Karolinska Institutet, Huddinge, Sweden; gLaboratory of Clinical
Chemistry and Hematology, UMC Utrecht; Department of Experimental Cardiology, UMC
Utrecht,
The Netherlands, Utrecht, Netherlands
Introduction: Extracellular vesicles (EVs) play a pivotal role in
intercellular communication through functional transfer of bioactive cargo, including
RNA
molecules. Despite increasing interest in EV-mediated RNA transfer, our understanding
of the
pathways and mechanisms regulating EV-mediated RNA delivery and processing is limited
due to
a lack of suitable readout systems. We recently developed a novel CRISPR/Cas9-based
reporter
system that allows study of EV-mediated RNA transfer at single-cell resolution. Here,
we
further validate this system by studying the role of known targets involved in EV
uptake and
intracellular membrane trafficking, and subsequently employ this system to uncover
various
novel genes that play a regulatory role in functional RNA transfer.
Methods: We employed a novel CRISPR/Cas9-based stoplight reporter system, in
which eGFP expression is activated upon functional delivery of targeting single guide
RNAs
(sgRNAs) stably expressed by donor cells. Intercellular functional RNA transfer was
assessed
by measuring eGFP expression in acceptor cells using fluorescence microscopy and flow
cytometry after direct co-culture, transwell co-culture, and upon addition of isolated
EVs.
Potential roles of various genes in intercellular RNA transfer were assessed by
RNAi-mediated target knockdown in acceptor cells, prior to co-culture experiments.
RNAi
knockdown was confirmed by qPCR analysis.
Results: A significant activation of eGFP expression was observed in acceptor
cells after direct co-culture and transwell co-culture with donor cells expressing
sgRNAs,
as well as after addition of EVs from cells expressing sgRNAs. Reporter activation
was
substantially decreased after knockdown of multiple targets involved in EV uptake
through
endocytosis and/or intracellular membrane trafficking. Based on these results, a potential
role of various novel genes in intercellular RNA transfer was studied in acceptor
cells.
These experiments uncovered various novel targets involved in ECM binding, endocytosis,
intracellular membrane trafficking, as well as various Rho GTPase interactors.
Summary/Conclusion: We previously demonstrated a CRISPR/Cas9-based reporter
system that allows the study of functional delivery of small non-coding RNAs with
single-cell resolution. Here, we show that this novel approach allows the study of
specific
genetic targets and pathways in EV-mediated functional RNA delivery, and unravel the
regulatory pathways that dictate the underlying processes.
Funding: This work was supported by the Biotechnology and Biological Sciences
Research Council (BB/M024393/1), the European Union’s Horizon 2020 Research and Innovation
Programme under grant agreement No. 721058, and the Dutch Research Council (NWO)
VI.Veni.192.174.
OT04.5
Quantitative characterization of extracellular vesicle uptake
and content delivery within mammalians cells
Gregory Lavieu
a, Emeline
Bonsergentb, Eleonora Grisardc and Clotilde Théryd
aINSERM U132/Institut Curie, Paris, France;
bInstitut Curie/Universite de Paris, Paris, France; cinstitut Curie,
Paris, France; dINSERM U932, Institut Curie, PSL Research University, Paris,
France
Introduction: Extracellular Vesicles (EVs), including exosomes, are thought to
mediate intercellular communication through the transfer of biomolecules from donor
to
acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been
unambiguously demonstrated, let alone quantified, and remains debated.
Methods: We developed a cell-based assay in which EVs containing
luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. Measurement
of
luciferase activity associated with acceptor cells revealed EV uptake efficacy. Additional
cell fractionation procedure that separates membranes from cytosol revealed the occurrence
of EV-content release within the cytosol of acceptor cells.
Results: Results from dose-responses, kinetics, and temperature-block
experiments suggest that EV-uptake is limited (1% spontaneous rate at 1h), does not
depend
on bona-fide EV-receptor, at least for the tested acceptor HeLa cells. Yet, further
characterization of this limited EV-uptake, through cell fractionation that separates
membranes from cytosol, revealed the occurrence of EV-content release within the cytosol
of
acceptor cells. Cytosolic release is inhibited by Bafilomycin-A and overexpression
of IFITM
proteins, which prevent virus content delivery.
Summary/Conclusion: Our results show that EV-content release requires
endosomal acidification and suggest the involvement of membrane fusion.
Funding: ANR18-CE15-0008-01 and ARC PJA 20171206453 and PGA1
RF20180206962.
OT05
Symposium Session 05: Single EV
Analysis
Chair: Shannon Stott - Department of Medicine,
Harvard Medical School
Chair: Joanne Lannigan – Flow Cytometry Core,
University of Virginia School of Medicine
OT05.1
Droplet-based Single Extracellular Vesicle Sequencing for Rare
Immune Subtype Discovery
Jina Ko
a, Yongcheng
Wangb, David Weitzc and Ralph Weisslederd
aMassachusetts General Hospital, Wyss Institute at Harvard
University, Cambridge, USA; bHarvard University, Boston, USA; cHarvard
University, Cambridge, USA; dCenter for Systems Biology, Massachusetts General
Hospital, Harvard Medical School, Boston, USA
Introduction: Glioblastoma is a highly malignant brain tumour with a poor
prognosis. Its ability to develop therapeutic resistance result in devastating clinical
outcomes. To solve the intractable problem, we need highly sensitive diagnostics that
can
detect the molecular changes during treatments. Extracellular vesicles (EVs) can be
a
potential biomarker to monitor treatments and the host cell EV mapping can better
reflect
molecular changes in the tumour immune microenvironment. We have developed a droplet-based
single EV protein sequencing platform that overcomes limitations of current bulk measurement
technologies, which make it difficult to discover a rare EV population in the presence
of
high background.
Methods: We multiplex protein measurements to profile hundreds of proteins at
a time by using an antibody-DNA conjugate and sequencing. We barcode each EV in droplets
and
make amplicons that are comprised of both EV barcodes and antibody barcodes for sequencing.
Barcoded antibodies are made using TCO-tetrazine click reaction and EVs are labelled
with
these barcoded antibodies. The labelled EVs are encapsulated into droplets with barcoded
beads that serve as a template for EV barcodes. We then perform extension to make
amplicons
that contain both EV barcodes and antibody barcodes for sequencing.
Results: We successfully fabricated barcoded beads using a split-pool approach
and validated by observing a fluorescence decrease of the SYBR Green after DNA strand
denaturation. We used a 3-channel droplet maker to encapsulate barcoded beads, single
EV,
and master mix into droplets. Close packing of barcoded beads allowed >95% encapsulation
into droplets. Both droplet and tube-based methods achieved a similar high amplification
efficiency (Ct < 30 for 300 EVs). We confirmed the amplicon size by running a gel,
which
showed the right amplicon size (~150 bp) from the droplet and tube prepared samples
and no
signal from the negative control.
Summary/Conclusion: The droplet-based single EV profiling platform has the
ability to identify rare immune EV subtypes in the peripheral blood, which would otherwise
be impossible to detect due to the co-presence of abundant normal EVs. This cutting-edge
technique has the potential to revolutionize treatment monitoring of high-cost
immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine
capabilities.
Funding: Schmidt Science Fellows, in Partnership with the Rhodes Trust
PO1 CA069246, RO1 CA204019, R21 CA236561
QuantBio graduate student award at Harvard University.
OT05.2
Advancing extracellular vesicle characterization with
quantitative single-molecule localization microscopy
Kathleen Lennona, Metztli Cisnerosa, Adam
Maddoxa, Devin L. Wakefielda, Matthew Brehovea, Saumya
Dasb, Kendall Van Keuren-Jensenc, Gagandeep Singhd and
Tijana Jovanovic-Talisman
a
aBeckman Research Institute, City of Hope, Duarte, USA;
bMassachusetts General Hospital, Harvard Medical School, Boston, USA;
cTranslational Genomics Research Institute, Phoenix, USA; dCity of
Hope, Duarte, USA
Introduction: Single-molecule localization microscopy (SMLM) can detect
individual biomolecules with nanoscale precision. Although the technique has been
routinely
used to quantify biomolecules in cells, several technical challenges have impeded
a
similarly robust quantification in extracellular vesicles (EVs) secreted by cells.
To
address this, we developed optimized analytical protocol followed by quantitative
SMLM to
robustly assess EVs derived from either cultured cells or patient biofluids.
Methods: Size exclusion chromatography was used to isolate EVs. EV membranes
were labelled with various fluorescent reagents; core beads or size exclusion columns
efficiently separated excess fluorescent molecules from labelled EVs. While imaging
conditions needed to be significantly optimized when EVs were detected with fluorescent
lipophilic membrane dyes, EVs were easily detected with lectins conjugated to fluorescent
dyes typically used for SMLM. To ensure that the SMLM imaging data was robustly quantified,
we optimized our analysis algorithms.
Results: We used our methodology to assess EVs isolated from plasma of
patients with pancreatic cancer. Informed consent was obtained from all subjects,
and the
study was approved by the Institutional Review Board. We determined the number of
isolated
EVs, their size, and the abundance of several biomarkers. According to our results,
patients
with pancreatic cancer exhibited a unique population of larger EVs containing epidermal
growth factor receptor and carbohydrate antigen 19–9.
Summary/Conclusion: The methodological advancement in analytical preparation,
SMLM imaging, and quantitative analysis can help identify sub-populations of EVs.
As
quantitative SMLM of EVs may represent a new diagnostic paradigm, our ultimate goal
is to
advance single molecule characterization of EVs for precision medicine.
Funding: UG3 TR002878 NIH/NCATS; SWIF Circle; Hirshberg Foundation for
Pancreatic Cancer Research; Beckman Research Institute, City of Hope; Irell & Manella
Graduate School of Biological Sciences.
OT05.3
An evaluation of four orthogonal single-particle analysis
platforms
Emily R. Mallick
a, Tanina
Araba, Yiyao Huangb, Zhaohao Liaoa, Zezhou
Zhaoa, Liang Dongc, Kenneth Pientac, Michael
Paulaitisdand Kenneth Witwere
aDepartment of Molecular and Comparative Pathobiology, Johns
Hopkins University School of Medicine, Baltimore, USA; bDepartment of Molecular
and Comparative Pathobiology Neurology, The John Hopkins University School of Medicine,
Baltimore, USA; cThe Brady Urological Institute, Johns Hopkins University School
of Medicine, Baltimore, USA; dThe Center for Nanomedicine at the Wilmer Eye
Institute, Johns Hopkins University School of Medicine, Baltimore, USA;
eDepartment of Molecular and Comparative Pathobiology and Department of
Neurology, The Johns Hopkins University School of Medicine, baltimore, USA
Introduction: In this study, we compared four orthogonal technologies for
sizing, counting, and phenotyping of EVs. The platforms were: single-particle
interferometric reflectance imaging senor (SP-IRIS) with optional fluorescence, nanoFCM
nanoflow (NF), nanoparticle tracking analysis (NTA) with fluorescence, and microfluidic
resistive pulse sensing (MRPS). Results from these platforms were compared with results
from
standard EV characterization techniques such as transmission electron microscopy (TEM)
and
western blot (WB).
Methods: Human T lymphocyte H9 (high CD81, low CD63) and promonocytic U937
(low CD81, high CD63) cells were chosen for their distinct tetraspanin profiles without
abnormalities that might result from genetic manipulation. EVs were isolated from
culture
conditioned medium (CCM) by differential ultracentrifugation (dUC) and size exclusion
chromatography (SEC) and characterized per MISEV2018 guidelines. Synthetic particles
(silica
and polystyrene spheres) with known concentrations and mixed size distributions were
also
tested.
Results: Particle counts from NF and MRPS were consistent, while NTA detected
approximately one order of magnitude lower for CCM derived EVs, but not for synthetic
particles. SP-IRIS events could not be used to estimate particle concentrations. For
sizing,
NF, MRPS, and SP-IRIS returned similar size profiles, with smaller sizes predominating
(per
power law distribution), but with sensitivity typically dropping off below diameters
of 60
nm. NTA detected a population of particles with a mode diameter above 100 nm. Additionally,
SP-IRIS, NF, and MRPS were able to identify at least three of four distinct size populations
in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping,
the SP-IRIS platform in fluorescence mode and NF were able to detect at least two
markers on
the same particle.
Summary/Conclusion: Based on the results of the study, we can draw conclusions
about existing single-particle analysis capabilities that may be useful for EV biomarker
development and mechanistic studies.
Funding: This project is funded by MH118164 and UG3CA241694.
OT05.4
Analysis of tetraspanin expression and spatial arrangement for
EV subpopulations using single particle interferometric reflectance imaging and
immunofluorescence.
Rachel Mizenko, Alyssa Powell and Randy
Carney
Department of Biomedical Engineering, University of California, Davis,
Davis, USA
Introduction: Protein expression on single extracellular vesicles (EVs) is of
great interest due to its importance to EV organotropism. Yet, most techniques rely
on bulk
characterization, or are severely restricted by the diffraction limit. The ExoView
R100
(NanoView Biosciences) combines interferometry, immunocapture, and immunofluorescence,
introduced as an alternative technique to multiplex protein detection on single EVs
below
the limit of diffraction. Here, we use this technique to characterize tetraspanin
multiplexing on EVs and to identify spatial patterning of tetraspanins using steric
hindrance of antibodies (Abs).
Methods: EVs were isolated from conditioned media from SKOV-3 cell culture or
human serum. EVs were incubated overnight on chips to allow immunocapture by anti-CD9,
anti-CD63, or anti-CD81. Chips were then incubated with three fluorescent Abs against
the
same epitopes and imaged on the ExoView R100. Following concentration optimization,
EVs were
tested after pre-incubating with carboxy-fluorescein diacetate succinimidyl ester
(CFSE) or
fluorescent Abs against tetraspanins.
Results: Using different concentrations of EVs, binding curves could be fit to
characterize binding kinetics of Abs. Maximum concentration of EVs could be identified
that
minimized fluorescent overlap. Bright-field interferometry (detection limit ~ 50 nm)
distinguished 10x fewer bound EVs than fluorescent detection, while pre-labelling
EVs with
CFSE produced 10x more detectable EVs than immunofluorescence. Interestingly, EVs
captured
by one tetraspanin did not necessarily show high fluorescent detection of the same
tetraspanin. Upon pre-incubating EVs with a single Ab, vastly different expression
profiles
were identified, indicating significant steric hindrance between Abs. Furthermore,
pre-incubating EVs with anti-CD9 Ab significantly decreased detection of CD81 with
less
impact on CD63. This discrepancy indicated possible spatial patterning of tetraspanins
with
CD9 and CD81 closely colocalizing on the EV surface.
Summary/Conclusion: This combination of interferometry, immunocapture, and
immunofluorescence produces unique information about size distribution of EVs and
single EV
protein profile. This data corroborates that EVs have distinct subpopulations of
tetraspanins and indicates that tetraspanins may be spatially patterned.
OT06
Symposium Session 06: MSC EV
Therapeutics
Chair: Andrew Hoffman, DVM, DVSc – University of
Pennsylvania School of Veterinary Medicine
Chair: Michael Davis – Emory
University
OT06.1
Regulation of liver homoeostasis, regeneration and diseases by
mesenchymal stem cell-derived apoptotic extracellular vesicles
Bingdong Sui
University of Pennsylvania, Philadelphia, USA
Introduction: Billions of cells undergo apoptosis and produce apoptotic
extracellular vesicles (ApopEVs) each day, whereas the roles of ApopEVs in regulating
the
organismal health and disease remain poorly understood. Mesenchymal stem cells (MSCs)
emerge
as critical contributors to tissue homoeostasis, while MSCs suffer from apoptosis
in
regenerative transplantation. In this study, we investigated the function and mechanisms
of
MSC-derived ApopEVs in regulating the organismal homoeostasis.
Methods: Fas mutant (Fasmut) and Caspase 3 knockout (Casp3-/-) mice were
applied for apoptotic and ApopEV deficiency. Mouse bone marrow MSCs were cultured
and
apoptosis was induced by Staurosporine (STS). MSC-derived ApopEVs were collected by
serial
centrifuges and were infused into mouse circulation via caudal vein. Tracing of ApopEVs
were
performed by radioisotope or fluorescent labelling. Liver homoeostasis was evaluated
at the
histological and functional aspects. Liver regeneration was induced by partial hepatectomy
(PHx). Acetaminophen (APAP) was used to establish acute liver drug injury. High-fat
diet
(HFD) was used to establish type 2 diabetes (T2D) and non-alcoholic fatty liver disease
(NAFLD).
Results: After systemic injection, MSC-derived ApopEVs migrate to liver and
can be uptaken by liver macrophages and hepatocytes. Fasmut and Casp3-/- mice develop
hepatomegaly with structural disorders, which particularly reveals hepatocyte
polyploidization. Furthermore, Fasmut and Casp3-/- mice demonstrate liver glucose
and lipid
metabolic disorders. Importantly, MSC-derived ApopEV infusion significantly rescues
structural and metabolic dysfunction in Fasmut and Casp3-/- mice. Mechanistically,
ApopEVs
use the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor
(SNARE) protein for interactions with recipient organelles thus transferring signalling
molecules. Moreover, MSC-derived ApopEV infusion promotes liver regeneration after
PHx,
prevents APAP-induced liver injury, and ameliorates NAFLD in T2D.
Summary/Conclusion: MSC-derived ApopEVs serve as crucial regulators of liver
homoeostasis, regeneration and diseases. These findings indicate potential significant
roles
of ApopEVs in maintaining the organismal health and in developing therapeutics for
diseases.
Funding: The Postdoctoral Innovative Talents Support Program of China
(BX20190380) and the General Program of China Postdoctoral Science Foundation
(2019M663986).
OT06.2
MSC-sEVs restore structural and mechanical properties in a
rabbit model of cartilage injury
Wei Seong Toh
a, Shipin
Zhangb, Francis Keng Lin Wongc, Barry Wei Loong Tand,
James Hoi Po Huie and Sai Kiang Limf
aFaculty of Dentistry, National University of Singapore,
Singapore. Department of Biomedical Engineering, Faculty of Engineering, National
University
of Singapore, Singapore. Tissue Engineering Program, Life Sciences Institute, National
University of Singapore, Singapore. Graduate School for Integrative Sciences and
Engineering, National University of Singapore, Singapore, Singapore, Singapore;
bFaculty of Dentistry, National University of Singapore, Singapore. Department of
Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore, Singapore, Singapore; cDepartment of Orthopaedic Surgery, Yong Loo Lin
School of Medicine, National University of Singapore, Singapore. Department of Orthopaedic
Surgery, Sengkang General Hospital, Singhealth, Singapore, Singapore, Singapore;
dDepartment of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National
University of Singapore, Singapore, Singapore, Singapore; eTissue Engineering
Program, Life Sciences Institute, National University of Singapore, Singapore. Department
of
Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore, Singapore, Singapore; fInstitute of Medical Biology, Agency for
Science, Technology and Research, Singapore. Department of Surgery, Yong Loo Lin School
of
Medicine, National University of Singapore, Singapore, Singapore
Introduction: We previously reported that small extracellular vesicles derived
from mesenchymal stem cells (MSC-sEVs) mediate osteochondral regeneration in rats.
However,
the therapeutic effects of these MSC-sEVs/exosomes in restoring the mechanical competence
of
the repaired cartilage for joint function in a clinically relevant animal model remain
to be
addressed. To investigate this, we compared the structural and mechanical properties
of the
repaired cartilage in a rabbit model after intraarticular administration of MSC-sEVs
and
hyaluronic acid (HA) with that of HA alone, which is widely used as
visco-supplementation.
Methods: Bilateral osteochondral defects were surgically created on 17
rabbits. Immediately after surgery and at days 7 and 14 post-surgery, 9 rabbits received
1-ml injections of 200 µg MSC-sEVs and HA in both knees, and 8 rabbits received 1-ml
injections of HA in both knees. At 6 and 12 weeks, macroscopic evaluation, histological
scoring and compressive testing at different points on the repaired cartilage were
performed.
Results: Defects treated with MSC-sEV/HA showed improvements with time in
macroscopic and histological scores and mechanical properties than defects treated
with HA
alone. In contrast, HA treated defects showed some repair at 6 weeks, but this was
not
sustained, as evidenced by significant deterioration of histological scores and a
plateau in
mechanical properties from 6 to 12 weeks. By 12 weeks, the MSC-sEV/HA repaired tissues
demonstrated significantly better macroscopic score (10.33 vs 7.5; P < 0.001) and
histological score (21.87 vs 11.11; P < 0.001). Mechanical strength as measured by
the
Young’s modulus was significantly higher in the MSC-sEV/HA repaired cartilage than
that in
HA repaired tissues [defect centre (13.41 vs 3.95MPa; P = 0.001) and overall periphery
(12.22 vs 3.37MPa; P = 0.012], and approximated that of the adjacent native cartilage.
Summary/Conclusion: Our findings demonstrated that MSC-sEVs and HA not only
improved tissue morphology of the repaired cartilage but also promoted functional
mechanical
competence. This study establishes a clinically translatable protocol for use of MSC-sEVs
for cartilage repair.
Funding: National Medical Research Council Singapore (NMRC/CNIG/1168/2017,
NMRC/CIRG/1480/2017 and NMRC/CNIG/1171/2017)
OT06.3
Mesenchymal stromal cell exosomes educate myeloid cell
populations to alleviate neonatal hyperoxia-induced lung injury
Gareth R. Willis
a, Monica
Reisa, Angeles Fernandez-Gonzaleza, Vincent Yeunga,
Xianlan Liub, S. Alex Mitsialisa and Stella
Kourembanasa
aHarvard University & Boston Children’s Hospital, Boston,
USA; bBoston Children’s Hospital, Boston, USA
Introduction: Mesenchymal stromal/stem cell (MSC)-exosome (MEx) treatment has
shown considerable promise in experimental models of bronchopulmonary dysplasia (BPD)
and
pulmonary hypertension (PH). Mechanisms by which MEx afford their beneficial effects
remain
incompletely understood and here, we embark into investigating them through assessment
of
MEx biodistribution and impact on immune cell heterogeneity.
Methods: Newborn FVB mice were exposed to hyperoxia (HYRX, 75% O2) at birth
and returned to room air at postnatal day (PN) 14. Mice received a bolus MEx dose
at PN4.
Adoptive transfer studies were used to determine the role of MEx-educated myeloid
cells in
vivo. Mice were harvested at PN4, 7, 14, or 28 to characterize MEx biodistribution
and for
assessment of pulmonary parameters.
Results: MEx therapy effectively ameliorated core features of HYRX-induced
neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular
remodelling and blood vessel loss. Exercise capacity testing and assessment of PH
showed
functional improvements following MEx therapy. Biodistribution studies demonstrated
that MEx
localize in the lung, where they interact with lung monocytes/macrophages. Whole lung
mass
cytometry (CyTOF) revealed that MEx treatment promotes a pro-homoeostatic shift in
lung
immune cell apportion, replenishing the early HYRX-induced depletion in pulmonary
CD45+ immune cells, restoring alveolar monocyte and macrophage populations and suppressing
cellular inflammation. Ex vivo and in vivo analysis showed that MEx promotes a
“pro-resolving” CCR2- monocyte phenotype. Notably, adoptive transfer of MEx-educated
bone
marrow-derived myeloid cells (BMDMy), but not naïve BMDMy, restored alveolar architecture,
blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss.
Summary/Conclusion: MEx treatment ameliorates core features of experimental
BPD, restoring lung architecture, decreasing pulmonary fibrosis and vascular
muscularization, ameliorating PH and improving exercise capacity. The beneficial actions
of
MEx are associated with modulation of immune cell phenotypes, arising from MEx-monocyte
interaction. Furthermore, adoptive transfer of MEx-educated BMDMy rescued, at least
in part,
alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary
blood
vessel loss.
Funding: This work was supported in part by an American Thoracic Society
Foundation Grant (GRW); The Little Giraffe Foundation (GRW); Charles H. Hood Foundation
Major Grants Initiative (SK), NIH R01HL146128 (SK) and United Therapeutics Research
Grant
(SK and SAM).
OT06.4
Immunomodulatory small extracellular vesicles derived from
mesenchymal stem cells: A potential cell-free therapy for acute and chronic pulmonary
vascular diseases
Alvin Tieu
a, Casey
Lansdellb, Duncan Stewartc, Dylan Burgerd and Manoj
Lalue
aClinical Epidemiology Program and Regenerative Medicine
Program, Ottawa Hospital Research Institute, Ottawa, Canada; bRegenerative
Medicine Program, Ottawa Hospital Research Institute, Ottawa, Canada; cOttawa
Hospital Research Institute, Ottawa, Canada; dChronic Disease Program, Ottawa
Hospital Research Institute, Ottawa, Canada; eClinical Epidemiology Program,
Ottawa Hospital Research Institute, Ottawa, Canada
Introduction: Vascular inflammation plays a critical role in acute respiratory
distress syndrome (ARDS) and pulmonary arterial hypertension (PAH). Despite decades
of
research, there is no curative therapy for either condition. Mesenchymal stem cells
(MSCs)
have shown preclinical efficacy, mediated by release of extracellular vesicles. Hence,
MSC-derived small extracellular vesicles (sEVs) can harness the benefits of MSCs with
advantages in cost and safety. This study aims to evaluate the immunomodulatory effects
of
sEVs in preclinical ARDS and PAH.
Methods: MSC-sEVs were characterized by nanoparticle tracking analysis,
electron microscopy and western blot. Live fluorescence imaging measured in vitro
and in
vivo distribution of sEVs. Using a lipopolysaccharide (LPS)-induced mouse model of
acute
lung injury (ALI), a time course study of inflammatory response guided endpoint analyses.
Cell count and cytokines were measured in bronchoalveolar lavage fluid (BALF) and
histological lung injury was assessed. In ALI mice, saline, MSCs, MSC conditioned
media or
sEVs were administered 0.5 h post-LPS. Using a monocrotaline (MCT)-induced rat model
of PAH,
animals received saline or sEVs at day 3. Haemodynamic changes and right ventricular
hypertrophy were evaluated at 3 weeks.
Results: MSC-sEVs were 72 nm in size with CD63/81 expression. PKH26-labelled
sEVs were taken up by endothelial cells. In the ALI time course study, cell count
and IL1b
in BALF peaked at 24h post-LPS, whereas IL6 peaked at 10h. Histology showed significant
intra-alveolar cell infiltrate at 72h. MSC conditioned media attenuated IL1b in BALF,
whereas a trend towards reductions in IL1b and cell count were seen from delivery
of MSCs
and sEVs. Using fluorescence imaging, lung accumulation of DiR-labelled sEVs was highest
when administered 1 h post-LPS as compared to 5 h, 10 h or 24h. For PAH rats, sEVs
reduced
right ventricular systolic pressure (51.4 ± 8.3 mmHg) as compared to control
(68.1 ± 0.4 mmHg; P = 0.012), whereas no changes were observed for right ventricular
remodelling.
Summary/Conclusion: These findings demonstrate the potential of MSC-sEVs to be
used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases.
Additional live and ex-vivo biodistribution studies will determine optimal timing
of sEV
administration, tissue distribution and clearance in both ALI and PAH.
OT06.5
Changes in extracellular vesicle protein cargo after
pro-inflammatory priming of umbilical cord mesenchymal stem cells
Mairead Hyland
a, Emma
Wilsonb, Aled Claytonc, Claire Mennand and Oksana
Kehoee
aKeele University, OSWESTRY, UK; bUniversity of
Chester, Chester, UK; cCardiff University, Cardiff, UK; dKeele
University and RJAH Oswestry, Oswestry, UK; eKeele University, Oswestry, UK
Introduction: Human Umbilical Cord Mesenchymal Stem Cells (UCMSCs) have been
shown to suppress inflammatory responses in studies of autoimmune diseases. These
therapeutic effects can be attributed to paracrine signalling, by which extracellular
vesicles (EVs) are one of the essential components. This study looks at how the culture
conditions of UCMSCs affects the type of EVs they secrete. It also aims to identify
an EV
population with an anti-inflammatory potential for the treatment of autoimmune diseases.
Methods: UCMSCs were isolated and culture expanded in a Quantum® cell
expansion system, then grown at 21%O2, 5%O2 and primed with a pro-inflammatory cocktail.
EVs
were isolated from UCMSC conditioned media by differential ultracentrifugation using
a
sucrose cushion and characterised by Transmission Electron Microscopy and Nanoparticle
Tracking Analysis. EV markers were analysed using a europium-based immunoassay, Macsplex
Exosome Detection kit and immunoblotting. A proximity-based extension assay was used
to
identify 92 inflammatory proteins in the EVs.
Results: There was no difference in EVs cultured at 21%O2, 5%O2 or with
pro-inflammatory conditions when analysed for size and morphology. All EVs displayed
the
tetraspanin markers (CD9/63/81) and internal proteins (Alix, Hsp70). EVs from primed
cells
showed a > twofold increase of 5 CC chemokines and a > sixfold increase in CXCL5 and
CSF-1. Protein cargo did not differ in EVs from 21%O2 and 5%O2.
Summary/Conclusion: This study showed that pro-inflammatory culture conditions
alter EV protein cargo, evidenced by the increased production of chemotactic and
angiogenesis associated proteins. Upcoming RNAseq analysis will show if UCMSC culture
conditions also affect miRNA expression in EVs. Ongoing functional studies will determine
how changes in EV cargo correlates with changes in T-cell proliferation and
polarisation.
Funding: This work is fund by the Orthopaedic Institute Ltd, Keele University
and the RJAH Orthopaedic Hospital Charity.
OT07
Symposium Session 07: Neurodegenerative
Diseases
Chair: Eva-Maria Albers Kramer – Institut für
Entwicklungs- und Neurobiologie Zelluläre Neurobiologie AG Extrazelluläre
Vesikel
Chair: Xandra Breakefield –
MGH
OT07.1
Alzheimer’s disease biomarkers in plasma extracellular vesicles
of neuronal origin correlate with brain pathology in mice
Francheska Delgado-Peraza
a, Erez
Eitanb, Carlos J. Nogueras-Ortiza, Dong Liuc, Mark P.
Mattsond, Nigel Greige and Dimitrios Kapogiannisa
aLaboratory of Clinical Investigation, National Institutes of
Ageing, Baltimore, USA; bNeuroDex, Natick, USA; cDepartment of
otorhinolaryngology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University,
Shanghai, China (People’s Republic); dJohns Hopkins School of Medicine,
Baltimore, USA; eNational Institute on Ageing/National Institutes of Health,
Baltimore, USA
Introduction: Multiple studies have shown that neuronal-derived extracellular
vesicles (NDEs) in blood contain Alzheimer’s disease (AD) biomarkers, especially Tau.
However, the convergent validity of Tau in blood NDEs in relation to brain pathology
is yet
to be determined. To address this, we measured total and phosphorylated Tau levels
in
matched NDE and brain tissue samples from AD mouse models.
Methods: We collected the cortex, hippocampus and plasma of 4 2xTg-AD, 15
3xTg-AD, and 9 Wild Type (total of 28 mice; 14 female, 14 male; age: mean = 8.17,
SD = 1.61,
5–11 months). Plasma samples were collected retro-orbitally for 5 weeks and at euthanasia
via heart puncture. NDEs from the pooled serial blood collections (NDE1) and the single
endpoint (NDE2) were immunocaptured by targeting the neuronal marker L1CAM. We measured
human total Tau and pThr181-Tau (p-Tau) in NDEs and cortex and hippocampus homogenates
using
a Luminex multiarray.
Results: Overall, there were strong positive correlations for both total Tau
and p-Tau between NDEs and brain tissues across mice types. Total tau in NDEs showed
positive correlations with levels in the cortex and hippocampus (r = 0.6885 and 0.7026,
p < 0.0001, cortex vs NDE1 and NDE2; r = 0.4958, p = 0.0073, hippocampus vs NDE1;
r = 0.6058, p = 0.0006, hippocampus vs NDE2). Levels of p-Tau in NDE1 showed positive
correlations with levels in the cortex (r = 0.4640, p = 0.0155) and hippocampus (r = 0.6086,
p = 0.0008); however correlations were not observed for NDE2 (r = 0.1152, p = 0.6273
vs
cortex; r = 0.0531, p = 0.7926 vs hippocampus).
Summary/Conclusion: Tau levels in circulating NDEs reflect levels in cortex
and hippocampus across AD model mice, supporting their convergent validity as “liquid
biopsy” biomarkers for AD.
Funding: This research was supported in part by the Intramural Research
Program of the National Institute on Ageing, National Institutes of Health.
OT07.2
Exosomal ceramide mediates neurotoxicity of Amyloid beta (Aβ) in
Alzheimer’s disease.
Ahmed Elsherbini
a, Simone
Crivellib, Alexander Kirovc, Michael Dinkinsd, Zhihui
Zhua, Haiyan Qina, Sanjib Karkia, Priyanka
Tripathia and Erhard Biebericha
aUniversity of Kentucky, Lexington, USA;
bUniversity of Kentucky, Lexington, USA; cAugusta University, Augusta,
USA; dAugusta University, Augusta, USA
Introduction: Amyloid beta is a pathologic hallmark of Alzheimer’s disease
(AD), however, the mechanism of Aβ neurotoxicity is not fully understood. It has been
reported that exosomes associate with Aβ, but it is not clear how this association
affects
Aβ neurotoxicity.
Methods: Here we utilized several techniques to isolate exosomes from the sera
of wild type (WT) and AD transgenic mouse model.
(5xFAD) as well as Alzheimer’s patients and healthy controls. We used Exoquick, Exoeasy,
sequential ultracentrifugation, and size exclusion chromatography. Particles’ size
and
number were characterized by nanoparticle tracking analysis (ZetaView).
Results: We report that the sphingolipid ceramide mediates neurotoxicity of
Aβ. We show that sera from AD transgenic mouse model (5xFAD) and AD patients, but
not the WT
or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are
enriched
with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle
tracking and cluster analyses. When taken up by Neuro2A cells and human iPS cell-derived
neurons, these astrosomes are shuttled to mitochondria where they induce mitochondria
clustering, evident by elevation of expression of the fission protein dynamin-related
protein1 (Drp1). Using proximity ligation assays, we show that Aβ forms a complex
with
voltage dependent anion channel 1 (VDA1), a protein that functions as a gatekeeper
for the
entry and exit of mitochondrial metabolites and is a key player in mitochondria-mediated
apoptosis. Complex formation colocalized with ceramide cotransported with Aβ by astrosomes.
The interaction between Aβ and VDAC1 leads to caspase3 activation and subsequently
apoptosis. Aβ-associated exosomes, not Aβ alone, induced neurite fragmentation and
neuronal
death, suggesting that association with astrosomes substantially enhances Aβ neurotoxicity
in AD. Interestingly, the novel ceramide analogue N-oleoyl serinol (S18) prevented
the
aggregation of exosomes, and Aβ association with astrosomes, and reduced Aβ interaction
with
VDAC1, suggesting that exosomal ceramide mediated binding of Aβ to astrosomes and
mitochondrial damage.
Summary/Conclusion: Our data suggest that the association of Aβ with ceramide
in astrosomes enhances Aβ interaction with VDAC1 and mediates Aβ neurotoxicity in
AD, which
can be prevented by novel ceramide analogues.
Funding: This work is supported by NIH R01AG034389 and R01NS095215, and VA 1
I01 BX003643.
OT07.3
Blood-derived extracellular vesicles in Multiple Sclerosis: the
effects of Particulate Matter exposure
Luca Ferrari
a, Mirjam
Hoxhab, Simona Iodicea, Federica Rotab, Jacopo
Marianic, Giulia Malluccid, Roberto Bergamaschid and
Valentina Bollatia
aEPIGET LAB, Department of Clinical Sciences and Community
Health, Università degli Studi di Milano, Milano, Italy; bEpiget Lab Department
of Clinical Sciences and Community Health, University of Milan, Milan, Italy, Milan,
Italy;
cEPIGET LAB, Department of Clinical Sciences and Community Health, Università
degli Studi di Milano, Milan, Italy; dIRCCS Fondazione Mondino, Department of
Neurology and Neurorehabilitation, Multiple Sclerosis Unit, Pavia, Italy
Introduction: Multiple Sclerosis is the most common chronic inflammatory
demyelinating disease of the central nervous system, affecting more than 2 million
people
worldwide. MS is a multifactorial, immune-mediated disease caused by complex genetic
and
environmental interactions. In recent years, extracellular vesicles (EVs) have been
described as powerful mediators of the modulation of biological processes (e.g. inflammatory
and immune response) following environmental exposures such as Particulate Matter
(PM), and
have been described altered in MS. We characterized EVs in 48 patients with MS and
20
healthy subjects matched for age and gender and evaluated the effects of PM exposure
on EV
release patients with MS compared with controls.
Methods: EVs isolated from blood samples were characterized by nanotracking
analysis and by flow cytometry after labelling with the following markers: CD14+ (monocyte),
CD61+ (platelet), CD66+ (neutrophil), CD25+ (T-reg), and CD105+ (endothelium). PM10
and
PM2.5 concentrations at the residency of each subject were obtained from the regional
air
quality monitoring network.
Results: We observed decreased concentrations of CD14+ (p < 0.05), CD61+
(p < 0.003), CD66+ (p < 0.009), CD25+ (p < 0.003), and CD105+ (p < 0.003) in
patients compared with controls. In cases, PM was inversely associated with CD14+ EVs
(PM2.5, β = −0.03; p < 0.05), CD66+ EVs (PM2.5 β = −0.03; p < 0.03), and CD25+ EVs
(PM10 β = −0.02; p < 0.04; PM2.5, β = −0.03; p < 0.02). On the contrary, in controls
PM was positively associated with CD14+ EVs (PM10 β = 0.02; p < 0.03; PM2.5, β = 0.03;
p < 0.02).
Summary/Conclusion: Our findings showed a different composition of
blood-derived EV subpopulations in patients compared with controls. Moreover, we observed
that patients and controls react differently to PM exposure in terms of blood-derived
EV
release, suggesting the involvement of this mechanism in the modulation of both inflammatory
and immune responses, and thus in MS pathogenesis.
OT07.4
Plasma neuronal and astrocyte-derived exosomes serve as
biomarkers of neurodegeneration and systemic bioenergetic effects in male Cynomolgus
monkeys self-administrating oxycodone
Ashish Kumara, Yixin Sua, David
Soto-Pantojaa, Jingyun Leea, Ravi Singha, Cristina
Furduia, Michael Naderb and Gagan
Deep
a
aWake Forest Baptist Medical Center, Winston-Salem, USA;
bWake Forest Baptist Medical Center, Winston-Salem, USA
Introduction: Opioid use disorder (OUD) is currently a health emergency in the
USA affecting millions of people. OUD is a complex issue requiring a multipronged
strategy.
At the biological level, there is an urgent need to understand the dynamic molecular
changes
and adverse effects associated with opioid addiction. Here, we aimed at identifying
the
biosignature of brain cells-derived exosomes associated with opioid addiction in a
non-human
primate (NHP) model of OUD in which Cynomolgus monkeys perform cognitive tasks and
self-administer (SA) intravenous oxycodone daily. We also characterized the systemic
adverse
effects of the brain cells-derived exosomes from drug-naïve and oxycodone SA monkeys.
Methods: We isolated total exosomes (TE) by ultracentrifugation and ExoQuick
methods from the plasma of male monkeys self-administrating oxycodone for 3 years
and naive
monkeys. Subsequently, from the TE population, we isolated neuron-derived exosomes
(NDE) and
astrocytes-derived exosomes (ADE) using surface biomarkers L1CAM (L1 Cell Adhesion
Molecule)
and GLAST (Glutamate Aspartate Transporter), respectively. This novel method involved
streptavidin coated magnetic beads and photo-cleavable (PC) biotin, providing us
biologically intact exosomes useful for co-culture studies. We characterized the exosomes
by
nanoparticle tracking analyses (NTA), Western blotting, flow cytometry, immunogold
labelling, transmission electron microscopy (TEM), ELISAs and mass spectrometry.
Respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment
was
performed by Seahorse XF.
Results: The quality of isolated exosomes (TE, NDE, and ADE) was confirmed by
NTA (size distribution and concentration), Western blotting (e.g. CD9) and TEM (size
and
shape). NTA did not show any significant difference in exosomes size and concentration
(number per ml) between control and oxycodone SA groups. Flow cytometry (e.g. L1CAM
and
GLAST) and immunogold labelling (CD9, CD63 and L1CAM) confirmed the purity of NDE
and ADE
isolated from TE. Proteomics analyses of TE, NDE and ADE identified several unique
proteins
present in exosomes from the oxycodone SA group. Interestingly, we observed significantly
higher expression of neurodegeneration markers neurofilament light protein (NFL) and
alpha-synuclein in NDE and ADE of oxycodone SA group compared to controls. Furthermore,
TE
treatment of H9C2 cardiac myoblasts and RAW264.7 monocytes significantly compromised
their
mitochondrial metabolism (basal and maximum respiratory capacity).
Summary/Conclusion: These results suggest the utility of plasma exosomes as
biomarkers for better understanding of the neurodegenerative and systemic effects
of
oxycodone addiction.
Funding: DA049267, DA017763.
OT08
Symposium Session 08: Bacterial and Fungal
Evs
Chair: Amy Buck – The University of
Edinburgh
Chair: Cherie Blenkiron – The University of
Auckland
OT08.1
Vesicles released during Mycobacterium tuberculosis infection:
immunomodulatory (glyco)lipids and role in host-pathogen interactions
Emilie Layre, Pierre Boyer and Jerome
Nigou
CNRS-Université Paul Sabatier, Toulouse, France
Introduction: The tuberculosis disease remains one of the top 10 causes of
death worldwide. Mycobacterium tuberculosis (Mtb) has evolved strategies to evade
immune
responses and to persist within the hostile intracellular environment of alveolar
macrophages. The current lack of efficient anti-tuberculosis strategies is largely
due to
our incomplete understanding of the host-pathogen interactions of Mtb infection. Vesicles
released by the bacillus itself (bacterial membrane vesicles, BMV) and by infected
cells
(host extracellular vesicles, HEV) have immunomodulatory properties in vitro and when
administered to animals. If vesicles likely play key role in host-pathogen interactions
of
the Tuberculosis infection, their content in bacterial factors, their uptake, trafficking
and interaction with host cells receptors remain incompletely deciphered.
Methods: BMV and HEV have been purified by combining differential
centrifugation, density gradient and exclusion chromatography. After characterization
by
microscopy, NanoSight and western blot, their content in bacterial (glyco)lipids has
been
characterized by the use of high sensitivity mass spectrometry-based lipidomic approach.
BMV
have been tested for their capacity to activate reporter cell lines of pattern recognition
receptors. In addition, fluorescent-labelled BMV have been used to study their uptake
by
host cells thank to super-resolution microscopy.
Results: We have undertaken to characterize the content, the trafficking and
interaction with pattern recognition receptors of BMV and HEV released during infection
by
mycobacteria of variable virulence. We have importantly optimized the purification
of BMV
showing that lipoproteins aggregates are co-purified with vesicles on density gradient.
SFC-MS lipidomic analyses allowed the characterization of the repertoire of immunomodulatory
bacterial lipids released by BMV and HEV, which excluded a continuum between these
two
release pathways. Preliminary, assays have shown that these vesicles are capable to
interact
with different pattern recognition receptors including TLR and lectins. Finally, we
have
been able to visualize fluorescent-labelled vesicles uptake by macrophages using
superresolution microscopy.
Summary/Conclusion: During M. tuberculosis infection, the bacillus as well as
infected cells release vesicles that harbour different content in immunomodulatory
bacterial
(glyco)lipids, including strain-specific lipids. These vesicles likely play important
role
in host-pathogen interactions by modulating immune response beyond the infected cells,
in
part through their interaction with different pattern recognition receptors.
Funding: Fondation pour la Recherche Medicale, Fondation FonRoga.
OT08.2
Blood-based detection of tuberculosis using outer membrane
vesicles
Nishal Shah
a, Jina Kob,
Takahiro Yanoa, Sruthi Ravimohana, Greg Bissona, Harvey
Rubina and David Issadorec
aUniversity of Pennsylvania, Philadelphia, USA;
bMassachusetts General Hospital, Wyss Institute at Harvard University, Cambridge,
USA; cDepartment of Bioengineering, University of Pennsylvania, Philadelphia,
USA
Introduction: Conventional diagnoses of Mycobacterium tuberculosis (Mtb) rely
on quantifying bacteria in sputum samples, which make it incapable of measuring the
body’s
total bacterial load and diagnosing patients that have difficulty producing sputum
– such as
children and those that are HIV-positive. Nanoscale (30–200 nm) outer membrane vesicles
(OMVs), which are shed from their bacterial cells of origin and circulate in the
bloodstream, have been found to contain rich molecular information from their mother
cells.
Despite the diagnostic potential, their nanoscale size in the presence of high background
has complicated the use of these promising biomarkers for clinical diagnosis of
tuberculosis.
Methods: Here we report two complementary approaches to systematically
discover and clinically detect Mtb-derived OMVs using protein and RNA biomarkers.
First, we
employ a digital droplet ELISA on whole, unprocessed samples to detect and quantify
the
presence of these OMVs using surface protein markers. Second, we have developed a
platform
to specifically enrich for Mtb-derived OMVs using our previously developed magnetic
nanopore
platform, wherein millions of nanofluidic devices are operated in parallel, increasing
throughput relative to a single nanofluidic device by a million-fold. Using this approach,
we identify RNAs that are specifically enriched in Mtb-derived OMVs and can be used
to
identify TB strain, infectious activity, and total body burden.
Results: Using these platforms, we enriched for Mtb-derived OMVs from plasma
and profiled their cargo, both proteins and RNA. We first determined a panel of protein
biomarkers for multiplexed detection of OMVs through a digital droplet sandwich ELISA.
We
then tested our protein markers on spiked plasma samples as models for clinical TB
samples.
Simultaneously, we performed RNA sequencing and discovered a panel of RNA biomarkers
that
are preferentially enriched in OMVs. We picked ten of the most highly-expressed RNA
biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore
platform.
Summary/Conclusion: These results demonstrate the capability of OMV biomarkers
in the development of novel liquid biopsy based Mtb diagnostics. Building on this
work, we
are working with clinical collaborators to test our assays on clinical samples from
Philadelphia and west Africa.
Funding: NIH
OT08.3
Mechanisms of innate immune regulation by exosomes released
during infection with pathogenic gram-negative bacteria
Adam Fleminga, Heather Hobbsa, Graham
Matulisa, Valentin Girouxa, Weidong Zhoub, Valerie
Calverta, Carolina Salvador-Moralesa, Nitin Agrawalc,
Emanuel Petricoina, Rekha Panchald, Sina Bavarid and
Ramin M. Hakami
a
aGeorge Mason University, Manassas, USA; bCenter
for Applied Proteomics and Molecular Medicine, George Mason University, Manassas,
VA,
Manassas, USA; cChildren’s National Hospital, Washington, USA;
dUSAMRIID, Frederick, USA
Introduction: A dearth of knowledge exists regarding the molecular mechanisms
by which host exosomes regulate immune response to infections caused by gram-negative
pathogens. To address this gap in knowledge, our laboratory has been using two
well-established model organisms; Yersinia pestis (Yp), and Burkholderia pseudomallei
(Bp).
Yp and Bp cause the emerging human diseases plague and melioidosis respectively. Currently,
no licenced vaccines or highly effective therapeutics are available for either disease.
Methods: EX were purified from naïve U937 monocytes (EXu) and infected U937
(EXi) by serial centrifugation followed by sucrose density gradient purification,
and
characterized by TEM, ZetaView nanoparticle tracking, and exosome markers (CD63, TSG101,
and
Flotillin-1). Immune responses of naïve U937 cells and response mechanisms were analysed
following treatment with equivalent amounts of EXi or EXu (as control). These included
macrophage differentiation assays, multiplex measurements of inflammatory cytokines,
bacterial clearance assays, quantitative protein microarray analysis of 173 host signalling
proteins, siRNA knockdown of EXi-induced cytokines in recipient cells, and mass spectrometry
(MS) analysis of EXi contents. For all assays, at least four biological replicates
were
performed.
Results: EXi induce monocyte differentiation to macrophages and dramatic
release of IL-6, IL-8 and IL-10 cytokines, effects that are also seen when monocytes
are
infected with the bacteria. The EXi also induce a substantial increase in the capacity
of
the recipient monocytes to clear bacteria in an IL-6-dependent manner. Specific host
signalling molecules are strongly modulated by the EXi, including p38, Jak2 and ALK
for
EXi-Yp, influencing the observed phenotypes. MS analysis showed lack of LPS in EXi-Yp
and
demonstrated the presence of specific bacterial proteins that have antigenic properties.
Summary/Conclusion: We have identified some of the molecular mechanisms by
which EXi assist the host in clearing infection. EXi prime distant naïve monocytes
through
modulation of distinct pathways such as p38 to mount immune responses similar to when
they
become infected. These include differentiation to macrophages and migration to infection
site for increased IL-6-dependent bacterial clearance.
Funding: NIH grant (1R15AI137981-01) and U.S. MRMC grant (W81WH-15-T0003)
OT08.4
Human pathogenic fungus Candida albicans induces
immunomodulatory TGF-β1-transporting vesicles from human immune cells
Luke D. Halder
a, Emeraldo
Joa, Mohammad Hasana, Marta Ferreira-Gomesb, Thomas
Krügerc, Martin Westermannd, Diana Palmea, Günter
Rambache, Niklas Beyersdorff, Cornelia Spethe, Ilse
Jacobsena, Olaf Kniemeyerc, Berit Jungnickelg, Peter
Zipfela and Christine Skerkaa
aLeibniz Institute for Natural Product Research and Infection
Biology – Hans Knöll Institute (HKI), Jena, Germany; bFriedrich Schiller
University, Berlin, Germany; cLeibniz Institute for Natural Product Research and
Infection Biology (HKI), Department of Molecular and Applied Microbiology, Jena, Germany,
Jena, Germany; dUniversity Hospital Jena, Jena, Germany; eMedical
University of Innsbruck, Innsbruck, Austria; fUniversity of Würzburg, Würzburg,
Germany; gFriedrich Schiller University, Jena, Germany
Introduction: Recruitment of monocytes to sites of infection is important in
restricting growth and invasion of various microorganisms such as pathogenic fungi
C.
albicans. Beside complement supported phagocytosis and extracellular trap formation,
human
monocytes secrete extracellular vesicles which are crucial in cellular communication
in
physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids.
The
current study attempts to shed light on immune evasion mechanisms by C. albicans via
extracellular vesicles.
Methods: Human monocytes were isolated by magnetic beads technique and
extracellular vesicles were isolated using polymer precipitation or ultracentrifugation
or
size exclusion chromatography. Vesicles were characterized by ELISA, LC/MS-based proteomics,
confocal laser scanning microscopy (CLSM) as well as electron- and dynamic light scattering
microscopy, etc. CRISPR-CAS9 based genome editing was performed to knockout CD11b
in human
monocytic THP-1 cells. Effect of isolated vesicles were determined by using proximity
ligation assay (PLA), ELISA, Western Blot, next generation RNA sequencing, QPCR,
Immunohistochemistry, etc.
Results: Here we show for the first time that human blood derived monocytes
alone and in a whole blood model strongly induced and released extracellular vesicles
in
response to the pathogenic fungus C. albicans. One induced population carried the
anti-inflammatory cytokine TGFβ-1. Release of these vesicles is triggered by binding
of
soluble β-glucan from C. albicans to the CR3 receptor on monocytes as demonstrated
by
CRISPR-CAS9 based CR3 genome editing in THP-1 cells, and by using CR3 knock out mice.
Isolated TGF-β1-transporting vesicles reduced the inflammatory response in human M1
macrophages and in a whole blood model. The anti-inflammatory effect by TGF-β1-transporting
vesicles is investigated in detail and results in inhibition of IL-1β gene
transcription.
Summary/Conclusion: Showing that human apoptotic bodies similarly induced
TGF-β1-transporting vesicles from human monocytes we hypothesize that C. albicans
hijacks
this new CR3-dependent anti-inflammatory vesicle pathway for immune escape.
Funding: This work was supported by the “Deutsche Forschungsgemeinschaft”
TransRegio Funginet 124 projects C4, C5, C6 and Z2.
OT09
Symposium Session 09: EV Biogenesis I
Chair: Guillaume van Niel – Institute of Psychiatry and Neurosciences of Paris U-1266
INSERM
Chair: Maureen Barr – Rutgers University
OT09.1
Fragmentation of extracellular ribosomes and tRNAs shapes the
extracellular RNAome
Juan P. Tosar
a, Mercedes
Segoviab, Fabiana Gámbaroc, Yasutoshi Akiyamad, Pablo
Fagúndeze, Bruno Costae, Tania Possic, Marcelo
Hillf, Pavel Ivanovg and Alfonso Cayotac
aFacultad de Ciencias – Universidad de la República,
Montevideo, Uruguay; bLaboratory of 9 Immunoregulation and Inflammation, Institut
Pasteur de Montevideo, Uruguay., Montevideo, Uruguay; cFunctional Genomics Unit,
Institut Pasteur de Montevideo, Uruguay, Montevideo, Uruguay; dDivision of
Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA, USA.
Department of Medicine, Harvard Medical School, Boston, MA, USA., Boston, USA;
eAnalytical Biochemistry Unit. Nuclear Research Center. Faculty of Science.
Universidad de la República, Uruguay., Montevideo, Uruguay; fLaboratory of
Immunoregulation and Inflammation, Institut Pasteur de Montevideo, Uruguay., Montevideo,
Uruguay; gDivision of Rheumatology, Immunology and Allergy, Brigham and Women’s
Hospital, Boston, MA, USA. Department of Medicine, Harvard Medical School, Boston,
MA, USA,
Boston, USA
Introduction: To date, most research involving extracellular RNAs has focused
in RNAs encapsulated inside extracellular vesicles (EVs) or in total unfractionated
biofluids. It is known that exRNAs also exist outside vesicles or in lipoprotein particles.
However, nonvesicular exRNAs remain widely uncharacterized despite being a feasible
source
of contaminants in EV preparations. Our interest in nonvesicular exRNAs arises from
the
observation that some small RNAs, such as specific tRNA-derived fragments, have much
higher
relative representation in this extracellular fraction. At least in part, this enrichment
seems to be a consequence of their differential extracellular stability.
Methods: To get a representative picture of the whole set of RNAs released to
the extracellular nonvesicular space by cultured human cells, we inhibited extracellular
degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media
and
studied the kinetics of RNA release and degradation. High-resolution iodixanol gradients
were used to separate EVs from extracellular RNPs or vesicle-free RNA. The conversion
rate
between parental ncRNAs and their fragments was studied by high-throughput sequencing
and
Northern blot.
Results: The inhibition of extracellular RNase activity revealed the presence
of full-length tRNAs and ribosomes in the extracellular space of a variety of malignant
and
non-malignant cell lines. Extracellular ribosomes co-isolate with EVs purified by
ultracentrifugation or size-exclusion chromatography, but not with EVs purified by
density
gradients.These ncRNAs are substrates of extracellular RNases, demonstrating an
extracellular biogenesis route for the formation of ncRNA-derived fragments, some
of which
achieve remarkable stability and can be detected in biofluids. We also highlight the
immunoregulatory potential of purified RNA-containing extracellular complexes.
Summary/Conclusion: In conclusion, ribonuclease inhibition dramatically shapes
extracellular RNA profiles and uncovers a population of extracellular ribosomes, tRNAs
and
other coding and noncoding RNAs which exists outside EVs. Although these RNAs are
prone to
degradation, some of their fragments can accumulate in cell culture media and in biofluids.
This dynamic view of exRNAs impacts our understanding of RNA secretion mechanisms
and may
offer a window to new molecules with biomarker potential. In contrast, EVs confer
an
RNase-protected environment and contain more full-length ncRNAs (tRNAs, YRNAs, 7SL
RNAs,
rRNAs depending on vesicle size) than their fragments.
Funding: ANII, Uruguay [FCE_3_2018_1_148745]. CISC-UdelaR, Uruguay
[MIA_PAS2019_2_99]. NIH, USA [RO1GM126150 and UG3CA241694, supported by the NIH Common
Fund,
through the Office of Strategic Coordination/Office of the NIH Director]
OT09.2
CD47 interactions with exportin-1 regulate targeting of
m7 G-capped RNAs to extracellular vesicles
Sukbir Kaur
a, Alejandra Cavazos
Saldanab, Abdel G. Elkahlounc, Jennifer D Petersend,
Anush Arakelyane, Satya P. Singhf, Lisa M Jenkinsg, Bethany
Kuob, Bianca Reginauldb, David G Jordanb, Andy D
Tranh, Weiwei Wuc, Joshua Zimmerbergd, Leonid
Margolisi and David D. Robertsb
aNational Cancer Institutes, Bethesda, USA;
bLaboratory of Pathology, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, USA, Bethesda, USA; cCancer Genetics
Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda,
USA, Bethesda, USA; dSection on Integrative Biophysics, Division of Basic and
Translational Biophysics, Eunice Kennedy-Shriver National Institute of Child Health
and
Human Development, National Institutes of Health, Bethesda, USA, Bethesda, USA;
eSection on Intercellular Interactions, Division of Basic and Translational
Biophysics, Eunice Kennedy-Shriver National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, Bethesda, USA; fInflammation Biology
Section, Laboratory of Molecular Immunology, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, USA., Bethesda, USA;
gLaboratory of Cell Biology, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Bethesda, USA, Bethesda, USA; hConfocal
Microscopy Core Facility, Center for Cancer Research, National Cancer Institute, National
Institutes of Health, Bethesda, USA, Bethesda, USA; iSection on Intercellular
Interactions, Division of Basic and Translational Biophysics, Eunice Kennedy-Shriver
National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, USA, Bethesda, USA
Introduction: CD47 is a ubiquitously expressed membrane protein that functions
as a receptor for thrombospondin-1 and the counter receptor for signal regulatory
protein-α
in phagocytes. High expression of CD47 is associated with a poor prognosis for some
cancers.
Conversely, CD47 blocking agents are in clinical trials for enhancing innate and adaptive
antitumor immunity in cancer patients. These studies suggest utility of CD47 as a
diagnostic
and prognostic biomarker and as a therapeutic target. CD47 is also expressed on
extracellular vesicles (EVs), and we reported that CD47 expression identifies a distinct
population of EVs from those that express the traditional EV markers CD63 or MHC1.
CD47-,
CD63- and MHC1-enriched vesicles contain distinct small RNA populations (PMID: 29416092),
and these differ in RNA content from EVs that lack any of these markers. The mechanisms
by
which CD47 directly or indirectly regulates which RNAs are packaged into EV remain
unknown.
Methods: To elucidate the mechanism by which CD47 regulates EV RNA composition
and function, we performed global miRNA microarray analysis between EVs produced by
wild
type and CD47-deficient T cells. Results were further validated using real-time PCR
and
RNA-immunoprecipitation. Interactions between CD47 and exportin-1/Ran complex was
identified
by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular
localization, flow cytometry, and confocal and electron microscopy.
Results: EV released from CD47-deficient human T cells and in cd47-/- mouse
plasma were enriched in 5ʹ-7-methylguanosine-capped microRNAs and mRNAs that depend
on the
exportin-1/RanGTP pathway. Knockdown of CD47 in wild type cells or thrombospondin-1
treatment correspondingly enhanced levels of capped-RNAs released in EV and re-expressing
CD47 in null cells decreased their levels. Mass spectrometry and co-immunoprecipitation
identified specific interactions of CD47 with components of the exportin-1/Ran nuclear
export complex and its known cargos and between the CD47 cytoplasmic adapter ubiquilin-1
and
the exportin-1/Ran complex. Interaction of CD47 with exportin-1 was inhibited by leptomycin
B, which inactivates exportin-1 and increased levels of cap-dependent RNAs in EV released
from wild type but not CD47-deficient cells.
Summary/Conclusion: These findings indicate that CD47-dependent
thrombospondin-1 signalling regulates cytoplasmic levels of cap-dependent RNAs in
T cells at
least in part through ubiquilin-1- and GTP-dependent physical interactions with the
exportin-1/Ran transport complex, which regulate levels of specific pre-miRNAs and
mRNAs
available for sorting into EVs.
Funding: This work was supported by the Intramural Research Program of the
NIH/National Cancer Institute (ZIA SC 009172).
OT09.3
Role of membrane protein palmitoylation in extracellular vesicle
biogenesis in squamous cell carcinoma
Mohammed Haque
a, Joseph P.
Flemmingb, Brianna L. Hill
c, James
Wahld and My Mahoneye
aThomas Jefferson University, Lansdale, USA;
bThomas Jefferson University, haddonfield, USA; cThomas Jefferson
University, Mohrsville, USA; dUNMC, Omaha, USA; eThomas Jefferson
University, Philadelphia, USA
Introduction: Desmoglein 2 (Dsg2), is a palmitoylated cadherin that is
involved in cell-cell adhesion. Interestingly, Dsg2 promotes mitogenic cell signalling
and
is upregulated in many cancers, including SCC, contributing to poor prognosis and
survivability. We recently demonstrated that Dsg2 promotes EV release, but the mechanism
by
which Dsg2 enhances EV biogenesis and role of palmitoylation is poorly understood.
Methods: Pharmacological drug inhibitors 2-bromopalmitate, GW4869, and
Bafilomycin A1 were used. Stable SCC cell lines were established by retrovirus infection
expressing GFP, wild type Dsg2/GFP, or palmitoylation deficient Dsg2cacs/GFP. EVs
were
isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed
by
NTA. Proteins associated with the endocytic pathway were analysed by immunofluorescence
and
imaged by confocal microscopy or immunoblotting and signals were quantitated using
ImageStudio.
Results: Here we demonstrate that the effect of Dsg2 on EV release was reduced
by the palmitoylation inhibitor 2-bromopalmitate. Furthermore, mutations that prevented
palmitoylation (Dsg2cacs) dramatically abrogated EV release by targeting of un-palmitoylated
Dsg2 to the lysosomes for degradation. Dsg2 increased expression and subcellular
localization of FLOT1, a membrane lipid raft protein critical for membrane invagination.
Dsg2 also altered membrane localization of several early (EPS15 and EEA1), but not
late
(Rab7, Rab11, and HRS), endocytic pathway proteins. Loss of palmitoylation in the
Dsg2cacs
mutants abrogated these effects. Finally, Dsg2-induced EV release was abrogated by
the
sphingomyelinase inhibitor GW4869 or augmented by the V-ATPase inhibitor Bafilomycin
A1.
Summary/Conclusion: The combined results of the drug treatments and functional
mutations of Dsg2 suggest that Dsg2 plays a critical role in EV biogenesis by modulating
proteins involved in early endosome sorting and is dependent on post-translational
palmitoylation.
Funding: NIH RO1
OT09.4
Translation initiation factor eIF4E reprograms the vesiculation
of cancer cells
Esterina D’Asti
a, Biljana
Culjkovic-Kraljacicb, Héloïse Chasséb, Jadwiga Gasiorekb,
Gavin Morrisb and Katherine Bordenb
aUniversity of Montreal, Laval, Canada;
bUniversity of Montreal, Montreal, Canada
Introduction: The translation initiation factor eIF4E (4E) is an oncogenic
protein that is upregulated in 30% of cancers including a subgroup of acute myeloid
leukaemia (AML) patients. EIF4E regulates post-transcriptional RNA processing including
the
nuclear export and/or translation of mRNA transcripts. In particular, it selectively
increases the expression of genes that have a prominent role in cancer progression
such as
MYC, cyclin D1, and MCL1. Furthermore, our lab pioneered studies demonstrating that
a subset
of 4E-high AML patients is clinically responsive to treatment with a 4E inhibitor
(Ribavirin) indicating the importance of 4E in AML progression and its relevance as
a
therapeutic target. We investigated an as yet unexplored perspective of 4E- whether
the
oncogenic role of 4E is in part mediated by its function as a master regulator of
vesiculation.
Methods: To assess mRNA export and identify 4E-bound mRNA targets that
correspond to vesiculation-related genes and associated cargo, we used cellular
fractionation and RNA immunoprecipitation techniques. To determine whether 4E regulates
the
number of extracellular vesicles (EVs) released as well as their protein and RNA cargo
we
used Nanoparticle Tracking Analysis (NTA) as well as mass spectrometry, antibody
microarrays, and RNA sequencing technologies.
Results: EIF4E upregulates cellular protein levels of the vesiculation marker
CD63 by increasing its nuclear export. In addition to increased cellular expression,
CD63,
CD81, CD9, and flotillin-1 proteins are elevated in EVs released from 4E-high cells.
This is
also associated with an increased release of vesicles that are 50–200 nm in size.
Currently,
we have validated the upregulation of several receptors and cytosolic proteins in
EVs
isolated from 4E-overexpressing cells that function in cell growth, migration, invasion,
and
stemness. The most abundant RNAs in our EV preparations are microRNAs (miRs) and we
have
confirmed downregulation of several of these.
Summary/Conclusion: Our work shows that 4E reprograms the vesiculation of
cancer cells changing the release and cargo of EVs. This may impact cellular communication
and tumour biology, which we are currently addressing in functional studies. We hope
that
these studies will highlight novel therapeutic strategies for AML patients.
Funding: Leukemia and Lymphoma Society, Cole Foundation
OT10
Symposium Session 10: EV-based Therapeutics I
Chair: Dolores Di Vizio – Cedars-Sinai Medical Center
OT10.1
Intranasal administration of neural stem cells-derived
extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid
plaques in a mouse model of Alzheimer’s disease
Leelavathi N. Madhu
a, Raghavendra
Upadhyab, Sahithi Attalurib, Maheedhar Kodalic, Laila
Melissaric, Andrew Vogelb, Bing Shuaib and Ashok K.
Shettyb
aInstitute of Regenerative medicine, College of Medicine,
Texas A&M University, College Station, USA; bInstitute of Regenerative
Medicine, College of Medicine, Texas A&M University, College Station, USA;
cInstitute for Regenerative Medicine, College of Medicine, Texas A&M
University, College Station, USA
Introduction: Cognitive and memory impairments worsen with time in Alzheimer’s
disease (AD), likely due to a progressive loss of hippocampal neurogenesis, and escalation
of neuroinflammation. These changes are also accompanied by increased deposition of
amyloid
plaques in the brain.
Methods: In this study, using the 5XFAD mouse model, we examined the efficacy
of extracellular vesicles (EVs) shed from the rat subventricular zone neural stem
cells
(SVZ-NSCs) for disease modification. We first purified EVs from the rat SVZ-NSC cultures
through ion-exchange chromatography and then administered intranasally to 3-months
old 5XFAD
mice (~75 billion/week for two weeks). Two months later, the functional effects of
EV
treatment were quantified through a series of behavioural tests, and animals were
euthanized
for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation,
and
amyloid plaque deposition.
Results: In comparison to AD mice receiving vehicle, AD mice receiving NSC-EVs
displayed improved cognitive function to discern minor changes in the environment
in an
object location test, better spatial recognition memory in an object-in-place test,
and
improved pattern separation ability in a pattern separation test. Besides, EV-treated
AD
mice displayed no anhedonia in a sucrose preference test. Analyses of neurogenesis
using the
birth-dating marker 5ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin
revealed maintenance of a higher level of hippocampal neurogenesis in AD mice receiving
EVs,
in comparison to vehicle-treated AD mice. Moreover, analyses of brain tissues from
EV-treated AD mice revealed decreased concentrations of oxidative stress markers
malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase
and
superoxide dismutase. Also, the concentration of proinflammatory cytokines tumour
necrosis
factor-alpha and interleukin-1 beta and the extent of amyloid plaques were significantly
reduced in EV treated AD mice. Immunohistochemical analysis showed reduced hypertrophy
of
astrocytes.
Summary/Conclusion: Intranasal administration of NSC-derived EVs restrains the
deterioration of cognitive and mood dysfunction of AD by maintaining higher levels
of
neurogenesis and curtailing the progression of neuroinflammation.
Funding: Supported by a grant from the National Institute of Neurological
Disorders and Stroke (1R01NS106907-01 to A.K.S.)
OT10.2
Protective effects of extracellular vesicles derived from
induced pluripotent stem cells in acute kidney injury model
Rafael Soares Lindoso
a, Federica
Collinob, Jarlene Alécia Lopesa, Marta Tapparoc, Giovane
Torteloted, Taís Kasai-Brunswicke, Gustavo Lopese,
Douglas Barrosoe, Renata Skovronovaf, Camila Wendte,
Kildare Mirandae, Benedetta Bussolatig and Adalberto
Vieyraa
aFederal University of Rio de Janeiro, Rio de Janeiro,
Brazil; bDepartment of Biomedical Sciences, University of Padua, Padua, Italy;
cDepartment of Medical Sciences, Molecular Biotechnology Center, University of
Torino, Turin, Italy; dDepartment of Pediatrics’ Section of Pediatric Nephrology,
Tulane University School of Medicine, New Orleans, USA; eInstitute of Biophysics
Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;
fDepartment of Molecular Biotechnology and Health Sciences, University of
Torino, Turin, Italy; gUniversity of Torino, Torino, Italy
Introduction: Induced pluripotent stem cells (iPSC) present a wide range of
applications, including cellular therapy. However, the tumorigenic potential is a
limitation
in the iPSC use. The extracellular vesicles (EV) have been shown to mimic the beneficial
effects of cells, as in the case of mesenchymal stromal cells, opening new perspectives
in
the use of iPSC in regenerative medicine. The aim of this study is to evaluate the
potential
of iPSC-EV in the treatment of kidney disease.
Methods: The iPSC were generated from skin fibroblast after informed consent
of healthy donors (CytoTune®-iPS 2.0 Sendai Reprogramming Kit – Protocol: Clementino
Fraga
Filho UH 38583914.7.0000.5257/933.018). The EV were isolated from iPSC supernatants
(cultured 24 h in mTeSR-1 medium) by ultracentrifugation (100,000 g for 2 h at 4°C).
Characterization of iPSC-EV was performed using ZetaView, TEM, ExoView™ Tetraspanin
Kit and
MACSPlex Exosome Kit. For in vitro injury, renal epithelial cells were cultured under
hypoxia (1% O2). For in vivo injury, male Wistar rats were submitted to bilateral
renal
arterial clamping (45 min) followed by reperfusion without or with injection of iPSC-EV
(Protocol approval: Federal University of Rio de Janeiro – 043/19). Kidney damage
was
assessed by histological and immunohistochemistry analyses (PCNA, TUNEL and ED-1).
Modulation of RNA levels was assessed by RT2 Profiler PCR array.
Results: The results show that iPSC-EV reduce renal cell death, tissue damage,
macrophage infiltration, promote mitochondria protection and ameliorate renal function.
The
iPSC-EV mechanism of action is related to the regulation of key genes known to prevent
damage caused by oxidative stress like GSTK1, SOD1, SOD3, TXN1 and TXNRD2. Characterization
of iPSC-EV showed that iPSC-EV can carry important molecules that can support renal
recovery
as EpCAM and Prominin-1.
Summary/Conclusion: iPSC-EV presents renoprotective properties, acting on
different aspects of AKI. This presents a new relevant application of iPSC as a source
of EV
for therapeutic purpose in kidney diseases.
Funding: This work was supported by National Institute of Science and
Technology for Regenerative Medicine REGENERA, Brazilian National Research Council;
Carlos
Filho Rio de Janeiro State Research Foundation; MIUR-local Research to BB; Starting
Grant
Padova to FC; the European Union’s Horizon 2020 research and innovation program to
BB.
OT10.3
Epigenetic regulation of foetal hypoplastic lungs by amniotic
fluid stem cell-derived extracellular vesicles
Lina Antounians, Huayun Hou, Cadia Chan,
Michael Wilson and Augusto Zani
The Hospital for Sick Children, Toronto, Canada
Introduction: Incomplete lung development, also known as pulmonary hypoplasia
(PH), is a recognized cause of neonatal death. We have previously shown that experimental
PH
can be rescued by the administration of extracellular vesicles derived from amniotic
fluid
stem cells (AFSC-EVs) through an RNA-mediated mechanism. This effect was not observed
with
EVs derived from mesenchymal stromal cells (MSC-EVs). The aim of this study was to
1)
evaluate which RNA species were responsible for PH rescue, and 2) to define the mechanism
behind this effect.
Methods: EVs were isolated and characterized from conditioned medium of rat
AFSCs and rat MSCs (control group) using ultracentrifugation. EVs were assessed for
size
(nanoparticle tracking analysis), morphology (TEM), and expression of CD63, Hsp70,
Flo-1,
and TSG101 (Western).
To identify the mediators of AFSC-EVs, we used DESeq (FDR<0.01) to differentially
analyse RNA from:
A) ASFC-EV and MSC-EV cargo, isolated with SeraMir and sequenced with NextSeq.
B) lung epithelial cells from rat PH lungs treated with vehicle or AFSC-EVs. Epithelial
cell RNA was isolated with miRvana and sequenced with NextSeq.
We correlated AFSC-EV cargo miRNA with validated mRNA targets that were downregulated
after
EV conditioning in lung epithelial cells.
Results: Of the RNA species contained in ASFC-EV and MSC-EV cargo, miRNAs were
the most proportionally different between the two EV populations. AFSC-EVs were enriched
for
miRNAs that are critical for lung development, such as miR17 ~ 92 and their paralogues
that
control lung branching morphogenesis. AFSC-EV administration to PH lung cells significantly
downregulated 35 genes, which formed 415 miRNA-mRNA reported interactions.
Summary/Conclusion: AFSC-EVs contain many RNA species in their cargo, but
miRNAs are the main effectors of their ability to rescue underdeveloped foetal lungs.
We
have identified for the first time that AFSC-EV biological effect on underdeveloped
foetal
lungs is in part due to the release of miR17 ~ 92 cluster.
Funding: CIHR-SickKids Foundation grant.
OT10.4
Bottom-up assembly of fully-synthetic extracellular
vesicles
Oskar Staufer
a, Franziska
Dietricha, Jochen Hernandeza, Martin Schrötera, Sebastian
Fabritzb, Heike Böhma, Ilia Platzmana and Joachim
Spatza
aMax Planck Institute for Medical Research, Department for
Cellular Biophysics, Jahnstraße 29, 69120 Heidelberg, Germany, Heidelberg, Germany;
bDepartment for Chemical Biology, Max Planck Institute for Medical Research,
Jahnstraße 29, 69120 Heidelberg, Germany, Germany, Germany
Introduction: Extracellular vesicles (EVs) are considered as key elements for
future therapeutic and diagnostic procedures. However, despite enormous research efforts
to
understand their physiological relevance and several greatly successful clinical trials,
EVs
are currently not authorized for clinical routines by American or European regulation
and
approval agencies. This is especially because therapeutic EVs are produced or isolated
from
cell cultures or biofluids, both of which are subjected to batch-to-batch variations
and
ill-defined contaminations. Therefore, complementary technologies that produce EVs
as
reproducible and defined as state of the art nanotherapeutics, would revolutionize
the
application of EVs in clinical settings and provide the scientific community with
a holistic
understanding of EV-mediated signalling processes. In our study, we achieve de novo
bottom-up assembly of fully synthetic EVs (fsEVs) that comprise identical physiological
and
therapeutic functionalities to natural EVs.
Methods: We applied droplet-based microfluidic synthesis to sequentially
amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that
display
analogous therapeutic capabilities to natural EVs. FsEVs were characterized by electron
and
confocal microscopy, dynamic light scattering and mass spectrometry and tested on
organotypic models or in vivo.
Results: Using previously described EVs as “nature-given” blueprints, we
assembled several fsEVs in their exact molecular composition. In particular, we produced
wound-healing promoting EVs composed of several exosomal proteins, lipids and microRNAs
and
showed that their therapeutic performance on human skin wounds is equivalent to that
of
natural EVs. Besides their high molecular complexity, being composed of dozens of
different
molecular building-blocks, the presented fsEVs are completely defined on a quantitative
level. Based on this, we achieved a stoichiometric understanding of cell-vesicle
interactions.
Summary/Conclusion: By applying bottom-up synthesis of fsEVs for quantitative
studies on EV signalling, we not only provide innovative and safe compounds for
EV-therapeutics but also a vastly new perspective on the application spectrum of
extracellular vesicles in fundamental research.
OT10.5
lncRNA EPHA6-1 in IFNβ-induced small extracellular vesicles
promotes cytotoxicity of NK cells by acting as a ceRNA for hsa-miR-4485 to up-regulate
NKp46 expression
Shuang Li
a, Anjing Zhub,
Shilin Lib and Limin Chen
c
aInstitute of Blood Transfusion, Peking Union Medical
College, Chengdu, China (People’s Republic); bInstitute of Blood Transfusion,
Chinese Academy of Medical Sciences Peking Union Medical College, Chengdu, China (People’s
Republic); cInstitute of Blood Transfusion, Chinese Academy of Medical Sciences
Peking Union Medical College, Chengdu, China (People’s Republic)
Introduction: Small extracellular vesicles (sEVs) contain functional molecules
from their cell of origin and can enter recipient cells for intercellular communication.
IFNβ has been shown to induce some lncRNAs to regulate host immune response and play
a major
role in the positive regulation of the activity of natural killer (NK) cells. Here,
we aim
to clarify whether IFNβ induced sEVs can regulate the cytotoxicity of NK cells by
transferring specific lncRNAs into NK cells.
Methods: EVs were purified from A549 with/without IFNβ treatment by serial
centrifugation followed by sucrose density gradient purification. ELISA assay were
performed
to demonstrate the cytotoxicity of NK cells. qPCR and Western Blot were used to verify
the
expression of NKp46.
Results: Surprisingly, IFNβ induced sEVs can strengthen the cytotoxicity of NK
cells. Through human transcriptome array (HTA) we found the expression levels of 69
lncRNAs
were significantly changed within sEVs isolated from A549 cells following IFNβ treatment.
Additionally we found a specific sEV cargo, linc-EPHA6-1, acted as a competing endogenous
RNA (ceRNA) for hsa-miR-4485 which subsequently up-regulate the natural cytotoxicity
receptor (NKp46) expression. Furthermore, we verified over-expression of linc-EPHA6-1
significantly enhance the cytotoxicity of NK cells against Zika virus-infected A549
cells.
Summary/Conclusion: Our results demonstrated that IFNβ-induced linc-EPHA6-1
wrapped in sEVs can regulate the cytotoxicity of NK cells. Our study provides a novel
link
between type I IFN and NK cells, which are two major players for the host innate immunity
against pathogen infections.
Funding: This work was financially supported by the Chinese Academy of Medical
Sciences Innovation Fund for Medical Sciences (2016–12 M-3-025 to LC); the National
Key
Research and Development Program from Ministry of Science and Technology of China
(2018YFE0107500 to LC) and the National Natural Science Foundation of China (NSFC
81702017
to S. Li).
OT11
Symposium Session 11: Viruses
Chair: Shilpa Buch – University of Nebraska Medical Center
Chair: Malene M. Møller – Department of Clinical Immunology, Aalborg University
Hospital
OT11.1
Unbiased quantitative proteomic analysis identifies novel
cellular components specifically incorporated into endogenous EV subtypes, HIV-1 particles
and virus-modified EVs
Lorena Martin-Jaulara, Nathalie Nevoa, Mercedes
Tkacha, Mabel Jouveb, Florent Dinglic, Damarys
Loewc, Kenneth Witwerd, Matias Ostrowskie, Georg
Bornerf and Clotilde Théry
g
aINSERM U932, Institut Curie, PSL Research university, paris,
France; bCNRS UMR3215, Institut Curie, PSL Research university, paris, France;
cInstitut Curie, PSL Research University, Laboratoire de Spectrométrie de Masse
Protéomique, paris, France; dDepartment of Molecular and Comparative Pathobiology
and Department of Neurology, The Johns Hopkins University School of Medicine, baltimore,
USA; eInstituto INBIRS, Universidad de Buenos Aires-CONICET, Buenos Aires,
Argentina; fDepartment of Proteomics and Signal Transduction, Max Planck
Institute of Biochemistry, Martiensried, Germany; gINSERM U932, Institut Curie,
PSL Research university, Paris, France
Introduction: HIV-infected T cells release simultaneously viral particles and
small extracellular vesicles (sEVs) including MVB-derived exosomes and plasma
membrane-derived EVs. sEVs and HIV share many physical and chemical characteristics,
which
makes their separation difficult. Although several approaches have been used to obtain
sEVs
free of virus they leave a majority of sEVs within HIV preparations. For this reason,
the
function of sEVs during HIV infection remains unclear.
Methods: We have developed a novel un-biased proteomic profiling approach to
identify specific markers of the virus or sEV subtypes released by a human T lymphoma
cell
line. Our approach was to combine differential centrifugation of medium/small EVs
contained
in the CCM with quantitative mass spectrometry to generate protein abundance profiles
across
the different sub-fractions. We generated an interactive database to define groups
of
proteins with similar profiles, suggesting their release in the same EVs.
Results: We thus identified different categories of EVs, which bear different
surface proteins, e.g. different combinations of T cell surface markers, integrins
or
tetraspanins. In EVs released by infected cells, we identified cellular proteins behaving
like HIV proteins, and several that changed behaviour after infection, either moving
towards
or away from the HIV cluster. We identified two cell-derived proteins that are included
in
the viral particles and one that is specific of non-viral sEVs that are modified by
infection, and analysed their respective roles in controlling EV composition or virus
infectivity.
Summary/Conclusion: Our approach presents a powerful tool for identification
of common cargoes of given EV subtypes, and could be now used to identify modifications
of
EV composition in any given physiological or pathological situation.
Funding: ANRS (2015–1); SIDACTION (17-1-AAE-1138); INCa (INCA-11548),
Fondation ARC (PGA1 RF20180206962); ANR (ANR-10-IDEX-0001-02 PSL*, ANR-11-LABX-0043,
ANR-18-CE13-0017-03); NIDA (DA040385, DA047807); NIH Common Fund (UG3CA241694),
DFG/Gottfried Wilhelm Leibniz Prize (MA 1764/2-1).
OT11.2
The encephalomyocarditis virus Leader modulates autophagic
pathways to promote the release of virions inside extracellular vesicles
Kyra A. Y. Defourny
a, Susanne G.
Van der Greinb, Huib Rabouwb, Frank J. M. Van Kuppeveldb
and Esther Nolte-’t Hoenc
aDept. Biomolecular Health Sciences, Fac. Veterinary
Medicine, Utrecht University, The Netherlands, Lathum, Netherlands; bDept.
Biomolecular Health Sciences, Fac. Veterinary Medicine, Utrecht University, The Netherlands,
Utrecht, Netherlands; cDept. Biomolecular Health Sciences, Fac. Veterinary
Medicine, Utrecht University, Utrecht, Netherlands
Introduction: Recent data indicate that naked viruses belonging to the
picornaviridae family can be released from host cells via enclosure in extracellular
vesicles (EV). EV cloak virus particles in a host-derived “envelope” and can thereby
affect
antiviral immune responses and disease severity. A better understanding of the formation
and
function of EV-enclosed viruses is therefore required. Previously, we showed the presence
of
the autophagosome marker LC3 in EV isolates from encephalomyocarditis virus (EMCV)
infected
cells, suggesting the involvement of a secretory autophagy pathway in EV-mediated
virus
release. However, little is known about the viral and host factors that regulate this
process. Here, we have assessed the role of the EMCV Leader, a viral protein that
is
dispensable for replication but is required for symptomatic disease.
Methods: Cells were infected with wildtype virus or a mutant carrying an
inactive Leader. EV produced during the infection were isolated using differential
ultracentrifugation and density gradient purification. EV were characterized by high
resolution flow cytometry and their infectivity determined using end-point dilution
assay.
In addition, the fate of autophagosomes in infected cells was monitored using a reporter
assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal
proteins.
Results: Inactivation of the EMCV Leader strongly reduced the release of
EV-enclosed virus. Whereas autophagosomes are typically degraded, we show this is
blocked by
the Leader. Instead, autophagosomes fuse with the plasma membrane, as indicated by
the
secretion of autophagy marker LC3 during infection with wildtype but not the mutant
virus.
Pharmacological reactivation of degradative autophagy in infected cells resulted in
a strong
reduction in the release of EV and EV-enclosed virus. Similarly, the reduction in
EV-enclosed virus release in the absence of the Leader could be partially reversed
by drugs
that promote the secretion of autophagosomes.
Summary/Conclusion: Our data supports a role for secretory autophagy in the
release of viruses in EV, a pathway that is regulated by the EMCV Leader. These findings
highlight an unconventional route for EV formation that intersects with autophagosomal
compartments and contributes to viral pathogenesis.
Funding: NWO ALWOP.351
OT11.3
ZIKV induced DEFA1B blocks ZIKV adsorption and retard cell cycle
by regulating ORC1 expression
Shuang Li
a, Shilin Lib
and Limin Chenc
aInstitute of Blood Transfusion, Peking Union Medical
College, Chengdu, China (People’s Republic); bInstitute of Blood Transfusion,
Chinese Academy of Medical Sciences Peking Union Medical College, Chengdu, China (People’s
Republic); cInstitute of Blood Transfusion, Chinese Academy of Medical Sciences
Peking Union Medical College, Chengdu, China (People’s Republic)
Introduction: Zika virus (ZIKV) causes a public health emergency of
international concern because of its correlation with microcephaly. During viral infection,
the innate immune response quickly to produce some endogenous functional molecules
which can
prevent viral invasion or replication. Extracellular vesicles (EVs) contain molecules
from
their cell of origin under virus infection and can enter recipient cells for intercellular
communication. Here, we aim to clarify whether ZIKV induced EVs can regulate viral
pathogenicity by transferring specific RNA.
Methods: EVs were purified from A549 with/without ZIKV infection by serial
centrifugation followed by sucrose density gradient purification. Human transcriptome
array
(HTA) was used to found RNA expression within EVs. Flow Cytometry was used to determine
cell
cycles. ZIKV replication was assayed by qPCR and Western Blot. Flow Cytometry was
used to
determine cell cycles.
Results: Through HTA we found the defensin alpha 1B (DEFA1B) expression was
significantly increased within EVs isolated from ZIKV infected A549 cells. Additionally,
we
found that the extracellular DEFA1B but not the intracellular DEFA1B exerts anti-ZIKV
effect
mainly before entry step. Surprisingly, up-regulate DEFA1B can retard cell cycles
of host
cell. We verified DEFA1B could bind with the origin recognition complex 1 (ORC1) which
is
required to start DNA replication during the cell cycle. Furthermore, up-regulate
DEFA1B
decreased the ORC1 level in nuclear. Interestingly, EVs with DEFA1B can internalize
into
recipient cells and inhibit their cell cycles.
Summary/Conclusion: Together, our results demonstrated that ZIKV infection can
induce DEFA1B wrapped in EVs, and DEFA1B not only exerts anti-ZIKV effect but also
regulate
cell cycles which may affect neurodevelopment. Our study provides a novel viewpoint
that
DEFA1B act as first-line anti-viral molecules during ZIKV infection also correlate
with
neurodevelopment by retarding cell cycles.
Funding: Academy of Medical Sciences Innovation Fund for Medical Sciences
(2016–12 M-3-025 to LC); the National Key Research and Development Program from Ministry
of
Science and Technology of China (2018YFE0107500 to LC) and the National Natural Science
Foundation of China (NSFC 81702017 to S. Li).
OT11.4
Extracellular vesicles mediate bacterial-immune cell
interactions during respiratory viral-bacterial co-infections
Sidney W. Lane
a, Matthew
Hendricksb and Jennifer Bombergera
aUniversity of Pittsburgh, Pittsburgh, USA;
bUniversity of Washington, Seattle, USA
Introduction: Respiratory infections are a major cause of morbidity and
mortality worldwide and host-derived extracellular vesicles (EVs) play important roles
in
mediating these infections. During respiratory infection, EVs are shown to have a
modulatory
effect: promoting or suppressing infection dependent on the pathogen and cell type.
In the
age of next-generation sequencing, we now appreciate that many respiratory infections
are
polymicrobial in nature, with viral-bacterial co-infections correlating with worse
disease
outcomes. Epidemiological studies correlate acute viral infections with the increased
likelihood and severity of both acute and chronic secondary bacterial infections;
however,
the exact mechanisms of these interactions remain poorly understood. EVs have been
understudied in the context of respiratory viral-bacterial co-infections; thus, their
role
in mediating these infections is relatively unknown. Unpublished data from the lab
shows
that in airway epithelial cells (AECs), viral infection induces the release of EVs
that
associate with Pseudomonas aeruginosa (Pa) and promote biofilm growth. Here, we aim
to
expand upon these findings and determine how AEC EVs mediate Pa-immune cell interactions
during respiratory viral-bacterial co-infection.
Methods: To determine how exposure to EVs impacts Pa-immune cell interactions,
EVs were isolated from the apical secretions of AECs and co-cultured with Pa. EV-treated
Pa
was then co-cultured with macrophages to evaluate EV impact on Pa uptake and survival.
Results: In preliminary experiments using control EVs, we observed that EVs
associate with Pa. Interestingly, during co-culture with macrophages, EV-treated Pa
are more
susceptible to phagocytosis in comparison to non-treated Pa. However, after 4 hours
of
co-culture with macrophages, EV-treated Pa are able to survive and replicate, while
non-treated Pa are effectively controlled by the macrophages.
Summary/Conclusion: These findings suggest that while Pa-EV association
promotes Pa uptake, it may ultimately enhance Pa immune evasion and survival. Ongoing
experiments in the lab are evaluating the mechanism of Pa-EV association and how EVs
from
virus-infected AECs affect the phenotypes observed with control EVs. Notably, this
is one of
few reports of a mammalian EV influencing the pathogenesis of a bacterium; thus, results
from these experiments will define the function of AEC EVs in regulating bacterial-immune
cell interactions during respiratory co-infections.
OT11.5
Using machine learning with neuronal EV target proteins and
clinical data to predict cognitive impairment in HIV infection
Lynn Pulliam
a, Michael
Listonb, Bing Sunc and Jared Narvidd
aUniversity of California, San Francisco, San Francisco, USA;
bVeteran Affairs, San Francisco, USA; cNCIRE, San Francisco, USA;
dUCSF, San Francisco, USA
Introduction: Objective biomarkers are needed to assess and predict neuronal
function and cognitive impairment. In people ageing with chronic infections, such
as HIV,
determining the mechanism of impairment will be important when therapies are available.
Methods: Sixty plasma samples from HIV-infected people were obtained from
NIH-sponsored AIDS banks. Clinical and epidemiological data were collected. All underwent
neuropsychological testing and 40 were considered impaired. Neuronal extracellular
vesicles
(nEVs) were isolated from plasma and assayed for high-mobility group box 1 (HMGB1),
neurofilament (NF-L) and phosphorylated tau-181 (p-tau) proteins.
Results: Using 3 different algorithms, Support Vector Machines (SVM) performed
the best with an area under the curve (AUC) value of 0.82 ± 0.16. Using 4 different
combinations of clinical data and the 3 nEV protein targets, selected clinical data
and
HMGB1 best predicted cognitive impairment (AUC = 0.82). The most important features
included
CD4 count, HMGB1, NF-L and education.
Summary/Conclusion: Specific clinical features plus nEV HMGB1, an inflammatory
marker, were the best predictors of cognitive impairment. Previous published data
showed nEV
p-tau-181 elevated in Alzheimer’s disease and in this study p-tau had no importance
in
assessing HIV-associated cognitive impairment. nEV target discovery can be improved
to
better identify neuronal damage, possibly to differentiate other neurodegenerative
diseases
and hopefully recovery after therapies are identified.
Funding: NIMH R21MH112483 (LP)
OT11.6
Who comes out first: Extracellular Vesicles (EV) or
Viruses?
Fatah Kashanchi
a, Yuriy
Kimb, Daniel Pintob, Gifty Mensahb, Maria
Cowenb, James Ericksonc, Michelle Pleetc, Catherine
DeMarinob and Renaud Mahieuxd
aLaboratory of Molecular Virology, School of Systems Biology,
George Mason University, Manassas, VA, Manassas, USA; bGeorge Mason University,
Manassas, USA; cGeorge Mason University, MANASSAS, USA; dSuperieure de
Lyon, Université, Lyon, France
Introduction: In recent years, we have been able to separate and characterize
Extracellular Vesicles (EVs) from several different viruses including HIV-1, HTLV-1,
Rift
Valley Fever Virus and Ebolavirus. However, to date it is not clear whether there
is a
timing difference between EV and virus release from infected cells.
Methods: EV isolation by Nanoparticle capture and differential centrifugation,
EV quantification by Nanoparticle tracking analysis, Western Blot, RT-qPCR, virus
rescue
assay.
Results: We have attempted to address the kinetics of EV and virus release
from multiple-infected cells using serum starvation experiments from infected (100%)
cells.
These infected cells were initially put in G0 quiescent stage using serum starvation.
Both
supernatants and cell pellets were collected post-induction release (20% FBS + PMA/PHA)
at
0, 3, 6, 12, and 24 hours and examined for the presence of EV, autophagy and viral
proteins
as well as viral RNA expression. Results from supernatants of uninfected cells showed
a peak
of tetraspanin proteins (CD63, CD81, and CD9) at 6 hours and a gradual decrease of
all EV
associated proteins by 24 hours. However, the EV from HIV-1 infected cells showed
all three
tetraspanins present at 3 hours and expression gradually increased up to 24 hours.
When
compared to HTLV-1 infected cells, the three tetraspanin proteins peaked at 6 hours
and
expression continued to decrease up to 24 hours. HTLV-1 infected cells also showed
a unique
pattern of CD81 expression. Autophagy associated proteins (LC3A, LC3B and p62) from
uninfected cells and HTLV-1 infected cells plateaued at 6 hours, whereas in HIV-1
infected
cells their expression continued to increase and peaked at 24 hours. HIV-1 viral proteins
(p24, gp120, Nef) expression was present at 6 hours and continued to increase and
peaked at
24 hours. HTLV-1 proteins (p19 and gp46/61) peaked at 6 hours and gradually decreased
overtime. HIV-1 and HTLV-1 RNA gene expression analysis was performed, and data correlated
with viral protein expression. Additionally, EVs release was quantified and showed
significant increase of EV concentration overtime in both uninfected and infected
samples.
Finally, experiments of infectivity from 6- and 24-hour supernatants were performed
on three
naive cells. HIV-1 supernatant 6- hour sample was found not to be infectious. However,
HIV-1
was successfully rescued from 24-hour sample.
Summary/Conclusion: Collectively, our data indicates that EV release may occur
prior to viral release in infected cells, thereby implicating a potentially significant
effect of EVs on uninfected recipient cells prior to subsequent viral infection.
Funding: This work was supported by National Institutes of Health (NIH) grant
NS099029.
OP1 = PT15 Oral with Poster Session 1: Lumgs Chair: Uta
Erdbrügger – University of Virginia Chair: Peter Kurre, MD – Comprehensive Bone Marrow
Failure Center, Children’s Hospital of Philadelphia; Perelman School of Medicine,
University of Pennsylvania
OP1.01 = PT15.01
Human urinary extracellular vesicles carry surface markers that
are indicative of haematopoietic origin
Veronika Mussack
a and Michael
Pfafflb
aTUM School of Life Sciences, Dept. Animal Physiology and
Immunology, Freising, Germany; bDivision of Animal Physiology and Immunology,
Technical University of Munich, Freising, Germany, Freising, Germany
Introduction: Urinary extracellular vesicles (uEVs) are important
intercellular communicators. By systems biology integration, uEVs prove to be relevant
in
genitourinary disease detection. However, it has recently been shown that labelled
EVs
administered to the circulation can be detected in the urinary system, as well. Thus,
this
pilot study aimed at phenotyping haematopoietic surface markers on uEVs to create
enough
plausibility for future non-invasive biomarker studies of circulation and immune disorders
that may translate into urine but are not yet timely recognized.
Methods: Urine was obtained from healthy men signing a written informed
consent (n = 31). Sampling was approved by the local ethics committee and in compliance
with
the Declaration of Helsinki. Cell-free urine was obtained by serial centrifugation
and
10 ml, each, were utilized for the MACSPlex Exosome Kit, human (Miltenyi Biotec).
The
manufacturer’s recommendations were followed to examine 37 distinct uEV surface markers
of
CD9+/CD63+/CD81+ vesicles in a multiplexed bead-based manner including respective
controls.
The Accuri C6 (BD) was utilized for data acquisition.
For further MISEV2018-compliant characterization, CD9+/CD63+/CD81+ uEVs were isolated
by
immunoaffinity and analysed by fluorescence nanoparticle tracking (f-NTA), transmission
electron microscopy (TEM) and western blotting (WB).
Urinary creatinine (Ucrea) was determined to control for variances in urinary dilutions
and
used for data normalization.
Results: Except CD209, all other 36 surface markers could be identified. The
most abundant markers were CD9 and CD63, which were detected in 93% of samples, followed
by
CD133/1 (84%), CD326 (81%), CD81 and CD24 (77%, each). CD3 (42%), CD9, CD45 (39%),
CD49e
(32%) and CD81 showed similar relative median fluorescent intensities (rMFI), while
CD63
yielded significantly higher (p = 0.009) and all other markers significantly lower
rMFI
(p < 0.011).
TEM and f-NTA revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 E + 10 particles/g
Ucrea. WB indicated uEV isolates that were positive for Alix, Syntenin, TSG101, CD9,
CD63
and CD81 without any uromodulin or calnexin contamination.
Summary/Conclusion: Our results imply that considerable quantities of
circulatory EVs are, indeed, filtered into urine and could serve as valuable non-invasive
biomarkers for systemic dysfunctions.
OP1.02 = PT15.02
Cardiovascular risk markers are strongly related to numbers of
circulating extracellular vesicles
Ruihan Zhou
a,Esra
Bozbasa, Plinio Ferreirab and Parveen Yaqooba
aUniversity of Reading, Reading, UK; bImperial
College London, London, UK
Introduction: Extracellular vesicles (EVs) are small plasma membrane-derived
vesicles released from various cells, which potentially affect many physiological
and
pathophysiological processes, and are emerging as a potential novel biomarker in
cardiovascular diseases (CVDs). However, there is little information about the association
of circulating EV levels with traditional cardiovascular risk markers and CVD risk
score.
Methods: • Subjects (n = 40) aged 40–70 yrs with moderate risk of CVDs were
recruited and assessed for body mass index (BMI), blood pressure (BP) and plasma lipid
profile (triacylglycerol, total cholesterol and high-density lipoprotein).
• EVs were isolated from platelet-free plasma by size exclusion chromatography and
analysed
by both Nanoparticle Tracking Analysis (NTA) and flow cytometry (FCM). NTA was used
to
measure the concentration and size distribution of EVs population, and EVs were phenotyped
by FCM via a 3-colour panel, which included Annexin V (for the majority of circulating
EVs),
CD41 (for platelet-derived EVs) and CD105 (for endothelial-derived EVs).
• The association between risk markers and EV numbers was examined by Pearson’s correlation
coefficient and stepwise multivariate regression model. Analysis of covariance (ANCOVA)
was
performed after adjustment for various variables to determine the correlation between
the
quartile range of EV numbers and 10-yr CVD risk detected by QRISK2.
Results: EV numbers, as determined by NTA, were strongly associated with BMI
(r = 0.602, p < 0.001), blood pressure (systolic BP: r = 0.359, p = 0.023; diastolic
BP:
r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). Plasma
total cholesterol level was positively associated with platelet-derived EVs, determined
by
FCM (r = 0.330, p = 0.038). A multivariate regression model demonstrated that plasma
triacylglycerol and diastolic BP independently predicted total EV numbers, with plasma
triacylglycerol concentrations explaining 49.4% of the variance for total EV numbers.
An
additional 9.3% of the variance in total EV numbers was predicted by diastolic BP.
ANCOVA of
the 10-yr CVD risk score in the quartile range of total EV numbers were positively
and
independently associated.
Summary/Conclusion: BMI, blood pressure, plasma triacylglycerol and total
cholesterol levels are strongly associated with EV numbers. Plasma triacylglycerol
and
diastolic BP independently predict circulating EV numbers. Elevated numbers of EVs
are
independently associated with 10-yr CVD risk.
Funding: This project is supported by Biotechnology and Biological Sciences
Research Council (BBSRC)-Diet and Health Research Industry Club (DRINC) in the UK.
OP1.03 = PT15.03
Extracellular vesicles from cardiosphere-derived cells
potentiate regulatory T cells
Akbarshakh Akhmerov, Geoffrey de Couto,
Russell G. Rogers, Ahmed Ibrahim, Weixin Liu, Lizbeth Sanchez and Eduardo Marbán
Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Introduction: Extracellular vesicles from cardiosphere-derived cells (CDC-EVs)
are known to be anti-inflammatory in various disease models. To further dissect the
mechanism, we examined the effects of CDC-EVs on T lymphocytes.
Methods: Naïve CD4 + T cells were isolated from secondary lymphoid organs of
Foxp3-RFP reporter mice, using magnetic-activated and fluorescence-activated cell
sorting.
Cells were subsequently polarized into effector subtypes (Th1, Th2, and Th17), as
well as
regulatory T cells (Tregs), and the effects of exposure to human-derived CDC-EVs on
proliferation and cytokine production were assessed. CDC-EVs were isolated from serum-free,
15-day conditioned medium, using ultrafiltration by centrifugation.
Results: After polarization and culture for 5 days, CDC-EVs resulted in
dose-dependent and cell-specific proliferative responses. Effector T cells (Th1, Th2,
Th17)
showed either no change in proliferation (Th1) or decrease in proliferation (Th2,
Th17),
compared to the vehicle control. In contrast, Tregs proliferated much more than control
(P < 0.0001). Next, we sought to characterize the changes in cytokine production by
each
effector T cell and Tregs. Compared to the vehicle control, exposure of polarized
effector T
cells to CDC-EVs had little effect on the expression of characteristic cytokine genes,
including Ifnγ and Tnfα (Th1), Il4 and Il13 (Th2), or Il17a and Il17 f (Th17). In
contrast,
exposure of Tregs to CDC-EVs resulted in ~1000-fold increase in expression of Il10,
a key
paracrine agent utilized by Tregs in suppression of inflammation. This response was
specific
to CDC-EVs insofar as it was not recapitulated with dermal fibroblast exosomes.
Concentrations of IL-10 in the culture media of CDC-EV-conditioned Tregs mirrored
the
increases in gene expression.
Summary/Conclusion: CDC-EVs potentiate Tregs by increasing their proliferation
and enhancing production of IL-10. This offers an attractive therapeutic approach
to
inflammatory diseases that relies on harnessing an endogenous mechanism of
immunosuppression.
Funding: NIH T32HL116273.
OP1.04 = PT15.04
Prostanoids impair platelet reactivity, thrombus formation and
platelet extracellular vesicle release in patients with pulmonary arterial
hypertension
Aleksandra Gąsecka
a, Marta
Banaszkiewiczb, Rienk nieuwlandc, Edwin van der Pold,
Najat Hajjie, Hubert Mutwilf, Sylwester Rogulaa, Wiktoria
Rutkowskaa, Szymon Darochag, Grzegorz Opolskia, Krzysztof
J. Filipiakf, Adam Torbickig and Marcin Kurzynag
a1st Chair and Department of Cardiology, Medical University
of Warsaw, Warsaw, Poland; bDepartment of Pulmonary Circulation, Thromboembolic
Diseases and Cardiology, Centre of Postgraduate Education Medical, European Health
Centre
Otwock, Poland, Warszawa, Poland; cDepartment of Clinical Chemistry, Amsterdam
UMC, University of Amsterdam, Amsterdam, the Netherlands, Vesicle Observation Center,
Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands;
dDepartment of Clinical Chemistry, Amsterdam UMC, University of Amsterdam,
Amsterdam, the Netherlands; Vesicle Observation Center, Amsterdam UMC, University
of
Amsterdam, Amsterdam, the Netherlands; Department of Biomedical Engineering and Physics,
Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands;
eLaboratory of Experimental Clinical Chemistry, Vesicle Observation Center,
Amsterdam UMC, Locatie AMC, University of Amsterdam, Amsterdam, Netherlands; f1st
Chair and Department of Cardiology, Medical University of Warsaw, Warszawa, Poland;
gDepartment of Pulmonary Circulation, Thromboembolic Diseases and Cardiology,
Centre of Postgraduate Education Medical, European Health Centre Otwock, Poland, Warsaw,
Poland
Introduction: Prostanoids (epoprostenol, treprostinil and iloprost) induce
vasodilation in advanced pulmonary arterial hypertension (PAH) but also inhibit platelet
activation, thereby increasing the risk of bleeding. Therefore, the platelet function
and
extracellular vesicle (EV) concentrations were measured in PAH patients treated with
prostanoids and compared to patients with PAH not receiving prostanoids.
Methods: Venous blood was collected from 42 patients treated with prostanoids
(study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with
prostanoids (control group; n = 38, 55 ± 19 years, 65% female). Platelet reactivity
was
analysed in whole blood by impedance aggregometry using arachidonic acid (AA; 0.5 mM),
adenosine diphosphate (ADP; 6.5 µM) and thrombin receptor-activating peptide (TRAP;
32 µM)
as agonists. In a subset of patients, concentrations of EVs from platelets (CD61+ and
CD62p+; PEVs), leukocytes (CD45+, LEVs) and endothelial cells (CD146+, EEVs) were
measured
in platelet-depleted plasma by flow cytometry (A60-Micro). Platelet-rich thrombus
formation
was measured using a whole blood perfusion system.
Results: Compared to the control group, patients treated with prostanoids had
lower platelet reactivity in response to AA and ADP (p = 0.01) and lower concentrations
of
PEVs and LEVs (p ≤ 0.05). Furthermore, thrombus formation was delayed (p ≤ 0.003)
and
thrombus size was decreased (p = 0.008) on prostanoids. Epoprostenol did not affect
platelet
reactivity in vitro, but decreased the concentrations of CD61+ PEVs (p = 0.04). In
contrast,
treprostinil and iloprost decreased both platelet reactivity in response to AA and
ADP
(p ≤ 0.05) and the concentrations of PEVs (p ≤ 0.08). All prostanoids delayed thrombus
formation and decreased thrombus size (p ≤ 0.04).
Summary/Conclusion: Patients with PAH treated with prostanoids have increased
risk of bleeding both due to impaired platelet aggregation, EV release and thrombus
formation, compared to patients not treated with prostanoids. Antiplatelet effect
of
prostanoids varies: whereas epoprostenol decreases the release of PEVs, treprostinil
and
iloprost impair platelet aggregation.
Funding: AG is supported by the National Science Centre, research project
PRELUDIUM 2018/31/N/NZ7/02260.
EvdP is supported by the Netherlands Organisation for Scientific Research – Domain
Applied
and Engineering Sciences (NWO-TTW), research programmes VENI 15924.
OP1.05 = PT15.05
Nanoflow cytometry identifies an imbalance of epithelium- and
neutrophil-derived extracellular vesicles in the airway environment of paediatric
cystic
fibrosis patients
Brian Dobosh, Vincent Giacalone, Milton Brown,
Lucas Silva, Lokesh Guglani and Rabindra Tirouvanziam
Emory University, Atlanta, USA
Introduction: Progressive lung disease is the leading cause of mortality in
cystic fibrosis (CF), a chronic condition characterized by recruitment of polymorphonuclear
neutrophils (PMNs) into the airways. Newly arrived PMNs are exposed to extracellular
vesicles (EVs) from the airway epithelium and PMNs recruited before them. In controlled
experiments, these EVs were necessary and sufficient to induce pathological changes
including reduced bacterial killing and immunosuppressive activities towards macrophages
and
T-cells. However, children with CF do not always show a high PMN presence in their
airways,
which suggests that the balance between PMN recruitment and the activity of other
cells is
still in flux in early stage disease.
Methods: We utilized spectral nanoflow cytometry to profile the single EV
content of the bronchoalveolar lavage fluid (BALF) from 17 CF children (<6 years of
age).
For nanoflow cytometry, EVs were stained with Di-8-ANEPPs, and with EpCAM, CD66b and
CD115
(to ascertain epithelial, PMN, and macrophage origins, respectively). Violet side
scatter
and/or fluorescence threshold triggering were used for EV detection.
Results: The ratio of neutrophil- to epithelial-derived EVs in CF BALF
correlated positively with the percentage of PMNs that are present in the airways
(p = 0.003, Spearman’s rho = 0.689). This ratio also correlated with the PRAGMA disease
score, which quantifies airway damage by chest computed tomography (p = 0.001,
rho = 0.857).
Summary/Conclusion: Using a method to quantify EVs from specific cell types in
vivo, we demonstrated that the ratio of PMN- and epithelial cell-derived EVs tracks
with
airway damage and neutrophil influx, suggesting a critical interplay between these
cells in
early CF disease. This EV-focused method can be applied to other diseases in which
sampling
cells is difficult. Future experiments will use CF BALF biobanks to strengthen data
presented here.
Funding: CF Foundation (TIROUV15A0), Emory Pediatrics Flow Core.
OP1.06 = PT15.06
The potential of crude extracellular vesicle microRNAs for the
diagnosis of community-acquired pneumonia and for the detection of pneumonia-related
sepsis as a severe secondary complication
Stefanie Hermann
a, Florian
Brandesb, Benedikt Kirchnerc, Dominik Buschmanna, Melanie
Borrmannb, Matthias Kleind, Stefan Kotschotee, Michael
bonine, Marlene Reithmairf, Gustav Schellingb and
Michael Pfaffl
c
aDivision of Animal Physiology and Immunology, School of Life
Sciences Weihenstephan, Technical University of Munich, Freising, Germany, Freising,
Germany; bDepartment of Anesthesiology, University Hospital,
Ludwig-Maximilians-University Munich, Munich, Germany, Munich, Germany; cDivision
of Animal Physiology and Immunology, Technical University of Munich, Freising, Germany,
Freising, Germany; dDepartment of Neurology, University Hospital,
Ludwig-Maximilians-University Munich, Munich, Germany, Munich, Germany; eIMGM
Laboratories GmbH, Planegg, Germany, Planegg, Germany; fInstitute of Human
Genetics, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany,
Munich, Germany
Introduction: Circulating cell-free microRNAs (miRNAs), often associated to
extracellular vesicles (EVs), are essential for cell-cell communication in the pathogenesis
of infectious pulmonary disorders. As early pneumonia diagnosis is often clinically
challenging, advances in disease detection could improve outcomes. We characterized
crude EV
miRNAs as potential biomarkers for community-acquired pneumonia and sepsis as a severe
secondary complication.
Methods: 142 individuals were enrolled into our study, subdivided into a
training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer
n = 20, pneumonia n = 18, sepsis n = 37). After precipitating crude EVs from sera
(miRCURY
Exosome Isolation Kit-Serum and Plasma) and extracting total RNA, small RNA sequencing
was
performed. miRNAs were selected as biomarker candidates by differential gene expression
analysis (DESeq2) and sparse partial-least-squares discriminant analysis (mixOmics).
Technical and biological validation was performed by reverse transcription quantitative
real-time PCR. Group classification was predicted by partial-least-squares discriminant
analysis. Gene targets and causal networks were identified by ingenuity pathway
analysis.
Results: Differential gene expression analysis revealed 29 significantly
regulated miRNAs in pneumonia compared to volunteers, and 25 miRNAs in pneumonia related
to
sepsis. Based on sparse-partial least discriminant analysis, group separation was
achieved
by 12 miRNAs as discriminators with high sensitivity and specificity (area under the
curve
of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992).
miR-193a-5p (log2FC = 1.86, padj = 1.49E-6) and miR-542-3p (log2FC = 1.67, padj = 3.29E-5)
differentiated between pneumonia and volunteers and miR-1246 (log2FC = −2.41,
padj = 1.78E-04) between pneumonia and sepsis. Expression levels of miR-193a-5p and
miR-1246
were related to disease severity. miR-542-3p was higher expressed in pneumonia compared
to
volunteers and had equal expression in patient groups. Prediction of group classification
in
the testing cohort was 73.33%. Signalling networks were constructed for “cellular
and
humoral immune response”, “antimicrobial response” and “pathogen influenced signaling”
involving the significantly regulated miRNAs.
Summary/Conclusion: Crude EV miRNAs are potentially novel biomarkers for
community-acquired pneumonia and may help to identify patients at risk for progress
to
sepsis allowing early intervention and treatment.
Funding: This work was supported by a grant from the German Federal Ministry
for Economic Affairs and Energy (protocol number ZF4247001MD6).
OP1.07 = PT15.07
microRNA exosome cargo from induced sputum: new tool for
approaching asthma research
David Sanz Rubio
a, Ana Rosa
Remachab, Laura Pastorb, Elisabeth Verab, Pablo
Cuberob, Jose María Marinc, Victoria Gilb and Marta
Fornerd
aIIS Aragón, HCU Miguel Servet, Zaragoza, Spain;
bIIS Aragón, HCU Miguel Servet, Zaragoza, Spain; cIIS Aragón, HCU
Miguel Servet, University of Zaragoza, Zaragoza, Spain; dCIBERES, HCU Miguel
Servet, Zaragoza, Spain
Introduction: It remains unclear the specific mechanisms that lead to airways
inflammation in asthma and the subsequent remodelling of the airways. Exosomes, small
extracellular vesicles, has become in an important mechanism of cell-to-cell communication
and participate in diverse biological processes including inflammation. In this study,
we
hypothesize that exosomes and their miRNA cargo play an important role in the
proinflammatory status of the upper airway of asthma patients, especially in those
patients
with severe asthma.
Methods: In a pilot study,3healthy subjects had induced sputum using standard
methods. After several centrifugation steps, we were able to isolate exosomes from
sputum
supernatant by both precipitation and Size Exclusion Cromatography (SEC). Exosome
size was
observed with Transmission Electron Microscopy (TEM) and the protein markers CD63
and CD81
were analysed by Western Blot (WB). Then, total RNAs were isolated from sputum exosomes
and
9 miRNAs (miR-103a-p, miR-191-5p, miR-320a, miR-200b-3p, miR-185-5p, miR-223-3p, miR-21-5p,
miR-155-5p, let-7 g-5p), were evaluated by RT-qPCR. After the optimization of the
methodology, 10 healthy adults subjects and 10 patients with persistent moderate-severe
asthma, matched by age and sex were selected and induced sputum was collected.
Results: Exosomes isolated with both methodologies (precipitation and SEC)
were observe under the TEM with a correct range of size. Furthermore, WB assay displayed
a
coherent protein profile for the exosome markers CD63 and CD81. However, SEC displayed
low
signal and the variability of between subjects was to higher. Using the optimized
method of
precipitation, we observed that after normalization, miRNA-320a showed a significant
increased (p = 0.02) in asthma patients compared to control. This miRNA has been linked
with
an active proinflammatory status.
Summary/Conclusion: Our results confirm the presence of exosomes in induced
sputum with promising applications in the field of asthma. The upregulation of exosomal
miR-320a, which is related with inflammation, suggest that exosomes could play a crucial
role in the chronic inflammation of airway described in asthma patients.
OP1.08 = PT15.08
Human nrf2-active multipotent stromal cell exosomes reverse
pathologic delay in the healing of cutaneous diabetic wounds
Joseph Kuhn
a, Absara
Hassanb, Sonali Sharmab, Jennifer Kwongb, Montaha
Rahmanb, Salma Adamb, Jasmine Leeb, Alvaro Villarreal
Ponceb and Piul Rabbanib
aNYU Langone Health, New york, USA; bNYU Langone
Health, New York, USA
Introduction: Multipotent stromal cells (MSCs) have attracted much attention
for their capacity to accelerate wound healing. Exosomes, nanosized extracellular
vesicles,
may be key to translating MSC therapy. We previously found that nuclear factor erythroid
2-related factor 2 (Nrf2) regulates MSC promotion of diabetic tissue repair. Here,
we
explore a novel role of Nrf2 in exosome biogenesis and investigate whether exosome
treatment
recapitulates the effects MSCs have on healing.
Methods: Exosomes were harvested by differential ultracentrifugation of
conditioned bone marrow derived MSC media. For Nrf2-active exosomes, MSCs were incubated
with potent Nrf2 activator, CDDO-Im. Exosomes and MSCs were vigorously characterized.
Full-thickness humanized-stented wounds were created on adult Leprdb/db diabetic mice
(db/db). Exosomes were injected intradermally and circumferentially to the wound margin.
Results: MSCs adopt an adherent fibroblast morphology, demonstrate robust
osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive MSC
markers (CD44, CD73, CD90, and CD105) and <5% express negative markers (CD45, CD31,
CD14,
CD19, or HLA-DR). Immunoblotting of MSC exosomes shows enrichment for positive exosomal
markers CD81, CD9 and TSG101. Nanoparticle tracking analysis (NTA) shows a nanoparticle
population with mean diameter of 168.0 ± 6.5nm. Transmission electron microscopy of
exosomes
reveals flattened cup-like spheres. NTA demonstrates that Nrf2-active human MSCs increase
exosome secretion by 54%, compared to Nrf2-baseline MSCs (p < 0.05). Both Nrf2-baseline
and Nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days
respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05). This reduction
eliminated the delay in closure time compared to wounds of C57/B6 mice. Nrf2-active
exosome
treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated
C57/B6 wounds. At day 10, exosome-treated db/db wounds have significant decreases
in
epithelial gap, expanded granulation tissue, and greater density of CD31+ vessels
compared
to vehicle-treated wounds.
Summary/Conclusion: Enhancing Nrf2 function in MSCs multiplies exosome yield.
Our results demonstrate exosome-based therapies hold tremendous promise and warrant
further
investigation for rapid translation.
Funding: PSF Pilot translational grant, WHS 3 M Fellowship, NYU CTSI
Translational Pilot Project
OP1.09 = PT15.09
Extracellular vesicles from adipose tissue end endothelial cells
of obese humans share miRNA cargos and increase prostate cancer aggressiveness in
conjunction with Twist1
Allison M. Mathiesen, Ryan Huyck, Bronson
Haynes, William McPheat and Anca D. Dobrian
Eastern Virginia Medical School, Norfolk, USA
Introduction: Obesity increases prostate cancer aggressiveness and adipose
tissue (AT) is a rich source of extracellular vesicles (EV) that have been shown to
contribute to pro-oncogenic effects in various malignancies. Twist1 is a key mediator
of
tumour cell metastasis.. The goal of this study was to determine molecular and phenotypic
changes of prostate cancer cells in response to EVs from obese human AT and the role
of
different levels of endogenous Twist1.
Methods: EV were harvested from human AT (ATEV) obtained from bariatric
subjects or from AT endothelial cells treated with proinflammatory cytokines (PIC-EV)
to
mimic the obese AT environment. EVs were isolated by ultracentrifugation and characterized
by electron microscopy, NTA and protein markers. We determined the effect of ATEV
and PIC-EV
on PC3-ML prostate cancer cells proliferation and invasion. EV miRNA cargo and transcriptome
of PC3-ML cells treated with ATEV or PIC-EV were assessed using NanoString. To establish
the
contribution of Twist to the EV-related phenotypic and molecular changes in recipient
cells,
we used PC3-ML lines stably overexpressing or deficient in Twist1.
Results: ATEV from obese subjects and EV-PIC from AT endothelial cells both
reduced invasion and increased proliferation in wild-type PC3-ML cells. A molecular
signature showing decreased expression of genes mediating invasion, adhesion and metabolism
supported these functional effects. Also ATEV and EV-PIC shared a subset of miRNA
that
target multiple MMPs, inhibit glycolytic genes and target cell cycle inhibitory genes.
PC3-ML overexpressing Twist1 showed an increase in both proliferation and invasiveness
and
this phenotype was supported by the transcriptomic analysis following EV treatment.
Summary/Conclusion: EV produced by obese AT or by AT endothelial cells share a
subset of miRNA that in conjunction with increased Twist1 expression contribute to
tumorigenesis and metastasis of prostate cancer cells in vitro.
Funding: American Heart Association
OF12
Symposium Session 12: Biodistribution & Antiviral Defence
Chair: Lorraine O’Driscoll – School of Pharmacy and Pharmaceutical Sciences, Trinity
College Dublin
Chair: Ana Claudia Trocoli Torrecilhas – Department of Biological Sciences,
Universidade Federal de Sao Paulo
OF12.1
Development of non-invasive clinically applicable in vivo
tracking of extracellular vesicles using magnetic resonance imaging (MRI)
Johnny Akersa, Paola Aguiarib, Hamsik
Soloyanb, Seda Mkhitaryanb, Gevorg Karapetyanb, Laura
Perinc, Mya Thuc and Sargis
Sedrakyan
c
aVisicell Medical, San Diego, USA; bChildren’s
Hospital Los Angeles, Los Angeles, USA; cChildren’s Hospital Los
Angeles-University of Southern California, Los Angeles, USA
Introduction: As researchers continue to explore the therapeutic potentials of
extracellular vesicles (EVs) for the treatment of many diseases, there is a growing
unmet
need for real-time in vivo monitoring of these therapeutic EVs after they are injected
into
a subject to understand their safety, targeting, and effectiveness. While current
optical
imaging solutions like bioluminescence and fluorescence are useful for EV tracking
studies
in animal models, there is limited utility in clinical applications. Here we present
a novel
EV tracking solution utilizing clinically applicable MRI technology.
Methods: To generate trackable EVs, cells were labelled with a clinically
applicable novel magnetic agent. EVs secreted by the labelled neural stem cells and
amniotic
fluid stem cells (AFSCs) were isolated by differential ultracentrifugation. The viability
and morphology of labelled-cells were evaluated, and the in vitro MR properties of
their
derived EVs were analysed by magnetometer. A proof of concept in vivo biodistribution
study
was conducted by injecting labelled EVs into WT and Alport mice (a model of chronic
kidney
disease) via retro-orbital and intra-cardiac routes and tracking them via MRI at 10
min and
3 hr post-injection.
Results: The magnetic label did not affect the physiological characteristics
of the cells. The MR detectability of labelled-EVs was confirmed by in vitro/ex vivo
MRI
phantoms. MRI studies showed that homing of AFSC EVs to the kidney injected
intra-cardiacally into Alport mice were more efficient versus the retro-orbital route,
and
Prussian blue staining of kidney sections confirmed the MR findings.
Summary/Conclusion: We have developed a clinically applicable novel magnetic
nanoparticle agent that can be used to label and track the biodistribution of EVs
in living
subjects using non-invasive, safe, and effective MRI technology that’s widely available.
This technology is highly adaptable and can be deployed in both preclinical and clinical
settings.
Funding: Children’s Hospital Los Angeles Small Animal Imaging Core Pilot
Grant
OF12.2
EV biodistribution studies with a novel non-invasive, dynamic in
vivo imaging method reveal diverse organ reactions to circulating EV: No EV retention,
EV
retention, and active EV recruitment
Thomas Viela, Dominique Charuea, Marie Le
Hoanga, Jean-Michel Guignerb, Bertrand Tavitiana, Chantal
Boulangera and Olivier P. Blanc-Brude
a
aUniversité de Paris, Paris Cardiovascular Research Center,
Inserm, Paris, France, Paris, France; bIMPMPC – Université Pierre & Marie
Curie, Paris, France
Introduction: A central question in EV biology is the fate of circulating EV.
This can be evaluated by developing non-invasive EV bioimaging techniques in mice
in order
to benefit from transgenic and knock-out models. Recent reports described EV biodistribution
in vivo using optical (fluorescence) and nuclear imaging. But the physicochemical
properties
of the probes impact EV integrity, labelling efficiency, background signals and observation
timecourse.
Methods: We developed the radiolabeling of red blood cells (RBC) and EV with
[18 F]fluorodeoxyglucose (18 F-FDG). We used RBC-derived EV in their native, intact
form,
without pre-experimental processing (no centrifugation or filtration). We tracked
18 F-FDG
in vivo by PET-Scan, within seconds of EV, RBC or free 18 F-FDG injection, and during
their
dissemination in blood and recruitment by organs over one hour. EV and RBC biodistribution
were confronted to the kinetics of free 18 F-FDG.
Results: We collected images of the biodistribution of RBC, and RBC-derived
EV. Nuclear imaging was well suited for accurate studies of EV organotropism, with
high
sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues
and an
inherent quantitative tomographic nature. EV-specific signals were mostly accumulated
within
minutes of injection (tail vein), in the spleen and liver, with a small part in the
bone
marrow (femurs). Signals in other compartments were largely transient and linked to
tissue
perfusion and blood volume. We selected the most drastic control conditions to secure
a
correct interpretation of the data. This made kidneys, hearts and brains unavailable
for
analysis. Hence the new approach came with limitations, but we describe how “free”
18 F-FDG
signals can be used to draw sound conclusions for EV.
Summary/Conclusion: We propose that three types of compartments coexist in
control mice at rest: Active EV-capturing organs with high capacity and specificity
including the spleen, and to a lesser degree the bone marrow; Passive EV-retaining
organs
with high capacity, including the liver; and EV-neutral organs where transient signals
only
mirror tissue perfusion. We also report how EV biodistribution patterns are altered
in
ageing animals, as an example. We hope that this novel, non-invasive, quantitative,
dynamic
whole-body imaging approach will help characterize native cell-derived EV and help
set
standards for the reproducibility of EV bioimaging in mice.
Funding: FRM grant “BiFace”, Inserm COPOC, CNRS.
OF12.3
A novel in vivo Drosophila model of vesicle-mediated
intercellular communication
Claire M. Hill
a, Elizabeth
Dellarb, Jana Terenovaa, Monika Gullerovaa, David R. F.
Carterc and Luis Alberto Baena-Lopeza
aUniversity of Oxford, Oxford, UK; bUniversity of
Oxford/Oxford Brookes University, Oxford, UK; cOxford Brookes University, Oxford,
UK
Introduction: Extracellular vesicles (EV) are important mediators of
intercellular communication; however, basic principles of EV biogenesis and loading
remain
largely unknown. A limited repertoire of tools has thus far made these processes challenging
to research. The development of an EV-transfer reporter in a genetically tractable
organism
such as Drosophila has allowed us to study mechanisms of cargo loading in vivo and
has
provided us with a platform to explore fundamental aspects of EV biology.
Methods: We have developed a bioinformatic pipeline to analyse the properties
embedded in the 3ʹUTR of mRNAs enriched in EVs released by Drosophila cells. In parallel,
we
have adapted a Cre-LoxP system for use in fruit flies that appears to be proficient
to
reveal the exchange of bioactive molecules between secretory and recipient cells.
Results: Taking advantage of computational methods, we uncovered sequence
motifs that preferentially appear in combinations along the 3ʹUTR. These sequence
motifs
occur within characteristic secondary structures, in a way that is more variable and
motif
dependent than previously reported. Identified motifs also show similarities to known
binding sites for RNA binding proteins; a feature potentially important for EV-loading.
In
parallel, we developed a Drosophila in vivo system to detect cell communication in
complex
tissues and between different cell types. Using this system, we studied the biological
significance of specific sequence motifs and identified their ability to modulate
mRNA
EV-transfer in a context dependent and evolutionarily conserved manner.
Summary/Conclusion: In summary, we have developed a novel tool to study cell
communication in complex tissues, and shown its effectiveness to study principles
of EV
biogenesis and loading. Beyond improving our understanding of EV biology and providing
a
novel tool to the scientific community, we hope this knowledge will pave the way to
harnessing EVs as a means of remotely manipulating cell communication in many biological
contexts.
Funding: CH and ED are supported by funding from the Biotechnology and
Biological Sciences Research Council (BBSRC) [grant number BB/M011224/1] as part of
the
Oxford Interdisciplinary Bioscience Doctoral Training Programme. DC is supported by
the
BBSRC (BB/P006205/1) and LAB-L by Cancer Research UK (C49979/A17516).
OF12.4
Dietary cross-species communication: context-dependent role of
bovine extracellular vesicles in cancer progression
Rahul Sanwlani
a, Monisha
Samuela and Suresh Mathivananb
aLa Trobe Institute for Molecular Sciences, La Trobe
University, Melbourne, Australia, Melbourne, Australia; bLa Trobe University,
Melbourne, Australia
Introduction: The idea of cross-kingdom, species and inter-individual transfer
of bioactive compounds via extracellular vesicles (EVs) is a recent avenue. However,
the
bioactivity and bioavailability of these dietary compounds upon consumption is highly
debated. It has been proposed that EVs from diet can absorbed by consuming organisms,
be
bioavailable in various organs and exert phenotypic changes. Milk is the most vastly
consumed beverage and is an abundant source of EVs that may act as signalosomes. Whether
these milk-derived EVs can serve as cross-species messengers and have a biological
effect on
host organism has been poorly understood.
Methods: Bovine milk-derived EVs were isolated by ultracentrifugation and
OptiPrep density gradient centrifugation. The EVs were characterised by TEM, NTA,
quantitative proteomics and RNA-Seq. EVs were orally administered to various mice
models of
colorectal, breast and pancreatic cancer. Primary tumour burden was monitored, and
the rate
of metastases was measured by imaging and qPCR. Immune cells were analysed by FACS.
Mechanistic insights were obtained using quantitative proteomics, confocal microscopy
and
biochemical experiments.
Results: We demonstrated that upon oral administration, bovine milk-derived
EVs were able to survive the harsh degrading conditions of the gut and be bioavailable
in
peripheral tissues. Interestingly, oral administration of milk-derived EVs reduced
the
primary tumour burden in various cancer models and attenuated cancer cachexia. Intriguingly,
despite the reduction in primary tumour growth, milk-derived EVs accelerated metastasis
in
breast and pancreatic cancer mice models. Timing of EV administration was critical
as oral
administration after resection of the primary tumour reversed the pro-metastatic effects
of
milk-derived EVs in breast cancer. Biochemical and quantitative proteomics analysis
highlighted the induction of epithelial-to-mesenchymal transition and senescence upon
treatment with milk-derived EVs.
Summary/Conclusion: Taken together, we were able to demonstrate the capacity
of bovine milk-derived EVs in mediating cross-species communication and regulating
cancer
progression in a context-dependent manner.
OF12.5
Bacterial membrane vesicles (MVs) – a bacterial innate defence
system against viral infection
Xiaomei Yan, Qian Niu and Ye Tian
Xiamen University, Xiamen, China (People’s Republic)
Introduction: In order to survive the constant onslaught of phage, bacteria
have evolved diverse defence mechanisms that act at every stage of the phage life
cycle. It
has been suggested that bacterial membrane vesicles (MVs) may play a key role in innate
bacterial defence against phage infection by acting as a decoy to prevent phage adsorption.
Nearly a decade has passed since MVs were first proposed as a decoy, but details of
how
bacteria utilize MVs to defend against phages remain poorly understood. Here we use
the
laboratory-built nano-flow cytometer (nFCM) to reveal details of the interaction between
MVs
and phages at the single-particle level, and to provide new insights into innate defence
mechanisms of MVs.
Methods: S. Typhimurium was used as the model system. Differential
ultracentrifugation and density gradient centrifugation were used to isolate and purify
MVs
and bacteriophage P22. Cryo-TEM was used to determine the morphologies of MVs and
phage P22.
The purity of MV isolates was validated by measuring the particle concentration before
and
after Triton X-100 treatment. Monodisperse silica nanoparticles were used as the size
reference standards to measure the size distribution of MVs via single-particle light
scattering detection. The purity of phage P22 was verified by concurrent detection
of side
scatter and fluorescence signals of single phages upon nucleic acid staining by SYTO
82.
Results: By incubating MVs and AF532-labelled P22, the number of phages
adsorbed on single MVs were accurately quantified. We found that S. Typhimurium and
MVs it
secretes express different affinity for phage P22 attachment. The binding ability
of P22 to
MVs is greater than that of bacteria. We confirmed that P22 can inject their nucleic
acids
into MVs, and these nucleic acids can be degraded by non-specific nucleases inside
MVs for
the first time. Besides, by labelling the nucleic acids of MVs with SYTO 82, we were
able to
distinguish three different subpopulations of MVs.
Summary/Conclusion: Taking advantage of the superior sensitivity of nFCM in
single-particle analysis, we developed a novel approach to the characterization of
the
interaction between MVs and phages. Our study revealed that bacteria produce MVs as
bait to
attract viral adsorption and nucleic acids injection.
Funding: This research was supported by the National Natural Science
Foundation of China (Grants 21934004, 21627811 and 21475112).
OF12.6
In vivo tracking of extracellular vesicles: comparison across
radioactive, fluorescence and bioluminescence approaches
Elisa Lázaro-Ibáñez
a, Farid N.
Faruqub, Amer Salehc, Andreia M. Silvaa, Khuloud
Al-Jamald and Niek Dekkera
aDiscovery Biology, Discovery Sciences, BioPharmaceuticals
R&D, AstraZeneca, Mölndal, Sweden; bInstitute of Pharmaceutical Science,
King’s College London, London, UK; cClinical Pharmacology & Safety Sciences,
Biopharmaceuticals R&D, AstraZeneca, Cambridge, UK; dInstitute of
Pharmaceutical Science, King’s College London, London, UK
Introduction: The development of EVs for therapeutic applications requires an
in-depth understanding of their in vivo biodistribution and pharmacokinetic profile.
In this
study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent
imaging technologies to identify the most suitable in vivo EV tracking method.
Methods: EVs were purified from Expi293 F cell supernatant by differential
centrifugation followed by iodixanol density gradient separation and further characterized
following MISEV guidelines. Engineered Expi293 F cells were used to generate EVs carrying
mCherry or NanoLuc (Nluc) proteins. The membrane of naïve EV was labelled with
Indium111(In111)-DTPA or Xenolight DiR post-EV isolation. CT26 tumour-bearing BALB/c
mice
were intravenously dosed with 1 × 1011 EVs followed by imaging at 1 h, 4h and 24h
using
SPEC/CT and IVIS systems. Tissue distribution and blood circulation profile of EVs
were
analysed from ex vivo samples up to 24h post-injection.
Results: Xenolight DiR and (In111)-DTPA were the most suitable EV labels for
live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification.
Nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal
for live imaging with limited sensitivity. mCherry EVs were found not suitable for
in vivo
tracking studies due to high background signal fluorescence. Ex vivo organ quantification
of
In111-DTPA and DiR showed that naïve EVs mainly accumulate in liver, followed by spleen,
kidneys, and lungs at 24h post-dose, with less than 5% EV exposure to the tumours.
Interestingly, Nluc-EVs accumulated mainly in the lungs, regardless of the small size
of the
particles injected and the absence of aggregation. Blood circulation profile of In111-DTPA
and Nluc EVs showed rapid clearance of vesicles from circulation, with 15% of injected
dose
detected in blood after 30 min and less than 5% after 16 h.
Summary/Conclusion: Radionuclide imaging is an excellent technology to detect
EVs in vivo and ex vivo with high resolution and sensitivity but requires advanced
infrastructure for radiolabeling. The optical methods have limited tissue penetration
and
sensitivity but can be improved with the right selection of the dye. These results
contribute to the understanding of the biodistribution and pharmacokinetics of EVs
and are
highly relevant to exploiting their potential for targeted delivery to diseased tissues
in
vivo.
OF13
Symposium Session 13: EV Characterization
Chair: Emanuele Cocucci – Comprehensive Cancer Center, Ohio State University,
USA
OF13.1
Analysis of extracellular vesicles by size exclusion
chromatography coupled with on-line fluorescence detection and microfluidic resistive
pulse sensing
Diana Kitkaa, Mihaly Juditha, Jean-Luc
Fraikinb and Zoltan Varga
c
aResearch Centre for Natural Sciences, Budapest, Hungary;
bSpectradyne, Torrance, USA; cBiological Nanochemistry Research
Group, Institute of Materials and Environmental Chemistry, Research Center for Natural
Sciences, Budapest, Hungary, Budapest, Hungary
Introduction: New methods for quantifying extracellular vesicles (EVs) in
complex biofluids are critically needed. We report the development of a new technology
combining size exclusion chromatography (SEC), a commonly used EV purification technique,
with fluorescence detection of specifically labelled EVs (Flu-SEC).
Methods: Flu-SEC was validated using red blood cell derived EVs (REVs). Size
and concentration measurements were performed by microfluidic resistive pulse sensing
(MRPS)
using the nCS1 instrument (Spectradyne LLC, USA). PE-CD235a (anti-Glycophorin A) and
Alexa647-WGA (wheat germ agglutinin) were used to label REVs. Flu-SEC experiments
were
performed on a liquid chromatography system using a Tricorn 5/200 glass column filled
with
Sepharose CL-2B gel (GE Healthcare).
Results: A log-normal size distribution was obtained for REVs with a mean
diameter of 163.5 ± 0.7 nm and standard deviation of 30.6 ± 0.6 nm. The concentration
of
REVs measured by MRPS was 3.14*10E11 particles/mL. The fluorescence chromatograms
of the REV
samples labelled with PE-CD235a and with Alexa647-WGA show the typical features of
the
separation of EVs from soluble proteins with SEC and enables the determination of
the
labelling efficiency of the markers. The linear range for quantification of EVs in
our
experiments spans over two orders of magnitude ranging from 10E9 particles/mL to 10E11
particles/mL. The LOD depends on the type of the label. In our experiments the lowest
LOD
was 10E9 particles/mL for Alexa647-WGA.
Summary/Conclusion: The results indicate that Flu-SEC is a quantitative
technique with very good linearity over a wide range of concentrations, though the
limit of
detection depends largely on the employed label (Sci.Rep. 9, 19868, 2019). Moreover,
the
ratio of EV-bound and free-antibody molecules can be also determined by Flu-SEC, which
can
be used to calculate the labelling efficiency of the used marker.
Funding: This work was supported by the National Research, Development and
Innovation Office (Hungary) under grant numbers PD 121326 and NVKP_16-1-2016-0007.
ZV was
supported by the Janos Bolyai Research Fellowship.
OF13.2
The CONAN assay: purity grade and concentration of EV microlitre
formulations by colloidal nanoplasmonics.
Paolo Bergesea, Andrea
Zendrinib
, Lucia Paolinic, Sara Busattod,
Annalisa Radeghierie, Miriam Romanoe, Marca H. M. Waubenf,
Martijn J. C. van Herwijnenf, Peter Nejsumg, Anne Boruph,
Andrea Ridolfii and Costanza Montisj
aConsorzio Interuniversitario Nazionale per la Scienza e la
Tecnologia dei Materiali, Florence, Italy; Department of Molecular and Translational
Medicine, University of Brescia, Brescia, Italy and Consorzio Sistemi a Grande Interfase,
Department of Chemistry, University of Florence, Sesto Fiorentino, Italy, Brescia,
Italy;
bDepartment of Animal Science, Food and Nutrition-Università Cattolica del
Sacro Cuore, Piacenza, Italy, Brescia, Italy; cDepartment of Molecular and
Translational Medicine, University of Brescia, Brescia, Italy, and Consorzio Sistemi
a
Grande Interfase, Department of Chemistry, University of Florence, Sesto Fiorentino,
Italy,
Brescia, Italy; dConsorzio Sistemi a Grande Interfase, Department of Chemistry,
University of Florence, Sesto Fiorentino, Italy and Department of Transplantation,
Mayo
Clinic, Jacksonville, FL, USA, Jacknsonville, USA; eDepartment of Molecular and
Translational Medicine, University of Brescia, Brescia, Italy and Consorzio Sistemi
a Grande
Interfase, Department of Chemistry, University of Florence, Sesto Fiorentino, Italy,
Brescia, Italy; fDepartment of Biomolecular Health Sciences, Faculty of
Veterinary Medicine, Utrecht University, Utrecht, Netherlands, Utrecht, Netherlands;
gDepartment of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus,
Q12 Denmark, Aahrus, Denmark; hDepartment of Clinical Medicine, Faculty of
Health, Aarhus University, Aarhus, Q12 Denmark, Aarhus, Denmark; iConsorzio
Sistemi a Grande Interfase, Department of Chemistry, University of Florence, Sesto
Fiorentino, Italy; Istituto per lo Studio dei Materiali Nanostrutturati (CNR-ISMN),
Bologna,
Italy and Department of Chemistry, University of Florence, Sesto Fiorentino, Italy,
Sesto
Fiorentino, Italy; jConsorzio Sistemi a Grande Interfase, Department of
Chemistry, University of Florence, Sesto Fiorentino, Italy and Department of Chemistry,
University of Florence, Sesto Fiorentino, Italy, Sesto Fiorentino (FI), Italy
Introduction: We will share our latest advancements in the use of colloidal
and nanoscience concepts and technologies to assign a purity grade to and quantify
microlitre volume solutions of extracellular vesicles (EVs). Control over such properties
is
constantly experienced by researchers to be critical for EV proper manipulation, engineering
and translation. However, the need for characterization methods that strike the balance
between robustness, working volume, cost and accessibility remains unmet.
Methods: The COlorimetric NANoplasmonic (CONAN) assay we developed consists of
a solution of gold nanoparticles (AuNPs) into which 1–2 μL of the EV formulation is
added.
The solution turns blue if the formulation is pure, while stays red if soluble exogenous
single and aggregated proteins (SAPs) are present. The colour shift is visible by
the naked
eye and can be quantified by conventional UV-Vis spectroscopy, providing a quantitative
index of purity and an estimation the EV molar concentration (particle number).
Results: The assay specifically targets SAPs, and not the EV-related proteins,
with a detection limit < 50 ng/μL (an order of magnitude higher resolution than the
Bradford protein assay). For pure solutions, the assay also allows for determining
the EV
number, as the colour shift is linearly dependent to the AuNP/EV molar ratio. Instead,
it
automatically reports if the solution bears SAP contaminants, thus avoiding counting
artefacts. Experiments, conducted on EV separated from Milk and Ascaris Suum culture
medium,
are repeatable, with an error below 20%.
Summary/Conclusion: CONAN proves to be robust and reliable, while displaying
appealing performances in terms of cost (inexpensive reagents, run by standard microplate
reader), working volumes (1–2 μL) and time (the procedure takes less than one hour).
The
ability to assign a quantitative purity grade is, up to date, a unique peculiarity
of this
assay. Finally, the assay is potentially extendable to all classes of natural and
artificial
lipid micro- and nanoparticles.
Funding: evFOUNDRY project, Horizon 2020 – Future and emerging technologies
(H2020-FETOPEN), ID: 801367.
OF13.3
Membrane-sensing peptides for extracellular vesicles
analysis
Marina Cretich
a, Roberto
Frigeriob, Alessandro Stradab, Greta Bergamaschib,
Marcella Chiaric and Alessandro Goric
aConsiglio Nazionale delle Ricerche (CNR), Istituto di
Scienze e Tecnologie Chimiche (SCITEC), Milano, Italy; bConsiglio Nazionale delle
Ricerche (CNR); Istituto di Scienze e Tecnologie Chimiche (SCITEC), Milano, Italy;
cConsiglio Nazionale delle Ricerche (CNR); Istituto di Scienze e tecnologie
Chimiche (SCITEC), Milano, Italy
Introduction: Small extracellular vesicles (sEV) present fairly distinctive
lipid membrane features in the extracellular environment. These include high curvature,
lipid packing defects and a relative abundance in lipids such as phosphatidylserine
and
ceramide. sEV membrane could be then considered as a “universal” marker, alternative
or
complementary to traditional characteristic surface-associated proteins.
Here we introduce the use of membrane sensing peptides as new, highly efficient ligands
to
directly integrate sEV capturing and analysis on a microarray platform.
Methods: We designed and synthesized membrane-sensing peptide ligands as
molecular baits for small EV and we demonstrate their use in a microarray platform
as
valuable alternative/complement to antibodies.
EVs from blood serum and plasma were isolated by ultracentrifugation, characterized
by TEM,
NTA, WB. Samples were analysed by label-free, single particle counting and sizing
on peptide
microarrays coupled to fluorescence co-localization immune staining with fluorescent
anti-CD9/anti-CD63/anti-CD81antibodies.
Results: Peptide microarrays were realized using a click-chemistry strategy
for optimal peptide surface orientation and used to analyse EVs from human blood.
Membrane
sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies.
In
addition to purified vesicles, peptide ligands were tested with pure serum showing
capacity
to capture EVs even from complex samples. In order to get insights into the EV-peptide
binding mechanism and verify whether it is directly mediated by the lipid membrane,
trypsin-treated EVs were captured on peptide microarrays demonstrating that binding
is not
directly mediated by surface associated proteins.
Summary/Conclusion: We introduced the use of membrane sensing peptides as a
novel class of molecular ligands for integrated sEV isolation and analysis, reporting
for
the first time on peptide microarrays for extracellular vesicles. Given their affinity
to
the membrane of small EV, these molecules can serve as general baits, enabling vesicles
capturing unbiased by differential surface protein expression. These new class of
molecular
probes may be integrated with the use of protein markers towards improved small EV
isolation
and characterization. Compared to proteins and antibodies, peptides are characterized
by low
cost of preparation, remarkable stability and ease of chemical manipulation, offering
virtually unlimited possibilities for experimental design. We anticipate that this
new class
of ligands, may greatly enrich the molecular toolbox for EV analysis.
Funding: HYDROGEX (Regione Lombardia&Fondazione Cariplo, grant n.
2018–1720) and INDEX (European Union’s Horizon 2020 research and innovation programme
under
Grant Agreement N° 766466) projects are acknowledged for partial financial support.
OF13.4
High-resolution size-based profiling and morphological analysis
of extracellular vesicles by scanning electron microscopy
Sara Cavallaro
a, Federico
Pevereb, Petra Håågc, Kristina Viktorssonc, Rolf
Lewensohnc, Jan Linnrosa and Apurba Devb
aKTH Royal Institute of Technology, Stockholm, Sweden;
bUppsala University, Uppsala, Sweden; cKarolinska Institute,
Stockholm, Sweden
Introduction: Extracellular vesicles (EVs) have been found to mediate
intercellular communication in physiological and pathological processes. Nevertheless,
the
understanding of EVs bio-functionality remains elusive, mainly because of their high
heterogeneity in molecular content, but also in size (30–2000 nm). Therefore, accurate
size
measurements of EVs are highly desired, particularly for exploiting their full
diagnostic/therapeutic potential.
Currently available techniques, such as Nanoparticle Tracking Analysis (NTA), cannot
accurately measure EVs smaller than 70 nm and are not capable to distinguish them
from
protein aggregates. On the contrary, electron microscopy (EM) techniques allow
high-resolution size-profiling and morphological analysis of EVs over their whole
size
range. However, their low throughput combined with several long preparatory steps
have
prevented EM from being routinely used for EV size profiling.
Methods: We shall present a method improvement in throughput and
reproducibility of EV size-analysis by scanning EM (SEM). The technique is based on
covalent
EV capture onto a silicon wafer, using the protocol reported by Cavallaro et al. up
to the
Glutaraldehyde step. After immobilization, Critical Point Drying (CPD) is performed
to
dehydrate EVs before SEM, while preserving their shapes.
Results: SEM images, showing the comparison in densities of EVs prepared by
covalent and non-covalent coupling to substrate, indicated a good capture efficiency
of our
covalent protocol. The size distribution analysis showed good agreement between NTA
and SEM
for EVs >80 nm. For smaller EVs, SEM is more sensitive than NTA, thus more suitable
to
check the purity of EV-isolation techniques. Last, atomic-force microscopy (AFM)
measurements was also used to validate our measurements.
Summary/Conclusion: To conclude, SEM can be used to accurately estimate EV
size distributions in a wide range, overcoming the well-known limitations of NTA.
Funding: Erling Persson Foundation
OF13.5
Detailed Characterization of how different extracellular
vesicles isolation methods results in different amount of Periostin protein
association.
Carolina Balbi
a, Edoardo
Lazzarinia, Emanuel Fertigb, Mihaela Gherghiceanub, Lucio
Barilea and Giuseppe Vassallia
aCardiocentro Ticino, Lugano, Switzerland; bVictor
Babes National Institute of Pathology, Bucharest, Romania
Introduction: Extracellular vesicles (EVs) are membrane vesicles secreted into
extracellular space, by almost all cellular populations, playing a major role in
cell-to-cell communication. It has been already demonstrated that changes in luminal
or
surface protein cargos of these vesicles, may reflect the status of producing cells.
For
this reason, EVs are considered as potential biomarkers in several types of diseases
ranging
from cancer diagnosis to heart rejection.
Periostin (POSTN) is a matricellular protein associated with EVs, and its level is
considered a possible biomarker, which indicate malignancy and poor clinical outcome
in
different types of cancer.
Here we extensively characterize the presence of POSTN associated on EVs, showing
how
different isolation methods can drastically affect the amount of POSTN content in
extracellular vesicles fraction.
Methods: Serial ultracentrifugation steps or size exclusion chromatography
were used to isolate EVs from primary culture of cardiac progenitor cells. EVs were
characterize, according to MISEV guidelines, by Western Blot, NTA, FACS and CryoTEM
analysis
methods. POSTN amount, associated with EVs, was analyses by Western Blot and ELISA.
Furthermore, functional tests were performed on H9C2 cardiomyoblast cell line, treated
with
the same amount of EVs from different isolation methods; cells response were analysed
by
Western Blot.
Results: EVs, from both the isolation methods, showed TSG101, Syntenin1, CD63
positivity while GRP94 was absent. NTA showed no differences, in terms of amount and
size of
particles. By FACS analysis EVs resulted enriched with CD63, CD9 and CD81. CryoTEM
showed a
similar morphology in the two preparations with presence of protein contaminant in
the
ultracentrifuge pellet. In vitro, H9C2 treated with EVs showed activation of pFAK
after 10ʹ
of treatment, this induction was 1.5 times higher in cells treated with EVs isolated
with
ultracentrifuge compared to EVs isolated with SEC, confirming a drastic effect of
POSTN
protein contamination. Furthermore, by Phospholipase-C treatment, we found that POSTN
is
bound to EVs surface through a GPI anchor.
Summary/Conclusion: These results suggest that selection of a proper isolation
method is critically relevant in EVs studies, in particular when protein analysis
is
considered. Different isolation methods dramatically influence protein amount in
extracellular vesicles and consequentially their function. Furthermore, in this study
we
show for the first time, that POSTN is actually bound to EVs surface and not carried
in
their lumen as previously believed.
Funding: This work has been supported by The Swiss National Science Foundation
under grant N° 310030_169194.
OF14 Symposium Session 14: Extracellular RNA
Chair: Takahiro Ochiya – Tokyo Medical University
Chair: Michael Pfaffl – Division of Animal Physiology and Immunology, Technical
University of Munich
OF14.1
Extracellular vesicles – a tattletale for rare gene editing
events
Koen Breyne
a, Stefano
Ughettob and Xandra O Breakefieldc
aMGH-Harvard Medical School, Charlestown, USA;
bMGH-Harvard Medical School, charlestown, USA; cMGH-Harvard Medical
school, charlestown, USA
Introduction: Since its discovery, gene editing has provided the ability to
meticulously change genes with a profound effect on both therapeutics and molecular
research. Even with new tools constantly being developed to increase efficiency and
precision of the technique, the repair mechanisms post-gene editing are still error
prone
making it critical to detect and/or select a desired gene-corrected cell clone. Since
the
contents of extracellular vesicles (EVs) reflect the cells that produced them, if
a gene
editing event occurs, the EV cargo should contain the gene corrected products, such
as a
protein or RNA species. The catch lays in the fact that EVs are by their nature very
heterogeneous and only a small fraction of the population may harbour the gene edited
products.
Methods: We designed a CD63 construct with a genomic DNA target sequence for
detection of a desired gene editing event. The gene editing target harbours a premature
stop
codon. Only when the desired gene editing event occurs to correct stop codon truncations
by
genomic missense or frameshift mutations, is a bioluminescent signal detected, as
it then
allows the CD63 tethered luciferase reporter to be translated.
Results: This reporter detects gene corrections 2 days post-introduction of
Cas9 and a sgRNA targeting the stop codon. Next-Generation Sequencing confirmed that
the
signal resulted from 1.12% gDNA changes. Our observations highlight the sensitivity
of our
system to detect even highly inefficient non-homologous end joining repair after a
double-strand break within the target DNA. The CD63 construct also contains a membrane
surface immune affinity tag to facilitate isolation of cells that encode the full
length
reporter, excluding the non-gene edited cells, without the need for single cell FACS.
Moreover, the latter tag enables isolation of a pure EV population from these corrected
cells to be isolated in a 1–2 hr procedure from the cell media. These EVs are detectable
with luminescence if the reporter is fully expressed in the target cells. Our data
shows
that with this construct, EVs can be selected out of a heterogeneous pool of EVs that
contain RNA solely expressed in the corrected cell. The latter observation allows
the EVs
derived from corrected cells to report on RNA derived from CRISPR/Cas9 events without
the
need for cell lysis and gene sequencing.
Summary/Conclusion: A CD63 transgenic reporter protein contained in the
membrane of cells and EVs may be used to detect and select out correctly gene edited
EV-donor cells early on, reducing effort in avoiding cells with off targets effects.
OF14.2
Y-RNA subtype ratios in plasma extracellular vesicles are cell
type specific and are candidate biomarkers for inflammatory diseases
Tom Driedonks
a, Sanne
Molb, Sanne de Bruinc, Anna-Linda Petersd, Xiaogang
Zhanga, Marthe Lindenbergha, Boukje Beugere, Anne-Marieke
Stalborchf, Thom Spaang, Esther de Jongh, Erhard van der
Vriesg, Coert Margadantf, Robin van Bruggene, Alexander
Vlaarc, Tom Groot Kormelinkh and Esther Nolte-’t
Hoeni
aDept. Biochemistry & Cell Biology, Fac. Veterinary
Medicine, Utrecht University, The Netherlands, Utrecht, Netherlands; bDept.
Biochemistry & Cell Biology, Fac. Veterinary Medicine, Utrecht University, The
Netherlands, Amsterdam, Netherlands; cDept. Intensive Care, Amsterdam University
Medical Centers, location AMC, Amsterdam, The Netherlands, Amsterdam, Netherlands;
dDept. Anesthesiology, University Medical Center Utrecht, Utrecht, The
Netherlands, Utrecht, Netherlands; eDept. of Blood Cell Research, Sanquin
Research, and Landsteiner Laboratory, Amsterdam University Medical Centers, Location
AMC,
Amsterdam, The Netherlands, Amsterdam, Netherlands; fMolecular Cell Biology lab,
Dept. of Molecular and Cellular Hemostasis, Sanquin Research, and Landsteiner Laboratory,
Amsterdam University Medical Centers, Location AMC, Amsterdam, The Netherlands, Amsterdam,
Netherlands; gDept. of Infectious Diseases & Immunity, Division of Virology,
Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands, Utrecht,
Netherlands; hDept. Experimental Immunology, Amsterdam University Medical
Centers, Location AMC, University of Amsterdam, Amsterdam, The Netherlands, Amsterdam,
Netherlands; iDept. Biomolecular Health Sciences, Fac. Veterinary Medicine,
Utrecht University, Utrecht, Netherlands
Introduction: Major efforts are made to identify changes in the microRNA
(miRNA) and messenger RNA content of patient plasma to discover novel disease-associated
biomarkers. MiRNA in plasma can be associated to various macromolecular structures,
including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein
particles (RNP). Besides miRNA, plasma contains various other non-coding RNA species,
of
which some are contained in EV. Members of the Y-RNA family have been detected in
EV from
various cell types and are among the most abundant non-coding RNA types in plasma.
We
previously showed that shuttling of full-length Y-RNA into EV is modulated by TLR-activation
of EV-producing immune cells. This suggested that Y-RNAs may have potential as biomarker
for
immune-related diseases.
Methods: We separated RNA-containing structures in plasma based on differences
in size, density, and resistance to protease/RNase treatment. Using RT-qPCR, we quantified
full-length Y-RNA subtypes (Y1, Y3, Y4) in EV from various blood-related cell types
cultured
with or without LPS-stimulation. Inflammation-induced changes in Y-RNA were assessed
in
plasma samples from a human endotoxemia study.
Results: Full-length Y-RNA in plasma was mainly found in EV (early
SEC-fractions, density 1.11–1.18 g/ml). In contrast, specific miRNAs were either enriched
in
LPP (e.g. miR-122), in both EV and LPP (e.g. miR-16 and miR-21), or in EV (e.g. miR-150).
EV-enclosed full-length Y-RNA was resistant to enzymatic degradation, while LPP-bound
miRNAs
were degradation sensitive. We discovered that EV released by different blood cell
types
varied in Y-RNA subtype ratios. These ratios remained stable upon LPS-stimulation
of the
EV-producing cells. In endotoxemia plasma samples, the neutrophil-specific Y4/Y3 ratios
and
PBMC-specific Y3/Y1 ratios changed significantly during systemic inflammation. Importantly,
the plasma Y-RNA ratios strongly correlated with the number and type of circulating
immune
cells during the inflammation process.
Summary/Conclusion: Cell type specific “Y-RNA signatures” in plasma EV can be
determined without prior EV-enrichment, and may be further explored as biomarkers
to
diagnose inflammatory responses or other immune-related diseases.
OF14.3
Mining public EV Small RNA-seq data with miREV – Insights into
potential reference transcripts and abundant miRNAs
Benedikt Kirchner
a, Alex
Hildebrandt
a, Esther Nolte-’t Hoenb and Michael
Pfaffla
aDivision of Animal Physiology and Immunology, Technical
University of Munich, Freising, Germany, Freising, Germany; bDept. Biomolecular
Health Sciences, Fac. Veterinary Medicine, Utrecht University, Utrecht, Netherlands
Introduction: Although recent endeavours such as EV-TRACK or the MISEV
guidelines have made real headway in the continuing effort of improved characterization
and
standardization of extracellular vesicles (EV) methodologies, comparability between
EV
studies is still hampered by the multitude of experimental setups scientists can choose
from. Different biological contexts or EV isolation methods will likely yield distinct
miRNA
profiles, which makes choosing suitable miRNAs as references or as potential biomarkers
from
literature difficult at best. At the same time, repositories for RNA-seq data harbour
a
wealth of EV-related data, which can be utilized in creating a comprehensive miRNA
overview
to detect influences of these different experimental procedures or disease contexts.
Methods: Recent literature as well as publicly available data bases (e.g. SRA,
ENA, GEO) were searched for EV-related Small RNA-Seq data sets comprising a large
variety of
different species, tissues and diseases. Raw RNA-Seq data was processed to exclude
poor
quality data and reads mapping to other RNA species (e.g. rRNA, tRNA) before generating
standardized miRNA read counts. To account for variable analysis setups, count lists
were
normalized by 6 commonly used normalization methods for abundance analyses and potential,
stably expressed reference transcripts were evaluated by 3 different established algorithms
(geNorm, BestKeeper, NormFinder).
Results: miRNA abundances and potential miRNA references are made available in
an easy-to-use web tool (http://141.40.217.80:3838/miREV/). In a first big feasibility
study, 654 data sets focusing on blood derived EVs were implemented. After strict
filtering,
407 data sets (of 654) and 326 candidate miRNAs (of 2632) remained. Applying normalization
methods and stability algorithms, 3 miRNAs turned out to be the most stable in blood
derived
EVs: miR-30d-5p, miR-140-5p, miR-23a-3p. In the near future, another 1,200 datasets
from
over 40 published EV studies will be implemented to further validate miREV applicability.
Results can be refined by the user by filtering for, amongst others, specific
disease/treatment context, tissues of origin or isolation methods to match individual
experimental setups as close as possible. All findings (normalized read counts as
well as
potential reference transcripts) are visualized to easily facilitate comparisons and
can be
downloaded as lists for further analysis.
Summary/Conclusion: miREV represents a new and comprehensive tool for
scientists working in EV transcriptomics. By summarizing publicly available small
RNA-seq
data sets, it assists in finding suitable miRNAs as qPCR references or for biomarker
research without the need to run prior RNA-seq yourself. In the future, the miREV
framework
can be easily expanded to include data from further species or experimental setups.
OF14.4
Presence of vault RNA and proteins indicates contamination of
extracellular vesicle preparations with vault particles
Xinming Liu, Daniel Lambert and Stuart
Hunt
The University of Sheffield, Sheffield, UK
Introduction: Extracellular vesicles (EVs) contain a variety of molecular
cargo such as proteins and nucleic acids. Vault particle components (vtRNA and proteins)
have been repeatedly reported in the literature as part of the EV cargo. Here, we
assessed
the presence of vault particle components in EVs prepared by commonly used isolation
methods
to determine whether they were bona fide EV cargo.
Methods: EVs were isolated by differential centrifugation, size exclusion
chromatography (SEC) and Dynabead immunocapture. Preparations were characterised by
TEM,
NTA, and western blotting for EV markers. Abundance of vtRNAs was determined by small
RNA
sequencing and qPCR. Major vault protein (MVP) abundance was determined by western
blotting.
RNase and proteinase K protection assays were used to investigate whether vault components
were protected by EV membranes.
Results: EVs isolated by all methods were positive for EV markers, such as
CD63, CD9 and TSG101. VtRNAs and MVP co-purified with EV markers by differential
centrifugation and SEC. RNase and protease treatment of EV preparations demonstrated
that
vtRNA and MVP were not protected within the EV membrane. Immunocapture of EVs already
pelleted by ultracentrifugation showed co-purification of MVP. Whereas, EVs captured
directly from conditioned medium were not MVP-positive.
Summary/Conclusion: Commonly used isolation techniques, such as differential
centrifugation and SEC, can lead to contamination of EVs with vault particle components.
The
current study highlights the importance of following the MISEV2018 guidelines when
determining the topology of EV-associated components, or if indeed they are EV cargo
or
contaminants that have been co-purifed.
Funding: Get-A-Head Charitable Trust
China Scholarship Council
OF14.5
Staphylococcus aureus secrete immunomodulatory RNA and DNA via
extracellular vesicles
Blanca V. Rodriguez and Meta Kuehn
Duke University, Durham, USA
Introduction: Bacterial-derived RNA and DNA can function as ligands for
intracellular receptor activation and induce downstream signalling to modulate the
host
response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory
RNA and DNA by human pathogens, such as Staphylococcus aureus, and their delivery
to
intracellular host cell receptors is not well understood. Recently, extracellular
membrane
vesicle (MV) production has been proposed as a general secretion mechanism that could
facilitate the delivery of functional bacterial nucleic acids into host cells. S.
aureus
produce membrane-bound, spherical, nano-sized, MVs packaged with a select array of
bioactive
macromolecules and they have been shown to play important roles in bacterial virulence
and
in immune modulation through the transmission of biologic signals to host cells. The
present
study sought to examine the nature of the association between nucleic acids and MVs
produced
by S. aureus. We also sought to analyse the immunostimulatory potential of MV-associated
RNA
and DNA, and to evaluate receptor-mediated recognition of MV-associated RNA and DNA
molecules by innate immune cells.
Methods: By following a stringent purification protocol, we characterized the
RNA and DNA content of MVs produced by actively growing S. aureus. Nuclease protection
assays were performed to determine whether MV-associated nucleic acids are protected
from
degradation. We assessed the immunomodulatory potential of MV-associated RNA and DNA
by
treating cultured mouse macrophages with MVs and measuring the induction of Interferon-β
mRNA using qPCR.
Results: We found that S. aureus secretes RNA and DNA molecules that are
mostly protected from degradation by their association with MVs. We demonstrate that
MVs can
be delivered into cultured macrophage cells through endocytosis and subsequently stimulate
a
potent IFN-β response in recipient cells via activation of endosomal Toll-like
receptors.
Summary/Conclusion: Our findings show for the first time an MV-mediated
pathway by which S. aureus-derived immunomodulatory nucleic acids are delivered to
host
cells and activate endosomal nucleic acid receptors. This study advances our understanding
of the mechanisms by which bacterial nucleic acids are trafficked extracellularly
to trigger
the modulation of host immune responses.
OF14.6
Urinary extracellular vesicles transcriptome in diabetic kidney
disease
Om Dwivedi
a, Maija
Puhkaa, Karina A. Barreiroa, Erkka Valob, Harry
Holthofera, Per-Henrik Groopc, Carol Forsblomb,
Tiinamaija Tuomid and Leif Groopa
aInstitute for Molecular Medicine Finland FIMM, University of
Helsinki, Finland, Helsinki, Finland; bFolkhälsan Institute of Genetics,
Folkhälsan Research Center, Helsinki, Finland, Helsinki, Finland; cAbdominal
Center Nephrology, University of Helsinki and Helsinki University Hospital, Finland,
Helsinki, Finland; dHospital District of Helsinki and Uusimaa (HUS), Helsinki,
Finland, Helsinki, Finland
Introduction: Urinary extracellular vesicle (UEV) transcriptome could
potentially reflect the kidney gene expression profile and serve as virtual/liquid
biopsy.
In order to explore this possibility, we performed mRNA sequencing of UEVs from individuals
with type 1 diabetes to assess whether it can capture a “kidney enriched genes” expression
signature that could lead to novel biomarker discovery for diabetic kidney disease.
Methods: The study included 75 type 1 diabetic individuals (41
normoalbuminuric, 15 microalbuminuric and 19 macroalbuminuric). Urine samples were
collected
either overnight (N = 38) or during 24-hours (N = 37) and UEVs were isolated from
20–30 ml
of urine by differential centrifugation. The EVs quality was ensured by Electron microscopy
(EM), western blotting and EV-RNAs – profiling with the Bioanalyzer. Isolated RNAs
were
subjected to RNA sequencing after cDNA library preparation (ultra-low amount protocols)
using HiSeq 2000 (Illumina) pair-end protocol. The association between kidney specific
gene
expression levels (>4 fold higher compared to other tissues, N = 53) and degree of
albuminuria or glomerular filtration rate was explored.
Results: Isolated EV quality appeared good by EM and Western blotting. RNA
quantity and quality were sufficient for sequencing of all samples with >5 million
pair
end reads. We detected on average expression of 12,642 genes. Principal component
analysis
(PC) of the expression of all genes did not reveal any systematic batch differences
between
the overnight and 24-hour urine collections. Comprehensive look-up of 53 kidney-enriched
genes revealed expression of >73% (total 39) of these genes in urine EVs with high
expression of five kidney-specific genes (SLC12A3, SLC12A1, NPHS2, AQP2 and SLC22A12).
PC
analysis combining the impact of 39 kidney-enriched genes revealed that most
macroalbuminuric patients clustered together along the PC1 axis, and the axis also
correlated with the albumin-to-creatinine ratio (p = 0.0004) explaining 11% of the
variance
(P = 0.005) in the whole data set. The PC1 axis also showed correlation with HbA1c
(P = 0.003), but not with diabetes duration, BMI, age and eGFR.
Summary/Conclusion: Our results show that urinary EV transcriptome can capture
kidney specific gene expression signatures suggesting its potential as a virtual kidney
biopsy.
Funding: BEAt-DKD, IMI-JU, GA115974 and Academy of Finland (317599)
OF15
Symposium Session 15: EV-based Therapeutics II
Chair: Chantal Boulanger – Université de Paris, Paris Cardiovascular Research Center,
Inserm, Paris, France
OF15.1
Engineering extracellular vesicles for targeted delivery of Cas9
to T cells
Devin M. Stranford
a, Lacy
Simonsb, Judd F. Hultquistb and Joshua N. Leonarda
aNorthwestern University, Evanston, USA;
bNorthwestern University, Chicago, USA
Introduction: Extracellular vesicles (EVs) are nanoscale lipid particles
secreted by all cells that mediate intercellular communication by transferring encapsulated
proteins and nucleic acids between cells. Intrinsic properties, such as non-toxicity
and
non-immunogenicity, and the ability to engineer EVs to incorporate desired cargo and
targeting moieties make them an attractive delivery platform for a wide range of
biomolecules. One such application is the use of EVs to deliver Cas9 ribonucleoprotein
for
targeted and translatable in vivo gene editing. Overcoming this delivery barrier could
enable novel therapeutic strategies, such as the inactivation of HIV proviral DNA
in T cells
to eliminate the latent viral reservoir.
Methods: We developed a novel system for actively loading functional Cas9
ribonucleoprotein into EVs and for engineering these vesicles to target delivery of
Cas9 to
recipient T cells. To promote specific interactions with recipient cells, we developed
a
novel, high-affinity EV targeting platform by displaying single-chain variable fragments
(scFvs) on the surface of the vesicles. To enhance vesicle uptake by T cells, we used
viral
glycoproteins to promote vesicle fusion at the cell surface.
Results: We demonstrated directed loading of Cas9 ribonucleoprotein into
engineered vesicle populations. We enhanced receptor-dependent EV targeting to T cells
~100-fold as compared to a non-targeted EV control. Our approach, as well as display
of
fusion-enhancing proteins, was successfully used to increase EV uptake by recipient
T cells,
which exhibit low basal rates of endocytosis and are difficult to deliver cargo to
by other
means.
Summary/Conclusion: The ability to direct EV uptake to desired cell types and
uptake pathways within those cells may ultimately enable functional delivery of biomolecular
cargo for a wide range of targeted therapies.
Funding: This work was supported by the Third Coast Center for AIDS Research
(CFAR), an NIH funded centre (P30 AI117943).
OF15.2
Exosomal AAVs in Myocardial Repair: Escaping Neutralizing
Antibody and Enhancing Delivery
Marta Adamiak
a, Shweta
Lodhaa, Morad Asadia, Yaxuan Lianga, Cherrie
Shermana, Erik Kohlbrennera, Dongtak Jeonga, Delaine
Ceholskia, Navneet Dograb, Nicole Duboisc, Roger
Hajjara and Susmita Sahooa
aCardiovascular Research Center, Icahn School of Medicine,
Mount Sinai, New York, USA; bDepartment of Genetics and Genomic Sciences,
Department of Pathology, Icahn School of Medicine, Mount Sinai, New York, USA;
cDepartment of Developmental and Regenerative Biology, Mindich Child Health and
Development Institute, Black Family Stem Cell Institute, Icahn School of Medicine,
Mount
Sinai, New York, USA
Introduction: Due to their safety profile, tissue tropism and long-term
transgene expression, adeno-associated viruses (AAVs) have become the vector of choice
for
human gene therapy. However, pre-existing neutralizing antibodies (NAbs) to many AAV
serotypes pose a critical challenge for the translation of gene therapies to clinic.
Here,
we describe the use of exosomal AAVs (eAAV) as a robust cardiac gene delivery system
that
enhance transduction efficiency while shielding from pre-existing humoral immunity
to the
viral capsid.
Methods: We developed an ultracentrifugation-based purification strategy to
obtain eAAV specimens from AAV-producing HEK-293 T cells, and used electron microscopy-based
visualization, confocal microscopy-based colocalization studies, qPCR, immunoblotting,
dynamic lights scattering, ExoView technology and protease assays to characterize
eAAV
morphology, contents and mechanism of action. We then evaluated efficiency of heart
targeting for eAAV9 or eAAV6 and standard AAV9 or AAV6 encoding for EGFP, mCherry
or firefly
luciferase in different human cell lines in vitro, in black mouse and in passive immunity
nude mouse model in vivo using flow cytometry, confocal microscopy, Langendorff perfusion
system and bioluminescence imaging. Last, to test therapeutic efficacy, eAAV9 or AAV9
vectors encoding for SERCA2a, a sarcoplasmic reticulum calcium adenosine triphosphatase,
were injected intramyocardially in post-myocardial infarction mice preinjected with
NAbs.
Echocardiography and histochemistry were used to evaluate cardiac function and
remodelling.
Results: We confirmed eAAV represent vesicular fractions that exhibit common
exosome morphology and antigen expression, along with the presence of virus particles,
and
demonstrated that eAAV infectious entry potentially involves trafficking via endocytic
compartments. Regardless of the presence or absence of NAbs, we showed in functional
studies
that eAAVs are more efficient in cell transduction in the same titre ranges as standard
AAVs. Remarkably, eAAV9-SERCA2a outperformed standard AAVs significantly improving
cardiac
function even in the presence of NAbs (%EF 55.14 ± 3.50 compared to 27.31 ± 1.63 at
6 weeks,
respectively).
Summary/Conclusion: Delivery of standard AAVs protected by carrier exosomes
(i.e. eAAVs) is a promising approach to evade pre-existing NAbs while still efficiently
transducing myocardium, which can be applied in the broader population of patients
with
myocardial infarction and may result in higher gene delivery efficacy.
OF15.3
Computational Modelling of Mesenchymal Stem Cell Exosomes
Predicts Cardiac Improvements in Preclinical and Clinical Models of Hypoplastic Left
Heart
Syndrome
Jessica R. Hoffman
a, Laxminarayana
Korutlab, Mohamed Abdullahc, Gregory Bittlec, Joshua
Hared, Joey Harmonb, Robert Hub, Rachana
Mishrac, David Moralesc, Sudhish Sharmac, Sunjay
Kaushalc, Prashanth Vallabhajosyulae and Michael
Davisa
aEmory University, Atlanta, USA; bUniversity of
Pennsylvania, Philadelphia, USA; cUniversity of Maryland School of Medicine,
Baltimore, USA; dUniversity of Miami, Miami, USA; eYale University,
New Haven, USA
Introduction: The palliation of hypoplastic left heart syndrome (HLHS), a
complex congenital heart disease, places significant demand on the right ventricle
(RV) to
sustain systemic circulation, resulting in high RV dysfunction-associated mortality.
Cell-based therapies may improve RV adaption to single ventricle physiology, specifically
through the release of pro-reparative exosomes. The transplantation of bone marrow-derived
mesenchymal stem cells (MSCs) in a pulmonary artery banding (PAB) swine model improves
RV
function and led to the ELPIS clinical trial: Allogeneic Human MSC Injection in Patients
with HLHS. Here, we model circulating MSC exosome cargo to predict RV functional recovery
in
preclinical PAB model and validate its translational potential in ELPIS subjects.
Methods: HLHS patients (n = 3) after Glenn procedure and swine (n = 3) after
PAB were given RV injections of allogeneic/xenogeneic MSCs. Donor-specific, HLA- I+,
exosomes were isolated from plasma. In swine, exosomes were collected and RV fractional
area
change (FAC) was measured post-MSC-injection. In the ELPIS patients, exosomes were
collected
and outcome measurements (FAC, stroke volume (SV), RV mass) were recorded 6 and 12-months
post-injection. Exosomal mRNA, microRNA (miRNA), and proteins were quantified and
partial
least squares regression (PLSR) reduced the dimensionality of the datasets to build
a swine
model, upon which ELPIS outcome predictions were made.
Results: Multiomics analysis of swine exosome cargo revealed miRNA to be the
largest contributor to overall variance. In swine and ELPIS patients, miRNAs were
similarly
expressed (98%, fold-change<2). PLSR reduced the dimensionality of the swine miRNA
dataset to 50 miRNAs with the highest weighted coefficients for changes in FAC. Pathway
analysis of miRNA targets revealed links to smooth muscle cell proliferation and cardiac
chamber development. Importantly, the swine miRNA PLSR model predicted ELPIS patient
improvements in FAC, SV, and RV mass with strong correlation (r > 0.9).
Summary/Conclusion: These findings support the use of: (1) swine PAB model for
RV failure in HLHS, (2) circulating donor-specific MSC-exosomal miRNA as a novel,
non-invasive biomarker of patient outcomes, and (3) computational modelling to predict
improvements and inform clinical trials.
Funding: 5R01HL145644-02
J.R.H. was supported by NIH training grant T32-GM008602
OF15.4
RNA nanoparticles as EV displaying ligands for specific cancer
targeting and efficient RNAi delivery in vivo
Zhefeng Li
a, Fengmei
Pib, Zhen Zhenga and Peixuan Guoa
aCenter for RNA Nanobiotechnology and Nanomedicine; College
of Pharmacy; Comprehensive Cancer Center; Dorothy M. Davis Heart and Lung Research
Institute; College of Medicine. The Ohio State University, Columbus, USA;
bExonanoRNA, Columbus, USA
Introduction: EVs have been shown promising potential as a drug delivery
vehicle, especially nucleic acid therapeutics. However, the overall short of specificity
to
target cancer cells has led to low therapeutic efficacy and potential toxicity. RNA
nanotechnology is the bottom-up self-assembly of nanometre-scale RNA architectures.
We
previously discovered a stable phi29 pRNA three-way junction (3WJ) motif and used
it to
construct multivalent RNA nanoparticles with high chemical and thermodynamic stability.
The
resulting arrow-shape RNA nanoparticles are homogenous, uniform in size and shape,
and can
harbour different functionalities while retaining their tertiary folding and independent
functionalities both in vitro and in vivo. This flexible platform using RNA nanotechnology
to achieve tumour-specific targeting has been demonstrated over the last decade. Here
we
introduce a strategy to take advantage of both EVs and RNA nanotechnology to develop
a
versatile platform for efficient target-specific delivery of siRNAs for cancer
treatment.
Methods: We design membrane-anchoring arrowtail 3WJ RNA nanoparticles to
display tumour targeting ligand (PSMA RNA aptamer or EGFR RNA aptamer or folate) on
BIRC5
siRNAs loaded EVs (Fig.1). Nanoparticles were characterized by Nanoparticles Tracking
Analysis (NTA), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS)
and
Atomic Force Microscopy (AFM). EVs were produced by Hollowfiber bioreactor and purify
by
Tangential Flow Filtration (TFF) follow by ultracentrifugation. Cell binding were
evaluated
by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity
reverse transcription-PCR (qRT-PCR). Formulated EVs were introduced to tumour (prostate,
triple negative breast cancer, colon PDX) xenograft mice by tail-vein injection and
evaluate
in vivo tumour inhibition.
Results: 1) We found the orientation of arrow-shaped RNA can be used to
control ligand display on EVs membranes for specific cell targeting. 2) By placing
membrane-anchoring cholesterol at the tail of the arrow results in display of RNA
aptamer or
folate on the outer surface of the EVs and enhance cancer cell binding and uptake.
3) Taking
advantage of the RNA ligand for specific targeting and EVs for efficient cytosolic
delivery,
the resulting ligand-displaying EVs or plant derived EVs-like nanovesicles were capable
of
specific delivery of siRNA to cells, and efficiently blocked tumour growth in three
cancer
models.
Summary/Conclusion: We developed an RNA-EVs based nanoparticles platform and
shown the flexibility for different cancer type treatment.
Related publications:
Pi F et.al. Nature Nanotechnology. 2018, 13:82.
Li Z et.al. Sci Rep. 2018, 8:14644
Zheng Z & Li Z et.al. J Control Release. 2019, 311:43.
OF16
Symposium Session 16: Cancer Biomarker
Chair: Carolina Soekmadji – QIMR Berghofer
OF16.1
Multiplex nanoscale flow cytometry to study the circulating
extracellular vesicle biome in patients with pancreatic cancer versus screen negative
controls
Mayu Moritaa, Thuy Ngob, Emek
Demira, Randall Armstronga, Scott Bornheimerc and
Terry K. Morgan
a
aOHSU, Portland, USA; bKnight CEDAR Technology,
OHSU, Portland, USA; cBD Biosciences, San Jose, USA
Introduction: Extracellular vesicles (EVs) contain plasma membrane surface
markers that provide insights into their cell source. Until now, our understanding
of the
circulating EV-biome has been limited by the lack of cell- and size-specific EV quantitation
methods. We have developed and validated a multiplex nanoscale flow cytometry approach
to
image cell- and size-specific EV populations using a novel human “EV-Lyoplate” with
3
differently coloured monoclonal antibodies per well in a 96 well plate format (n = 242
separate antibodies with isotype, stained PBS, unstained plasma, and quant-beads controls
per plate). We hypothesized that platelet poor plasma samples from patients diagnosed
with
pancreatic cancer would have significantly different EV-biome profiles than screen
negative
study subjects.
Methods: Study subjects were enrolled and sampled before clinically scheduled
endoscopic ultrasound-guided biopsy (EUS-FNA) procedures to screen patients with symptoms
of
pancreatic duct obstruction who later had at least two years of clinical follow up,
including surgical resection in cases of pancreatic neoplasia (n = 36) or at least
one
follow up clinic visit to confirm resolution of symptoms. Blood samples were uniformly
collected, processed, and banked per ISEV recommended guidelines. Uniform machine
(FACSymphony) settings to standardize light scatter and fluorescence detection were
based on
commercially available beads (eg. Megamix). Samples were coded and randomized for
testing
and results were reported as the mean cell- and size-specific EV events/ul of plasma.
Results: Clinical outcomes confirmed 10 cases of cancer and 26 screen negative
controls. Principle component analysis suggested that a number of different cell-
and
size-specific EVs were significantly more common in the cancer cases (adjusted p-value
<
0.05, with AUCs >0.80), including Epcam+/CD63+ events likely from cancer cells and
CD41+/CD62p+/CD9+ microvesicles from platelets, among others.
Summary/Conclusion: In this proof of principle study employing an EV-Lyoplate
design and nanoscale flow cytometry, we could reliably discriminate the EV-biomes
in
patients with cancer from negative controls. Ongoing studies will determine whether
these
discriminators will be validated in larger cohorts and provide at least non-inferior
predictive value compared with the current gold standard clinical testing assay
(EUS-FNA).
Funding: Knight Cancer Institute, Oregon Health & Science University
OF16.2
Analysis of surface markers as biomarkers for small cell lung
cancer
Patricia Midori Murobushi Ozawa, Sarah M.
Groves, Vito Quaranta and Alissa Weaver
Vanderbilt University, Nashville, USA
Introduction: Small Cell Lung Cancer (SCLC) is an aggressive tumour type,
usually metastatic at diagnostic leading to poor overall survival. Interestingly,
SCLC
tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem
to
drive tumour survival. Since the subtype of SCLC may have prognostic significance,
the aim
of this study was to identify surface marker proteins as biomarkers of SCLC.
Methods: A linear discriminant analysis (LDA) model, implemented in Python via
Sci-kit learn, was used to choose the best 4 markers for distinguishing subtypes.
This
analysis was based on RNA-seq data from a previous study. In order to identify EV-based
biomarkers that would identify SCLC EVs and not normal EVs, we excluded from this
analysis
proteins without a verified transmembrane domain and proteins associated with EVs
expected
to be present in white and red blood cells, and endothelial cells (according to Exocarta
and
Vesiclepedia databases). We also prioritized proteins that could be pan markers for
SCLC and
that might have prognostic significance. To validate our findings, we performed Western
blotting and Flow cytometry in SCLC cell lines from different subtypes.
Results: Our RNA analysis indicated that the best 4 surface markers to
distinguish SCLC subtypes were CEACAM5, FAM174A, LRFN1, EPHA2. Immunoblot analysis
validated
CEACAM5 and EPHA2 but not FAM174A or LRFN1. We also found that NCAM1, a commonly used
SCLC
marker, only marks some of the subtypes. For further analysis, we chose proteins with
antibodies validated for flow cytometry as our chosen biomarker platform. Flow cytometry
analysis of CD24 is suitable as a pan-SCLC marker. However, the expression of NON-NE
cell
lines was decreased compared to RNA-seq data.
Summary/Conclusion: Protein analysis of CEACAM5 and EPHA2 corresponded to
RNA-seq data. NCAM was not detected as a pan marker for all SCLC subtypes. However,
we could
see CD24 expression in all SCLC subtypes, indicating it may be a useful pan marker
for SCLC.
Future studies will be performed to validate the expression of other surface markers
in
cells, purified EVs, and plasma of SCLC patients.
Funding: NIH U01 CA224276 and NIH U54 CA217450.
OF16.3
Leukobiopsy – exploiting extracellular vesicle-mediated
leukocyte sequestration of cancer-specific signatures
Janusz Rak
a, Shipa
Chennakrishnaiaha, Laura Monterminib, Brian Meehanb,
Thupten Tseringb, Saro Aprikiana and Dongsic Choib
aMcGill University, Montreal, Canada; bRIMUHC,
Montreal, Canada
Introduction: In cancer, extracellular vesicles (EVs) act as a unique exit
mechanism for mutant and oncogenic macromolecules (proteins, RNA and DNA) en route
from
malignant cells to blood1. While this process has inspired major liquid biopsy efforts,
the
biology of circulating EVs that carry oncogenic mutations (oncosomes)2 is still poorly
characterized. It is also unclear what part (if any) of the tumour-related cell free
DNA
(ctDNA)3,4, a major liquid biopsy analyte, is linked to circulating EVs and what is
their
fate, receptacles and biological activity.
Methods: We employed as series of cancer cell lines carrying mutations in
major oncogenes (HRAS, HER2, EGFRvIII). EV-DNA was analysed by digital droplet PCR
(ddPCR),
along with nuclear anomalies in donor cells (DAPI, Electron Microscopy) and transfer
of DNA
to recipient cells of endothelial (HUVEC, MMBEC), astrocytic (NHA) or myeloid (HL60)
origin.
Blood underwent fractionation into red blood cells (RBC), white blood cells (WBC),
platelets
(PLT), EVs (100,000g ultracentrifugation) and soluble plasma (SUP)5.
Results: HRAS-mediated cellular transformation (in RAS-3 cells) triggers
profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm
and released as cargo of EVs. Oncogenic DNA is detectable in blood fractions of tumour
bearing mice. While EVs, ctDNA and PLTs contain intermediate levels of mutant DNA,
RBCs
contain only traces of this material. The highest HRAS copy number per ml of blood
is found
in WBCs (monocytes and neutrophils), which contain more cancer DNA/cell than liver,
spleen
and bone marrow. Depletion of neutrophils using anti-Ly6G antibody results in an increase
in
EV- and ctDNA-associated mutant DNA in blood, suggesting the role of these cells in
regulating the circulating levels of cancer cell-derived particles. Uptake of DNA-containing
EVs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties.
These cells also retain cancer-specific transcripts and other cargo. Finally, normal
astrocytes treated with oncogenic EVs also exhibit phenotypic changes and signs of
genomic
instability including formation of micronuclei.
Summary/Conclusion: We propose that the process of leukocyte sequestration of
circulating particles containing tumour-related nucleic acids renders these cells
potentially usable as a novel liquid biopsy platform (LEUKOBIOPSY) in cancer. 1.
PMID:18425114; 2. PMID:27680302; 3.PMID: 25086355; 4. PMID:31469961; 5. PMID:29971917
Funding: Canadian Institutes of Health Research (CIHR) Foundation Grant
(FDN143322) to JR.
OF16.4
Whole-transcriptomic profiling of circulating small
extracellular vesicles derived RNAs for early detection of colorectal carcinoma and
adenoma
Li Min
a, Shengtao Zhua,
Xiang Liub, Da Qina, Qingdong Guoa, Rui Weia,
Libo Zhaob, Guanyi Kongb and Shutian Zhanga
aDepartment of Gastroenterology, Beijing Friendship Hospital,
Capital Medical University, National Clinical Research Center for Digestive Disease,
Beijing
Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive
Disease, Beijing, 100050, P. R. China., Beijing, China (People’s Republic); bEcho
Biotech Co., Ltd, Beijing, China., Beijing, China (People’s Republic)
Introduction: Early diagnosis of colorectal cancer (CRC) and precancerous
adenoma patients is of vital importance. Previously we profiled small extracellular
vesicles
(sEVs) derived miRNAs isolated from plasma, proposed a new promising biomarker category
of
CRC patients. Here we further gave a full landscape of circulating sEVs derived RNAs
to
explore and evaluate sEVs based RNA biomarkers for early detection of both CRC and
adenoma
patients.
Methods: Plasma sEVs were isolated from 60 participants, including 31
early-stage CRC patients, 19 adenoma patients, and 10 normal controls (NC), and
characterized according to MISV2018 guideline. The total sEVs derived RNA expression
profile
of all participants was investigated by next-generation sequencing (NGS). Weighted
gene
coexpression network analysis (WGCNA) was performed to categorize differentially expressed
RNAs, and t-distributed stochastic neighbour embedding (tSNE) was adopted to distinguish
CRC, adenoma, from NC samples with the top-ranked genes in WGCNA modules. RT-qPCR
validation
was performed in a cohort of 120 additional participants.
Results: A total of 1615 RNA species (including miRNAs, mRNAs, and lncRNAs)
were found differentially expressed between plasma sEVs in CRC and NC participants.
Additionally, 888 RNA species were differentially expressed between plasma sEVs in
adenoma
and NC participants. 1160 RNA species were differentially expressed between plasma
sEVs in
CRC and adenoma participants. WGCNA categorized all RNAs into 6 modules, which exhibited
different expression trends during the carcinogenesis of CRC. A 60-gene combined tSNE
model
consists of the top 10 genes in each module could perfectly classify CRC, adenoma,
and NC
samples. A 6-gene combined tSNE model consists of the top 1 gene in each module could
roughly distinguish CRC and adenoma from NC, with only 1 sample misclassified. RT-qPCR
assays also confirmed the potential classification ability of those genes in another
validation cohort of 120 participants.
Summary/Conclusion: Circulating sEVs have a distinct RNA profile in both CRC
and adenoma patients as compared to normal controls. sEVs derived RNA biomarker combination
could for Early Detection of Colorectal Carcinoma and Adenoma.
Funding: This work was supported by grants from the Beijing Nova Program
(Z191100001119128); National Natural Science Foundation of China (81702314); Beijing
Municipal Science and Technology Project (Z191100006619081); Beijing Municipal
Administration of Hospitals’ Youth Programme (QML20180108); The Digestive Medical
Coordinated Development Center of Beijing Municipal Administration of Hospitals
(XXZ0201).
OF16.5
RNA signatures from blood and urine EVs display tissue-lineage
from prostate cancer patients
Navneet Dogra
a, Mehmet
Ahsenb, Tina Chenb, Reena Olsenc, Dan Hanb,
Stacey M. Giffordd, Benjamin Wunschd, Rachel Weilb, Melissa
Smithb, Joshua Smithd, Sungchel Kimd, Kamala
Bhattb, Sujit Nairb, Bojan Losice, Robert
Sebrab, Adam Margolinc, Carlos Cordon-Cardob, Ashutosh
Tewaric and Gustavo Stolovitzkyc
aDepartment of Genetics and Genomic Sciences, Department of
Pathology, Icahn School of Medicine, Mount Sinai, New York, USA; bIcahn School of
Medicine at Mount Sinai, New York City, USA; cIcahn School of Medicine at Mount
Sinai, New York, USA; dIBM, Yorktown Heights, USA; eIcahn School of
Medicine at Mount Sinai, New York, USA
Introduction: Although the concept of systematic “liquid biopsy” using bodily
fluids is simple and elegant, the path of clinical reality has been challenging. Recently,
numerous tissue-specific biomarkers have been discovered in EVs derived from blood,
urine,
cerebrospinal fluid, cell culture media, and a variety of other fluids. However, tracing
the
lineage of EVs to their tissue of origin remains challenging due to their minute amount
of
cargo and unavailability of matching biopsied tissue and bodily fluids from the same
patient.
We recently demonstrated in three separate publications (Dogra et. al; Smith et. al;
Murillo et. al), a new device (nanoDLD) for EV isolations, it’s comparison with current
technologies, bioengineered vesicles, and a detailed study of RNA types present in
small/large vesicles, lipoproteins, and Ago2 protein in different biofluids. In the
present
study, we aim to investigate the lineage of prostate derived EVs in biofluids.
Methods: Using our chip technology, we have isolated exosomes from prostate
cancer cell lines and patient tissue, blood and urine samples. After exosome isolation,
small RNA libraries were prepared, and sequencing is carried out at Icahn School of
Medicine
and New York Genome Center using illumine sequencer HISeq2500. Our nanofluidic pillar
array
is manufactured in an SiO2 mask using optical contact lithography and deep ultraviolet
lithography.
Results: Our study revealed i) RNA markers, which are exclusive to their
prostate tissue of origin and are secreted in EVs; ii) approximately 1–3% of prostate
tissue-specific RNA were discovered in EVs; iii) Over 77% (17 of 22 RNA) of literature
curated prostate-specific RNA signatures were detectable in serum and urine EVs from
PCa
patients; iv) EVs contained over 50–70% of noncoding RNA (40–60% was miRNA), while
tissue
predominantly yielded rRNA (>60%); v) Finally, gene set analyses generated that over
90%
of EVs RNA were enriched for signalling pathways, yielding miRNA-associated, non-canonical
Wnt signalling, and androgen receptor pathways. This study enables us to noninvasively
monitor prostate tissue-specific biomarkers, identify tumour-specific RNA, and potentially
may benefit in liquid biopsy by avoiding unnecessary surgical procedures.
Summary/Conclusion: In summary, we have investigated patient matched tissue,
serum, and urine derived EVs in prostate cancer. We present a set of 68 prostatic
RNA in
EVs, which are enriched in noncanonical Wnt signalling, and androgen receptor pathways.
This
study enables us to noninvasively track prostatic biomarkers, identify tumour specific
RNA,
and potentially may benefit in liquid biopsy by avoiding unnecessary surgical
procedures.
OF16.6
A multi-model, liquid biopsy approach for diagnosing and staging
pancreatic adenocarcinoma
Zijian Yang
a, Michael
LaRiviereb, Jina Koc, Jacob Tillb, Theresa
Christensenb, Stephanie Yeeb, Taylor Blackb, Kyle
Tienb, Andrew A. Linb, Hanfei Shenb, Andrew
Adallahb, Mark O’Harab, Charles Vollmerb, Bryson
Katonab, Ben Stangerb, David Issadored and Erica
Carpenterb
aUniversity of Pennsylvania, Philadelphia, USA;
bUniversity of Pennsylvania, philadelphia, USA; cMassachusetts General
Hospital, Wyss Institute at Harvard University, Cambridge, USA; dDepartment of
Bioengineering, University of Pennsylvania, Philadelphia, USA
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the third largest
contributor to cancer-related death in the USA. Since there is not yet a feasible
technology
to diagnose PDAC early in the disease, 80% of patients are diagnosed at an advanced
stage.
Moreover, for patients with confirmed PDAC, standard imaging method has low sensitivity
to
detect early metastatic disease, which complicates the selection of therapy.
To address these challenges, there has been great interest in developing
minimally-invasive, extracellular vesicle (EV) based blood tests for PDAC. To this
end, we
have integrated measurements of tumour derivedEV RNA cargo with circulating proteins
and
cell free DNA (cfDNA), and use machine learning algorithms to distill this multiplexed
diagnostic to 1. diagnose PDAC patients from healthy and disease controls and 2. distinguish
PDAC patients with distance sites of metastasis to guide their treatment. We make
use of our
lab’s magnetic nanopore isolation technique to specifically enrich for tumour derived
EVs
directly from patient plasma.
Methods: We have developed a high throughput nanofluidic sorting platform,
which immunomagnetically isolates individual EVs from plasma using magnetic nanostructures.
However, our architectures is uniquely designed for massive parallelization allowing
high
throughput, robust processing of mL of plasma in minutes. We performed sequencing
on a
discovery set of patients and controls (N = 29). Subsequently, we trained our panel
of
biomarkers using a training set of N = 47. Finally, we validated the performance of
our
platform using an independent blinded test set of N = 145.
Results: The results of a blinded test set achieved an accuracy = 92% and an
AUC = 0.95 on binary classification of PDAC patients versus those that were healthy
or
disease controls. In addition, we achieved an AUC = 0.85 and accuracy = 89% with sensitivity
of 90% and specificity of 88% on detecting occult metastasist.
Summary/Conclusion: We developed a highly sensitive pancreatic cancer
diagnostics by combining our nanomagnetic isolation platform for tumour-derived EV
isolation, RNA sequencing, and machine learning. We isolated tumour-derived EVs and
profiled
their RNA cargo, combined with cfDNA and CA19-9 for pancreatic cancer diagnosis. The
predictive panels successfully distinguished non-cancer patients from PDAC patients,
and
no-distant metastasis patients(M0) from distant metastasis patients(M1) for appropriate
treatment. The resulting AUC and accuracy from the independent blinded test set outperformed
any individual biomarker, showing both the benefits and the robustness of combining
multiple
orthogonal biomarkers for PDAC diagnosis.
OF17
Symposium Session 17: Heart, Lung, and Vessels
Chair: J. Brian Byrd – University of Michigan
OF17.1
The proteome of circulating large EVs in diabetes and
hypertension
Dylan Burger
a, Ozgun
Varolb, Maddison Turnerb and Christopher Kennedyb
aChronic Disease Program, Ottawa Hospital Research Institute,
Ottawa, Canada; bOttawa Hospital Research Institute, Ottawa, Canada
Introduction: Both hypertension and diabetes exhibit significant molecular
changes to the vasculature that are associated with increased cardiovascular risk.
Here we
examined the protein composition of large EVs (L-EVs) isolated from the plasma of
hypertensive, diabetic and healthy mice to identify common and disease-specific molecular
changes.
Methods: We examined circulating L-EVs isolated from transgenic mice
expressing active human renin in the liver (TtRhRen, a model of hypertension), OVE26
type 1
diabetic mice, and their wild-type (WT) littermates. At 20 weeks of age mice were
sacrificed
and blood samples were obtained by cardiac puncture. L-EVs were isolated from platelet-free
plasma via differential centrifugation and protein content was assessed via mass
spectrometry (MS).
Results: TtRhRen mice exhibited increased blood pressure compared with OVE26
mice or their WT littermates. (144.2 ± 7.6 vs 123.5 ± 4.9 [OVE26] vs. 114.6 ± 5.7 mmHg
[WT],
p < 0.05). MS identified 297 independent proteins with at least 2 peptides per protein.
Of these, 163 proteins were found in all groups studied, 48 were exclusive to WT mice,
23
were exclusive to OVE26 mice and 4 were exclusive to TtRhRen mice. In addition, 22
proteins
were observed with >1.5 fold change (FC) compared to wild-type mice, and 68 proteins
were
reduced by >50%. Amongst the top ten differentially expressed proteins, fibrinogen
was
upregulated in both OVE26 and TtRhRen mice compared with wild-type controls. Similarly
Trem-like transcript 1, sarcoplasmic/endoplasmic reticulum calcium ATPase 3 and junction
plakoglobin were all downregulated in both OVE26 mice and TtRhRen mice suggesting
molecular
changes common to both conditions.Conversely, arginase was up-regulated in diabetic,
but not
hypertensive mice while carboxypeptidase was up-regulated in hypertensive but not
diabetic
mice.
Summary/Conclusion: Taken together, these results show that the protein
composition of circulating L-EVs is altered in diabetes and hypertension and that
both
common and disease-specific changes may be detected. Further analysis of these changes
may
lead to the identification of novel pathways associated with the pathogenesis of vascular
injury in hypertension and diabetes.
Funding: This study was supported by grants (to DB) from the Canadian
Institutes of Health Research, an Ontario Early Researcher Award, and the Canada Foundation
for Innovation.
OF17.2
Understanding the role of endothelial cell-derived apoptotic
bodies in inflammatory signalling and cell clearance in an atherosclerosis model of
inflammation.
Amy A. Baxter
a, Georgia
Atkin-Smithb, Stephanie Paonec, Mark Hulettd and Ivan
Poond
aLa Trobe University, Thornbury, Australia; bLa
Trobe University, Walter & Eliza Hall Institute for Medical Research, Bundoora,
Australia; cSt Pancras Clinical Research, London, UK; dLa Trobe
University, Bundoora, Australia
Introduction: Apoptotic bodies (ApoBDs) are a class of large (~1-5um) EVs
formed during apoptotic cell disassembly, that are becoming increasingly recognized
as
potential mediators of intercellular communication, e.g. via the transfer of proteins
and
other cargoes to target cells. During the inflammatory vascular disease atherosclerosis,
endothelial cell (EC) apoptosis contributes to loss of barrier function and promotes
the
formation of plaques in regions of EC damage. Although, experimentally, ECs generate
an
abundance of ApoBDs, a specific role for EC-derived ApoBDs (EC-ApoBDs) in the progression
of
atherosclerosis remains poorly defined.
Methods: In the present study, a detailed in vitro characterization of EC
disassembly was performed via flow cytometry, confocal live cell imaging and cytokine
profiling, followed by function analyses of EC-ApoBDs using a murine in vivo model
of dead
cell clearance.
Results: Characterization of EC disassembly revealed that ApoBD formation in
ECs is regulated by rho-associated, coiled-coil-containing protein kinase 1 (ROCK1),
a
process that can be pharmacologically inhibited using a ROCK-1 inhibitor, thereby
providing
tools for functional in vivo studies. The specific cargo and role in clearance of
EC-ApoBDs
were then investigated. Profiling of EC-ApoBDs was performed via cytokine antibody
array to
reveal that EC-ApoBDs generated under inflammatory conditions contain high levels
of
pro-inflammatory cytokines including MCP-1 and IL-8, suggesting a potential role for
EC-ApoBDs in the propagation of inflammation during vascular disease. Furthermore,
the
ability of EC-ApoBDs to be cleared from the vasculature via phagocytosis was investigated,
revealing that EC-ApoBDs can travel to distal organs to undergo clearance.
Summary/Conclusion: These findings provide important insights into the
potential functions of EC-ApoBDs generated under both non-inflammatory and inflammatory
conditions and may contribute to future studies involving the therapeutic targeting
of EC
disassembly for the treatment of atherosclerosis.
Funding: This work was supported by grants from the National Health &
Medical Research Council of Australia (GNT1141732, GNT1140187)
OF17.3
Adipose mesenchymal stromal cell derived EVs foster cardio-renal
protection in the DOCA-salt hypertensive rat model
Rafael Soares Lindosoa, Jarlene Alécia Lopesa,
Renata Binatob, Eliana Abdelhayc, Christina Moeda Takiyad,
Giovanni Montini, Benedetta Bussolatie, Adalberto Vieyraa and
Federica Collino
f
aFederal University of Rio de Janeiro, Rio de Janeiro,
Brazil; bNational Institute of Cancer, rio de janeiro, Brazil;
cNational Institute of Cancer, Rio de Janeiro, Brazil; dFederal
University of Rio de Janeiro, Rio de Janeiro, Brazil; eUniversity of Torino,
Torino, Italy; fDepartment of Biomedical Sciences, University of Padua, Padua,
Italy
Introduction: Cardio-renal syndromes (CRS) are disorders of the heart and
kidneys whereby “acute or chronic dysfunction in one organ may induce dysfunction
of the
other”. Stem cell-derived extracellular vesicles (EVs) mediates the protection of
the kidney
from development of chronic kidney disease (CKD). We here investigated the potential
of
adipose-mesenchymal stromal cells derived EVs (ASC-EVs) as therapeutic tools for the
treatment of CRS.
Methods: Adult Wistar rats were uninephrectomized and treated with a high-Na+
diet and deoxycorticosterone-acetate (DOCA-salt) for 8-weeks (038/15; A02/16-61-15).
EVs
were isolated by ultracentrifugation method. EV dimension, concentration and surface
markers
were characterized by NTA, cytofluorimetric analysis and transmission electron microscopy.
To characterize the role of EVs in CRS, DOCA-salt rats were injected weekly with ASC-EVs.
Systolic blood pressure was measured by the tail-cuff method. Plasma creatinine and
urinary
protein excretion were determined by colorimetric assays and microalbuminuria by immune
turbidimetric assay. qRT-PCR and western blot were conducted to evaluate fibrosis
and
inflammatory-related genes and proteins in the kidney and heart of DOCA-salt rats.
Immunohistochemistry was used to confirm matrix accumulation (a-SMA) and immune infiltrate
(CD68+ cells).
Results: Multiple administration of ASC-EVs in DOCA-salt rats induced a
protective effect on the kidney, by reducing tubular and vascular damage. Kidney function
was also conserved by EV treatment as detected by the normal glomerular filtration
rate and
the absence of proteinuria with respect to DOCA-salt untreated rats. EV administration
significantly decreases the pro-inflammatory molecules MCP-1 and PAI1 and reduce the
recruitment of macrophages in the kidney. The mitigation of the inflammatory response
by
ASC-EV infusion consequentially affected the development of fibrosis, as detected
by the
decrease in collagens (Col1A1, Col4A1) and fibronectin (FN) expression in respect
to
DOCA-salt animals. ASC-EVs were able to act in multiple organs, preventing fibrosis
and
inflammation also in the heart, therefore alleviating blood pressure rise during the
8-weeks
of treatment in DOCA-salt rats.
Summary/Conclusion: Our results indicate that ASC-EV administrations in
hypertensive-induced CKD rats promote protection from renal damage, reduction of the
inflammatory response and prevention of interstitial fibrosis in the kidney. ASC-EVs
are
also able to protect the cardiac tissue and to control blood pressure increase, displaying
complex and multiorgan beneficial effects.
Funding: Supported by the Brazilian National Research Council (CNPq) and the
Carlos Filho Rio de Janeiro State Research Foundation (FAPERJ).
OF18
Symposium Session 18: EV Biogenesis II
Chair: Suresh Mathivanan – La Trobe University
Chair: Clotilde Théry, PhD – INSERM U932, Institut Curie, PSL Research
University
OF18.1
Glycolytic restraint in alveolar macrophages is critical for
vesicular secretion of suppressor of cytokine signalling 3
Mikel D. Haggadone
a, Jennifer
Spetha, Hanna Honga, Eric Zhangb, Costas
Lyssiotisa and Marc Peters-Goldena
aUniversity of Michigan Medical School, Ann Arbour, USA;
bUniversity of Michigan, Ann Arbour, USA
Introduction: Alveolar macrophages (AMs) tonically secrete extracellular
vesicles (EVs) containing suppressor of cytokine signalling 3 (SOCS3) protein. Uptake
of
SOCS3-containing EVs by alveolar epithelial cells is critical for restraint of
cytokine-induced Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3)
signalling to promote homoeostasis in the distal lung. At steady state, AMs exhibit
suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function.
Whether this glycolytic restraint is critical for AM secretion of SOCS3 is unknown.
In fact,
to our knowledge, metabolic control over release of any EV cargo has never been explored
in
any cellular context.
Methods: Immortalized mouse AMs (MH-S) were treated with various doses of
2-deoxy-D-glucose (2-DG) and oligomycin, inhibitors of glycolysis and oxidative
phosphorylation, respectively. Primary rat AMs collected by lung lavage were treated
with an
aqueous extract of cigarette smoke (CSE) with or without 2-DG. Metabolic activity
was
measured by Seahorse assay, EVs were quantified by nanoparticle tracking analysis,
and
vesicular (>100-kDa) SOCS3 secretion was determined by western blot of conditioned
medium. Additionally, AMs collected from wild-type (WT) and LSL-KrasG12D mice bearing
lung
tumours 16 weeks after intrapulmonary Ad-Cre were cultured ex vivo in the presence
or
absence of 2-DG. Vesicular (>100-kDa) SOCS3 secretion was measured by ELISA.
Results: In a dose-dependent manner, oligomycin inhibited, whereas 2-DG
enhanced, SOCS3 and EV release by MH-S cells. Treatment of rat AMs with CSE (1%) attenuated
secretion of SOCS3, an effect that coincided with increases in glycolytic activity,
and
co-treatment of AMs with 2-DG abrogated the inhibitory effect of CSE on SOCS3 release.
Finally, AMs collected from LSL-KrasG12D mice exhibited a deficiency in SOCS3 secretion
relative to WT AMs, an effect that was reversible by overnight culture in the presence
of
2-DG.
Summary/Conclusion: In tandem, our data generated using in vitro and in vivo
approaches demonstrate that AM secretion of vesicular SOCS3 is down-regulated by glycolysis.
We speculate that metabolic control over release of EV cargoes is a phenomenon of
broad
biologic relevance within and outside of the lung.
Funding: National Science Foundation Graduate Research Fellowship DGE 1256260
(MH) and National Institutes of Health R35 HL144979 (MP-G)
OF18.2
Cyanobacterial extracellular vesicles are an alternative
secretion mechanism to deal with copper-induced stress
Steeve S. Lima
a, Joaquin
Giner-Lamiab, Francisco Florencioc, Narciso Coutod,
Phillip Wrighte, Paula Tamagninif and Paulo Oliveirag
aI3 s/IBMC (Porto, Portugal), Santa Maria da Feira, Portugal;
bCentre for Plant Biotechnology and Genomics, Madrid, Spain; cPlant
Biochemistry and Molecular Biology, Universidad de Sevilla, Sevilla, Spain;
dDepartment of Chemical and Biological Engineering, University of Sheffield,
Sheffield, UK; eSchool of Chemical Engineering and Advanced Materials, Newcastle
University, Newcastle, UK; fFaculdade de Ciências, Departamento de Biologia,
Universidade do Porto; i3 S/IBMC, Porto, Portugal; gi3 s/IBMC, Porto,
Portugal
Introduction: Bacterial extracellular vesicles (EV) are described to play
roles in defence and resistance, pathogenesis and stress responses. Cyanobacteria
pioneered
oxygenic photosynthesis, and are the ancestors of modern chloroplasts. We previously
described that by deleting the gene encoding TolC (ΔtolC) in the model cyanobacterium
Synechocystis sp PCC6803 (S6803), a key player in protein-mediated secretion systems,
a
hyper-vesiculating phenotype could be obtained. The goal of this work was to understand
why
ΔtolC hyper-vesiculates.
Methods: Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) was used
for quantitative proteomic analyses of total cell extracts. EV were isolated as follows:
cells were separated from the extracellular medium (EM) by centrifugation (4400 g,
10 min)
and filtration (0.2 µm pore-size filters). Cell-free EM was concentrated using centrifugal
filters (MWCO of 100 kDa), and later ultracentrifuged for 3 h at 100000 g. The final
EV
fraction was suspended in growth medium. EV characterization was performed using TEM,
DLS,
Nanosight, and by the detection and quantification of LPS (lipopolysaccharides). Detection
of specific proteins in EV was carried out by Western blot. Copper (Cu) levels were
quantified by atomic absorption spectrometry (AAS).
Results: A large-scale quantitative proteomic analysis was performed,
resulting in the identification of several metal-related proteins with differential
regulation in S6803 ΔtolC. Both wild-type (WT) and ΔtolC cells were then challenged
with
different metals. Compared to the WT, ΔtolC showed impaired growth only when exposed
to Cu,
a co-factor for several proteins with roles in primary metabolism. The intracellular
Cu
levels were quantified and ΔtolC accumulates threefold more Cu than WT cells. We then
asked
whether the hyper-vesiculating phenotype observed could be linked to the stress induced
by
Cu accumulation. In EV isolated from ΔtolC we detected the metallochaperone CopM,
a
periplasmic Cu-binding protein involved in Cu-resistance mechanisms in S6803. In addition,
Cu could also be detected in isolated ΔtolC-EV. In addition, more EV were detected
when
S6803 WT cells were challenged with Cu, in a Cu-concentration dependent manner.
Summary/Conclusion: These results support the idea that bacterial EV represent
an alternative Cu-secretion mechanism to deal with Cu-induced stress.
Funding: FCT PhD grant SFRH/BD/130478/2017;
FEDER-COMPETE2020-POCI-FCT project: POCI-01-0145-FEDER-029540.
OF18.3
Ciliary EV cargo sorting and biogenesis in living
animals
Juan Wang and Maureen Barr
Rutgers University, Human Genetics Institute of NJ, Piscataway, USA
Introduction: Extracellular vesicles (EVs) function in intercellular
communication. Despite their physiological importance and biomedical relevance, knowledge
of
EV fundamental biology is not well understood, in part due to a lack of tractable
animal
systems. Our analysis of environmentally-released C. elegans ciliary EVs provides
strong
evidence that nematodes package cargo in EVs that mediate inter-organismal communication,
in
analogy to intercellular signalling in mammals. We predict that conserved mechanisms
underlie EV cargo sorting, biogenesis and signalling.
Cilia act as cell towers to both receive extracellular signals and to send information
via
ciliary EVs. Ciliary defects result in human ciliopathies including autosomal dominant
polycystic kidney disease (ADPKD). ADPKD is a life-threatening disease that affects
1/800
and is caused by mutations in PKD1 and PKD2, which encode polycystin-1 and −2. In
C. elegans
and humans, the polycystins are architecturally similar, act in the same genetic pathway,
function in a sensory capacity, localize to cilia, and are shed in EVs, suggesting
ancient
conservation. Moreover, ciliary EV biogenesis and shedding is an evolutionary conserved
process from algae to worms to humans. By studying how cilia make and receive EVs,
we aim to
uncover fundamental principles of how cells communicate using EVs.
Methods: To study ciliary EV cargo sorting and biogenesis, we use
genetically-encoded fluorescent-tagged EV cargo and superresolution Zeiss Airyscan
confocal
microscopy in living animals.
Results: We find that cargoes are sorted into distinct populations. In cilia,
kinesin-2 motors and kinesin-3 KLP-6/KIF28 transport different EV cargoes to the ciliary
tip
and generate an EV cargo enrichment zone. From here, EVs are shed and released into
environment in a spatially and temporally regulated manner. Ciliary EV biogenesis
and
release is regulated by mechanical pressure and pH. Our work reveals – at the single
cell
level – that different EVs are made in response to environmental stimuli, which may
be
important for EV signalling properties.
Summary/Conclusion: Cells exploit the spatially-restricted cilium and its
sophisticated transport system to generate distinct populations of ciliary EVs. How
these
ciliary EV communicate cellular messages awaits decoding.
Funding: NIH DK059418 & DK116606 (MB), KUMC PKD Center (JW)
OF18.4
Comparative proteomics from different cancer cell types reveals
novel mechanisms of Rab11-exosome subtype regulation
Pauline Mariea, Shih-Jung Fana, John
Masona, Claudia Mendesa, Mark Wainwrighta, Adrian
Harrisb, Clive Wilsona and Deborah C I
Goberdhan
a
aUniversity of Oxford, Oxford, UK; bUniversity of
Oxford, University of Oxford, UK
Introduction: We recently demonstrated that recycling endosomes marked by
Rab11a generate exosome subtypes distinct in cargos and functions from late endosomes,
which
we collectively term Rab11-exosomes. These exosomes are preferentially released from
cancer
cells in response to metabolic stress and promote adaptive changes in a xenograft
model.
Here we use comparative EV proteomics in HCT116 colorectal and HeLa cervical cancer
cell
lines to identify Rab11-exosome signature proteins and screen for functional effects.
Methods: We analysed EV preparations by mass spectrometry using Tandem Mass
Tag® labelling to identify changes in EV protein cargo in response to glutamine depletion.
Candidate genes were subsequently knocked down in Drosophila secondary cells, which
permit
visualisation of Rab11-exosome biogenesis using fluorescence microscopy, and in human
cancer
cell lines.
Results: We show that accessory ESCRT-III proteins, CHMP5, CHMP1 and IST1, are
enriched on glutamine-depletion-induced EVs and play a selective and conserved role
in
generating Rab11-exosomes. They are, however, not required to traffic ubiquitinated
cargos
into late endosomes and lysosomes. ESCRT-0 components, thought to regulate trafficking
of
ubiquitinated cargos into intraluminal vesicles, are also required to make Rab11-exosomes.
In flies the ESCRT-0, Hrs, localises to the limiting membrane of Rab11-endosomes.
Comparative proteomics reveals other proteins enriched in Rab11-exosomes, which also
appear
to be needed to mediate this novel exosome formation mechanism.
Summary/Conclusion: We conclude that Rab11-exosome subtypes are formed via a
distinct mechanism requiring accessory ESCRT-III components, suggesting a route to
selectively target these exosomes.
Funding: Cancer Research UK [C19591/A19076], the CRUK Oxford Centre
Development Fund [C38302/A12278], BBSRC [BB/N016300/1, BB/R004862/1], John Fell Fund,
Oxford, Wellcome Trust (MICRON; #091911, #107457), Royal College of Surgeons.
OF18.5
Three-dimensional culture models to elucidate exosomal
biology
Kathleen M. McAndrews
a, Fei
Xiaoa, Valerie LeBleua and Raghu Kallurib
aUT MD Anderson Cancer Center, Houston, USA;
bDepartment of Cancer Biology, Metastasis Research Center, University of Texas MD
Anderson Cancer Center, Houston, USA
Introduction: The tumour microenvironment consists of a complex network of
host cells embedded within extracellular matrix. Communication between these cellular
compartments is critical for tumour progression and exosomes have emerged as important
regulators of intercellular communication. While a number of studies have implicated
exosomes in cancer progression, mechanisms controlling exosome transfer are not well
understood. We developed three-dimensional (3D) culture models to evaluate the role
of cues
provided by the extracellular matrix in exosome release and uptake.
Methods: Exosomes were isolated from cells in two- and three-dimensional
culture via ultracentrifugation and characterized by Nanosight, Qubit protein
quantification, and flow cytometry analysis of exosome markers. Exosomes were labelled
with
fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry.
Results: Cells cultured in 2D display decreased exosome release and increased
uptake compared to 3D cultured cells. Exosome release in 3D culture was inhibited
with the
exosome release inhibitors brefeldin A and GW4869, but was not significantly altered
by
knockout of Rab27B. In addition, disruption of polarity signals provided by 3D culture
did
not impact exosome release or uptake in 3D, but induction of oncogenic HRAS increased
both
secretion and uptake of exosomes through activation of PI3 K signalling.
Summary/Conclusion: Release and uptake of exosomes is altered in 3D
environments. These studies help provide insight into exosome production and uptake
in vivo
and have potential implications for therapeutically targeting exosome release and
the
development of exosome based therapeutic delivery vehicles.
OF18.6
Extracellular vesicles derived from R345 W-Fibulin-3 retinal
pigment epithelial cells promote epithelial-mesenchymal transition
Mi Zhou, Stephanie Grillo, Sarah Weber,
Yuanjun Zhao and Jeffrey Sundstrom
Penn State Hershey Medical Center, Hershey, USA
Introduction: Previous studies in our lab found that expression of
R345 W-Fibulin-3 induces RPE to undergo EMT. The purpose of current study was to
characterize the extracellular vesicles (EVs) in RPE cells expressing WT-Fibulin-3
versus
RPE cells expressing R345 W-Fibulin-3 and investigate the effects of these EVs on
RPE cell
differentiation.
Methods: ARPE-19 cells were infected with lentivirus with luciferase-tagged
wild-type (WT)-Fibulin-3 or luciferase-tagged R345 W-Fibulin-3. EVs were isolated
from the
media of ARPE-19 cells by conventional ultracentrifugation or density gradient
ultracentrifugation. Transmission electron microscopy (TEM) and Cryogenic electron
microscopy (cryo-EM) were performed to study the morphology of the EVs. The amount
and size
distribution of EVs were analysed by Nanosight Tracking Analysis (NTA). EV protein
concentrations were quantified using the DCTM Protein Assay (Bio-Rad). EV cargo were
analysed by unbiased proteomics using LC-MS/MS with subsequent pathway analysis (Advaita).
Migration ability was evaluated in ARPE-19 cells with or without the exposure of EVs
by
conducting scratch assays.
Results: Morphologically, TEM imaging showed concave-appearing vesicles and
cryo-EM imaging showed spherical vesicles with two subpopulations of EVs: a small
group with
diameters around 30 nm and a large group with diameters around 100 nm. Moreover, TEM
and
cryo-EM showed an increased amount of small EVs (~30 nm) in the mutant group compared
to the
WT group. This result was further confirmed by NTA showing that, in the mutant group,
the
particle size distributions were smaller than the WT EVs. No significant differences
were
shown in EV protein concentrations per particle between WT and mutant groups. Our
previous
data suggest that the expression of R345 W-fibulin-3 causes RPE cells to undergo EMT
as
evidenced by upregulated EMT drivers and an increased migration ability. Proteomic
studies
showed that EVs derived from ARPE-19 cells overexpressing WT-fibulin-3 contain critical
members of sonic hedgehog signalling (SHH) and ciliary tip components, whereas EVs
derived
from RPE cells overexpressing R345 W-Fibulin-3 contain EMT mediators, indicating that
EV
cargo reflects the phenotypic status of their parental cells. EV transplant studies
showed
that exposing native RPE cells to mutant RPE cell-derived EVs containing EMT drivers,
including TGF-β-induced protein (TGFBI), VIM, and SMAD4, leads to an enhanced migration
ability of RPE cells in a dose-dependent manner.
Summary/Conclusion: The expression of R345 W-Fibulin-3 promotes EMT in RPE
cells and leads to the secretion of EVs containing EMT drivers. EVs derived from RPE
cells
overexpressing R345 W-Fibulin-3 are sufficient for inducing EMT in native RPE cells.
OP2 = PF17 Oral with Poster Session 2: Cancer and Technology
Chair: Lizandra Jimenez – Postdoctoral Research Fellow, Vanderbilt University Chair:
Susmita Sahoo – Cardiovascular Research Center, Icahn School of Medicine, Mount
Sinai
OP2.01 = PF17.01
Development of scalable processes to produce therapeutic
mesenchymal stromal cell-derived extracellular vesicles and their
characterization
Raquel M. S. Cunha
a, Elga
Vargasb, Filipa Piresc, Cecília Caladod, Joaquim
Cabrale, Cláudia Silvae and Ana
Fernandes-Platzgummere
aInstituto Superior Técnico, University of Lisbon, Lisbon,
Portugal; bFaculty of Medicine, National University of Colombia, Bogotá, Bogotá,
Colombia; cInstituto Superior de Engenharia de Lisboa, Lisboa, Lisboa, Portugal;
dInstituto Superior de Engenharia de Lisboa, Lisboa, Portugal;
eDepartment of Bioengineering and iBB – Institute for Bioengineering and
Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal,
Lisboa,
Portugal
Introduction: Despite of high expectations, mesenchymal stromal cell
(MSC)-based therapies still lack efficacy, partially due to loss of cell viability
and
function upon administration. MSC-derived extracellular vesicles (MSC-EV) emulate
the
regenerative potential of MSC, shifting the field towards cell-free therapies. Clinical
applications require the establishment of a scalable and GMP-compliant processes for
the
production and isolation of MSC-EV, combined with robust characterization platforms.
Methods: To develop a well-established process for the production of
therapeutic MSC-EV, we compared different MSC sources (bone marrow, adipose tissue,
umbilical cord matrix), culture media compositions (DMEM supplemented with foetal
bovine
serum (Thermo Fisher Scientific), DMEM supplemented with human platelet lysate (AventaCell
Biomedical) and StemPro MSC SFM Xeno Free medium (Thermo Fisher Scientific)) and culture
parameters (oxygen tension and shear stress) in two different culture platforms (2D
static
tissue culture flask vs 3D dynamic spinner vessels). Subsequently, MSC-EV were isolated
by
ultracentrifugation or a commercially available isolation kit and characterized according
to
ISEV guidelines.
Results: MSC derived from different sources/donors were able to grow under
normoxia and hypoxia in 2D T-flasks and 3D spinner vessel culture systems, while maintaining
their immunophenotype and differentiation potential, according to the minimal criteria
defined by the ISCT. The time point for pre-conditioning and collection of conditioned
medium for MSC-EV isolation was also optimized for both 2D and 3D culture systems.
MSC-EV
were characterized according to MISEV 2018 guidelines, using techniques as NTA, protein
and
lipid quantification, western blot, imaging and Fourier-Transform Infrared Spectroscopy
(FTIR). The results indicate that MSC-EV derived from different sources/donors have
similar
size distribution, however, EV yields tend to be higher for the 3D culture system.
Of
notice, several spectral regions were identified by FTIR, enabling the detection of
differences in the biomolecules present in MSC-EV, MSC-conditioned media and cells
produced
under different conditions.
Summary/Conclusion: In summary, this study contributes to the establishment of
a scalable process for MSC-EV production.
OP2.02 = PF17.02
Evaluation of three different isolation methods for small
extracellular vesicles from human plasma in prostate cancer diagnosis
Bairen Pang
a, Ying Zhua,
Jie Nia, Xupeng Baia, Julia Beretova, Valerie
Wasingera, David Maloufb, Joseph Buccib, James
Thompsonb, Peter Grahamb and Yong Lia
aUNSW Sydney, Sydney, Australia; bSt George
Hospital, Sydney, Australia
Introduction: Extracellular vesicles (EVs) have great potential in prostate
cancer (PCa) diagnosis and progression monitoring to complement the inaccurate prostate
specific antigen (PSA) screening and invasiveness of tissue biopsy. However, current
methods
cannot isolate pure EVs and therefor EVs characteristics remain largely unknown. In
order to
develop an accurate approach for EV isolation, we aimed to compare three emerging
methods
with different characteristics of small EVs (sEVs) from human PCa plasma samples and
to
choose the best one for diagnostic and functional studies
Methods: PCa patients and age-matched healthy controls (HC) plasma (n = 6 in
each group) were used to isolate sEVs with 3 different isolation methods including
commercial ExoQuick Ultra Kit, qEV 35 and qEV 70 size exclusion chromatography (SEC).
Isolated sEV were characterized by nanoparticle tracking analysis, immunoblotting,
cyrogenic
electron microscopy, flow cytometry (FC) and proteomics analysis. For FC characterizing
surface marker expression, the sEVs were further purified by CD9 and CD81 commercial
immunoaffinity magnetic beads . Lipoprotein was captured by streptavidin biotinylated
ApoB
magnetic beads to measuring the lipoprotein contamination
Results: The sEV size, morphology, surface protein and protein cargo with
proteomics were analysed between the three isolation methods. sEVs isolated from SEC
methods
had a lower particle size, protein amount, protein/sEV marker ratio and ApoB+/sEV
marker
ratio than those from ExoQuick Ultra method. In addition, sEVs isolated from qEV35
demonstrated a significantly higher sEV content, more up-regulated and down-regulated
PCa
proteins from proteomics but lower sEV marker/protein ratio and a higher protein
contamination than those from qEV70. Furthermore, sEV marker signal also showed a
good
correlation with particle numbers instead of protein content in all the methods
Summary/Conclusion: qEV 70 method demonstrated better performance in isolating
relatively pure sEVs from human plasma; qEV35 has the better performance in isolating
samples with higher sEV content; ExoQuick Ultra isolated samples with closely sEV
content to
the qEV35 but with the highest non-sEV protein contaminations. People can choose higher
sEV
content or higher sEV purity according to the downstream analysis
Funding: St. George Hospital Cancer Research Trust Fund, UNSW
Sydney-University International Postgraduate Award, Cancer Institute NSW Early Career
Fellowship
OP2.03 = PF17.03
Multiplexed surface protein profiling of tumour-derived
extracellular vesicles by an electrokinetic sensor
Sara Cavallaro
a, Vasiliki
Arapib, Lorenca Berishac, Petra Håågb, Christiane
Stillera, Kristina Viktorssonb, Rolf Lewensohnb, Amelie
E. Karlströma, Jan Linnrosa and Apurba Devd
aKTH Royal Institute of Technology, Stockholm, Sweden;
bKarolinska Institute, Stockholm, Sweden; cPolitecnico di Torino,
Turin, Italy; dUppsala University, Uppsala, Sweden
Introduction: Small extracellular vesicles (sEVs) (30–200 nm in diameter) are
secreted by most cells, including tumour cells. They have attracted interest as biomarker
for cancer diagnostics based on liquid biopsies, because they are abundant in body
fluids
and their content (proteins, RNAs and other cargos) reflects their parent cells. Moreover,
sEVs may also be used for treatment monitoring, as recent studies suggested that the
expression levels of certain markers may change during therapy, reflecting tumour
response.
For cancer diagnostics and therapeutic purposes in clinical settings, it is important
to
have a device which allows multiplexed measurements, in order to scan a large number
of
markers simultaneously and compare the expression levels of different patients, or
same
patients at different treatment stages, in a time efficient manner.
Methods: Herein, we propose a multiplexed platform for label-free detection
and surface protein profiling of sEVs. The technique is based on the electrokinetic
phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta*
change
upon sEV binding on functionalized microcapillary surfaces. For the purpose, we used
sEVs
derived from lung cancer cells. In its current form, the platform can measure up to
5
channels simultaneously, however, it can be further expanded.
Results: Having demonstrated that our electrokinetic sensor successfully
detects sEVs in a specific way, we tested its ability to measure the expression level
of
membrane proteins. The analysis showed that it could detect differences in the expressions
of EGFR on sEVs, with a sensitivity of 10%. We then extended the platform for multiplexed
analysis, by connecting and measuring four capillaries, functionalized with different
capture probes, simultaneously. For the purpose, we targeted specific tumour markers,
i.e.
EGFR, and exosomal tetraspanin family proteins, such as CD9 and CD63. The results
showed
successful multiplexed EV detection.
Summary/Conclusion: Being the sensor suitable for multiplexed sEV detection,
we shall present our investigation on a set of pleural effusion samples collected
from a
cohort of lung-cancer patients with different genetic makeup.
Funding: Erling Persson Foundation
OP2.04 = PF17.04
Optimized immunocapture methods for the direct detection of EV
tumour associated proteins in biological fluids: playing around with biophysics
Carmen Campos Silva
a, Yaiza Cáceres
Martellb, María del Puerto Moralesc, Estela Sánchez
herrerod, Atocha Romero Alfonsod, Ricardo Jara Acevedoe,
María Yáñez Móf and Mar Valés Gómezb
aSpanish National Centre for Biotechnology, Madrid, Spain;
bSpanish National Centre for Biotechnology (CNB-CSIC), Madrid, Spain;
cInstituto de Ciencia de Materiales de Madrid (ICMM-CSIC), Madrid, Spain;
dInstituto de Investigación Sanitaria Hospital Universitario Puerta del Hierro,
Madrid, Spain; eImmunostep S. L. Salamanca, Spain; fCentro de Biología
Molecular Severo Ochoa (CBM-SO), Madrid, Spain
Introduction: Extracellular vesicles (EVs) are released to biological fluids
from different tissues and organs and they contain molecules proposed as biomarkers
for
multiple pathological conditions. However, most EV biomarkers have not been validated
due to
the lack of sensitive techniques compatible with high-throughput analysis required
for
routine screenings. Using immunocapture techniques, combining antibodies against
tetraspanins and candidate tumour-specific markers we have recently optimized several
assays
that greatly facilitate EV characterization.
Methods: We have improved flow cytometry and ELISA assays, increasing
substantially the sensitivity for EV detection. Using DLS, EM and analytical
ultracentrifugation, we have characterised the biophysical basis of this enhancement.
The
final methodology can be performed in any laboratory with access to conventional flow
cytometry or ELISA reader.
Results: Using combinations of antibodies specific for the tetraspanins CD9,
CD63 and CD81, it is possible to detect EVs in minimal volumes of urine and plasma
samples
without previous enrichment. Additionally antibodies against other less abundant markers,
like the epithelial marker EpCAM, have been used to capture and identify EVs directly
in
minimal volumes of urine or plasma with sensitivity higher than Western Blot analysis
of
isolated EVs. Furthermore, we demonstrate that additives altering the biophysical
properties
of an EV suspension, increased detection of tumour antigens in these immune-assays.
Summary/Conclusion: The development of sensitive, high-throughput methods,
easily translatable to clinical settings, as ELISA and flow cytometry described here,
opens
a new avenue for the systematic identification of any surface marker on EVs, even
scarce
proteins, using very small volumes of minimally processed biological samples. These
methods
will allow the validation of EV biomarkers in routine liquid biopsy tests.
Funding: MINECO, IMMUNOTHERCAN, TENTACLES, Immunostep
OP2.05 = PF17.05
Normalized extravesicular protein expression profiles on
antibody microarrays reveal protein associations in EVs of organotropic and metastatic
breast cancer cell lines
Molly L. Shen, Rosalie Martel, Lucile
Alexandre, Philippe Decorwin-Martin, Grant Ongo, Andy Ng, Lorenna Oliveira and David
Juncker
McGill University, Montreal, Canada
Introduction: When EV subpopulations are enriched on antibody microarrays and
probed for their surface proteins, the detection signal is biased towards abundant
subpopulations as it is dependent on both the protein expression level and the number
of EVs
captured. To address this challenge, we developed a novel normalization approach allowing:
1) the estimation of a target signal independent of EV subpopulation size through
dye-based
EV quantification, and 2) the assessment of subpopulation target enrichment relative
to the
population average by leveraging TIM4 as an unbiased, lipid-based EV capture. Here,
we
investigated the expression of cancer-associated proteins, particularly
metastasis-associated integrins (ITGs), in breast cancer EVs with varying metastatic
potential and organotropism.
Methods: The relative protein enrichment profiles for various EV
subpopulations were established from EVs of SkBr3 (HER2+), T47D and MCF-7 (ER+PR+),
BT549
and MDA-MB-231 (triple negative) breast cancer cell lines, as well as five
MDA-MB-231-derived cell lines of four different organotropisms (brain, bone, lung,
liver)
using our custom antibody microarrays with our normalization approach.
Results: As expected, HER2 was broadly detected in HER2+ SkBr3 EVs.
Interestingly, HER2- T47D and MCF-7 EVs also expressed HER2 where it was highly enriched
in
its EpCAM+ subpopulations. ITG α6, β3 and β4 were only found in triple negative and
organotropic EVs with ITG β3 and β4 differentially enriched based on the organotropism.
The
population average of MDA-MB-231 and lung-tropic EVs had high expression of ITG β4,
where
subpopulations of CD44+ EVs showed positive enrichment while CD9+ and CD63+ EVs showed
negative enrichment. ITG α5, β3 and β4 were absent in the bone-tropic CD81+ EV
subpopulation, a profile atypical in other organotropisms. Lastly, EGFR was negatively
enriched in Tetraspanin+ subpopulations in MDA-MB-231 EVs, but positively enriched
in these
subpopulations in organotropic EVs, especially for brain-tropism.
Summary/Conclusion: Following normalization, we were able to quantify specific
protein associations, uncovering a multitude of co-enrichment profiles that characterize
specific metastatic and organotropic cell lines. Notably, we found enrichment signatures
that distinguish between different organotropisms derived from the same parental cancer
line.
Funding: This research was supported by the Natural Sciences and Engineering
Research Council of Canada (NSERC) and the Genome Canada Distruptive Innovation program.
OP2.06 = PF17.06
Heparan sulphate proteoglycans are required for EV-mediated
delivery of multiple growth factors
Sara Veiga, Alex Shephard, Alex Cocks, Aled
Clayton and Jason Webber
Cardiff University, Cardiff, UK
Introduction: The tissue microenvironment surrounding tumours is complex and
the cross-talk between cancer and non-cancer cells is essential for tumour growth
and
progression. We have previously shown that heparan sulphate proteoglycans (HSPGs),
on the
surface of prostate cancer EVs, are required for delivery of TGFβ and initiation of
a
disease-supporting fibroblast phenotype. However, HSPGs are known to bind numerous
growth
factors, so here we have explored the repertoire of such proteins tethered to EVs
by
HSPGs.
Methods: EVs were isolated from DU145 prostate cancer cell conditioned media
by ultra-centrifugation onto a sucrose cushion. Vesicular HSPGs were modified either
by
removal of heparan sulphate (HS) glycosaminoglycan (GAG) chains using the enzyme Heparinase
III (HEPIII), or attenuation of HSPG core protein expression using shRNAs to knockdown
specific HSPGs within the parent cell. Differences in proteins present in control
vs
modified EVs were identified by a sensitive protein array, based on proximity-ligation
technology, and selected targets validated by ELISA. Functional delivery of growth
factors
by EV-associated HSPGs to recipient fibroblasts is being explored using a variety
of in
vitro techniques.
Results: Proteome analysis identified 49 targets that bind to HS-GAG chains,
and also 108 different proteins that showed altered expression following the loss
of one or
more HSPGs from EVs. Using ELISA, we have been able to quantify selected candidates
on wild
type vesicles, some of these are lost following HS-digestion. We were also able to
validate
proteins on HSPG-deficient vesicles. Gene ontology analysis suggests that EV HSPG-mediated
delivery of growth factors is important for control of processes such as angiogenesis,
tumour invasion and immune regulation. Functional validation of proteins identified
is
ongoing.
Summary/Conclusion: Here we demonstrate that HSPGs play a key role in loading
of EVs with a complex assortment of growth factors, and therefore subsequent EV-mediated
growth factor delivery. We anticipate that loss or damage of EV-associated HSPGs will
result
in attenuation of EV induction of a tumour-supporting fibroblast phenotype.
Funding: Cancer Research Wales
OP2.07 = PF17.07
Robust exosomal biomarker panel discovery in ovarian cancer
using machine learning approaches and studying miRNA & miRNA-target
interactions
Paritra Mandal
a, Tyler
Sloneckib, Brian C. Deanb, R. Kenneth Marcusb, William
Bridgesb and Terri F. Bruceb
aClemson University, Pendleton, USA; bClemson
University, Clemson, USA
Introduction: Ovarian cancer (OC) is the fifth leading cause of cancer-related
death in women, partly due to difficulty in early diagnosis. Extracellular vesicles
(EVs)
show promise for use in early diagnostics of OC. Here, EVs from cervical mucus (CM)
of
ovarian cancer patients were used for discovery of OC biomarkers for diagnostics.
Machine
learning was used to mine EV miRNA data to develop an OC biomarker panel (validation
via The
Cancer Genome Atlas). Examination of the miRNA targets reveal that the panel is a
sufficiently accurate predictor of OC.
Methods: EVs from the CM of 48 patients (15 high-grade serous, 24 low-grade, 7
benign) were isolated for small RNA-sequencing. The top differentially expressed miRNAs
were
used in a random forest and “voom” (variance modelling at the observational level)
model.
Unsupervised approaches were used and then vetted against patient symptomology data.
A TCGA
ovarian cancer dataset (n = 100) was used for validation.
Results: An OC biomarker panel of 10 microRNAs (voom: 96.55% accuracy; random
forest: 88% accuracy) was generated. The panel consists of members from the mir-200
family
and the mir-16 family, among others. The miRNA targets are associated with molecular
functions and pathways specific in OC progression.
Summary/Conclusion: Our method has identified EV miRNA biomarkers that may be
crucial for early, non-invasive detection of OC. Data science has been used to develop
a
feedback system integrating biochemical experiments, smaller datasets, and previously
available data to identify and verify a biomarker panel for OC diagnostics.
Funding: Support from the National Science Foundation, Eppley Foundation for
Scientific Research, Gibson Foundation, Prisma Health System and ITOR Biorepository
are
gratefully acknowledged.
OS19
Symposium Session 19: Neurologic Mechanism
Chair: Efrat Levy – Center for Dementia Research, Nathan S. Kline Institute Chair:
Shanthini Sockanathan – Johns Hopkins University
OS19.1
Extracellular vesicle associated microRNA-29a elicits microglial
activation and synaptodendritic injury with chronic methamphetamine exposure
Dalia Moorea, Alexander Clarkb, Farah
Shahjinc, Niming Wud, Subhash Chandd, Katherine
Odegaardd, Austin Gowend, Rick Bevinse, Gurudutt
Pendyalad, Howard Foxd and Sowmya V.
Yelamanchili
f
aUniversity of Nebraska Medical Center, Omaha, USA;
bCreighton University, Omaha, USA; cUNMC, Omaha, USA;
dUniversity of Nebraska Medical Center, Omaha, USA; eUniversity of
Nebraska at Lincoln, Lincoln, USA; fDepartment of Anesthesiology, Omaha, USA
Introduction: Methamphetamine (MA) and related amphetamine compounds, which
are potent psychostimulants, are among the most commonly used illicit drugs. Neuroimaging
studies have revealed that chronic MA abuse can indeed cause neurodegenerative changes
in
the brains of human MA abusers including prominent microglial activation throughout
the
brain. It is still unclear how chronic inflammation caused by MA abuse leads to long-term
damage to the brain. With this in mind, we are particularly interested in studying
the role
of extracellular vesicles (EVs) in eliciting chronic inflammation in MA exposed brains.
In
the present study, we focus on the role of a miRNA, miR-29a-3p (miR-29a) in chronic
MA
exposure. Here, we present novel data that shows for the first time how chronic MA
impacts
not only the biogenesis but also the EV associated miRNA cargo thereby affecting the
overall
health of the neurons and glial cells in the brain.
Methods: – Density gradient centrifugation for isolation of Brain-derived
Vesicles
- Characterization of BDEs by Western Blotting, Nanoparticle tracking analysis and
Transmission Electron Microscopy
- Quantitative RT-PCR
- Digital droplet PCR
- Confocal Imaging of dendritic spines and synapses
Results: In the present study, we show from both in vivo and in vitro studies
that chronic methamphetamine (MA) treatment alters EV biogenesis and microRNA (miRNA)
cargo.
Brain-derived EVs (BDE) isolated from frontal grey tissue of rhesus macaques that
were
administered MA in a chronic regimen revealed a significant increase in both number
and
size. Further analysis revealed increase in biogenesis genes and increased levels
of miRNA,
miR-29a-3p (miR-29a). In situ hybridization of the frontal brain area revealed that
miR-29a
was exclusively expressed in microglia and neurons. Further, in vitro studies revealed
that
EV associated miR-29a elicited not only neuronal damage but also was able to activate
microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory
cycle. Finally, we show that an anti-inflammatory drug was able to rescue inflammation,
miR-29a levels and synaptodendritic injury.
Summary/Conclusion: In summary, our results present for the first time show
that chronic MA exposure in the brain affects EV biogenesis and miRNA expression.
We further
confirm that miR-29a can serve as potential marker to diagnose synaptic deficits for
chronic
MA addiction in humans. Finally, we reveal that anti-inflammatory drug could rescue
the EV
biogenesis and reduces the secretion of miR-29a, thereby rescues synaptodendritic
injury.
Our data further supports the use of the anti-inflammatory drugs as therapeutic
interventions for MA addiction.
Funding: NIDA funding # R01DA042379
OS19.2
Blood-borne and brain-derived ectosomes/microparticles in
morphine-induced anti-nociceptive tolerance
Deepa Ruhela, Veena Bhopale, Ming Yang, Kevin Yu, Eric Weintraub,
Aaron Greenblatt and Stephen R. Thom
University of Maryland School of Medicine, Baltimore, USA
Introduction: Opioid pain treatment is impeded because chronic administration
decreases analgesia, a condition called tolerance that prompts dose escalation contributing
to morbidity and mortality. Inflammatory interleukin (IL)-1β is required for tolerance
development, so we hypothesized that pro-inflammatory extracellular vesicles (EVs)
play a
role.
Methods: EVs with opioid administration were assayed in mice and humans.
Annexin V-positive, 0.1–1 µm diameter microparticles (MPs) were assessed by flow cytometry
in murine and human blood and in murine deep cervical lymph nodes that drain brain
glymphatics. Blood-borne exosomes (<100 nm) were assayed by tunable-resistance pulse
sensing (TRPS). Anti-nociceptive tolerance following morphine administration to mice
was
assessed by speed of tail removal from warm water.
Results: Repetitive morphine dosing of mice to induce anti-nociceptive
tolerance increased blood-borne MPs by eightfold, and by tenfold in cervical lymph
nodes.
MPs expressed proteins specific to neutrophils, microglia, astrocytes, neurons and
oligodendrocytes. IL-1β content of MPs increased 68-fold. Administration of an IL-1β
antagonist to mice diminished blood and glymphatic MPs elevations and abrogated tolerance
induction. Intravenous polyethylene glycol Telomer B that lyses MPs and intraperitoneal
methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation
factor-2 to inhibit toll-like receptors, inhibited MPs elevations and tolerance. Neutropenic
mice did not develop anti-nociceptive tolerance, elevations of blood-borne MPs or
cervical
node MPs expressing microglial proteins. Elevations of blood-borne exosomes were not
identified based on TRPS analysis. Patients entering treatment for opioid use disorder
exhibited similar MPs elevations as do tolerant mice.
Summary/Conclusion: Neutrophil-derived MPs containing IL-1β are required for
morphine-induced anti-nociceptive tolerance.
Funding: This project was supported by Grant N00014-16-1-2868 from the Office
of Naval Research and an unrestricted grant from the National Foundation of Emergency
Medicine.
OS19.3
EVs are a conveyor of toxic dipeptide repeat proteins in C9orf72
ALS/FTD models
Maria Elena Cicardi and Davide Trotti
Thomas Jefferson University, Philadelphia, USA
Introduction: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative
disease characterized by loss of motor neurons. In ALS, motor symptoms initiate focally
and
then progress gradually, distal from the initial focus. Abnormal forms of ALS-associated
proteins are physically exchanged between neuronal cells. Pathogenic ALS proteins
like SOD1,
FUS and TDP43 are transmitted between cells by assisted mechanisms, mainly extracellular
vesicles (EVs), spreading toxicity and misfolding of native proteins within the recipient
cells. An intronic G4C2 aberrant nucleotide repeat expansion in C9orf72 gene is the
most
common genetic cause of ALS. Translation of this expanded region occurs by a process
called
repeat associated non-AUG (RAN) translation that produces five dipeptide repeats proteins
(DPRs), polyGA, polyGP, polyGR, polyPA and polyGA. PolyGA, polyGR and polyPR are associated
with toxicity in neurons. In this work we study the recruitment of these aberrant
proteins
into extracellular vesicles (EVs) and the potential role of these EVs in spreading
toxicity
between cells of the central nervous system.
Methods: To isolate the EVs from cell culture media we isolated by
ultracentrifugation the larger vesicles at 21,000xg and the smaller EVs at 100,000xg.
Number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack
analysis (F-NTA) and by Cytoflex. The protein content of the vesicles was analysed
by
western blot (WB). To evaluate the potential toxicity of the EVs, a transwell system
(TW)
was employed. Neuron viability was assessed using live imaging techniques.
Results: NSC34 were transfected with reporter constructs expressing DPRs
tagged with GFP protein. By F-NTA, Cytoflex and WB analysis we assessed that all the
five
DPRs were loaded in both the large and the small vesicles isolated from cell culture
medium.
By TW, NSC34 transfected with the DPRs were put in contact with primary cortical neurons
(CNs) transfected with synapsin driven Td-Tomato for live imaging purposes. We observed
that
polyGR+ NSC34 were able to cause a significant decrease in CNs viability. We also
observed
that polyGR+ EVs associated toxicity was directly dependent on polyGR length. This
effect
was reverted reducing the number of polyGR+ EVs treating NSC34 with GW4869. To understand
the downstream effect of polyGR+ EVs in recipient cells we studied TDP43 mislocalization,
RAN-translation and activation of the integrated stress response, finding a dysregulation
of
all these potentially toxic pathways in neurons treated with polyGR+ vesicles.
Summary/Conclusion: Concluding, DPRs are actively secreted in EVs and polyGR+
vesicles cause the activation of toxic mechanisms in the recipient cells, possibly
contributing to the spreading of ALS.
Funding: NIH 080–19250-S31201
OS19.4
Amniotic fluid stem cell derived extracellular vesicles modulate
pathogenic immune responses in experimental autoimmune encephalomyelitis
Giorgia Manni
a, Rita
Romanib, Marco Gargaroc, Luisa Pascuccid, Pierluigi
Orvietanie, Paolo Puccettif, Vincenzo Nicola Talesag and
Francesca Fallarinoh
aDepartment of Experimental Medicine, Section of
Pharmacology, University of Perugia, Perugia, Italy; bDepartment of Experimental
Medicine, Sec. Biology, Perugia, Italy; cDepartment of Experimental Medicine, Sec
Pharmacology, Perugia, Italy; dDepartment of Veterinary Medicine, Perugia, Italy;
eDepartment of Experimental Medicine, Sec. Phisiolgy and Biochemistry, Perugia,
Italy; fDepartment of Experimental Medicine, Sec. Pharmacology, Perugia, Italy;
gDepartment of Experimental Medicine, Sec. of Biology, Perugia, Italy;
hDepartment of Experimental Medicine, Sec. of Pharmacology, Perugia, Italy
Introduction: Pregnancy is the a condition that profoundly mitigates symptoms
of multiple sclerosis (MS) a complex disease characterized by immune dysfunction and
neurodegeneration affecting 2.3 million people worldwide. Serum exosomes, released
by
specific cells during pregnancy, modulate the immune and central nervous system function
and
contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis
(EAE), an induced preclinical model of MS. Extracellular vesicles (EVs) are the new
means
for communication among cells. The aim of our study was to characterize the ability
of Human
Amniotic Fluid Stem Cells-derived EVs (HASC-EVs) to antigen presenting cell function
thus
correcting immune dysfunction in EAE.
Methods: Amniotic Fluids were obtained from human 16–17-week pregnant women.
HASC-EVs were collected by ultra-centrifugation. EVs were characterized for their
specific
proteins, lipids and nucleic acids expression. The ability of EVs to modulate immune
responses was performed in vitro, testing the ability of EVs to induce a tolerogenic
phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential
to
suppress EAE, induced by immunization C57/B6 female mice with MOG35-55 peptide.
Results: We found that HASC-EVs expressed high levels of Galectin-1 and
promoted a significant increase of the immunoregulatory enzyme indoleamine 2,3-dioxygenase-1
enzyme in DCs. Moreover in in vivo experiments administration of HASC-EVs significantly
reduced disease severity in EAE. Such effect was associated with reduced neurological
deficits and suppression of pathogenic T helper 17 (Th17) cells, and increased percentage
of
regulatory T cells (Treg-Foxp3+) cells.
Summary/Conclusion: Our findings unravel immunoregulatory effects of EVs
secreted by HASCs. EVs may represent a novel cell-free immune regulatory and regenerative
therapeutic approach that can potentially mitigate immune dysfunction and promote
remyelination.
OS19.5
Association of neuronal-derived extracellular vesicles cargo
with cognitive decline in late middle life
Erden Eren
a, Jack Huntb,
Michelle Shardellc, Sahil Chawlaa, Joyce Trana, Jeffrey
Gud, Nick Vogtb, Sterling Johnsonb, Barbara B.
Bendlinb and Dimitrios Kapogiannise
aNational Institute on Aging, NIH, Baltimore, USA;
bWisconsin Alzheimer’s Disease Research Center, University of Wisconsin,
Madison, USA; cUniversity of Maryland, Baltimore, USA; dNIA/NIH,
Baltimore, USA; eLaboratory of Clinical Investigation, National Institutes of
Aging, Baltimore, USA
Introduction: Alzheimer’s disease (AD) is characterized by a long preclinical
stage during which phosphorylated Tau pathology spreads in the brain leading to clinical
symptoms. Pathogenic Tau spreads, in part, via Extracellular vesicles (EVs). We and
others
have demonstrated that Tau cargoes of neuronal-derived EVs (nEVs) from blood can serve
as
biomarkers for AD. We aimed to examine whether nEV Tau cargo can predict cognitive
decline
in late middle age by leveraging samples from participants in the Wisconsin Registry
for
Alzheimer’s Prevention (WRAP) study.
Methods: We blindly immunoprecipitated nEVs using antibody against neuronal L1
cell adhesion molecule (L1CAM) from serum samples of 146 WRAP participants who were
cognitively unimpaired at baseline (mean age 62.4 ± 6.3 years old; 71.2% females;
42.5%
APOE4 carriers), of whom half subsequently developed cognitive decline. We measured
phosphorylated (p181 and p231) and total Tau in nEVs using electrochemiluminescence
assays.
We used linear regression models to identify differences between cognitive status
groups
including age, sex ApoE status and the cognitive status*age interaction in the model.
Results: At baseline, we found trends for higher p181-(p = 0.07) and p231-Tau
(p = 0.05) levels in future decliners compared to stable participants. Further, there
were
significant cognitive status*age interactions for pTau231 (p < 0.01), total Tau
(p < 0.001) and pTau181 (p < 0.05) with higher levels with increasing age in future
decliners
Summary/Conclusion: nEV Tau cargo differs between late middle-aged individuals
at risk for AD with and without future cognitively decline even before decline occurs,
presumably due to subclinical spread of Tau pathology. Further nEV biomarker development
may
allow preclinical AD diagnosis.
Funding: This research was supported in part by the Intramural Research
Program of the National Institute on Aging, NIH, and the WRAP grant R01AG027161.
OS19.6
Extracellular vesicle release from the choroid plexus visualized
using ExoMap-2 Mice
Christie D. Fowler
a, Valeria
Lallaia, Francis Fordjourb, Lane Christensonc and Stephen
Gouldb
aUniversity of California, Irvine, Irvine, USA;
bJohns Hopkins University, Baltimore, USA; cUniversity of Kansas
Medical Center, Kansas City, USA
Introduction: In the brain, circulating extracellular vesicles (EVs) in the
cerebrospinal fluid (CSF) contain a variety of signalling factors, including proteins,
enzymes, and RNA transcripts. While EVs have been implicated in many cell-to-cell
signalling
contexts, the vast majority of these studies are based on findings derived from cell
culture
conditions. Thus, the ability to identify cell type-specific EV release from cellular
subpopulations within the brain represents a critical barrier in the field.
Methods: To address this knowledge gap, we utilized a novel transgenic mouse
model to determine the release of cell-type specific EVs. Here we report the ExoMap-2
mouse,
which is designed to express an exosomal green fluorescent protein in response to
expression
of Cre recombinase. Specifically, the ExoMap-2 transgene was inserted at the mouse
H11 locus
and consists of (i) a broadly expressed CAG promoter/enhancer, (ii) a floxed ORF encoding
MTS-tdTomato, (iii) an ORF encoding the exosomal protein AcylTyA fused to mNeonGreen
(mNG),
and (iv) a 3ʹ UTR containing the WPRE element and polyadenylation signal from the
bovine
growth hormone gene.
Results: Intracranial ventricular injections of the viral vector AAV-TTR-Cre,
which drives Cre recombinase expression from the choroid plexus-specific promotor
of the
transthyretin gene, leads to AcylTyA-mNG expression in the choroid plexus. Moreover,
we
observed that these mice released mNeonGreen-positive EVs into the cerebrospinal fluid
and
also visualized the vesicles in the blood. Furthermore, these mice displayed an accumulation
of AcylTyA-mNG fluorescence in the medial habenula.
Summary/Conclusion: The results indicate that choroid plexus-derived EVs are
trafficked to the CSF and the medial habenula, and more generally, that the ExoMap-2
mouse
can be used to follow the trafficking of tissue-specific EVs into biofluids and between
tissues in vivo.
Funding: Supported by the NIH (U19CA179563 to SG, DA039658 to CDF and HD082484
to LKC)
OS20
Symposium Session 20: Cancer: Pathogenesis and Treatment
Chair: Mary Bebawy, PhD – The University of Technology Sydney
Chair:
Janusz Rak – McGill University
OS20.1
Extracellular vesicles containing oncogenic mutant β-catenin
activate Wnt signalling pathway in the recipient cells
Pamali Fonseka
a, Hina
Kalrab, Lahiru Gangodab and Suresh Mathivananb
aLa Trobe Institute for Molecular Sciences, La Trobe
University, Melbourne, Australia; bLa Trobe University, Melbourne, Australia
Introduction: Large-scale colorectal cancer (CRC) sequencing studies have
shown that 93% of all tumours had at least one mutation in proteins implicated in
the Wnt
signalling pathway. Mutations in
β-catenin have often been associated with the constitutive activation of Wnt signalling
pathway and has been established as a major driver of CRC. One of the proposed mechanisms
of
activating Wnt signalling involves extracellular vesicles (EVs) as cellular couriers
to
transfer Wnt ligands from one cell to another. However, the association of oncogenic
mutant
β-catenin with EVs has not been studied. Subpopulations of cancer cells with different
mutational loads and behavioural variations lead to intra-tumour heterogeneity
Methods: Integrative proteogenomic analysis showed the secretion of mutant
β-catenin via EVs. EVs were isolated by ultracentrifugation and OptiPrep density gradient
centrifugation. SILAC-based quantitative proteomics analysis, immunofluorescence,
biochemical analysis, qPCR and xenograft models were employed to unveiling the role
of EVs
carrying mutant β-catenin.
Results: An integrative proteogenomic analysis identified the presence of
mutated β-catenin in EVs secreted by colorectal cancer (CRC) cells. Follow up experiments
established that EVs released from LIM1215 CRC cells stimulated Wnt signalling pathway
in
the recipient cells with wild type β-catenin. SILAC-based quantitative proteomics
analysis
confirmed the transfer of mutant β-catenin to the nucleus of the recipient (RKO CRC)
cells.
In vivo tracking of DiR labelled EVs in mouse implanted with RKO CRC cells revealed
its bio
distribution, confirmed the activation of Wnt signalling pathway in tumour cells and
increased the tumour burden.
Summary/Conclusion: Overall, for the first time, this study reveals that EVs
can transfer mutant β-catenin to the recipient cells and promote cancer progression.
OS20.2
Salivary exosomes – Carrier of high risk human papillomavirus
causing oropharyngeal cancer
Chamindie Punyadeera
a, Kai
Tangb, Liz Kennyc, Brett Hughesc, Sarj Vasanic
and Yunxia Wand
aQUT, Kelvin Grove, Australia; bQUT, brisbane,
Australia; cRBWH, Brisbane, Australia; dQUT, Brisbane, Australia
Introduction: There has been a significant increase in incidence of human
papillomavirus 16 (HPV16) driven oropharyngeal cancer (OPC) in developed countries.
There is
evidence that HPV alters the molecular cargo of exosomes released by OPC. Emerging
evidence
suggests that HPV integration within the human genome is associated with both genomic
and
transcriptomic alterations. Consistent with previous studies, the genomic viral–cellular
junctions were identified using DIPS-PCR method in 15 (88%) saliva samples collected
from
HPV16-driven OPC.
Methods: Morphology and molecular features of exosomes derived from three
different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and
salivary
oral rinses were examined using Transmission electron microscopy (TEM), nanoparticle
tracking (NTA) and western blot analysis. HPV-16 DNA detection in salivary exosome
was
determined by using qPCR method. Proteome profile of salivary exosomes derived from
both
cancer-free controls and HPV16-driven OPC patients was characterized using liquid
chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS).
Results: We demonstrate that unstimulated saliva had greater abundance of
exosomes when compared to the other sampling methods. Three common exosome markers
(CD9,
CD63 and CD81) were higher in unstimulated saliva. Only salivary exosomes derived
from
HPV-driven OPC patients had a detectable level of HPV-16 DNA. The proteomic signature
of
salivary exosome was significantly (p < 0.01) different between cancer-free controls
and
HPV-driven OPC. We found elevated protein abundance of five main glycolytic enzymes
(i.e.
Phosphoglycerate Kinase 1 (PGK1), Glyceraldehye-3-phosphate dehydrogenase (GAPDH),
Aldolase
(ALDOA) and Lactate dehydrogenase A (LDHA) in salivary exosomes derived from OPC patients,
suggesting a functional role of salivary exosome in the reciprocal interplay between
HPV-driven OPC and glucose metabolism.
Summary/Conclusion: Our data suggest that the development of a low-cost
non-invasive saliva-based test using both salivary exosomal DNA and protein may offer
an
opportunity to detect HPV-driven OPC, that may be clinically useful in managing these
patients.
Funding: Cancer Australia Grant
OS21
Symposium Session 21: EV Signalling
Chair: Wei Guo – University of Pennsylvania
OS21.1
Continuous in vivo release of mast cell derived extracellular
vesicles from an implanted device spreads pro-inflammatory response in mice
Krisztina V. Vukman
a, Gábor
Seregélyesb, Tamás Visnovitza, Péter Lőrinczc, Anna
Koncza, Andrea Ferenczd, Daniella Fehérd, Krisztina
Juhosd, Barabara Sódara, Krisztina Pálóczia, Eszter
Tótha, András Försönitsa, Delaram Khamaria, Alicia
Galinsogaa and Edit Buzáse
aDept. of Genetics, Cell- and Immunobiology, Semmelweis
University, Budapest, Hungary, Budapest, Hungary; b1Dept. of Genetics, Cell- and
Immunobiology, Semmelweis University, Budapest, Hungary, Budapest, Hungary;
cDepartment of Anatomy, Cell and Developmental Biology, Eötvös Loránd University,
Budapest, Hungary, Budapest, Hungary; dDepartment of Surgical Research and
Techniques, Semmelweis University, Budapest, Hungary, Budapest, Hungary;
eSemmelweis University, Department of Genetics, Cell and Immunobiology, MTA-SE
Immune-Proteogenomics Extracellular Vesicle Research Group, Budapest, Hungary and
HCEMM_SE
Extracellular Vesicle Research Group, Budapest, Hungary
Introduction: Mast cells are important players of the immune system and they
secrete a wide range of mediators during bacterial infections. Mast cells are also
able to
release extracellular vesicles (EVs). Here, we report that mast cells communicate
with each
other in vivo by EVs.
Methods: We isolated bone marrow-derived and peritoneal mast cells from
GFP-transgenic and wild type mice. EVs were separated from the conditioned media of
these
cells cultured in the presence or absence of lipopolysaccharide (LPS). EVs were
characterised according to the MISEV2018 guidelines by flow cytometry, electron and
fluorescent microscopy, TRPS, the SPV lipid and the BCA protein assays. Separated
EV-s were
cultured with naïve mast cells, and tumour necrosis factor (TNF)-α production was
tested by
ELISA and intracellular flow cytometry. GFP+ mast cells were seeded in diffusion chambers
which were implanted into the peritoneal cavities of mice enabling us to investigate
the
continuous in vivo release of EVs. Uptake of GFP+ EVs and TNF-α expression of peritoneal
mast cells were tested by flow cytometry and fluorescent microscopy.
Results: Here, we showed that bacterial LPS-sensing mast cells release EVs
that in turn, induce TNF-α expression in resting MCs in vitro. Moreover, we confirmed
that
EVs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory
response by inducing TNF-α secretion in peritoneal mast cells.
Summary/Conclusion: EV communication between members of the mast cell network,
play an important role in spreading and escalating pro-inflammatory responses to immune
stimuli. Our data may provide an explanation how the relatively rare tissue resident
mast
cells can play key roles in diseases such as autoimmune arthritis.
Funding: Hungarian Scientific Research Fund (OTKA PD112085), National Heart
Program (NVKP_16-1-2016-0017), VEKOP-2.3.2–16-2017-000002, 2.3.3–15-2017-00016,
H2020-MSCA-ITN-2017-722148 TRAIN EV, Higher Education Excellence Program FIKP, European
Union’s Horizon 2020 Research and Innovation Programme (No 739593), János Bolyai Research
Fellowship of the Hungarian Academy of Sciences.
OS21.2
Small extracellular vesicles modulated by the αVβ3 integrin
reprogramme recipient cells towards an aggressive neuroendocrine cancer
phenotype
Fabio Quaglia
a, Shiv Ram
Krishna, George Daaboulb, Srawasti Sarkea, Peter A.
McCuea, William Kellya, Qin Liuc and Lucia R.
Languinoa
aThomas Jefferson University, Philadelphia, USA;
bNanoView Biosciences, Boston, USA; cWistar Institute, Philadelphia,
USA
Introduction: The ability of small extracellular vesicles (sEVs) to
reprogramme cancer cells is known. Integrins, receptors for extracellular matrix proteins,
are major players in mediating sEV functions. Previously, we have reported that the
αVβ3
integrin is detected in sEVs of prostate adenocarcinoma (PrCa) cells and transferred
into
recipient cells in a paracrine fashion; however, its role and expression have never
been
explored in the most aggressive forms of PrCa, such as neuroendocrine PrCa (NEPrCa).
NEPrCa
does not express androgen receptor (AR) but does express neuron-specific proteins,
such as
Aurora kinase A, Synaptophysin and Neuron specific enolase, that activate pro-tumorigenic
pathways independently from the AR.
Methods: We isolated sEVs from PrCa C4-2B cells using iodixanol density
gradients and characterized them by immunoblotting and ExoView. The experiments were
performed in vivo by injecting subcutaneously, in nude mice, DU145 cells treated with
sEVs
expressing or lacking the αVβ3 integrin, and in vitro, by testing anchorage-independent
growth of different cell lines treated with the same sEVs. Discarded human tissues
from PrCa
metastasis were analysed by immunohistochemistry (IHC).
Results: We demonstrate that a single treatment of PrCa cells with sEVs
significantly stimulates tumour growth and anchorage-independent growth. Moreover,
we show
that one treatment with sEVs, shed from C4-2B cells that express αVβ3, but not from
the
control cells that lack αVβ3, induces differentiation of PrCa cells towards a neuroendocrine
phenotype and downregulates AR. Finally, our IHC analysis shows co-expression of αVβ3
integrin and Synaptophysin in NEPrCa metastatic lesions.
Summary/Conclusion: In conclusion, our current study shows, for the first
time, that αVβ3 integrin expression in donor cells generates sEVs that reprogramme
recipient
cells towards an aggressive tumour phenotype.
Funding: This study was supported by NCI-P01-140043, R01-224769 to LRL.
OS21.3
Exosomes in filopodia formation
Caitlin McAtee
a, Daisuke
Hoshinob, Nan Hyung Hongc, Bong Hwan Sungd, Ariana von
Lersnera, Andries Zijlstrae and Alissa Weaverf
aVanderbilt University, Nashville, USA; bKanagawa
Cancer Center Research Institute, Yokohama, Japan; cFred Hutchinson Cancer
Research Center, Seattle, USA; dDepartment of Cell and Developmental Biology,
Vanderbilt University, Nashville, USA; eVanderbilt University Medical Center,
Nashville, USA; fDepartment of Cell and Developmental Biology, Vanderbilt
University School of Medicine, Nashville, USA
Introduction: Exosomes are small extracellular vesicles (SEVs) that carry a
variety of cargoes and have been shown to promote tumour cell motility and metastasis.
Cell
motility is influenced by dynamic formation and stability of filopodia: actin-rich
protrusions that extend from the leading edge and perform directional sensing. Filopodia
regulators such as fascin are upregulated in multiple epithelial cancers and can promote
invasive phenotypes. However, how filopodia are induced and controlled by extracellular
factors is poorly understood. Here, we describe a role for SEVs in regulating filopodia
formation and tumour cell motility.
Methods: We utilized B16F1 melanoma cells and HT1080 fibrosarcoma cells for
fixed- and live-cell imaging to quantify filopodia numbers and dynamics in control
and
exosome-deplete conditions. iTRAQ proteomics was used to identify SEV protein cargoes
that
contribute to filopodia formation. In vivo experiments were performed using a chick
embryo
model for metastasis.
Results: Inhibition of exosome secretion in cancer cell lines, via Rab27a or
Hrs knockdown, led to decreased filopodia numbers. Specificity to SEVs was demonstrated
by
rescue experiments in which purified SEVs but not large EVs rescued the filopodia
phenotypes
of exosome-inhibited cells. Live imaging of Hrs-KD cells revealed that exosome secretion
regulates formation and stability of filopodia. Proteomics data and molecular validation
experiments identified the TGF-beta coreceptor endoglin as a key SEV cargo regulating
filopodia formation, cancer cell motility, and metastasis.
Summary/Conclusion: In this study, we identified exosomal endoglin as a
regulator of filopodia formation and in vivo metastasis. These data are relevant to
cancer
as endoglin expression is altered in many cancers. In addition, endoglin is the disease
gene
for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. Overall,
our
data implicate SEV-carried endoglin as a key cargo regulating filopodia.
OS21.4
Astrocyte-derived EV-mediated blood-brain barrier
disruption
Shilpa Buch, Ke Liao, Susmita Sil, Fang Niu
and Guoku Hu
University of Nebraska Medical Center, Omaha, USA
Introduction: The breach of the Blood-brain barrier (BBB), resulting in
ensuing neuroinflammation, is a key feature of HIV-associated neurological disorders
(HANDs). While combination antiretroviral therapy (cART) has successfully suppressed
peripheral viraemia, cytotoxicity associated with the presence of viral Tat protein
in
tissues such as the brain, remains a significant concern. Our previous study has
demonstrated that HIV-1 Tat can induce disruption of BBB by downregulation of tight
junction
(TJ) proteins in Human Brain Microvascular Endothelial cells (HBMECs) and that this
is
regulated by the autophagic pathways.
Methods: EVs were isolated from HIV Tat-stimulated mouse/human primary
astrocytes using the standard differential ultracentrifugation method and characterized
by
transmission electron microscopy, NanoSight & western blot analyses. Among the various
miRs dysregulated in HIV Tat -stimulated astrocyte EV cargo, miR-7 was found to be
upregulated by realtime PCR. Confocal microscopy identified uptake of astrocytic EVs
by
HBMECs. Functional assessment of astrocytic EV uptake by HBMECs involved cell permeability
using transepithelial electrical resistance as well as trans-well endothelial cell
monolayer
permeability assays.
Results: HIV-1 protein Tat-mediated induction of microRNAs (miRs) in
astrocyte-derived extracellular vesicles (ADEVs) regulated the permeability of BBB
by
targeting the expression of TJ proteins in the HBMECs. Exposure of HBMECs to Tat-ADEVs
resulted in down-regulation of the tight junction protein Claudin 5, resulting in
increased
endothelial cell monolayer paracellular permeability. Microarray data of Tat-ADEVs
demonstrated upregulation of several miRs compared to that of controls, among which
upregulated miR-7 was identified to target the TJ proteins using ingenuity pathways
analysis. Increased expression of miR-7 was validated in Tat exposed astrocytes and
Tat-ADEVs. ADEVs loaded with miR-7 oligos showed similar effects as that observed
with
Tat-ADEVs in inducing permeability in HBMECs. Increased expression of miR-7 with
downregulation of claudin-5 was also recapitulated in microvessels isolated from the
brains
of Doxycycline-inducible HIV-1 Tat transgenic mice (iTat) mice and in lysates isolated
from
the frontal cortices of SIV+ macaques/HIV+ autopsied brains.
Summary/Conclusion: Our findings demonstrated that Tat-ADEVs containing miR-7
as an important mediator underlying Tat-mediated disruption of the BBB.
OS22
Symposium Session 22: EVs as Delivery Vehicles
Chair: Steven M. Jay – Assistant Professor, University of Maryland, College
Park
OS22.1
Single particle Raman spectroscopy: an emerging tool to quantify
composition of engineered extracellular vesicles
Randy Carney
a, Marissa
Taubb, Rachel Mizenkoa and Dina Phama
aDepartment of Biomedical Engineering, University of
California, Davis, Davis, USA; bUniversity of California, Davis, Fremont, USA
Introduction: Endogenous exosomes and related extracellular vesicles (EVs) are
potent nanoparticles released by all cells tested to date. The exploitation of their
unique
scaffolding for engineering next-generation drug delivery systems represents a major
area of
academic and commercial interest. The lag in exploiting this potential is in part
due to our
inability to measured extent and efficiency of modification, e.g., composition and
drug
loading. Here we report a robust pipeline of optical tweezing combined with Raman
spectroscopy to molecularly characterize engineered EVs and quantitatively assess
extent of
drug loading at single particle resolution.
Methods: EVs derived from cell culture and isolated by ultracentrifugation
were fused with synthetic liposomes to create engineered EVs (eEVs). These eEVs were
formed
via well-established vesicle fusion techniques, namely (1) mechanical extrusion, (2)
freeze-thawing, or (3) probe-tip sonication. Prior to formation, calcein was encapsulated
in
the liposomes and used as a surrogate for soluble drug loading. Laser trapping Raman
spectroscopy (LTRS) was used to optically trap single EVs, before and after synthetic
manipulation. Raman spectral analysis was used to assess trapped eEVs compared to
pure
standards to quantify ratiometric variation in chemical composition.
Results: Raman laser trapping experiments confirmed that each formation method
results in largely varying (1) extent of fusion between EVs and synthetic calcein-loaded
liposomes, (2) efficiency of calcein loading, and (3) particle size. We could also
quantify
the molar amounts of liposome vs. EV molecules for single particles, revealing a great
amount of variation from particle to particle. Functional membrane proteins we left
intact
to varying degree across fusion methods.
Summary/Conclusion: Given the rising importance of analytical tools able to
characterize extent of molecular loading for engineered EVs, we believe this technology
will
be very useful, thus warrants further investigation for eEV characterization across
a
variety of clinical applications.
Funding: Randy Carney, PhD was supported by a Research Scholar Grant,
RSG-19-116-01-CDD, from the American Cancer Society.
OS22.2
Extracellular vesicles containing host restrictive factor IFITM3
inhibited Zika virus infection of foetuses in pregnant mice through trans-placenta
delivery
Allen Z. Wu
Nanjing University, Nanjing, China (People’s Republic)
Introduction: Zika virus (ZIKV) infection can lead to neurological
complications and foetal defects, and has attracted global public health concerns.
Effective
treatment for ZIKV infection remains elusive and a preventative vaccine is not available
yet. Therapeutics for foetus need to overcome blood brain barriers to reach placenta
and
require higher safety standard.
Methods: In the present study, we engineered mammalian extracellular vesicles
(EVs) to deliver a host restrictive factor, interferon-induced transmembrane protein
3
(IFITM3), for the treatment of ZIKV infection.
Results: Our results demonstrated that the engineered IFITM3-containing EVs
(IFITM3-Exos) were overall safe to the animals and suppressed ZIKV viraemia by 2 log10 s
in
the pregnant mice. Moreover, the engineered EVs effectively delivered IFITM3 protein
across
placental barrier and suppressed overall ZIKV viraemia in the foetuses to the basal
level
with significant reduction of viraemia in key foetal organs as measured by Q-PCR.
Mechanistic study showed that IFITM3 was delivered to the endosomes/lysosomes where
it
inhibits viral entry to the host cells.
Summary/Conclusion: Our study demonstrates that exosomes can act as a cross
placenta drug delivery vehicle to foetus and IFITM3, an endogenous restriction factor
that
is highly expressed in placenta, is a potential treatment for ZIKV infection during
pregnancy.
Funding: The Major Research and Development Project from the National Health
Commission (Grant# 2018ZX10301406), National Science Foundation of China (Grant# 31970149),
the Key Project of Research and Development of Ningxia Hui Autonomous Region of China
(Grant# 2017BN04)
OS22.3
Engineering extracellular vesicles with altered cellular tropism
for targeted payload delivery in vivo
Monique Kauke, Nikki Ross, Dalia Burzyn,
Shelly Martin, Ke Xu, Nuruddeen Lewis, Charan Leng, Su Chul Jang, Stephanie Yu, Kevin
Dooley, Sriram Sathyanarayanan and Jonathan Finn
Codiak BioSciences, Cambridge, USA
Introduction: Extracellular vesicles (EV) are natural and abundant
nanoparticles capable of transferring complex molecules between neighbouring and distant
cell types. Translational research efforts have focused on co-opting this communication
mechanism to deliver exogenous payloads to treat a variety of diseases. Important
strategies
to maximize the therapeutic potential of EVs include payload loading, functionalization
of
the EV surface with pharmacologically active proteins, and delivery to target cells
of
interest.
Methods: Through comparative proteomic analysis (LC/MS) of purified EVs, we
identified several highly enriched and EV-specific proteins, including a transmembrane
glycoprotein (PTGFRN) belonging to the immunoglobulin superfamily. Leveraging PTGFRN
as a
scaffold for surface display, we generated EVs with functional targeting ligands,
including
single domain antibodies (sdAbs), single chain variable fragments (scFvs), single
chain Fabs
(scFabs), and receptor ligands, on the surface to direct EV uptake to cell types of
interest. Biological activity of these engineered EVs was assessed in an array of
in vitro
and in vivo assays and compared to untargeted controls.
Results: We engineered EVs displaying anti-Clec9A scFabs to target
conventional type 1 dendritic cells (cDC1s), anti-CD3 scFabs to target T cells, and
CD40
ligand to target B cells. In mice, systemic administration of anti-Clec9A EVs resulted
in a
75% increase in the percentage of cDC1 cells that take up EVs over controls. Anti-CD3
EVs
resulted in both an increase in the percentage of EV positive T cells (3.75 and 3-fold
for
CD4+ and CD8+) and the number of EVs per cell (15 and 7-fold for CD4+ and CD8+) in
the
blood. Furthermore, in primary mouse dendritic cells, anti-Clec9A EVs loaded with
STING
agonist achieved a 15-fold greater pathway induction compared to untargeted controls.
Preliminary in vivo data suggest that anti-Clec9A EVs reduce the required STING agonist
dose
10-fold to achieve efficacy and induce anti-tumour responses, compared to control
EVs.
Summary/Conclusion: These results demonstrate the potential of our EV
engineering platform to generate novel EV therapeutics targeted to cell types of interest
for pharmacologic payload delivery.
OS22.4
A novel method for the delivery of cell-free therapy to foetuses
with congenital anomalies: a proof of principle study
Lina Antounians, Louise Montalva, Gabriele
Raffler, Maria Sole Gaffi and Augusto Zani
The Hospital for Sick Children, Toronto, Canada
Introduction: Antenatal cell-based therapies are currently considered invasive
for the foetus. A promising cell-free strategy that holds great regenerative potential
for
several organs is the administration of stem cell derived EVs, whose cargo contains
bioactive molecules that epigenetically regulate target cells. Herein, we aimed to
1) assess
the ability of EVs to reach foetal organs when administered to the mother intravenously
or
intra-amniotically; 2) compare these administration routes on normal foetuses and
foetuses
with a congenital anomaly.
Methods: EVs were isolated from rat amniotic fluid stem cell conditioned
medium using ultracentrifugation. EVs were assessed for size (nanoparticle tracking
analysis), morphology (TEM), and expression of CD63, Hsp70, Flo-1, and TSG101 (Western).
We
injected rat dams with EVs stained by ExoGlow™-Vivo or saline (control) via maternal
tail
vein (IV) or intra-amniotically (IA) at E20.5. IA and IV injections were performed
on dams
carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic
hernia. After 24h, dams and pups were sacrificed. 3D high-sensitivity optical
reconstructions of whole foetuses or micro-dissected foetal organs were imaged using
the
IVIS® Spectrum imaging system. EV fluorescence signal was compared between normal
(n = 27)
and nitrofen-exposed (n = 45) foetuses.
Results: Both IV and IA injection routes were successful in delivering EVs to
foetal organs. No fluorescent signal was detected in saline only control. IA injections
yielded higher signal than IV, and EVs reached more organs with IA than IV injections.
IA
injected EVs were detected in the lungs, gastrointestinal, and urinary tract of normal
and
nitrofen-exposed foetuses. Nitrofen exposed foetuses had higher signal than normal
foetuses.
Summary/Conclusion: This proof of concept study shows that antenatal
administration of stem cell EVs is feasible with different routes. Although maternally
administered EVs cross the placenta, IA injection is more effective at reaching foetal
organs. Further studies are underway to reproduce these findings in experimental models
of
various congenital anomalies.
Funding: CIHR-SickKids Foundation grant
OS22.5
Microalgae as a novel and renewable bioresource of extracellular
vesicle
Antonella Bongiovanni
a, Giorgia
Adamob, Sabrina Picciottoc, David Fierlid, Maria Elena
Baroned, Anita Aranyosd, Rachel Parkesd, Svenja
Morsbache, Samuele Raccostaf, Darja Božičg, Antonella
Cusimanob, Christopher Stanlyh, Daniele P Romancinob,
Rita Carrottaf, Carolina Paganinii, Umberto Capasso
Palmieroi, Vincenzo Martoranaf, Rosina Notof, Giovanna L.
Liguorij, Annamaria Kisslingerk, Laura Corcueral, Elia Di
Schiavih, Veronika Kralj-Igličg, Ales Igličg, Katharina
Landfestere, Paolo Arosioi, Gabriella Pocsfalvih, Mauro
Mannof and Nicolas Touzetd
aInstitute for Research and Biomedical Innovation (IRIB) –
National Research Council (CNR), palermo, Italy; bInstitute for Research and
Biomedical Innovation (IRIB) – National Research Council (CNR), Palermo, Italy;
cInstitute for Research and Biomedical Innovation (IRIB) – National Research
Council (CNR); University of Palermo, Palermo, Italy; dInstitute of Technology
Sligo (ITSligo), Sligo, Ireland; eMax-Planck Institute for Polymer Research,
Mainz, Germany; fInstitute of Biophysics (IBF) – National Research Council (CNR),
Palermo, Italy; gUniversity of Ljubljana, Ljubljana, Slovenia;
hInstitute of Biosciences and BioResources (IBBR) – National Research Council
(CNR), Naples, Italy; iETH Zurich, Zurich, Switzerland; jInstitute of
Genetics and Biophysics (IGB) – National Research Council (CNR), Naples, Italy;
kInstitute of Experimental Endocrinology and Oncology (IEOS), Naples, Italy;
lZabala Innovation Consulting, Navarra, Spain
Introduction: Safe, efficient and specific nano-delivery systems are essential
to the current cosmetic, nutraceutical and therapeutic medicine sectors. The ability
to
optimise the bioavailability, stability, and targeted cellular uptake of bioactive
molecules
while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost
priority. VES4US is a European project, which aims to develop an innovative platform
for the
efficient production of extracellular vesicles (EVs) from microalgae, which constitute
a
promising renewable bioresource (www.ves4us.eu). Here we present characteristics of
EVs from
several microalgal lineages, which offer the opportunity for a potentially developing
a new
and scalable tailor-made biogenic nanotechnology.
Methods: We cultivated a number of EV-producing microalgal species and
developed protocols for EV isolation both at laboratory (differential ultracentrifugation)
and pilot scales (tangential flow filtration). The physico-chemical characterization
of
microalgal EVs was carried out according to the minimal information for studies of
extracellular vesicles 2018 (MISEV-2018 guidelines): biochemical methods to verify
the
presence of specific EV-biomarkers, tuned for microalgal EVs; dynamic light scattering
(DLS)
and nanoparticle tracking analysis (NTA) to assess the particles number and size
distribution; electronic scanning microscopy (SEM), atomic force microscopy (AFM),
and cryo
transmission electron microscopy (cryo-TEM) for imaging analyses; bilayer-specific
fluorescence staining (F-NTA) to test the purity of EV preparation.
Results: We identified microalgae as a novel natural source of EVs that could
constitute a cost-effective and sustainable way of mass-producing them. We screened
20
strains of microalgae and generated an “EV Identity Card” for each, which contained
a
variety of EV features relating to their biophysical, biochemical and biological
characteristics in line with the MISEV-2018. Our approach will next focus on the scalable
production, surface functionalization and bio-engineering of selected microalgal EVs.
At the
same time, their bioactivity will be explored using both in vitro and in vivo biological
models.
Summary/Conclusion: The VES4US consortium is investigating the potential of
microalgae as novel EV bioresources. This research will attempt to bioengineer novel
naturally-derived nanocarriers, microalgal EVs, suitable for the development of future
cosmetics, nutraceutical or therapeutic formulations.
Funding: This project has received funding from the European Union’s Horizon
2020 research and innovation programme under grant agreement No 801338.
OS22.6
Sequence-specific RNA trafficking to Extracellular Vesicles is
conserved across cell types
Andreia M. Silva
a, Olga
Shatnyevaa, Elisa Lázaro-Ibáñeza Molly Stevensb and Niek
Dekkera
aDiscovery Biology, Discovery Sciences, BioPharmaceuticals
R&D, AstraZeneca, Mölndal, Sweden; bDepartment of Materials, Department of
Bioengineering and Institute for Biomedical Engineering, Imperial College London;
Department
of Medical Biochemistry and Biophysics, Karolinska Institutet, London, UK
Introduction: Extracellular vesicles (EV) are an attractive biological vehicle
for drug delivery, such as therapeutic RNA. Loading of cargo RNAs into EV during their
biogenesis can be achieved by hijacking the physiological pathways of intracellular
RNA
trafficking. Several sequences have been identified that act as a zipcode for preferential
RNA targeting into EV (EV-tropic) or for retention in parental cells (cell-tropic).
In this
work, we aimed to compare the EV-tropic capacity of specific RNA sequence motifs in
promoting loading into EV, across different cell models representing the main cell
types
found in the body.
Methods: Immune, epithelial and mesenchymal cell lines were transiently
transfected with xenogeneic C. elegans microRNAs (miRNAs) containing EV-tropic or
cell-tropic sequences and grown in culture. EV were isolated from the supernatant
by
differential (ultra)centrifugation. RNA was extracted from both cell pellets and isolated
EV
fraction, and target miRNAs were quantified by digital droplet PCR. Distribution of
cargo
miRNA across cells and EV was also analysed for chimeras of EV- and cell-tropic
sequences.
Results: The miRNAs containing an EV-tropic sequence were highly enriched on
the EV fraction, with 1000–10,000 higher levels than in parental cells. Contrarily,
cell-tropic miRNAs were only 10–100 times higher in EV. No significant differences
were
observed in the EV loading efficiency for the various EV-tropic motifs tested. Mutations
in
the EV-sorting motif resulted in reduced EV loading. EV-tropic sequences consistently
promoted miRNA loading into EV across all the cell models evaluated, suggesting conserved
biological mechanisms.
Summary/Conclusion: We showed that RNA loading into EV is dependent on the
presence of defined EV-tropic RNA motifs, and that sorting mechanisms are conserved
across
the major cell types tested. The highest loading efficiencies resulted in 0.001 miRNA
copies
per particle on average, suggesting a limited scope for EV-tropic motifs for therapeutic
RNA
loading into EV.
Funding: AS, OS and ELI are fellows of the AstraZeneca PostDoc Programme.
OS23
Symposium Session 23: EVs in Immunology and Inflammation
Chair: Hang Yin – Tsinghua University
Chair: Bahnisikha Barman – Postdoctoral Research Fellow, Vanderbilt
University
OS23.1
Pro-inflammatory cytokine-mediated alterations in beta cell
extracellular vesicle cargo and function enhance activation of the CXCL10-CXCR3 axis
in
diabetes
Naureen Javeed
a, Tracy
Hera, Matthew Browna, Patrick Vanderboomb, Aoife
Eganb, Adrian Vellab, Ian Lanzab, Tushar
Patelc and Aleksey Matveyenkoa
aDepartment of Physiology and Biomedical Engineering, Mayo
Clinic, Rochester, USA; bDivision of Endocrinology, Diabetes, and Metabolism,
Mayo Clinic, Rochester, USA; cDepartment of Transplantation, Mayo Clinic,
Jacksonville, USA
Introduction: Coordinated activity between pancreatic islet cells is critical
for the regulation of glucose homoeostasis. Chronic exposure to diabetogenic factors
such as
pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes.
Extracellular vesicles (EVs) derived from cytokine-exposed β-cells modulate physiological
and pathological responses to β-cell stress. However, the mechanisms governing this
process
remain largely unknown. We set out to test the hypothesis that β-cell failure in diabetes
is
mediated in part through β-cell autocrine release of pro-inflammatory EVs which promote
inflammation and inhibit β-cell function.
Methods: Pro-inflammatory cytokine-exposed EVs (cytoEVs) were generated using
conditioned media from mouse Min6 β-cell line treated with diabetogenic cytokines
(TNFα,
IL-1β, IFNγ, 48h). EVs were also isolated from human type 2 diabetic (T2DM) and lean
non-diabetics (LND) plasma. GW4869 (N-SMase inhibitor) was used in the presence of
cytokines
to determine the effect of reduced EV concentrations on the restoration of β-cell
function.
Proteomic and RNA-Seq analysis was conducted on Min6 β-cell cytoEV (vs. control EV)
and
cytoEV treated mouse islets, respectively.
Results: Assessment of EV concentrations from cytoEV and human T2DM plasma
revealed a ~ twofold increase (p < .05, vs. control (ctl) and LND EV). Immunofluorescence
staining of CD9 and CD63 expression was significantly elevated in human T2DM pancreas
(p < .05, vs. LND). While acute inhibition of EV formation with GW4869 (5 µM) showed
significant restoration in β-cell function (glucose stimulated insulin secretion assay,
GSIS) in cytokine-exposed mouse and human islets (~7 and 2 fold vs. cytokines alone,
p < .05). Moreover, functional assessment of mouse islets exposed to cytoEV (48h)
resulted in suppression of GSIS (~55%, vs. untreated, p < .05). Identification of
cytoEV
content through proteomic analysis revealed a significant upregulation of the chemokine,
CXCL10 (~40 fold vs. ctlEV) and RNA-Seq analysis of cytoEV treated mouse islets depicted
a
marked upregulation of transcripts associated with CXCL10-CXCR3 signalling (p < .001)
and
downstream pathways (e.g. NFκB; p = .016 and JAK/STAT; p = .021). Furthermore, inhibition
of
cytoEV (GW4869) with cytokines markedly decreased CXCL10 (~30%) and CXCR3 receptor
(~65%)
expression in Min6 β-cells.
Summary/Conclusion: These data suggests that cytokines elevate CXCL10
expression in β-cell EV to enhance inflammation-induced diabetes. This is mediated
through
EV-autocrine release of CXCL10 consequently activating CXCR3 signalling and downstream
pathways to impair β-cell function in diabetes.
OS23.2
Synergy between 15-lipoxygenase and secreted PLA2 promotes
inflammation by formation of TLR4 agonists from extracellular vesicles
Mateja Manček Keber
a; Van Thai
Haa; Duško Lainščeka; Bernd Gesslbauerb; Eva
Jarcc; Tuulia Hyötyläinend; Nejc Ilce; Katja
Lakotaf; Matija Tomšičf; Fons A. J. van de Loog; Valery
Bochkovh; Toni Petanc; Roman Jeralaa
aNational Institute of Chemistry, Ljubljana, Slovenia;
bUniversity of Graz, Graz, Austria; cJožef Stefan Institute,
Ljubljana, Slovenia; dÖrebro University, Örebro, Sweden; eUniversity
of Ljubljana, Ljubljana, Slovenia; fUniversity Medical Centre Ljubljana,
Ljubljana, Slovenia; gRadboud University Medical Center, Nijmegen, Netherlands;
hUniversity of Graz, Ljubljana, Slovenia
Introduction: Damage associated molecular patterns (DAMPs) are endogenous
ligands that induce innate immune response, thus promoting sterile inflammation. During
oxidative stress, stress-derived EVs (stressEVs) were found to activate Toll-like
receptor 4
(TLR4), but the activating ligands were not fully determined. Additionally, several
enzymes,
among them 15-lipoxygenase (15-LO) and secreted phospholipase A2 (sPLA2) are induced
during
inflammation and were suggested to promote DAMP formation.
Methods: StressEVs were produced from HEK293 cells exposed to 10uM A23187 and
isolated with ultracentrifugation. 20:4 lysoPI was oxidized for 10 min with 15-LO.
Additionally, synEVs were prepared from phospholipids (PLs), oxidized with 15-LO and
hydrolysed with sPLA2. Activity was measured by qPCR and ELISA on wt and TLR4-KO
macrophages. 15-LO oxidized 20:4 lysoPI was analysed by mass spectrometry. sPLA2 activity
was measured in synovial fluid from rheumatoid and gout patients using fluorometric
assay.
K/BxN serum transfer induced arthritis model on wt and TLR4 KO mice (C57Bl/6 mice)
with
sPLA2-IIA injection was used (approval no. U34401-14/2019/8 by MKGP of Slovenia).
Results: StressEVs released after oxidative stress were found to activate TLR4
with a gene profile different from bacterial lipopolysaccharide (LPS). StressEVs,
15-LO
oxidized synEVs, but only 15-LO oxidized lysoPLs activated cytokine expression through
TLR4/MD-2. Hydroxy, hydroperoxy and keto products of 20:4 lysoPI oxidation were determined
by MS and they activated the same gene pattern as stressEVs. Furthermore, sPLA2 activity,
which we detected in the synovial fluid from patients, promoted formation of TLR4
agonists
after 15-LO oxidation. Injection of sPLA2-IIA into mice promoted K/BxN serum induced
arthritis in TLR4-dependent manner.
Summary/Conclusion: Both 15-LO and sPLA2 are induced during inflammation,
therefore these results imply the role of oxidized lysoPLs in stressEVs in promoting
sterile
inflammation through TLR4 signalling. The formation of TLR4 agonists is enzyme driven
so it
provides an opportunity for therapy without compromising innate immunity against
pathogens.
Funding: H2020-MSCA-ITN project TOLLerant (grant no. 642157), Slovenian
research agency (project no. J3-9257 to MMK, research core no. P4-0176 to RJ).
OS23.3
Monocytes traffic extracellular vesicles to damaged muscle and
adopt a novel immunophenotype to support muscle regeneration
Russell G. Rogers, Akbarshakh Akhmerov, Weixin
Liu, Lizbeth Sanchez and Eduardo Marbán
Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Introduction: Extracellular vesicles (EVs) are secreted membrane vesicles that
carry bioactive molecules such as miRNAs, mRNAs, proteins, and lipids to modify recipient
cell behaviour. We recently demonstrated EVs secreted by cardiosphere-derived cells
(CDC-EVs) augment endogenous muscle regeneration in mdx mice, a model of Duchenne
muscular
dystrophy, when delivered intravenously. In parallel, macrophages preferentially accumulate
surrounding small regenerating myofibers in CDC-EV treated mdx muscle. However, it
is
currently unclear how intravenous CDC-EVs home to dystrophic muscle and exert their
therapeutic bioactivity.
Methods: Fluorescently-labelled and unlabelled CDC-EVs were infused into the
contralateral femoral vein of wild-type mice with unilateral muscle injury induced
by BaCl2.
Injured and uninjured muscles were dissected 24h following infusion and subjected
to optical
imaging, immunohistochemistry, and confocal microscopy. This experiment was repeated
using
clodronate liposomes to deplete endogenous monocytes/macrophages. Next, RNA-seq was
preformed on bone marrow-derived M1, M2, and CDC-EV (MCDC-EV) polarized macrophages
from mdx
mice. Conditioned media (CM) from these macrophages were tested in an in vitro model
of
myogenesis. Lastly, small RNA-seq was performed on EVs secreted by M1, M2, and MCDC-EV
macrophages.
Results: When delivered intravenously, CDC-EVs naturally home to injured, but
not uninjured, skeletal muscle. CDC-EVs were detected in the interstitium adjacent
to
non-muscle cells, macrophages, and within surviving myofibers. After depletion of
monocytes/macrophages by clodronate liposomes, the presence of CDC-EVs in the injured
muscle
was attenuated. Bioinformatic analyses indicate CDC-EVs confer a novel immunophenotype
to
mdx macrophages with features of both M1 and M2. Indeed, MCDC-EV CM promotes myoblast
proliferation and supports myogenic differentiation. Interestingly, MCDC-EV EVs have
a
unique miRNA signature and contain several miRNAs with known roles in myogenesis.
Summary/Conclusion: These data indicate circulating monocytes traffic CDC-EVs
to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration.
We propose MCDC-EV macrophages mediate their pleiotropic effects via paracrine factors,
possibly including EVs.
Funding: NIH R01HL124074 to EM
OS23.4
Microglial derived extracellular vesicles activate autophagy and
mediate multi-target signalling to maintain cellular homoeostasis
Bram Van den Broeka, Isabel Pintelonb, Ibrahim
Hamada, Mansour Haidarc, Niels Hellingsc, Jerome
Hendriksc, Markus Kleinewietfeldc, Jean-Pierre
Timmermansd, Vincent Timmermanb, Veerle Somersc, Luc
Michielsc and Joy I. Irobi
c
aHasselt university, Diepenbeek, Belgium; bAntwerp
university, Antwerpen, Belgium; cHasselt University, Diepenbeek, Belgium;
dAntwerp University, Antwerpen, Belgium
Introduction: Microglia, the immunocompetent cells of the CNS, play an
important role in maintaining cellular homoeostasis in the CNS. These cells secrete
immunomodulatory factors including nanovesicles and participate in the removal of
cellular
debris by phagocytosis or autophagy.
The contribution of microglial-derived extracellular vesicles (M-EVs) to the maintenance
of
CNS homoeostasis is unclear. In addition, knowledge of canonical signalling pathways
of
inflammation and immunity gene expression patterns in human microglia exposed to M-EVs
is
scarce.
Methods: Here, we analysed the effects of M-EVs produced in vitro by either
TNFα-activated or non-stimulated microglia BV2 cells. We showed that M-EVs are internalized
by both mouse BV2 and human C20 microglia and that the uptake of M-EVs in microglia
induced
autophagic vesicles at various stages of degradation including autophagosomes and
autolysosomes. Consistently, exposure of microglia to M-EVs increased the protein
expression
of the autophagy marker, LC3B-II, and promoted autophagic flux in live cells. To elucidate
the biological activities occurring at the transcriptional level in C20 microglia
exposed to
M-EVs, the gene expression profiles, potential upstream regulators, and enrichment
pathways
were characterized using targeted RNA sequencing.
Results: Inflammation and immunity transcriptome gene panel sequencing of both
activated and normal microglia exposed to M-EVs showed involvement of several canonical
pathways and reduced expression of key genes involved in neuroinflammation, inflammasome
and
apoptosis signalling pathways compared to control cells.
Summary/Conclusion: We demonstrate that in vitro produced microglial EVs are
able to influence multiple biological pathways and promote activation of autophagy
in order
to maintain microglia survival and homoeostasis.
Funding: This work was financed by Hasselt University and by EFRO through the
Interreg V Grensregio Vlaanderen Nederland project Trans Tech Diagnostics.
OS23.5
Evaluation of plasma extracellular vesicles as biomarkers for
longevity
Xin Zhang
a and Virginia
Krausb
aLaboratory Medicine Center, Nanfang Hospital, Southern
Medical University, Guangzhou, Guangdong, 510515, P. R. China, Guangzhou, China (People’s
Republic); bDivision of Rheumatology, Duke Molecular Physiology Institute, Duke
University School of Medicine, Durham, USA
Introduction: Extracellular vesicles (EVs) have emerged as key indicators and
effectors of ageing. Although plasma concentrations of EVs decline with age, the EV
biomarkers associated with ageing and longevity are not fully understood. Recently,
our
group found an age-related decline of plasma EVs associated with immune cells during
normal
human ageing. Our study aims to evaluate the association of plasma EVs with longevity.
Methods: Plasma samples were selected from the Established Populations for
Epidemiologic Studies of the Elderly study subjects (n = 48): half dying within 2 years
(short-lived group) and half surviving ≥10 years (long-lived group) after the blood
draw;
all matched for age (median age 77.3 ± 1.7 years, range 72–80), gender (50% female),
and
race (50% White/50% Black). The samples were acquired under donor consent and IRB
approval
of Duke University. EVs were separated from the plasma samples, and profiled based
on the
surface markers of haematopoietic stem cells (HSCs), mesenchymal stem cells, immune
cells,
skeletal muscles, cardiac muscles and adipocytes (CD81, CD9, CD29, CD63, CD8, CD4,
CD68,
CD14, CD56, CD15, CD19, CD235a, CD41a, CD34, CD31, HLA-ABC, HLA-G, HLA-DRDPDQ, CD90,
CD73,
CD105, M Cadherin, RYR1, RYR2, FABP4, DLK1). The percentages of EVs expressing each
tested
molecule were determined using a high-resolution multicolour BD LSR Fortessa X-20
Flow
Cytometer as we recently reported. GraphPad Prism 8.0 software was used for statistical
analysis.
Results: We found significantly increased percentages of CD9+, HLA-ABC+,
CD31+ and CD41a+ large EVs (1000–6000 nm) in the long-lived compared to the short-lived
group. None of the tested surface marker expressing medium (100–1000 nm) or small
(<100 nm) EVs showed differential percentages between the short- and long-lived
groups.
Summary/Conclusion: EVs carry surface markers from their parent cells. CD9 is
expressed by HSCs and immune cells. CD9 regulates homing of human cord blood CD34+ HSCs,
and
delivers a potent CD28-independent costimulatory signal to activate T cells. HLA-ABC,
the
key human immunogen, is expressed by nucleated cells and platelets. CD31 is expressed
by
HSCs, immune cells and epithelial cells, and CD31+ plasma EVs declined with age in
healthy
people. CD41a is expressed by HSCs, megakaryocytes and platelets, and is functionally
relevant for HSC maintenance and haematopoietic homoeostasis. Our preliminary data
suggest
that HSCs and immune cell associated plasma EVs (CD9+, HLA-ABC+, CD31+, CD41a+ large
EVs)
inform on health status related to longevity.
Funding: National Institute on Ageing grant 1R56AG060895-01
OS24
Symposium Session 24: Advances in Separation and Concentration II
Chair: Lei Zheng – Department of Laboratory Medicine, Nanfang Hospital, Southern
Medical University
OS24.1
Scaling-up the manufacturing of well-characterized mesenchymal
stromal cell-derived extracellular vesicles for biomedical applications
Ana Fernandes-Platzgummer
a, Sara
Rosab, Ricardo Silvaa, Raquel MS Cunhac, Miguel Almeida
Fuzetad, Cecília Caladoe, Carla Carvalhoa, Joaquim
Cabrala, Ana Azevedoa and Cláudia Silvaa
aDepartment of Bioengineering and iBB – Institute for
Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa,
Lisboa,
Portugal, Lisboa, Portugal; bDepartment of Bioengineering and iBB – Institute for
Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa,
Lisboa,
Portugal, lisboa, Portugal; cInstituto Superior Técnico, University of Lisbon,
Lisbon, Portugal; dDepartment of Bioengineering and iBB – Institute for
Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa,
Lisboa,
Portugal; Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de
Lisboa,
Lisboa, Portugal, Lisboa, Portugal; eInstituto Superior de Engenharia de Lisboa,
Lisboa, Portugal
Introduction: It is anticipated that stem/progenitor cells-derived
extracellular vesicles (SPC-EVs) will rapidly progress towards clinical studies, and
the
development of reproducible, efficient, scalable and cost-effective process for their
production is expected to boost the therapeutic applications of EVs-based products.
In
addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations
could
result in substantial improvements for SPC-EVs production in terms of reproducibility,
stability and quality, while ensuring the approval of regulatory agencies. The main
goal of
this work is to develop a full-controlled manufacturing platform for the SPC-EVs
production.
Methods: Human mesenchymal stromal cells (MSC) were expanded in a xeno-free
microcarrier-based bioreactor culture system operating in fed-batch feeding mode and
after
10 days the conditioned medium was collected. Different methods for SPC-EV
isolation/purification from the MSC-derived conditioned medium, including chromatography
were compared and the the quality of the final product obtained was characterized
by
different methods according to MISEV, including nanoparticle tracking analysis, lipidomics
and Western blot. Moreover Fourier-Transform InfraRed (FTIR) spectroscopy was evaluated
in
terms of its implementation as a standard technique for the identification and
characterization of EVs.
Results: After 10 days of MSC expansion under dynamic conditions, we collected
1.3 L of conditioned medium with approximately 0.5 million EVs/MSC. A combination
of a
pretreatment with a nuclease for the digestion of DNA/chromatin with a purification
using
strong anion exchange chromatography led to the best results so far in terms of EVs
isolation. Of notice, by FTIR spectroscopy, it was possible to define ratios of spectral
bands, that can be used as biomarkers, enabling the discrimination of EVs chemical
fingerprint in function of the culture conditions tested.
Summary/Conclusion: The platform established herein could be applied to the
production of well-characterized SPC-EVs targeting their biomedical use in different
settings (e.g. as drug delivery systems), as well as EVs from other parental cells
lines
(i.e. dendritic cells) in therapeutic settings as cancer.
Funding: Fundação para a Ciência e Tecnologia (SFRH/PD/BD/128328/2017,
PTDC/EQU-EQU/31651/2017, UIDB/04565/2020).
OS24.2
Ultrasensitive protein detection for quantification of
extracellular vesicles in human biofluids enables comparison of isolation
techniques
Dmitry Ter-Ovanesyan, Maia Norman, Wendy
Trieu, Roey Lazarovits, George Church and David Walt
Wyss Institute, Boston, USA
Introduction: Extracellular vesicles (EVs) are released by all cells into
biofluids and hold great promise as reservoirs of disease biomarkers. One of the main
challenges in studying EVs and using them in diagnostics is a lack of suitable methods
to
quantify EVs that are sensitive enough and can differentiate EVs from similarly sized
lipoproteins and protein aggregates. We propose using ultrasensitive single molecule
array
(Simoa) assays to quantify EVs by immuno-isolating and detecting EV transmembrane
proteins
in microwell arrays.
Methods: We developed single molecule array (Simoa) assays using the Quanterix
HD-X Analyser for the quantification of EVs using the tetraspanins CD9, CD63, and
CD81.
Simoa allows for the detection of single proteins using arrays of femtoliter wells,
turning
ELISA into a digital immunoassay. We then used these assays, together with an additional
assay for albumin, to compare commonly used EV isolation methods from plasma and
cerebrospinal fluid (CSF): ultracentrifugation, precipitation (ExoQuick), and size
exclusion
chromatography (SEC) using the Izon qEV columns. We further used these assays to rapidly
optimize and improve SEC by comparing different SEC resins and column dimensions in
both
plasma and CSF.
Results: In comparing our Simoa assays to traditional ELISA with the same
antibodies, we found that the Simoa assays were more than 100 times more sensitive,
detecting the tetraspanins in samples where the proteins were undetectable by ELISA.
Given
the high dynamic range and high-throughput capabilities of Simoa, we were able to
comprehensively compare relative EV yields and EV purity for different isolation methods
of
EVs from plasma and CSF. We provide average tetraspanin and albumin levels to directly
compare the methods. We also tested different SEC resins and provide data for custom
SEC
columns that outperform Izon qEV and allow for fine tuning of different ratios of
EVs to
albumin.
Summary/Conclusion: Our results highlight the utility of quantifying EVs using
ultrasensitive Simoa assays for tetraspanins. We were able to rapidly Simoa to rapidly
evaluate different EV isolation methods in CSF and plasma. In general, the experimental
framework we present could be easily applied to evaluate new EV isolation methods,
or
applied to any other biological fluid. Thus, we think Simoa is a powerful new tool
for
relative EV quantitation.
Funding: Open Philanthropy Project (OPP)
OS24.3
Combinatorial antibody microarray profiling of intra- and
extravesicular proteins in colorectal cancer cell line extracellular vesicles
Rosalie Martel, Molly L. Shen, Philippe
Decorwin-Martin, Lucile Alexandre, Lorenna Oliveira, Andy Ng and David Juncker
McGill University, Montreal, Canada
Introduction: The protein profile of extracellular vesicle (EV) subpopulations
has been shown to contain valuable disease information, notably in cancer. Currently,
techniques aiming to find EV proteins that associate together mainly focus on transmembrane
proteins, while methods that also probe cytosolic proteins generally resort to a combination
of affinity capture, elution, and lysis, which limits throughput. To allow the
high-throughput analysis of both membrane and cytosolic EV proteins, we optimized
a Total
Extracellular Vesicle Antibody Microarray (tEVAM) incorporating fixation and heat-induced
epitope retrieval (HIER), then leveraged it to perform combinatorial protein profiling
of
EVs from colorectal cancer (CRC) cell lines HT29 and SW403.
Methods: Arrays of IgGs targeting surface protein markers were incubated
overnight with EVs purified from cancer cell line supernatants. HIER optimization
was
carried out through variation of buffer contents, presence or absence of prior
permeabilization, as well as incubation time and temperature, for a total of 38 conditions.
A431 EVs, previously profiled with other methods, were used as a model during the
optimization. Cytosolic protein HSP90 and membrane marker EGFR, both with high expression
in
A431 EVs, were probed and the results used to compare HIER conditions. Following HIER
treatment, protein targets were detected through incubation with primary antibodies
and
fluorescent secondary antibodies or streptavidin. The resulting optimized tEVAM workflow
was
used to phenotype HT29 and SW403 EVs through probing of trios of surface (2) and internal
(1) protein targets.
Results: The selected tEVAM protocol successfully maximized HSP90 signal while
minimally affecting EGFR detection, enabling simultaneous analysis of surface and
internal
proteins. Profiles of more than 450 combinations, featuring integrins, claudins, cytokines,
and other key actors of cancer-relevant pathways, were obtained for HT29 and SW403
EVs,
revealing co-expression patterns that highlight the biomolecular heterogeneity both
within
and between CRC cell line EVs.
Summary/Conclusion: Using tEVAM, intra- and extravesicular proteins can be
detected simultaneously in EVs immobilized based on surface protein content, yielding
extensive combinatorial protein profiles with significance for health and biomarker
research.
Funding: This work was supported by the Fonds de recherche du Québec – Nature
et technologies (FRQNT) and the Genome Canada Disruptive Innovation program.
OS24.4
Characterization of EVs using orthogonal techniques identifies
discrete EV populations from a mouse dendritic cell line
Bryce Killingsworth
a, Timothy
Traynorb, Joshua A. Welshc, Aleksandra Dakica, Jason
Savagea, Kevin Camphausend, Kenneth Aldapea and Jennifer
Jonesa
aLaboratory of Pathology, National Cancer Institute, National
Institutes of Health, Bethesda, USA; bLaboratory of Pathology, National Cancer
Institute, National Institutes of Health, Gaithersburg, USA; cLaboratory of
Pathology, National Cancer Institute, National Institute of Health, Bethesda, USA;
dRadiation Oncology Branch, National Cancer Institute, National Institutes of
Health, Bethesda, USA
Introduction: Extracellular vesicles (EVs) have the potential to serve as
valuable biomarkers for patient response to cancer therapy. However, development of
robust
EV-based clinical assays relies on knowledge of EV concentration and diameter distribution.
Many different methods exist to measure the size and concentration of EVs, and each
method
exhibits strengths and limitations. It is important to use orthogonal methods for
determination of these important properties of EV preparations. Here, we use dendritic
cell-derived EVs to demonstrate that some EV analysis methods can give a biased
interpretation of both diameter and concentration. Through comparison, we highlight
why
orthogonal assays are essential in providing measurement reliability.
Methods: DC2.4 mouse dendritic cells were cultured in flasks containing a
total of 1.2 L of EV-depleted media (10% FBS, centrifuged 18 hr. x 100,000 g.) When
cells
reached 80% confluency, conditioned media was collected, depleted of debris with two
10 min.
x 2,500 g spins, and concentrated down to ~5 mL using a Pall Jumbosep 100 kDa MWCO
filter.
The EV concentrate was purified from protein using an Izon qEV-10 column, with 5 mL
fractions collected. The protein content of the EV-containing fractions was analysed
by
A280, Pierce BCA, and bioanalyzer. The diameter distribution of the EVs was determined
by
nanoparticle tracking analysis (NTA), resistive pulse sensing (RPS), flow cytometry
(FCM),
and electron microscopy (EM.) Concentration was compared using NTA, RPS, and FCM.
EVs were
further analysed by protein mass spectrometry and RNA sequencing.
Results: We have identified two distinct populations of EVs with our DC2.4
preparation, one highly abundant population with a power-law distribution, whose peak
diameter is below 60 nm, and a second, less abundant population with a peak diameter
at
approximately 140 nm. These two distinct populations and their relative concentration
were
not detectable with all analysis techniques. Based on cross-platform measurements,
these
populations appear to have distinct compositions that warrant further investigation.
Summary/Conclusion: The use of orthogonal methods allowed the detection of two
discrete populations of EVs which was not possible on some platforms and would have
resulted
in a biased perspective of the sample composition. This work has highlighted the need
for
orthogonal measurements to be conducted by pairing techniques that do not have the
same
biases.
Funding: BK, TT, JAW, AD, JS, and JCJ were supported by the Intramural
Research Program of the National Institutes of Health (NIH), National Cancer Institute,
and
Center for Cancer Research. JCJ acknowledges NIH ZIA BC011502, NIH ZIA BC011503, NIH
U01
HL126497, NIH R01 CA218500, NIH UG3 TR002881, and the Prostate Cancer Foundation.
OS24.5
Hybrid plasmonic biomaterial nanofilter scaffold for cancer EV
diagnostics based on surface-enhanced Raman scattering (SERS)
Hanna J. Koster
a, Tatu
Rojalina, Juanjuan Liub, Sebastian Wachmann Hogiub and
Randy Carneya
aDepartment of Biomedical Engineering, University of
California, Davis, Davis, USA; bDepartment of Bioengineering, McGill University,
Montreal, Canada
Introduction: Extracellular vesicles (EVs) are nano-sized vesicles shed by all
cells that serve vital roles in cell-to-cell communication. Tumour-associated EV
subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules),
enabling minimally invasive spectroscopic analysis for a wide variety of cancers.
Here, we
use surface-enhanced Raman spectroscopy (SERS) in combination with a novel plasmonic
substrate for global chemical composition analysis of cancerous and non-cancerous
populations of EVs to determine distinguishing surface characteristics.
Methods: EVs were isolated from ovarian cancer (OvCa) patient serum samples by
differential ultracentrifugation. A new hybrid nanoplasmonic scaffold comprised of
a
microscale biosilicate diatoms embedded with silver nanoparticles (AgNPs) was used
for SERS
measurements. The substrate was incubated with cysteamine to positively-charge the
AgNPs
(responsible for the SERS enhancement) so that EVs could attach (EVs are naturally
anionic).
In a typical experiment, 40 μL of ~108 particles/mL EVs per sample were incubated
with the
porous substrate surface, which was inverted on a glass cover slip for Raman interrogation.
Principle component analysis (PCA) was used to compare the spectra and determine
distinguishing characteristics between populations from tumour and non-tumour sources.
We
also trypsinized EVs before SERS analysis to see the extent of influence the surface
molecules play in localizing the EVs to the AgNP “hot spots.”
Results: A total of 8 clinical samples (7 OvCa and 1 non-malignant control)
were tested in combination with OvCa SKOV-3 cell line EVs. Simple PCA was able to
separate
clinical samples according to disease subtype and major peaks were identified to provide
chemical content analysis. Each sample exhibited inherent heterogeneity but clustered
together in a distinguishable way from the others.
Summary/Conclusion: Despite innate heterogeneity within single samples (i.e.,
EVs isolated from a single patient sample), EVs isolated from clinical samples could
be
easily distinguished from each other using our hybrid SERS substrate, with minimal
sample
processing, a label-free approach, and only a few microlitres of sample. Our study
using
this novel plasmonic material demonstrates its potential for use as a component in
next-generation diagnostic platforms.
Funding: Ovarian Cancer Education & Research Network (OCERN)
OS24.6
Laser Trapping Raman spectroscopy (LTRS) of single vesicles can
distinguish extent of lipoprotein contamination
Marissa Taub
a, Rachel
Mizenkob, Dina Phamb and Randy Carneyb
aUniversity of California, Davis, Fremont, USA;
bDepartment of Biomedical Engineering, University of California, Davis, Davis,
USA
Introduction: Single-particle analysis is critical for understanding
extracellular vesicle (EV) heterogeneity. Yet such techniques remain technically challenging
due to low detection sensitivity and presence of variable amounts of “contaminants,”
including lipoproteins. The high degree of structural similarity between EVs and
lipoproteins in size, density, and chemical composition, results in their co-isolation
using
any of the standard EV isolation techniques. Here we introduce Laser Trapping Raman
spectroscopy (LTRS) as a well-suited, label-free, and non-destructive tool to distinguish
EVs from various lipoprotein species at single particle resolution.
Methods: EV samples were isolated from SKOV-3 cell culture supernatant by
differential ultracentrifugation and their Raman spectra measured. As the most abundant
lipoproteins in EV isolations from human biofluids are sub-micron low density lipoprotein
(LDL), very low density lipoprotein (VLDL), and high density lipoprotein (HDL) particles,
these were purchased as pure components and also measured by LTRS. LDL and VLDL were
then
spiked-in to isolated EVs to mimic “contaminated” post-isolation EV samples. Raman
spectra
were analysed by principal component analysis (PCA) using a custom MATLAB script.
Results: LDL and VLDL have been observed to adhere to EV surfaces in vitro
after standard isolation techniques. We could readily distinguish pure VLDL, LDL,
and HDL
standards according to their Raman spectra. PCA revealed distinction of SKOV-3 EVs
from both
LDL and VLDL. PCA also differentiated SKOV-3 EVs incubated with LDL from SKOV-3 EVs
incubated with VLDL. Extent of LDL and VLDL adherence to EVs could be observed and
quantified.
Summary/Conclusion: Through Raman and PCA, classes of lipoprotein and EVs can
be identified and quantified when co-incubated. LTRS is a quantitative single-EV analysis
technique that can be used to differentiate between lipoprotein classes and EVs when
incubated together. This technique allows for analysis of EVs where standard isolation
methods fall short.
OS25
Symposium Session 25: Neurologic and Ageing Mechanism
Chair: Nicole Noren Hooten, MD, PhD – National Institute on Ageing, National
Institutes of Health
Chair: Cathryn L. Ugalde – Department of Biochemistry and Genetics, La Trobe Institute
for Molecular Science, Australia
OS25.1
Fibroblast growth factor 2-mediated regulation of neuronal
exosome release depends on VAMP3/cellubrevin in hippocampal neurons.
Thomas Koeglsperger
a, Rohit
Kumara, Qilin Tanga, Stephan A. Müllera, Pan
Gaoa, Mahlstedt Dianaa, Yi Tana, Klingl
Andreasb, Kai Bötzelc, Stefan F. Lichtenthalera and
Günter Höglingera
aGerman Center for Neurodegenerative Diseases (DZNE), Munich,
Germany; bDepartment of Biology, Ludwig Maximilian University, Munich, Munich,
Germany; cDepartment of Neurology, Ludwig Maximilian University, Munich, Munich,
Germany
Introduction: Extracellular vesicles (EVs) are endogenous membrane-derived
vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons,
thereby
promoting neuronal survival and plasticity in the CNS and contributing to neurodegenerative
conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the
specific
molecular factors that regulate neuronal EV uptake and release are currently unknown.
Methods: We used a combination of patch-clamp electrophysiology and
pH-sensitive dye imaging to examine stimulus-evoked EV release in individual neurons
in real
time.
Results: Whereas spontaneous electrical activity and the application of a
high-frequency stimulus (HFS) induced a slow and prolonged fusion of multivesicular
bodies
(MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor
bFGF
(basic fibroblast growth factor) greatly increased the rate of stimulus-evoked MVB-PM
fusion
events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis
of
neuronal EVs demonstrated bFGF to increase the abundance of the v-SNARE vesicle-associated
membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking-down VAMP3 in
cultured
neurons attenuated the effect of bFGF on EV release.
Summary/Conclusion: Our results determine for the first time the temporal
characteristics of MVB-PM fusion in hippocampal neurons and reveal a new function
for bFGF
signalling in controlling neuronal EV release.
Funding: Parkinson Fonds Deutschland
Hilde Ulrichs Stiftung
Friede Springer Stiftung
Lüneburg Heritage
OS25.2
The well-chaperoned extracellular vesicle: their presence in
neuropathologies
Xiaoli Yua, Anthony Fringuelloa, Steven G.
Griffithsb, Brooke Metzgerc and Michael W.
Graner
d
aUniversity of Colorado Denver, Anschutz Medical Campus,
Department of Neurosurgery, Aurora, USA; bX0S0ME, Moncton, Canada;
cIllinois College, Jacksonville, USA; dUniversity of Colorado Anschutz
Medical Campus, Aurora, USA
Introduction: Heat shock proteins (HSPs) function as chaperones under both
normal and pathologic conditions. As chaperones they assist in protein folding, in
holding
protein complexes for current or future activation, and in degradation of senescent
proteins
for recycling of components and display for immune surveillance. During stressful
situations, HSP quantities and/or activities increase as cells and tissues seek protection
from insults. These insults can result in the cell surface display of HSPs, which
can then
lead to the surface display of HSPs on extracellular vesicles (EVs). HSPs present
on the
cell surface or in the extracellular space are regarded as “danger signals” in an
ancient
biologic paradigm. HSP-accessorized EVs may act as “danger boli”, carrying not only
the
HSPs, but hundreds of components of the stressed parental cell, capable of prompting
differential responses depending on the status of the recipient cell.
Methods: Clarified/filtered plasma from patients suffering from neurologic
maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed
to
bind HSPs. The EVs congeal under these conditions and are pelleted (microfuge) and
washed
with increasing-stringency buffers. We lysed the EVs and subjected them to metabolomic
analyses (focused on lipids) or assayed them on phosphokinase arrays.
Results: We show that EVs from the blood of patients suffering from brain
tumours, or from TBI, or from MS, possess distinct metabolomes compared to blood EVs
from
healthy donors. We found hundreds of differentially-expressed lipids amongst the patients
vs
the healthy donors. The levels of annotation and identification for these compounds
ranges
from level 4 (low, no matches in databases) to level 2 (high, annotation matches to
known
database components). In addition, we found differences in phosphorylated kinases
as cargo
in these EVs between patients with matched primary vs recurrent gliomas, and among
TBI/stroke patients compared to healthy donors.
Summary/Conclusion: HSP-accessorized EVs present different metabolomic and
phosphokinase content which may serve as biomarkers in a “liquid biopsy” setting,
but may
also play roles in the pathobiology of neurologic diseases.
Funding: NIH/NIBIB R01EB016378
NIH/NIGMS R01GM129046
NIH/NIMH R21MH118174
OS25.3
Methamphetamine use disorder uniquely plasma extracellular
vesicle miRNA expression
Ursula S. Sandau
a, Erika
Dugganb, Xiao Shic, Tracy Swansonc, Marilyn
Huckansd, Sierra Smitha, Jennifer Loftise, Aaron
Janowskyf, John Nolanb and Julie A. Saugstada
aOregon Health and Science University, Portland, USA;
bScintillon Institute, San Diego, USA; cVA Portland Health Care
System; Oregon Health and Science University, Portland, USA; dVA Portland Health
Care System; Methamphetamine Abuse Research Center, Portland, USA; eVA Portland
Health Care System; Methamphetamine Abuse Reaseach System, Portland, USA; fVA
Portland Health Care System; Methamphetamine Abuse Reaseach System; Oregon Health
and
Science University, Portland, USA
Introduction: Methamphetamine (MA) has deleterious effects to both peripheral
organs and the central nervous system. The rewarding properties and addictive potential
of
MA are correlated with increased synaptic dopamine availability following alterations
in
dopamine and vesicular monoamine transporter function. In rodents, MA alters brain
miRNA
expression and the miRNA content of serum extracellular vesicles (EV). Here we examined
plasma EVs isolated from human subjects actively using MA (MA-ACT) for size, concentration,
protein markers, and miRNA content.
Methods: Plasma samples from 10 MA-ACT, and 10 controls (CTL) were obtained
from the Methamphetamine Abuse Research Center. Plasma EVs were evaluated by vesicle
flow
cytometry (VFC) for size, concentration, and surface protein markers. VFC antibodies
included markers for a pool of tetraspanins (CD9, CD63, and CD81), platelet EVs (CD41),
pro-coagulant EVs (AnnV), and red blood cell EVs (CD235). Next plasma EV isolated
by size
exclusion chromatography were analysed by qPCR on TaqMan® Array Human MicroRNA A + B
Card
Set v3.0. Fold change was calculated by ΔΔCq between MA-ACT and CTL for miRNA expressed
in ≥
60% of samples in at least 1 group. We identified the top 20% of ranked miRNA by
F-statistic; of these, the miRNA of interest for MA-ACT were identified by at least
a (i)
1.2 fold change in expression, (ii) area under the receiver operating characteristic
curve
of 0.75, and (iii) Glass’s ∆ of 1. For miRNA of interest correlations to additional
MA
variables were conducted, along with Ingenuity Pathway Analysis of predicted gene
targets.
Tobacco use was controlled for.
Results: VFC data show that the size (~110 nm) and concentration (~7.5 x 1010
particles/ml) of all plasma EVs is comparable between MA-ACT and CTL groups. In addition,
the plasma EVs primarily consist of tetraspanin+, AnnV+, or CD41+ EVs, and to a much
lesser
extent CD235+ EVs. Of the 207 miRNA expressed in MA-ACT and/or CTL plasma EVs, there
were
110 miRNA that have at least a 1.2 fold increase or decrease in MA-ACT. 10 miRNA were
identified to be of interest in MA-ACT based on fold change, effect size and diagnostic
potential, compared to CTL. Further, 5 of the 10 miRNA correlate with a MA associated
variable, including frequency of use and age of first use. Together the 10 miRNA best
cluster subjects based on MA-ACT status, not tobacco use. Finally, the predicted gene
targets of the 10 miRNA are associated with canonical pathways linked to MA.
Summary/Conclusion: EV miRNA expression in MA-ACT subjects was unique to CTL
participants, suggesting that MA may affect EV communication among cells. The differential
miRNA expression also implicates a role for EVs in behavioural and physiological effects
specific to MA, and suggests that there may be changes in expression of miRNA that
are
relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated
addiction
disorders.
OS25.4
Bone marrow-derived extracellular vesicles may alter the ageing
phenotype of murine haematopoietic stem cells.
Sicheng Wena, Jill Kreilingb, Mark
Doonerc, Elaine Papac, Michael Del Tattoc, Yan
Chengc, Mandy Pereirac, Peter Quesenberryc and
Laura R. Goldberg
d
aRhode Island Hospital/Brown University, providence, USA;
bBrown University, Providence, USA; cRhode Island Hospital,
Providence, USA; dBrigham and Women’s Hospital, Boston, USA
Introduction: Extracellular vesicles (EVs) have been implicated in many
age-related diseases, but their role in natural ageing of haematopoietic stem cells
(HSCs)
is unclear. We tested the hypothesis that bone marrow-derived EVs (BM-EVs) can modulate
the
ageing HSC phenotype.
Methods: We flushed bone marrow from old (24–26-month old) and young (6-8-week
old) C57/BL6 mice and collected BM-EVs by differential centrifugation (2000 × g for
30 min,
supernatant collected and centrifuged 100,000 × g for 1 hour, BM-EV pellet collected
and
quantified by Nanoparticle Tracking Analysis). We injected old mice with 2 x 10^9
young
BM-EVs via tail vein, daily x 3 days. Control mice were injected with age-matched
BM-EVs or
vehicle alone. We euthanized the mice one month post-injection, harvested whole bone
marrow
(WBM) and highly purified HSCs (Lineage negative/c-Kit+/Sca-1+/CD150+; LSK-SLAM) and
tested
stem cell function in competitive bone marrow transplants (4–5 recipients/group).
Results: At 6 months post-transplant, WBM from old mice exposed to young
BM-EVs exhibited a statistically significant decrease in engraftment when compared
to WBM
exposed to age-matched BM-EVs (percent average donor chimerism ± SEM: 15% ± 5% (young
EVs)
vs. 61% ± 14% (old EVs)). For LSK-SLAM from old mice, we observed a trend towards
decreased
engraftment when exposed to young BM-EVs and a trend towards increased engraftment
potential
when exposed to old BM-EVs (percent average donor chimerism ± SEM: 7% ± 4% (young
EVs), 27%
± 10% (old EVs), 15% ± 2% (vehicle)). These findings are consistent with our previous
data
showing that, in contrast to highly purified HSCs which develop impaired stem cell
function
with ageing, total un-separated old WBM actually has increased engraftment capacity
when
compared to young WBM. Of note, we found that the classic myeloid skewing by old LSK-SLAM
was partially reversed by exposure to both young and old BM-EVs. Finally, consistent
with
the known increase in highly purified HSCs with age, our preliminary data showed that
old
mice exposed to young BM-EVs had an approximately 7-fold decrease in the number of
LSK-SLAM
cells in marrow, indicating that BM-EVs may influence age-related changes in HSC number.
Summary/Conclusion: These preliminary data suggest BM-EVs may play a role in
modulating HSC ageing phenotypes, potentially altering engraftment capacity, lineage
fate,
and LSK-SLAM population size. Future studies delineating the molecular mechanisms
underlying
these EV-mediated effects could provide key insights into normal haematopoietic ageing.
Funding: This work was supported by the NIH grants 5P20GM119943, DK112808-01A1
and by the Savit Foundation.
OP3 = PS15 Oral with Poster Session 3: Neurological & ID
Chair: Andrew Hoffman, DVM, DVSc – University of Pennsylvania School of Veterinary
Medicine Chair: Sophie Rome – INRAE, Department of Human Nutrition
OP3.01 = PS15.01
Mitovesicles: a new extracellular vesicle of mitochondrial
origin altered in ageing and neurodegeneration
Pasquale D’Acunzo
a, Tal
Hargashb, Yohan Kimb, Rocío Pérez-Gonzálezc, Chelsea
Millerb, Melissa J. Alldredb, Chris Goulbourneb, Hediye
Erdjument-Bromaged, Monika Pawlikb, Mitsuo Saitoe, Mariko
Saitof, Stephen D. Ginsbergb, Thomas Neubertg and Efrat
Levyb
aCenter for Dementia Research, Nathan S. Kline Institute, New
York, USA; bCenter for Dementia Research, Nathan S. Kline Institute, Orangeburg,
USA; cCIBERNED, Hospital de la Santa Creu i Sant Pau (Barcelona, Spain),
Orangeburg, USA; dDept. Cell Biology, New York University School of Medicine, New
York, USA; eDivision of Analytical Psychopharmacology, Nathan S. Kline Institute,
Orangeburg, USA; fDivision of Neurochemistry, Nathan S. Kline Institute,
Orangeburg, USA; gCell Biology, New York University School of Medicine, New York,
USA
Introduction: Brain extracellular vesicles (EVs) are heterogenous and include
previously described microvesicles and exosomes. Herein we characterized a formerly
unappreciated population of mitochondria-derived EVs that we term “mitovesicles”.
Mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative
disorders as Down syndrome (DS). Hence, we examined mitovesicle levels and cargo under
these
conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial
stressors.
Methods: Employing a high-resolution density gradient, distinct and novel
populations of EVs were isolated from murine and human DS and diploid control post-mortem
brains or from cell media. Morphometric EV features were analysed by nanoparticle
tracking
analysis and cryogenic electron microscopy, while EV constituents were characterized
by
Western blotting, mass spectrometry, lipid profiling and mitochondrial RNA qPCR.
Results: We identified a population of double-membrane, electron-dense brain
EVs containing multiple mitochondrial markers (“mitovesicles”) that are highly distinct
from
microvesicles and exosomes. Proteomic data show that mitovesicles contain a unique
subset of
mitochondrial proteins while lacking others, such as Tom20. Mitovesicles have a lipid
composition that is unlike that of previously described EVs and is consistent with
mitochondrial origin. Functionally, the complex-III inhibitor antimycin-A stimulated
in
vitro mitovesicle release into the cell media, suggesting an interrelationship between
mitochondrial dysfunction and mitovesicle biology. In mouse brains, mitovesicle levels
increased with age and were found to be higher in DS compared to diploid controls.
Mitochondrial RNA and protein levels were also altered in DS compared to diploid
controls.
Summary/Conclusion: We describe a previously unidentified type of
metabolically competent EVs of mitochondrial origin that we designate mitovesicles.
Our data
demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal
conditions and are modified during pathophysiological processes in which mitochondrial
dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player
in
mitochondria quality control and may have a role in the trans-cellular tissue response
to
oxidative stress.
Funding: AG017617
AG056732
OP3.02 = PS15.02
Reducing extracellular vesicle release with a novel neutral
sphingomyelinase 2 inhibitor for the treatment of Alzheimer’s disease
Carolyn Tallon
a, Kristen
Hollingerb, Benjamin Bellb, Michal Salac, Ranjeet
Dashb, Ajit Thomasb, Amrita Datta Chaudhurib, Asit
Kumarb, Lyndah Lovellb, Ying Wub, Rana Raisb,
Norman Haugheyd, Radim Nenckae, Camilo Rojasb and Barbara
Slusherb
aJohns Hopkins University, Baltimore, USA; bJohns
Hopkins University School of Medicine, Baltimore, USA; cAcademy of Sciences of
the Czech Republic, Prague, Czech Republic; dDepartment of Neurology, Johns
Hopkins University School of Medicine, Baltimore, USA; eJohns Hopkins University
School of Medicine, Prague, Czech Republic
Introduction: Alzheimer’s disease (AD) is a devastating neurodegenerative
disease leading to progressive memory loss and ultimately death with limited therapeutic
options. Growing evidence supports the theory that toxic proteins, like tau and amyloid,
may
propagate from diseased cells by packaging toxic proteins into extracellular vesicles
(EVs)
and releasing them to infect other cells. One enzyme involved in the biogenesis of
EVs is
neutral sphingomyelinase 2 (nSMase2), which catalyzes the hydrolysis of sphingomyelin
to
produce phosphorylcholine and ceramide. Several groups have reported improved cognition
and
reduced tau propagation when nSMase2 is pharmacologically inhibited or genetically
knocked
down in AD mouse models. Unfortunately, current nSMase2 inhibitors are not suitable
for
clinical development due to poor solubility and inadequate pharmacokinetic profiles.
Methods: Our group carried out a high-throughput screening campaign followed
by extensive medicinal chemistry efforts leading to the discovery of phenyl
(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b] pyridazin-8-yl) pyrrolidin-3-yl)
carbamate (PDDC), an orally active, nM potent inhibitor with excellent selectivity
and brain
penetration. We tested PDDC’s ability to inhibit exosome release in cultured primary
glial
cells as well as an in vivo model of acute EV release. We then treated 5XFAD mice
with
10 mg/kg of PDDC daily for six months and monitored their behaviour in the fear conditioning
assay.
Results: PDDC dose dependently reduced EV release from cultured primary glial
cells and significantly reduced plasma EV numbers in an in vivo model. Following chronic
treatment with PDDC, 5XFAD mice demonstrated significantly improved cognitive function
in
the fear conditioning assay.
Summary/Conclusion: These promising findings are currently being expanded
using mouse models of tau propagation. If successful, these data would support PDDC
as a
novel compound for targeting the pathological spread of tau as a therapeutic for AD.
OP3.03 = PS15.03
Profiling EVs in the anterior cingulate cortex of individuals
with major depressive disorder
Pascal Ibrahim
a, Zahia
Aouabedb, Corina Nagyb and Gustavo Tureckic
aIntegrated Program in Neuroscience, McGill University,
Montreal, Canada; bDouglas Hospital Research Centre, Montreal, Canada;
cDepartment of Psychiatry, McGill University, Montreal, Canada
Introduction: Major Depressive Disorder (MDD) is one of the leading causes of
disability worldwide, affecting 20% of the population. The environment has been thought
to
play a role in the disease development, resulting in biological changes mediated by
epigenetic mechanisms. MicroRNA’s (miRNA) are well known epigenetic regulators that
are
disrupted in the depressed brain, and they are packaged into extracellular vesicles
(EVs).
EVs have emerged as means of intercellular communication, a process that is also disrupted
in MDD. They are thought to transfer miRNA between cells, which can alter gene expression
in
recipient cells. Therefore, we hypothesize that EV cargo is altered in MDD subjects
compared
to healthy controls (HC). The aim is to extract EVs from human post-mortem anterior
cingulate cortex, a region previously associated with depression, and profile the
miRNA
cargo and compare it between MDD subjects and HC.
Methods: Post-mortem human brain tissue from the anterior cingulate cortex of
20 MDD subjects and 20 HC was mildly dissociated in the presence of collagenase type
III.
Residual tissue, cells, and large vesicles were eliminated, and EVs were isolated
using size
exclusion chromatography. The quality was assessed by western blots and transmission
electron microscopy (TEM). RNA was extracted and a small-RNA library was constructed
and
sequenced using the Illumina Platform. Differential expression analysis was then
performed.
Results: Western blots showed little to no Endoplasmic Reticulum (Calnexin),
Golgi (BiP), or mitochondrial (VDAC) contamination, along with enrichment of the exosomal
marker CD9. TEM images showed the typical cup-shaped morphology with sizes mostly
between 30
and 200 nm. Preliminary sequencing results revealed that miR-33a-5p, which is predicted
to
target glutamate receptors, is downregulated in EVs from MDD subjects.
Summary/Conclusion: High quality EV extractions can be obtained from
post-mortem brain tissue using our method. This will be the first study to profile
brain-derived EV miRNA in the context of depression. Future studies will be needed
to
determine the effect of the different levels of miR-33a-5p. This could provide novel
mechanistic insights into the pathophysiology of MDD and will serve as a starting
point to
examine the potential role of EVs in MDD pathology.
Funding: Réseau québécois sur le suicide, les troubles de l’humeur et les
troubles associés (RQSHA) Student Award
McGill Faculty of Medicine Internal Studentship Award (Max E. Binz Fellowship)
OP3.04 = PS15.04
Combining nanomagnetic isolation and artificial intelligence to
guide the treatment of traumatic brain injury
Zijian Yang
a, Kryshawna
Beardb, David Meaneyb, Danielle Sandsmarkb, Ramon
Diaz-Arrastiaa and David Issadorec
aUniversity of Pennsylvania, Philadelphia, USA;
bUniversity of Pennsylvania, philadelphia, USA; cDepartment of
Bioengineering, University of Pennsylvania, Philadelphia, USA
Introduction: Traumatic brain injury (TBI) is characterized by diverse primary
mechanisms of injury that lead to the development of secondary pathological cascades
that
drive neurological deficit post-TBI. Inability to separate patients based on the presence
of
these different endophenotypes represents a major challenge for diagnosis and treatment
of
TBI.
Extracellular vesicles including exosomes isolated from patient plasma have emerged
as
promising potential biomarkers for TBI due to their ability to cross the BBB into
systemic
circulation with molecular cargo intact for analysis. We have developed a novel microfluidic
platform for rapid isolation of brain-derived EVs providing a tool with which the
biochemical state of neurons and glia can be directly assessed post-TBI. We used the
ultra-sensitive, single molecule array (SIMOA) to quantify concentrations of 7 protein
biomarkers from the plasma and brain derived EVs from mild TBI patients and controls.
By
combining multiple protein biomarkers, we could discriminate mTBI patients from controls
in
both the training and the blinded test set.
Building on this work, we are also characterizing single EV heterogeneity of neuron
derived
EVs by developing novel droplet based digital assay for single EV quantification at
ultra-low concentration. Droplet based assay for single EV analysis would potentially
be
very informative for early disease diagnosis and therapy decision.
Methods: Our microfluidic platform for EV isolation consists of tracked-etched
membranes with millions of nanopores (600 nm), coated with a magnetic film (NiFe)
to
precisely capture immunomagnetically labelled brain-specific EVs from plasma. Single
molecule array (SIMOA) was used to quantify concentrations of the 7 protein biomarkers
(Tau,
UCHL-1, NFL, GFAP, IL6, IL10, and TNF) in the plasma and brain-derived exosomes of
mild TBI
(mTBI) patients and controls. To identify single EV, we applied droplet based enzyme-linked
immunosorbent assay and encoded the fluorescent signal for single EV quantification
within
parallelized microfluidic platform.
Results: We report that concentrations of plasma and exosome GFAP, NFL, and
UCHL1 were elevated in mTBI patients compared to controls (p < 0.05), and that each
of
these biomarkers are uncorrelated with one another. Discrimination of mTBI patients
from
controls was most accurate when machine learning algorithms on the panel of biomarkers.
Specifically, combining plasma NFL, GFAP, IL6 and TNF- with Tau from GluR2+ EVs showed
88%
accuracy with 80% sensitivity and 100% specificity.
Summary/Conclusion: This data suggests that neuron-derived exosomes contain
information that characterizes the injured and recovering brain. It also suggests
that
analysis of a panel of biomarkers from a combination of both blood and exosomal compartments
could lead to more accurate diagnosis of mTBIs.
OP3.05 = PS15.05
L1CAM is not associated with extracellular vesicles in
cerebrospinal fluid or plasma
Maia Norman, Dmitry Ter-Ovanesyan, Wendy Trieu
and David Walt
Wyss Institute, Boston, USA
Introduction: Neurons in living psychiatric and neurological patients are
inaccessible for cell type specific analysis of RNA and protein. Our understanding
of these
diseases instead relies upon imperfect sources of biochemical information such as
post-mortem brain tissue analysis and animal models. Furthermore, there is a paucity
of
biochemical assays available to diagnose and manage brain diseases. Extracellular
vesicles
(EVs) present an opportunity to noninvasively sample the contents of neurons in
cerebrospinal fluid (CSF) and plasma. In order to isolate neuron-derived EVs (NDEVs),
a cell
type specific transmembrane protein is necessary for immunocapture. L1CAM, a protein
abundant on the surface of neurons, has been used extensively in the literature for
NDEV
isolation. However, L1CAM exists in humans in several isoforms without a transmembrane
domain, and as such it can be secreted as a free protein. Additionally, the ectodomain
of
L1CAM can be cleaved off of the cell surface in physiological processes. It remains
to be
demonstrated whether the L1CAM found in CSF and plasma is EV associated, or if it
is instead
a spliced or cleaved isoform behaving as a free protein.
Methods: Using Single Molecule Arrays (Simoa), a digital form of ELISA, as
well as Western blotting, we quantify EV markers (CD9, CD63 and CD81) as well as L1CAM
and
Albumin. We use these assays to determine in which fractions of size exclusion
chromatography (SEC) and density gradient the L1CAM appears. We also immunocapture
L1CAM
from CSF and plasma and perform Western blots for the internal and external domains
of
L1CAM.
Results: Simoa and Western blot analysis of SEC and density gradient fractions
demonstrated that while the EV markers peaked all together, L1CAM eluted in the free
protein
fractions along with Albumin in both CSF and plasma. When immunoprecipitation was
performed,
Western blotting revealed different isoforms of L1CAM in CSF and plasma.
Summary/Conclusion: Our data utilize a multitude of distinct techniques that
converge to demonstrate that L1CAM is not associated with EVs in CSF or plasma. Furthermore,
our data suggest that the isoforms present in CSF and plasma are distinct, which indicates
that the L1CAM in plasma is likely not coming from the brain. This data call into
question
the utility of L1CAM as a NDEV marker and point to the need to find novel candidates
for
immunoprecipitation of NDEVs.
Funding: Chan Zuckerberg Initiative
OP3.06 = PS15.06
An in vitro and in vivo perspective on the role of
erythrocyte-derived extracellular vesicles in Parkinson’s disease pathology
Helena L. Denis
a, Melanie
Alpaughb, Manon Leclercc, Vicky Caronc, Michel
Panissetd, Alexandre Prate, Frédéric Calonc, Éric
Boilardf and Francesca Cicchettib
aCentre de Recherche du CHU de Québec and Faculté de
médecine, Département de psychiatrie & neurosciences, Université Laval, Québec, QC,
Canada, Québec, Canada; bCentre de recherche du CHU de Québec; Faculté de
médecine, Département de psychiatrie & neurosciences, Université Laval, Québec, QC,
Canada, Québec, Canada; cCentre de recherche du CHU de Québec; Faculté de
Pharmacie, Université Laval, Québec, QC, Canada, Québec, Canada; dCentre de
recherche du CHU de Montréal; Département de médecine de Université de Montréal, Montréal,
Québec, Canada, Montréal, Canada; eCentre de recherche du CHU de Montréal;
Département de médecine de Université de Montréal, Montréal, Québec, Canada, Montréal,
Canada; fCentre de recherche du CHU de Québec; Faculté de médecine, Département
de microbiologie-infectiologie et d’immunologie, Université Laval, Québec, QC, Canada,
Québec, Canada
Introduction: In Parkinson’s disease (PD), α-synuclein (α-Syn) aggregates
known as Lewy bodies (LB) are present in both the central and peripheral nervous system.
Furthermore, data showing that α-Syn can spread from PD patients to transplanted tissue
has
led to a new theory postulating that pathological forms of α-Syn can drive disease
by
“infecting” healthy cells and corrupting normal proteins. The exact routes and mechanisms
involved in such spreading are yet to be fully understood but it is known that α-Syn
can be
secreted from cells and transported via extracellular vesicles (EV). EV derived from
erythrocytes (EEV) are of particular interest in this regard as they have been shown
to
contain α-Syn.
Methods: We first optimized a protocol for the isolation of fluorescently
labelled human EEV. The capacity of these EEV to cross the blood-brain barrier (BBB)
was
then evaluated in vitro using a Boyden chamber composed of primary human brain endothelial
cells. Next, EEV were added to a more complex and physiologically relevant 3D human
BBB
model including iPSC-derived brain microvascular endothelial cells. In both in vitro
protocols, flow cytometry was performed on media collect from each compartment to
determine
the number of EEV. Immunofluorescence was performed to assess the localization of
fluorophore tagged EEV. We are also using an in vivo paradigm for the extraction and
testing
of EEV spread and an in situ cerebral perfusion (ISBP) model in WT mice to investigate
if
and how EEV cross the BBB using confocal microscopy.
Results: In both in vitro models, flow cytometry analyses showed that
fluorescently tagged EEV added to the luminal side traversed the endothelial cell
barrier.
Confocal analysis revealed that some EEV could also be found within endothelial cells
themselves. Ongoing experiments are being conducted in our newly developed 3D BBB
to further
confirm these results. Our preliminary in vivo experiments showed that fluorescently
labelled beads, similar in size to EEV, used in the ISBP experiments are detectable
in the
brain parenchyma of injected WT mice using confocal microscopy. Preliminary work also
includes ISBP injections of EEV in 6-month-old WT mice, (n = 6/groups) derived from
PD
patients (at different stage of the disease) and a healthy individual as a control.
Summary/Conclusion: Our preliminary data suggests that EEV can indeed move
across the BBB in both in vitro and in vivo experimental setups. Ongoing experiments
will
determine the dynamics and processes involved in this transport and whether EEV can
precipitate and/or exacerbate disease-related features.
Funding: FRQS
OP3.07 = PS15.07
Exosomes from N-Myc amplified neuroblastoma cells induce
migration and confer chemoresistance to non-N-Myc amplified cells: implications of
intra-tumour heterogeneity
Pamali Fonseka
a, Michael
Liemb and Suresh Mathivananb
aLa Trobe Institute for Molecular Sciences, La Trobe
University, Melbourne, Australia; bLa Trobe University, Melbourne, Australia
Introduction: Neuroblastoma accounts for 15% of childhood cancer mortality.
Amplification of the oncogene N-Myc is a well-established poor prognostic marker for
neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour
aggression and resistance to treatment, the role of N-Myc in the aggressiveness of
the
disease is poorly understood. Exosomes are released by many cell types including cancer
cells and are implicated as key mediators in cell-cell communication via the transfer
of
molecular cargo. Hence, characterising the exosomal protein components from N-Myc
amplified
and non-amplified neuroblastoma cells will improve our understanding on their role
in the
progression of neuroblastoma.
Methods: In this study, comparative proteomic analysis, nanoparticle tracking
analysis, transmission electron microscopy, RNAi-based knockdown, migration and cellular
survivability assays were performed to understand the role of exosomes isolated from
cells
with varying N-Myc amplification status.
Results: Label-free quantitative proteomic profiling revealed 968 proteins
that are differentially abundant in exosomes released by the N-Myc amplified and
non-amplified neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment
of proteins involved in cell communication and signal transduction in N-Myc amplified
exosomes. Treatment of less aggressive SH-SY5Y cells with N-Myc amplified SK-N-BE2
cell-derived exosomes increased the migratory potential, colony forming abilities
and
conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from
N-Myc
knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced
apoptosis.
Summary/Conclusion: These findings suggest that exosomes could play a pivotal
role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between
cells.
OP3.08 = PS15.08
Dissecting the heterogeneity of extracellular vesicle
sub-populations at single vesicle level
Anudeep Yekula
a, Koushik
Muralidharana, Bob Carterb and Leonora Balajc
aMassachusetts General Hospital, Harvard Medical School,
Boston, USA; bMassachusetts General Hospital, Cambridge, USA;
cMassachusetts General Hospital and Harvard Medical School, Cambridge, USA
Introduction: Quantification and characterization of single extracellular
vesicles (sEVs) based on surface markers can aid in dissecting the heterogeneous landscape
of EV subpopulations. We and others have demonstrated the potential of imaging flow
cytometry (IFC) to perform sEV characterization. We recently showed release of
protoporphyrin (PpIX) positive sEVs by 5-aminolevulinic acid (5-ALA) dosed glioma
cells, in
vitro and in vivo. Rickfels et al. also used IFC to demonstrate the enrichment of
CD63+/CD81+ EVs in the plasma of glioma patients. Herein, we performed in vitro studies
to
characterize EV subfractions using 5-ALA as well as EV and CNS specific surface markers.
Methods: We use IFC to characterize EVs released by glioma using 5-ALA,
fluorescently labelled EV (CFDA-SE, CD81) and glioma specific (tenascin C and epidermal
growth factor receptor vIII, EGFRvIII) markers. Furthermore, we characterized EVs
released
by EGFRvIII positive glioma cells treated with dexamethasone, a steroid commonly used
in
glioma patients, to determine the effect of steroids on EV release. EVs were quantified
by
IFC and results were confirmed by qPCR for the levels of EGFRvIII mRNA.
Results: Firstly, we optimized protocols to label glioma sEVs using
fluorescently labelled EV markers (CFDA-SE, CD81) and tumour specific markers (tenascin
C
and EGFRvIII). Of the total EVs (CFDA-SE), we demonstrate that 58% are tenascin C
positive,
2.7% are EGFRvIII positive and 1.6% are 5-ALA positive. There was only a minor overlap
(<16%) between the sub-populations. Finally, we show that dexamethasone treated glioma
cells release lower total EVs (2.5-fold), tumour specific EVs (2.8-fold; EGFRvIII),
EGFRvIII
mRNA compared to mock treated cells.
Summary/Conclusion: We demonstrate the potential of IFC to monitor sEVs
released by glioma cells exposed to different stimuli. This allows the characterization
of
EV sub-populations providing a working model to understand the dynamics of tumour
EVs at a
single vesicle level.
Funding: This work is supported by grants U01 CA230697 (BSC, LB), UH3 TR000931
(BSC), P01 CA069246 (BSC).
OP3.09 = PS15.09
Proteomic analysis of EVs from the filamentous fungal plant
pathogens Fusarium graminearum and Fusarium oxysporum f. sp. vasinfectum
Donovan Garcia-Ceron
a, Charlotte
Dawsonb, Mark Bleackleyc and Marilyn Andersona
aLa Trobe University, Melbourne, Australia;
bCambridge University, Cambridge, UK; cIncannex, Melbourne,
Australia
Introduction: F. graminearum (Fgr) and F. oxysporum f. sp. vasinfectum (Fov)
are severe fungal pathogens of cereals and cotton, respectively. Fgr and Fov cause
economic
losses and threaten food and fibre supplies worldwide. Understanding host-pathogen
interactions is crucial for developing new strategies for disease control. We are
determining whether extracellular vesicles (EVs) have a role in the interaction between
fungal pathogens and their host plant.
Methods: We isolated EVs from Fgr and Fov by size-exclusion chromatography and
characterized them by NTA and TEM. EVs from Fgr and Fov are between 100–300 nm and
have
morphology similar to EVs reported for other fungi. We performed label-free quantitative
proteomics to describe the protein cargo of EVs from Fgr and Fov, including a comparative
study of EVs from Fov grown on different media: Czapek Dox (CD) and Saboraud’s Dextrose
Broth (SDB).
Results: A total of 658 proteins were detected in Fgr EVs and, according to
prediction software EffectorP, 12.5% of these were potential effectors. Similarly,
70% of EV
proteins do not contain signal peptide indicating that packaging into EVs is a novel
mechanism of secretion for these proteins. Notable Fgr EV proteins include lipases,
proteases and synthases for toxins and chitin. Fov produced EVs in similar quantities
in
both growth media tested, but EV protein cargo differed between them. There was a
39%
overlap in proteins identified in the 465 CD and the 658 SBD EV proteins. In general,
EV
proteins were involved in metabolism, cell wall architecture and oxidoreduction, with
15.4%
and 12.9% of potential effectors, respectively. Polyketide and toxin synthases, proteases
and effectors were present in both types of Fov EVs.
Summary/Conclusion: This new fungal EV isolation method was rapid, yielded
high-quality EVs, and did not submit particles to high centrifugal forces. Our data
revealed
that both Fgr and Fov produce EVs enriched with proteins that could alter host immune
responses or facilitate fungal infection. Furthermore, the protein composition of
Fov EVs
was dependant on culture conditions. This supports a potential role for fungal EVs
in
disease progression in plants and provides the foundations to pursue the role of EVs
in
plant-fungal interactions with the potential to identify new targets for disease
control.
Funding: Australian Research Council DP160100309
OP3.10 = PS15.10
Trypanosoma cruzi releases different types of extracellular
vesicles that distinctly modulate host cells
Yifat Ofir- Birin
a, Rafael Pedro
Madeirab, Sergio Schenkmanb, Ori-Yam Revachb, Ori
Avinoamb, Yael Fridmann-Sirkisb, Ziv Poratb, Mattia
Morandib, Neta Regev-Rudzia, Ana Claudia Torrecilhasb and
ana Claudia Trocoli Torrecilhas
b
aWeizmann Institute of Science, Rehovot, Israel;
bUniversidade Federal de Sao Paulo, Sao Paulo, Brazil
Introduction: Extracellular Vesicles (EV) released by infective forms of
Trypanosoma cruzi, the agent of Chagas’ disease, modulate inflammatory response of
macrophages through the activation of Toll 2 receptor (TLR2) via mitogen-activated
protein
kinase pathway. This induces the production of nitric oxide (NO) and expression of
the
cytokines TNF-α, IL-12 and IL-6, which could explain the inflammation observed in
experimental Chagas’ disease, and eventually in the progression of human disease.
EVs
released by the parasite are heterogeneous and it is unknown which factor, or factors
present in the different vesicle populations act during the interaction with host
cells.
Objectives. The goal of the present work was to characterize and isolate the different
populations of EVs released by T. cruzi and test their effects on macrophages.
Methods: EV released by trypomastigotes forms of T. cruzi (Y strain) were
purified by Asymmetric flow field-flow fractionation (AF4) and characterized by
Nanoparticles tracking analysis (NTA). The different populations of EVs were incubated
with
host human monocytes cells (THP-1) and cytokines production determined by ELISA and
qPCR.
The different EV populations were also incubated with LLCMK-2 epithelial cells and
the
infection by T. cruzi determined.
Results: We found two distinct populations of EVs. A population with 50 to 50
nm (EV1) and another with 100 to 120 nm (EV2). EV1 induced more TNF-alpha, IL-6, IP-10
and
CCL20 than EV2. It was also more effective in promoting T. cruzi infection in epithelial
cells.
Summary/Conclusion: T. cruzi released two EV populations that affects
differently host cells. Identification of these EVs composition might help to better
understand the role of EVs in the modulation of T. cruzi infection
Funding: FAPESP, CNPq and CAPES
OP3.11 = PS15.11
Commensal bacterial extracellular vesicles act as carriers for
norovirus
Sutonuka Bhar, Melissa Jones, Annalise
Galbraith and Mariola Edelmann
University of Florida, Gainesville, USA
Introduction: Human norovirus (HuNoV) are one of the most common causes of
gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have
a
massive economic impact resulting in approximately 60 USD billion each year in healthcare
costs and missed worker productivity. Development of anti-viral therapies for HuNoV
has been
hampered by the lack of robust in vitro cultivation systems. Several cell types support
viral replication but only produce modest amounts of virus due to unknown reasons,
making
these systems insufficient for use in drug development and infectivity assays.
Noroviruses are known to attach to gram-negative enteric bacteria and this facilitates
infection in vitro. However, the microbiome- norovirus-host communication link is
missing.
Noroviruses infect immune cells present in lamina propria during acute infection,
but
bacteria themselves are large enough to cross the mucosal and the tight epithelial
barrier
which separates gut lumen from lamina propria. We hypothesized that binding of noroviruses
to bacteria enhances extracellular vesicles (EV) production. Because commensal bacterial
EVs
by themselves do not have any detrimental effects on host cells, we believe using
EVs in in
vitro culture will enhance norovirus infection, thus producing higher titre of viruses
for
vaccine and anti-viral drug development.
Methods: Attachment assay: Purified norovirus was incubated with Enterobacter
cloacae, Lactobacillus acidophilus and Bacteroides thethiotaomicron, and grown to
produce
EVs. The attachment was confirmed via qPCR.
Isolation of EVs: Clarified media supernatants were subjected to ultracentrifugation
at
varying speeds and 0.2um filtration. Co-purification of norovirus with the EVs was
checked.
EV quantification and characterization: EV total protein content was measured by microBCA.
The number of vesicles were quantified by Nanoparticle tracking analysis. Scanning
and
Transmission electron microscopy was performed to check quality of EV preparation
and
determine if virus was attached to the vesicles. Internal EV protein content was evaluated
using MS-HPLC. The EVs were also check for infectivity via TCID50 assay.
Results: Incubation of noroviruses with commensal bacteria resulted in
significant increases in production of EVs compared to uninfected controls. Murine
norovirus
(MNV), used as a surrogate, was found to be associated with EVs. EM analysis determine
association of viruses with the bacteria as well as the MVs, while also showing certain
surface structural changes in virus attached bacteria compared to mock bacteria. The
EVs
were found to cause infection in naive macrophages.
Summary/Conclusion: Changes in EV production and content by bacteria exposed
to noroviruses will provide insight into its pathogenesis and possible solutions to
the low
viral output from HuNoV culture systems.
OP3.12 = PS15.12
Detection of bacterial extracellular vesicles in blood from
healthy volunteers
Kylie Krohmaly
a, Claire
Hoptayb, Andrea Hahna and Robert Freishtata
aChildren’s National Hospital, Washington, USA;
bChildrens National Hospital, Washington, USA
Introduction: Bacteria constitutively produce biologically active
extracellular vesicles (EVs), which contain RNA, DNA, and/or proteins. Bacteria use
these
EVs for communication with other bacteria and recent research suggests bacterial EVs
can
also affect host cells. Given these findings, it is necessary to examine the role
of
bacterial EVs in human disease. Current methods of bacterial EV isolation from human
specimens cannot distinguish between bacterial species. However, there is utility
in
examining EVs from specific species, as bacterial species and their EVs may have unique
contributions to human disease. Our objective was to isolate circulating EVs specifically
from Escherichia coli (EEVs) and Haemophilus influenzae (HEVs), two known colonizers
and
pathogens in the gut and airway, respectively.
Methods: Total EVs were isolated from the blood of six healthy volunteers via
precipitation and size exclusion chromatography. EVs were then selected via a novel
latex
bead-based fluorescent antibody construct targeting species-specific outer membrane
proteins. We used flow cytometry to evaluate the isolated EVs.
Results: The constructs were saturated with EEVs at an antibody concentration
of 11.5 µg/mL of plasma, as geometric means ≥11.5 µg/mL were nearly equal. HEVs were
detected at 48 µg/mL of plasma, but saturation is yet to be determined. EEVs were
imaged by
a FEI Talos F200X electron microscope and measured between 40–90 nm, and HEVs were
between
60–160 nm. Both types of EVs were spherical.
Summary/Conclusion: Using this novel technique, we were able to isolate,
detect, and visualize EEVs and HEVs. This technique enables the study of specific
bacterial
EVs. In the future, EV contents will be assayed. Furthermore, this technique will
be
modified so that specific bacterial EVs from body fluids can be used for downstream
functional applications. This is the first time that bacterial EVs from targeted bacterial
species have been detected in blood from healthy humans.
OS26
Symposium Session 26: Scientific Collaboration and Outreach
Chair: An Hendrix – Laboratory of Experimental Cancer Research, Department of Human
Structure and Repair, Ghent University, Belgium; Cancer Research Institute Ghent,
Belgium
Chair: Cecilia Lässer – Postdoc, Krefting Research Centre, Institute of Medicine,
Sahlgrenska Academy at University of Gothenburg
OS26.1
HEVI NZ: A hub for extracellular vesicle investigations in New
Zealand supports research and education
Cherie Blenkiron, Colin L. Hisey, Vanessa
Chang and Lawrence W. Chamley
The University of Auckland, Auckland, New Zealand
Introduction: New Zealand (NZ) has a population of just 4.8 Million people
with a remote geographical location in the Pacific Ocean. Its unique culture, food-based
industries and ethnic population make NZ an invaluable place for extracellular vesicle
research into all areas. However, as for many places in the world, standardization
of
methodologies, training and access to appropriate equipment is challenging.
Methods: The Hub for Extracellular Vesicle Investigations (HEVI) is a virtual
research centre established in 2017 with three-year seed funding from a University
of
Auckland Strategic Research Initiatives Fund. Two staff members are employed to support
training, education and optimization of methods. The HEVI is guided by a governance
group
providing valuable input from Australasian experts in EVs.
Results: Since 2017 the HEVI has organized 2 research symposia, 2 hands-on
training days, hosted 2 international students as well as providing one-on-one training
for
41 individuals from universities and institutes across NZ. Training is provided on
multiple
isolation and characterization methods and tailored to individuals access to essential
equipment without bias towards individual manufacturers or techniques. Travel funding
has
supported 38 people to attend conferences and workshops for the purposes of education,
networking and research dissemination. The HEVI also provides support for project
design
with 10 grants awarded to HEVI members and a number of manuscripts in submission for
publication.
Summary/Conclusion: Establishment of a local research hub has aided
recruitment of new researchers to the field as well as supporting established researchers
through standardization of methodology and provision of expert staff to enable development
of new techniques tailored to project needs. Over the next year we aim to secure ongoing
funding for this valuable resource, strengthen a national network and link with the
Australasian and other EV societies for further training opportunities.
Funding: Vice Chancellors Strategic Research Initiatives Fund, The University
of Auckland
OS26.2
EMBO practical course “extracellular vesicles: from biology to
biomedical applications”
Esther Nolte-’t Hoen
a, Guillaume
van Nielb, Cecilia Lässerc and An Hendrixd
aDept. Biomolecular Health Sciences, Fac. Veterinary
Medicine, Utrecht University, Utrecht, Netherlands; bInstitute of Psychiatry and
Neurosciences of Paris U-1266 INSERM, Paris, France; cKrefting Research Centre,
Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg,
Sweden;
dLaboratory of Experimental Cancer Research, Department of Human Structure and
Repair, Ghent University, Belgium; Cancer Research Institute Ghent, Belgium, Ghent,
Belgium
Introduction: The EMBO practical course “Extracellular Vesicles: From Biology
to Biomedical Applications” is organized each year by a group of 4 researchers active
in the
EV field in collaboration with the EMBL Advanced Training Center in Heidelberg. The
course
focuses on training PhD students and post-doctoral researchers who enter or are already
active in the field of EV research. Given the large number of methods and protocols
that is
being used by researchers in the EV field, the organizers aim to provide practical
guidance
to new researchers and teach them appropriate skills.
Methods: Participants obtain theoretical knowledge and hands-on experience on
different EV purification and characterization techniques, such as fluorescent labelling,
density gradient centrifugation, size exclusion chromatography, electron microscopy,
flow
cytometry and nanoparticle tracking analysis and on databases like EV-TRACK and FunRich.
In
addition, the organizers and invited lecturers from several different research areas
explain
which strategies are used to understand the role of EV in biomedical applications
and give
an overview of the current state of the field of EV research.
Results: The course therefore covers a broad range of topics important in the
EV field, including heterogeneity in EV subpopulations, mechanisms of EV cargo selection,
EV
biogenesis, pre-analytical variables, therapeutic and diagnostic use of EV, and in
vivo
functions of EV. Group discussions are facilitated and stimulated via assignments
to analyse
data obtained during the practicals and to critically evaluate literature. Participants
also
have the opportunity to present their own research during poster presentations and
ask for
feedback from organizers and invited lecturers.
Summary/Conclusion: Among the participants selected for the course, a large
geographical distribution is reached, including researchers from the newer EU member
states
and from outside of Europe, to ensure a broad geographical distribution of the knowledge
gained during this course.
OS26.3
1st Lugano ExoDay – technical challenges in exosome research.
First extracellular vesicles workshop in the Southern Switzerland
Daniele D’Arrigo
a, Carolina
Balbib, Giona Pedriolic, Elena Vacchic, Vanessa
Biemmid, Giorgia Mellic, Giuseppe Vassallib, Matteo
Morettie, Paolo Paganettic and Lucio Barileb
aRegenerative Medicine Technologies Lab, RMTL-EOC, Lugano,
Switzerland; bCardiocentro Ticino, Lugano, Switzerland; cLaboratory
for Biomedical Neurosciences, LBN-EOC, Taverne-Torricella, Switzerland;
dCardiocentro Ticino, Taverne-Torricella, Switzerland; eRegenerative
Medicine Technologies Lab, RMTL-EOC, Taverne-Torricella, Switzerland
Introduction: On 25 October 2019, we organized the 1st Lugano ExoDay, first
initiative in the Southern Switzerland to bring together resident researchers and
European
experts in the field of Extracellular Vesicles (EVs). The workshop, centred on “Technical
challenges of extracellular vesicle research” aimed to highlight technical requirements
and
advances in the EVs area, focusing on isolation, characterization and tracking.
Methods: The workshop started with a lecture by Dr. Cecilia Lässer, from the
University of Gothenburg. The rest of the workshop was divided in two working groups
(WG),
each introduced by a keynote lecture followed by presentations by young researchers
and a
round-table discussion. WG1, introduced by Dr. Mercedes Tkach, from the Institute
Curie in
Paris, focused on recent advances on EVs characterization and isolation. WG2 was centred
on
EVs tracking and introduced by Dr. Frédérik Verweij, from the Institute of Psychiatry
and
Neuroscience of Paris.
Results: Dr. Lässer opened the workshop with a comprehensive review and
introduced recent developments in the EVs field. The first WG discussed different
isolation
methods, focusing on ultracentrifugation, size exclusion chromatography and
immunoprecipitation-based techniques. Supported by the keynote speakers, the participants
agreed that the best approach to optimize the isolation process consists in the combination
of different techniques. WG2 shared insights about new strategies to visualize and
tracking
EVs, focusing on how to improve the routinely approaches used, defining optimal criteria
for
EVs labelling and imaging. All the participants had an in-depth overview on the requirements
and the state-of-the-art techniques currently in use for the isolation, characterization
and
tracking of EVs.
Summary/Conclusion: The transferable knowledge acquired during the workshop
ensures participants to remain up-to-date with the advances in the field of EVs. As
our
ultimate goal is to create a competence centre in Southern Switzerland around the
field of
EVs, the workshop was an invaluable opportunity to intensify collaborations between
resident
laboratories and broaden the scientific exchange with laboratories of the experts
hosted
during the event. Given the success of this first workshop we are already working
to prepare
the second edition and make the event a recurring appointment.
Funding: Supported by the Swiss National Science Foundation
OS26.4
The role of core facilities and emerging technologies in
maximizing rigour and reproducibility of EV quantification and characterization and
following MISEV guidelines
Rachel DeRita
a and Andrew
Hoffmanb
aThomas Jefferson University, Philadelphia, USA;
bUniversity of Pennsylvania School of Veterinary Medicine, Philadelphia, USA
Introduction: It remains very clear in the field of extracellular vesicle (EV)
research that the rapid rate of increase in publications and expansion of interdisciplinary
clinical EV interest has created the need for increased standardization and access
to the
appropriate technologies to uphold these standards. As the first core facility in
the USA
with the sole intention of creating a space where users can both isolate and characterize
EVs, we provide a central location for the facilitation of EV research via access
to
multiple technologies (both established and emerging) such as resistive pulse sensing,
nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography,
flow cytometric analysis of EVs and additional immune or fluorescence-based EV
characterization techniques.
Methods: We surveyed a group of leading scientific investigators and
researchers in varying stages of their scientific careers in the Mid-Atlantic region
of the
US. The survey data demonstrate applications of greatest current and future interest
to be
employed in a shared lab resource.
Results: The current demand is highest for isolation services,
ultracentrifugation and NTA, with a gradually increasing demand for immunophenotying
analyses such as the ExoView chip array, fluorescent NTA and flow cytometry. We additionally
present strategies and data-based examples of how shared resource facilities can facilitate
multifactorial and rigorous EV characterization in accordance with MISEV guidelines,
and
encourage collaboration among EV researchers.
Summary/Conclusion: In order to answer the larger remaining questions in the
EV field such as the isolation of specific EV subsets, EV tracking between cells and
the use
of EVs for biomarker discovery and drug delivery, it is essential that shared resource
facilities interact not only with investigators, but with each other to integrate
the
necessary resources to progress.
OS26.5
Programme to assess the rigour and reproducibility of
extracellular vesicle-derived analytes for cancer detection
Matthew Young and Sudhir Srivastava
National Cancer Institute, Rockville, USA
Introduction: Cancer cells release more EVs than normal cells and EVs secreted
from tumour cells can promote tumour progression, survival, invasion and angiogenesis.
The
EV cargo may mirror the altered molecular state of the cell of origin. Therefore,
EVs have
potential for the development of non-invasive markers for early detection of cancers.
EVs
and their cargo also have the potential to be multiplexed with other molecular markers
or
screening modalities (e.g., imaging) to develop integrated molecular-based computational
tools for the early detection of cancer.
One challenge with using EVs as a biomarker is the lack of robust and reproducible
methods
for the isolation of a pure vesicular population. There is a lack of clear consensus
for an
optimal method of isolation of a pure EV population that is devoid of contamination
with
similar-sized vesicles of different origins. There is also a lack of standards to
ensure
rigour reproducibility.
Methods: The current funding opportunity announcement (FOA), PAR20-053, is
promoting research on the isolation and characterization of extracellular vesicles
(EVs) and
their cargo for the discovery of biomarkers to predict cancer and cancer risk.
Results: The previous cycle of this FOA, PAR16-267/277, successfully funded 7
R01 and 4 R21 grants. These awards are focused on proteomics profiling of EVs, effect
of
methodological and biological variability, asymmetric-flow field-flow technology,
therapeutic monitoring, LSS and SERS lab on a chip optical spectroscopic, EVs in
obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular
heterogeneity, urinary EV DNA, and EV markers in paediatric cancers.
Progress from these awardees have shown separation of two discernible exosome
subpopulations and identified a distinct nanoparticle, the exomere (Nature Cell Biology,
2018); and have shown that large-EVs contain the entire genome of the cell of origin,
including cancer-specific genomic alterations (Journal of Extracellular Vesicles,
2019).
Protocols that critically evaluate and refine the existing methodologies to improve
utilization of EVs in clinical use have been shared (Nature Protocols, 2019).
Summary/Conclusion: Drs. Sudhir Srivastava and Matthew Young are the program
directors for the PAR which began accepting applications on 5 January 2020. This and
other
EV funding opportunities will be discussed.
Funding: This is a Funding Opportunity Announcement offered by the National
Cancer Institute
Late-Breaking Oral Presentations
OS27
Symposium Session 27: Late Breaking: EV Biology and Biomarkers
Chair: Xiaomei Yan – Professor, Xiamen University
Chair: Eduard Willms – Post Doc, La Trobe University
OS27.1
Detecting tumour-associated membrane receptors on extracellular
vesicles via immuno-PCR with Affibody-DNA-Conjugates
Christiane Stiller
a, Petra
Håågb, Elizabeth Paz Gomeroa, Vasiliki Arapib, Kristina
Viktorssonb, Rolf Lewensohnb and Amelie E. Karlströma
aKTH Royal Institute of Technology, Stockholm, Sweden;
bKarolinska Institute, Stockholm, Sweden
Introduction: Early detection of cancer as well as monitoring cancer treatment
are important to improve cancer care. Diagnostics for cancer are mainly based on tissue
biopsies and re-biopsy during treatment is challenging. Moreover, current diagnostics
are
expensive, time-consuming and have low-throughput. Therefore, liquid biopsies are
expected
to bring the next breakthrough in cancer diagnostics. In liquid biopsies tumour-secreted
material is isolated from body fluids and subsequent analyses thereof allow for non-invasive
diagnostics. One type of tumour-secreted materials are extracellular vesicles (EVs),
which
are schedded from tumour cells. EVs are surrounded by a lipid bilayer, whichs composition
resembles the plasma membrane of their parental cell. As many tumours are driven by
over-expression or upregulation of transmembrane proteins e.g. growth factor receptors,
detection of the later on EVs holds promise for early tumour detection and treatment
monitoring.
Methods: For the immuno-PCR EVs were first affinity-captured on magnetic
beads, allowing immobilization of purified EVs as well as EVs secreted into cell culture
medium or spiked into plasma. Afterwards each sample was divided and affibody-DNA-conjugates
directed against different targets were added. Affibodies are small affinity proteins,
which
often are developed as high affinity binders for tumour imaging, making them suitable
probes
in the presented assay. After washing, the bead-EV-affibody-DNA-complexes were analysed
for
the immobilized DNA-amount via qPCR.
Results: Via the presented immuno-PCR EVs secreted from the non-small cell
lung cancer cell line H1975 as well as the ovarian cancer cell line SKOV3 were analysed.
The
immuno-PCR method allowed the detection of the tumour-associated membrane receptors
epidermal growth factor receptor (EGFR), receptor tyrosine-protein kinase ERBB2/Her2
and
insulin-like growth factor 1 receptor (IGF1R). Different levels of membrane receptors
depending on the EV source and concentration were detected.
Summary/Conclusion: The presented immuno-PCR showed to be a comparably fast
and robust method for detection of tumour-associated membrane receptors on EVs derived
from
cancer cell lines with medium through-put and is currently further developed into
a method
for liquid biopsy for non-small cell lung cancer patients.
Funding: Erling Persson Foundation. Swedish and Stockholm Cancer Society
OS27.2
Heterogeneity in EV cargo expression measured by single vesicle
flow cytometry
John Nolan and Erika Duggan
Scintillon Institute, San Diego, USA
Introduction: Introduction. EVs produced by cells can originate from different
cellular compartments and EVs in complex biofluids may originate from many different
cell
types. Traditional biochemical analysis, which reports on the total composition of
all EVs
in a sample can’t adequately resolve this heterogeneity. Single vesicle analysis methods
can, if they have the necessary specificity, sensitivity and speed. Flow cytometry
(FC) is
capable of rapid and quantitative analysis of individual particles, but conventional
FC-based assays lack the specificity and sensitivity to measure individual EVs. Assays
that
combine sensitive instruments with EV-selective sample staining can measure individual
EVs
with accuracy and precision. To better understand the nature and origins of EV diversity,
we
used single vesicle FC (vFC) to quantitatively measure vesicle number, size, and surface
cargo expression on individual EVs.
Methods: Methods. EVs in culture supernatants (293 T, A431, U87, THP-1,
SH-SY5Y) were used neat or enriched by standard methods including differential
centrifugation or ultrafiltration. EVs from platelets (PLT) and red blood cells (RBC)
were
induced by ionophore treatment of washed cells, and measured in diluted supernatant.
EVs
were stained with a membrane-selective dye and fluorescence-labelled antibodies using
a
commercial vFC assay kit (Cellarcus Biosciences), measured using a commercial flow
cytometer
(CytoFlexS, Beckman Coulter), and data analysed using FCS Express v6 (De Novo Software).
Vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate
vesicle and bead-based standards and essential controls performed as recommended by
the
MIFlowCyt-EV reporting guidelines.
Results: Results. To assess the compositional heterogeneity of EVs, we first
characterized the expression of tetraspanins (TSs; CD9, CD63, CD81, CD82, CD151, CD53,
CD231) on EVs released from cultured cell line and primary cell cultures. We find
quantitative differences in the expression of TS on EVs from different cell types
that
generally reflected the expression on the cell of origin, with most EV types expressing
detectable amounts at least one of the common TS molecules (CD9, CD63 or CD81) but
generally
not all three. In EVs from some cell types, TS expression was uniform across the EV
population (CD9 on EVs), but EVs from other cell types differentially expressed TSs,
with
some EVs expressing no detectable TS (RBC EVs). Intracellular cargo labelled genetically
using fluorescent proteins (eGFP or mNeonGreen) or fluorogenic enzyme substrates (CFSE)
were
measured in individual EVs and revealed distinctive associations between EV surface
and
internal cargo.
Summary/Conclusion: Conclusions. High resolution measurement of cargo on/in
individual EVs can help interpret EV heterogeneity in terms of cell of origin, signals
carried, and effects on target cells.
OS27.3
Integrated omics reveal conserved and divergent modulation of
cardiovascular disease by tissue-entrapped extracellular vesicles
Mark C. Blaser
a, Fabrizio
Buffoloa, Arda Halua, Florian Schlottera, Hideyuki
Higashia, Lorena Pantano Rubinob, Louis A. Saddica,
Samantha Atkinsa, Maximillian A. Rogersa, Tan H. Phama,
Eugenia Shvartzc, Galina K. Sukhovac, Silvia Monticoned,
Giovanni Camussie, Simon C. Bodyf, Jochen D.
Muehlschlegelg, Peter Libbyh, Sasha A. Singha, Masanori
Aikawaa and Elena Aikawaa
aCenter for Interdisciplinary Cardiovascular Sciences,
Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical
School,
Boston, MA, 02115, U.S.A., Boston, USA; bThe Picower Institute for Learning and
Memory, Massachusetts Institute of Technology, Boston, MA, 02139, U.S.A., Boston,
USA;
cCenter for Excellence in Vascular Biology, Brigham and Women’s Hospital,
Harvard Medical School, Boston, MA, 02115, U.S.A., Boston, USA; dDivision of
Internal Medicine and Hypertension, Department of Medical Sciences, University of
Torino,
Torino, Italy, Torino, Italy; eDepartment of Medical Sciences, University of
Torino, Turin, Italy; fBoston University School of Medicine, Boston, MA, 02118,
U.S.A., Boston, USA; gCenter for Perioperative Genomics, Department of
Anaesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard
Medical School, Boston, MA, 02115, U.S.A., Boston, USA; hCenter for Excellence in
Vascular Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA,
02115,
U.S.A.A, Boston, USA
Introduction: Fewer than 50% of patients develop both vascular and valvular
calcification, implying differential pathogenesis. While circulating extracellular
vesicles
(EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs are implicated
in
early mineralization but their contents and function are unstudied. We developed an
innovative method to isolate and study EVs from fibrocalcific tissue and investigated
entrapped EV cargoes in human cardiovascular diseases.
Methods: Human carotid artery endarterectomies and stenotic aortic valves were
obtained from 27 donors under IRB-approved informed consent. Tissues underwent enzymatic
digestion, ultracentrifugation, and a 15-fraction OptiPrep density gradient. Global
proteomics was performed on intact tissue, each OptiPrep fraction, and EV-enriched
pooled
fractions; the latter also underwent miRNA-seq. Fractionated samples were also studied
by
CD63 immunogold electron microscopy (TEM) and nanoparticle tracking analysis (NTA).
High
confidence miR targets were predicted by TargetScan, pathway analyses utilized the
BioCarta/KEGG/Reactome databases, and protein-protein interaction networks were built
in
STRING.
Results: Vesicle-associated pathways were increased 5.1x (p < 0.01; 15/30
vesicle-related top GO terms) in proteins common to intact arteries and valves (n = 1,411).
Proteomics found 24 EV markers to be highly enriched in the four least-dense OptiPrep
fractions of arteries and valves, while extracellular matrix and mitochondria were
predominant in denser fractions, as confirmed by TEM/NTA. Proteomics and miRNA-seq
of tissue
EVs quantified 1,104 proteins and 123 miR cargoes linked to 5,182 target genes. Pathway
networks of proteins and miR targets common to artery and valve tissue EVs revealed
a shared
regulation of Rho GTPase and MAPK intracellular signalling cascades. 179 proteins
and 5 miRs
were significantly altered between artery and valve EVs (q < 0.05); multi-omics
integration found that EVs differentially modulated cellular contraction and p53-mediated
transcriptional regulation in vascular and valvular tissue.
Summary/Conclusion: Our findings delineate a novel strategy for studying
tissue-entrapped EV protein and miR cargoes and identify critical roles that tissue-resident
EVs play in mediating cardiovascular disease.
Funding: This study was supported by a research grant from Kowa Company (MA)
and NIH grants R01 HL136431, R01 HL141917 and R01 HL147095 (EA).
OS27.4
MiR-20a regulates exogenous CD82 expression on proliferation,
invasion, migration and angiogenesis of gastric cancer
Tingting Guo
Zhengzhou University, Zhengzhou, China (People’s Republic)
Introduction: To investigate the possible mechanism of MiR-20a regulating the
expression of exosome CD82 on proliferation, invasion, migration and angiogenesis
of gastric
cancer, and to study the application value of CD82 in the early diagnosis and prognosis
of
gastric cancer.
Methods: The gastric cancer cell line MGC-803 was used as the research object.
The exosomes were extracted from the culture supernatant of MGC-803 by exosome extraction
kit. The extracted exosomes were identified by transmission electron microscopy and
Western
blotting. The expression of CD82 in exosomes was detected by ELISA. The expression
of CD82
in exosomes and CD82 in whole blood and serum were detected by Western Blot. They
were
randomly divided into blank group (MOCK) and miR-20a lentivirus experimental group
(miR-20a
group). The lentivirus control group (miR-20a/Con) was transfected into cells. qRT-PCR
was
used to verify the status of miR-20a after transfection; Western-blot was used to
detect the
expression of CD82 and downstream ERK1/2, AKt and m TOR proteins; MTT assay, cell
colony
formation assay, Transwell migration assay for cell proliferation, invasion, and migration.
A nude mouse xenograft model was constructed to observe the growth of transplanted
tumours,microvessel density (MVD) was detected by immunofluorescence, and distant
metastasis
was recorded.
Results: The expression of CD82 in exosomes was detected by ELISA and Western
Blot. The expressions of CD82, Akt, ERK1/2 and m TOR in miR-20a group were significantly
lower than those in miR-20a/Con and MOCK groups. CD82 protein is positively correlated
with
downstream protein levels.The growth rate and cell invasion ability of miR-20a group
were
significantly lower than those of miR-20a/Con group and MOCK group. The weight of
the nude
mice in the MOCK group and the miR-20a/Con group decreased, while the weight loss
in the
miR-20a group was not significant. The tumours in the miR-20a/Con group and the MOCK
group
showed invasive growth accompanied by abundant microvessels, while the miR-20a group
had
smaller tumour volume and uniform cell distribution. Only a small amount of
microangiogenesis was observed, and no obvious necrotic area was observed.
Summary/Conclusion: miR-20a affects the proliferation, invasion, migration and
angiogenesis of gastric cancer mediated by Akt/ERK/m TOR signalling pathway by regulating
the expression of exosome CD82.
OS27.5
Streamlined detection and quantification of plasma extracellular
vesicles and their protein cargo by high-performance nanoscale flow cytometry and
label-free mass spectrometry
Samuel Tassi Yunga
a, Randall
Armstrongb, Austin Gowera, Meghan Fitzgeralda, Matthew
Changa, Ashok Reddyc, Mark Floryb and Owen
McCartyd
aKnight Cancer Institute, Oregon Health & Science
University, Portland, USA; bOHSU, Portland, USA; cProteomics Shared
Resources, OHSU, Portland, USA; dDepartment of Biomedical Engineering, Oregon
Health & Science University, Portland, USA
Introduction: Nanoscale flow cytometry (FC) and mass spectrometry (MS) are
useful for profiling EV surface proteins and performing bulk EV proteomics, respectively.
This study sought to develop pre-analytical and analytical pipelines for EV protein
profiling that are applicable to clinical studies.
Methods: To optimize plasma EV detection and quantification by FC,
modifications of instrument settings and serial dilutions of platelet-free plasma
(PFP) and
antibodies were tested for improved separation of signal from noise and reduction
of event
coincidence and swarming. The high-performance flow cytometry (HPFC) platform was
used to
assess the effect of time (1, 2, 3, 5, 9, 24, 48 or 120hrs) between blood draw (into
ACD,
NaCit, EDTA or heparin) and blood processing, on ex-vivo release of EVs from blood
cells.
Label-free MS was used to examine the intensity and breadth of identified proteins
in plasma
EVs purified using several density and size separation methods, either manually or
automated, along with various buffer conditions.
Results: EV event aborts were minimized at a PFP dilution, prior to staining,
of 1:10 and by using a narrow cytometer window extension. Target EV signals were distinct
from noise and were Triton X-100 labile. The most significant changes in plasma EVs
were
associated with platelet-derived fractions, use of heparin and >5-hour delay before
blood
processing. Yet, platelet EV numbers did not significantly change for up to 24 hrs
in
citrated and EDTA plasma. Higher overall coverage of known EV proteins and a fivefold
increase in number of uniquely identified proteins were observed in MS profiling of
EVs
prepared by a combination of ultracentrifugation (UC) and manual size-exclusion
chromatography (SEC) compared to preparation by FPLC on Capto Core 700/Superose 6
resins.
UC/SEC was better than direct SEC at reducing contamination by excipient plasma proteins.
Column buffers with trehalose increased EV protein recovery while adding protease
inhibitors
had minimal effect.
Summary/Conclusion: With our optimized HPFC protocol, we established that
blood EV numbers remain stable for up to 24 hrs in ACD or EDTA and that UC+SEC with
trehalose-containing buffer result in high canonical EV protein recovery. We are applying
these workflows to investigate cancer-associated changes in plasma EV protein cargo.
Funding: This project was supported by funding from the Cancer Early Detection
Advanced Research (CEDAR) center at Oregon Health & Science University’s Knight Cancer
Institute.
Support for this project also came from the Proteomics Shared Resources of Oregon
Health
& Science University
OS27.6
The value of exosomes as a potential biomarker for Devil Facial
Tumour Disease.
Camila Espejo, Richard Wilson, Greg Woods and
Bruce Lyons
University of Tasmania, Hobart, Australia
Introduction: The Tasmanian devil (Sarcophilus harrisii), the largest living
carnivorous marsupial is endangered because of two transmissible cancers: Devil Facial
Tumour Disease (DFTD) one and two. Current efforts to manage DFTD are hindered by
the lack
of a preclinical diagnostic test for DFTD. Detecting DFTD infection is only possible
once
tumours are noticed, too late to stop DFTD progression. A preclinal test could tell
us about
unknown components of DFTD pathogenesis, such as latent period and host-tumour dynamics.
Exosomes are extracellular vesicles released by most types of cells under both
physiological and pathological conditions. Exosomes have utility as diagnosis and
prognosis
biomarkers in a range of diseases, including cancers. The aim of this study is to
investigate exosomes-based approaches towards a preclinical and progression biomarker
for
DFTD 1 and 2 in Tasmanian devils.
Methods: Exosomes were isolated from three different DFTD-1, DFTD-2 and devil
fibroblast cell lines by size-exclusion chromatography. Likewise, exosomes were isolated
from plasma of healthy and diseased devils. To determine the size and morphology of
exosomes, samples were imaged with transmission electron microscopy. Exosomes isolated
from
cell lines and devil plasma were analysed with mass spectrometry to characterise proteins
and determine their differential expression between the cell origins, and healthy
and
diseased animals.
Results: This study identified the presence of myelin proteins in exosomes
from DFTD cells relative to fibroblasts, which are diagnostic of DFTD. Additionally,
we
found that exosomes derived from DFTD-2 abundantly express the inhibitory checkpoint
molecule CD200 relative to exosomes from DFTD-1 cells and devil fibroblasts, indicating
a
potential candidate for a differential diagnosis between tumours. Moreover, exosomes
from
DFTD cells present a greater amount of proteins related with metastasis in comparison
with
fibroblast exosomes, such as integrins. Finally, we report the protein expression
profile of
exosomes from healthy and diseased devils, showing clear differences between them
and the
presence of immunosuppressive and metastasis proteins in animals in late stages of
the
disease.
Summary/Conclusion: DFTD-exosomes may provide a non-invasive diagnosis tool to
detect early stages of DFTD in Tasmanian devils to facilitate the prevention of the
disease.
Furthermore, DFTD-exosomes may have utility as a prognosis biomarker, determining
late
stages of the disease using a simple a blood test, which would facilitate monitoring
of wild
populations. This project will provide long-term benefits for the future of the devils
and
encourage exosome-based solutions for other future wildlife disease outbreaks.
Funding: National Geographic Early Career Grant
Holsworth Wildlife Research Endowment Grant
Dr Eric Guiler Tasmanian Devil Research Grant
OS28
Symposium Session 28: Late Breaking: Therapeutics, Biogenesis and
Biodistribution
Chair: Charles Lai – Ministry of Science and Technology, Taiwan
Chair: Ann
M. Wehman – Junior Group Leader, Rudolf Virchow Center at the University of
Würzburg
OS28.1
Absolute quantification of Extracellular vesicles in vivo by a
sensitive bioluminescence system.
Dhanu Gupta
a, Xiuming
Liangb, Joel Nordinb and Samir El-Andaloussic
aDepartment of Laboratory Medicine, Karolinska Institutet,
Sweden, Huddinge, Sweden; bClinical Research Center, Department for Laboratory
Medicine, Karolinska Institutet, Stockholm, Sweden, Stockholm, Sweden;
cDepartment of Laboratory Medicine, Clinical Research Center, Karolinska
Institutet, Huddinge, Sweden
Introduction: Despite the increased understanding of EVs, from involvement in
disease pathophysiology to therapeutic delivery, improved molecular tools to track
biodistribution are largely lacking. Current approaches used for EV labelling lacks
sensitivity and specificity. Here, we have explored bioluminescent labelling of EVs
to
achieve a highly sensitive system for absolute in vivo quantification and tracking
of
exogenous EVs at low cost and in a high throughput manner.
Methods: EV-producing cells were genetically engineered to express various
tetraspanin-luciferase fusion proteins. EVs purified by UF-SEC from these cells were
characterized by NTA, multiplex bead-based array, TEM and WB, followed by luciferase
assay
to determine the labelling efficiency. For in vitro applications cell lysate from
treated
cells or the conditioned medium were subjected to luciferase assay. For in vivo applications
two different methodologies were applied to determine biodistribution; either by
non-invasive real time in vivo imaging using IVIS or by luciferase assay on harvested
tissues for absolute quantification of injected EVs.
Results: We initially performed a systematic comparison of five different
luciferases for endogenous labelling of EVs and identified NanoLuc and ThermoLuc as
lead
candidates. We applied this technology to monitor in vitro cellular uptake and observed
cell
type differences in cellular uptake of engineered EVs. In addition, we also observed
an
effect of different culturing conditions on exocytosis kinetics. For in vivo application,
we
applied the NanoLuc labelling strategy to determine the pharmacokinetics and effect
of
different routes of injection on EV distribution. Our results indicated a rapid uptake
profile of administered EVs in different tissues with liver, spleen, and lungs being
the
primary recipients. We also observed similar results upon tracking in vivo biodistribution
in real time immediately after administration. Finally, we show how different subpopulations
of EVs differ in their in vivo biodistribution.
Summary/Conclusion: Overall, NanoLuc and ThermoLuc labelling of EVs holds
great potential for various in vivo and in vitro applications. In addition, it can
enable
the simultaneous detection of different sub-populations of EVs in vivo, which may
aid in our
understanding of different sub-populations and their behaviour in vivo. Apart from
monitoring therapeutic EVs, with one simple modification this platform offers great
potential for tracking tumour derived EVs both in vivo and in vitro and thus could
aid in
the development of anti-tumour therapies.
OS28.2
Biofunctional peptide-modified extracellular vesicles for cell
targeting, macropinocytosis induction, and effective intracellular delivery
Ikuhiko Nakase
Department of Biological Science, Graduate School of Science, Osaka
Prefecture University, Sakai-shi, Japan
Introduction: [INTRODUCTION] In our research group, developing therapeutic
techniques based on extracellular vesicles (exosomes, EVs) by effective usage of peptide
chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been
focused.
In this presentation, modification techniques using biofunctional peptides such as
arginine-rich cell-penetrating peptides [1], artificial coiled-coil peptides with
receptor
targeting [2], and cell-penetrating sC18 peptides [3] derived from cationic antimicrobial
protein, CAP18 for cancer targeting with macropinocytosis induction, on the EV membranes
will be introduced. I will also show effects of lyophilization of the peptide-modified
EVs
on their biological activity [4].
Methods: [METHODS] CD63 (EV marker)-GFP-fusion protein expressed EVs were used
for cellular EVs uptake assessments. All biofunctional peptides were synthesized by
Fmoc
solid-phase method.
Results: [RESULTS] Macropinocytosis with actin reorganization has been shown
to be crucial for cellular EV uptake [5]. We developed the methods for modification
of
arginine-rich CPPs or sC18 peptides on EV membranes using chemical linker techniques,
and
for example, arginine-rich CPPs modification can induce proteoglycan-clustering (e.g.
syndecan-4) and macropinocytosis signal transduction [1]. The artificial leucine zipper
peptide-modified EVs recognize the peptide-tagged epidermal growth factor receptor
(EGFR) on
targeted cells, leading to macropinocytotic cellular EV uptake [2]. In addition,
lyophilization is a useful technique for long term storage, however, we found that
lyophilization negatively affected biological functions of encapsulated proteins in
the EVs
after their cellular uptake [4].
Summary/Conclusion: [CONCLUSION] These techniques and findings will contribute
to development for the EV-based intracellular delivery systems.
Reference: [1] Sci. Rep. 6, 34937 (2016), [2] Chem. Commun. 53, 317 (2017), [3]
ChrmMedChem. 12, 42 (2017) [4] Anticancer Res. 39, 6701 (2019), [5] Sci. Rep. 5, 10300
(2015)
OS28.3
Multi-compartmented microvesicles: novel extracellular secretory
organelles that release exosomes and extracellular vesicles
Jennifer D. Petersen
a, Sukhbir
Kaurb, Elena Mekhedovc, David D. Robertsd and Joshua
Zimmerberga
aSection on Integrative Biophysics, Division of Basic and
Translational Biophysics, Eunice Kennedy-Shriver National Institute of Child Health
and
Human Development, National Institutes of Health, Bethesda, USA, Bethesda, USA;
bLaboratory of Pathology and Laboratory of Experimental Carcinogenesis, Center
for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,
USA; cSection on Integrative Biophysics, Division of Basic and Translational
Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development
NICHD, Bethesda, USA, Bethesda, USA; dLaboratory of Pathology, Center for Cancer
Research, National Cancer Institute, National Institutes of Health, Bethesda, USA,
Bethesda,
USA
Introduction: Extracellular vesicles (EV) bud from the plasma membrane (PM) as
microvesicles (MV) or arise from the fusion of multivesicular bodies (MVB) with the
PM to
release intralumenal vesicles (ILV) as exosomes. The variety of bioactive molecules
carried
by EV imparts diverse functionality to EV in intercellular signalling. The biogenesis
and
extracellular release of these specialized messenger organelles is not well understood.
To
investigate, we studied endothelial cells that line the inside of blood vessels, known
to
release EV that support angiogenesis.
Methods: Cultured human umbilical vein endothelial cells (HUVEC) were examined
by thin-section electron microscopy (EM), serial sectioning and immunogold labelling
to
study the structure and composition EV release sites. To obtain optimal views of cellular
ultrastructure, cells were preserved by fast-freezing and a freeze-substitution.
Results: A potential release site was identified in EM thin sections as a
discrete domain, up to several microns long, on the otherwise smooth HUVEC PM, where
numerous bulbous membrane protrusions with thin necks were clustered. The cytoplasm
in these
protrusions was enriched with MVB and other vesicles and appeared to be on the verge
of
pinching off to release multi-compartmented MV (MCMV). Consistent with this notion,
in the
neighbouring extracellular space, a plethora of MCMV of 300–1200 nm with ultrastructural
features matching the bulbous protrusions were observed, supporting the concept that
MCMV
bud from the release site. Serial sections confirmed that these extracellular MCMV
were
independent of cells and not linked by nanotubes or other processes. Remarkably, fusion
of
MVB with the MCMV membrane was directly observed, presumably caught in the act of
releasing
ILV (exosomes) from the MCMV. Immunogold labelling for EV markers is being used to
identify
proteins enriched at release sites and on released MCMV.
Summary/Conclusion: In summary, 1) MCMV bud from localized sites on the
endothelial PM, 2) MCMV contain MVB, and 3) fusion of MVB to MCMV to release exosomes
occurs
extracellularly. MCMV can now be evaluated as a potential source of exosome and EV
release
that occurs after budding from the cell of origin, adding new layers of regulation
to when,
where and how EV are assembled and released.
Funding: This work was supported by the Division of Intramural Research of the
NIH.
OS28.4
One size does not fit all: overcoming barriers to successful
discovery and scaled manufacturing of therapeutic extracellular vesicles
Jieun Leea, Wei Guob, Hal
Sternbergc, Mike Westd and Dana
Larocca
d
aStem cell team, Seoul, Republic of Korea;
bUniversity of Pennsylvania, Philadelphia, USA; cAgeX Therapeutics
Inc, Alameda, USA; dAgeX Therapeutics Inc., Alameda, USA
Introduction: Extracellular vesicles have tremendous intrinsic therapeutic
potential. However, the limited availability of production cell lines presents a barrier
to
scaled EV production and novel EV discovery. Indeed, EV sources have been largely
confined
to a handful of cell types with the vast majority consisting of MSC EVs. To overcome
this
limitation, we developed a diverse library of hundreds of clonally pure and scalable
progenitor cell lines that provides an alternative resource for EV drug discovery
and
production.
Methods: We harnessed the capacity of human pluripotent stem cells (hPSC) to
differentiate into virtually any cell type by subjecting hPSC to a wide variety of
media and
culture conditions to maximize the diversity of partially differentiated cells. The
resulting heterogeneous “candidate cultures” were plated at clonal density and further
selected for self renewing and scalable clones. Transcriptomic analysis indicated
>200
distinct progenitor lines. Cell fate potentials were mapped by screening for cell
type
specific marker expression in various differentiation conditions. EVs were produced
using
cGMP methods (TFF and SEC) and characterized by NTA, TRPS, surface marker analysis,
RNA and
protein content. Bioactivity assays included proliferation, migration, vascular tube
network
formation, senolysis, and oxidative stress.
Results: The progenitor library contained >200 distinct lines with diverse
lineage fates including various types of bone, cartilage, muscle,and fat cells, as
well as
all blood vessel cell types. The lines displayed much longer replicative lifespans
(70–100pd) than primary cell lines like MSC. Clonal purity minimized phenotypic drift
resulting in maintenance of cell identity, genome integrity, differentiation capacity
and
bioactive EV production over extended culture. EVs were highly diverse in their RNA
and
protein cargo and bioactivity displaying various degrees of migratory, proliferative,
angiogenic and senolytic activity. Library screening identified EVs with higher angiogenic
potency than primary adult stem cell EVs.
Summary/Conclusion: We demonstrated scalable and stable production of
bioactive EVs from a large progenitor cell library. Library screening resulted in
discovery
of novel angiogenic and senolytic EVs having diverse RNA and protein cargo. We are
currently
creating a corresponding library of progenitor cell EVs to accelerate discovery of
novel EVs
and their production cell lines.
Funding: The initial establishment of the cell library was funded in part by
grants from the California Institute of Regenerative Medicine and National Institutes
of
Health.
OS28.5
Structural insight to interactions of extracellular vesicles and
membrane active anticancer-antimicrobial peptides
Tamas Beke-Somfai
a, Imola
Szigyarto, Priyanka Singhb, Mayra Quemé-Penac, Szilvia
Bőszed, Tünde Juhászc, Ference Zsilac, Zoltan
Vargae and Mihaly Judithc
aPI, Budapest, Hungary; bMs, Budapest, Hungary;
cResearch Centre for Natural Sciences, Budapest, Hungary; dEötvös
Loránd University, Budapest, Hungary; eBiological Nanochemistry Research Group,
Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences,
Budapest, Hungary, Budapest, Hungary
Introduction: Besides extreme potential in biomedical applications,
extracellular vesicles (EVs) are also promising candidates to expand biophysical
understanding of membrane active biomolecules. Their complex bilayer composition allows
the
better understanding of adsorbed proteins and protein coronas as well, which sets
of
macromolecules will likely be key for advanced EV targeted delivery. Considering cargo,
membrane active peptides are interesting as these can be both drugs to be delivered,
but can
also facilitate cargo insertion through lipid bilayers. However, at present very little
is
understood regarding interactions between the peptides and the EV lipid bilayer, and
between
peptides and membrane associated proteins on EVs.
Methods: We have recently demonstrated, that EV membrane adsorbed proteins and
their interactions can be studied by techniques such as polarized light spectroscopy,
microfluidic resistive pulse sensing measurements and freeze-fraction transmission
electron
microscopy [1]. Furthermore, initially we studied several peptides with known antimicrobial
properties and found that these strongly interact with the EV surface proteins, resulting
in
efficient removal of some from the lipid bilayer [2].
Results: Here we present investigation of further EV-peptide interactions also
focusing on anticancer peptides, which may be promising drug candidates for targeted
delivery. These studies allowed to gain insight to novel functions of several peptides,
such
as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed
understanding on how EV protein coronas, or EV bilayers are affected, to such extent
that
they cannot exert their potential function as delivery systems.
Summary/Conclusion: The above interactions are expected to be interesting both
for applicability, i.e. for selecting suitable compounds for EV processing, and also
for
curiosity-driven understanding of peptide functions, and EV-biomolecule interactions.
Based
on these we promote that peptide – EV interactions will receive increased focus in
EV-engineering.
References
[1] I. Cs. Szigyártó, R. Deák, J. Mihály, S. Rocha, F. Zsila, Z. Varga, T. Beke-Somfai,
ChemBioChem, 2018, 19, 545–551
[2] P. Singh, I. Cs. Szigyártó, M. Ricci, F. Zsila, T. Juhász, J. Mihály, Sz. Bősze,
É.
Bulyáki, J. Kardos, D. Kitka, Z. Varga, T. Beke-Somfai, Frontiers in Chemistry,
submitted
Funding: This work was funded by the Momentum programme (LP2016-2), by the
National Competitiveness and Excellence Program (NVKP_16-1-2016-0007) and
BIONANO_GINOP-2.3.2–15-2016-00017.
OS29
Symposium Session 29: Late Breaking: Central Nervous System EVs
Chair: Saumya Das – Massachusetts General Hospital, Harvard Medical School
Chair: Mikin Patel – Graduate student, Department of Biological Sciences, Vanderbilt
University
OS29.1
A specific non-coding RNA in extracellular vesicles from induced
neuronal cells and epigenetic regulation of neurotransmission
Ellen Tedforda, Norhidayah Badyab, Conor
Laingc and Glenn McConkey
c
aCambridge University, Cambridge, UK; bU of Leeds,
Leeds, UK; cUniversity of Leeds, Leeds, UK
Introduction: Our late-breaking finding is the identification of a non-coding
RNA (ncRNA) in extracellular vesicles (EVs) from neuronal cells that is a natural
antisense
transcript for the DBH gene and associated with epigenetic changes and gene silencing.
DNA
methylation in neurons is involved in memory and neurological disorders (Science 2018
361
(6409)). Earlier work found that during chronic brain infection with Toxoplasma gondii
induced a decrease in norepinephrine levels and expression of the host DBH gene; and
the
decrease is correlated with behaviours linked to noradrenergic signalling (Infect
Immun.
2019 87(2); Infect Immun. 2016 84(2861)). DBH catalyzes the production of norepinephrine
from dopamine in noradrenergic neurons. We found that EVs from infected cultures suppress
transcription of the DBH gene and hypermethylation of the gene in noradrenergic cells
in
vitro. In this study, we identify a ncRNA in the EVs from infected neuronal cells.
Methods: Neuronal cells were induced by infection with Toxoplasma gondii and
EVs purified on sucrose gradients. EVs were characterised by electron microscopy and
used to
treat rat and human neuronal cells and expression levels of DBH mRNA and nascent DBH
gene
transcription were measured. Induced EVs were injected into the locus coeruleus of
rats and
DBH gene expression was monitored. RNA purified from EVs was screened for natural
antisense
transcripts (NATs) by strand-specific RT-PCR.
Results: We found that EVs purified from infected neuronal cultures induced
transcriptional gene silencing (TGS) and DNA methylation of DBH in recipient neuronal
cells.
The induced EVs down-regulated DBH gene expression >200-fold and induced DNA
hypermethylation of the DBH gene. This could be induced in the brains of recipient
rats by
intracerebral injection of EVs. Using a panel of strand-specific primers, antisense
transcripts for the DBH gene were identified in infected cells. This permitted us
to examine
the RNA in purified EVs and identify a lncRNA in EVs selective for EVs from infected
cultures.
Summary/Conclusion: This is the first study to find a specific
neurotransmitter antisense lncRNA in EVs associated with transcriptional gene silencing
and
epigenetic changes in the gene. This represents a different type of neuron-to-neuron
signalling than the classic chemical and electrical neurotransmission. The findings
will
enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy,
drug
addiction) and how memory works.
OS29.2
Human CD4 + T regulatory-derived extracellular vesicles and
associated microRNAs: role in cell-to-cell communication and involvement in the loss
of
immune tolerance during multiple sclerosis
Silvia Garavellia, Claudio Procaccinia,
Alessandra Colamatteob, Fortunata Carbonea, Elena
Tagliabuec, Donatella Carpid, Laura Cantonee, Valentina
Bollatie, Ilaria Giustif, Vincenza Dolog, Sarah
Grossih, Paola Campomenosih, Dario Di Silvestrei,
Pierluigi Maurii, Annibale Pucac, Fabio Buttarij, Diego
Centonzej, Veronica De Rosaa, Giuseppe Matareseb and
Paola de Candia
c
aLaboratorio di Immunologia, Istituto di Endocrinologia e
Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), Naples, Italy,
Naples, Italy; bTreg Cell Laboratory, Dipartimento di Medicina Molecolare e
Biotecnologie Mediche, Università Degli Studi di Napoli “Federico II”, Naples, Italy,
Naples, Italy; cIRCCS MultiMedica, Milan, Italy; dJoint Research
Centre, Ispra, Varese, Italy, Varese, Italy; eEPIGET LAB, Department of Clinical
Sciences and Community Health, Università degli Studi di Milano, Milano, Italy;
fDepartment of Life Health and Environmental Sciences, University of L’Aquila,
Italy, L’Aquila, Italy; gDepartment of Life, Health and Environmental Sciences,
University of L’Aquila, L’Aquila, Italy; hUniversita’ degli Studi dell’Insubria,
Dipartimento di biotecnologie e scienze della vita, Varese, Italy, Varese, Italy;
iIstituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche
(ITB-CNR), 20090 Segrate, Milano, Italy, Milan, Italy; jUnit of Neurology and
Unit of Neurorehabilitation, IRCCS Istituto Neurologico Mediterraneo (INM) Neuromed,
Pozzilli, IS, Italy, Isernia, Italy
Introduction: An impairment of immune tolerance is a determining factor in
multiple sclerosis (MS) and dysregulation of CD4 + T regulatory (Treg) cell function
is
believed to be a major pathogenic factor. MicroRNAs (miRNAs) released by Treg cells
in
association with extracellular vesicles (EVs) have been shown to participate in the
block of
pathological immune responses by inhibiting the growth and cytokine production of
CD4 + T
conventional (Tconv) cells, but the molecular mechanism is still poorly characterized.
Aim
of the present work was to evaluate whether Treg cell-derived EV-associated miRNA
signature
is dysregulated in MS and whether this defect may play a role in the development of
autoimmunity.
Methods: Human Treg cells isolated from blood of naïve to treatment
relapsing-remitting MS patients and healthy controls were in vitro stimulated and
released
EVs were isolated by size exclusion chromatography and characterized by nanoparticle
tracking analysis, electron microscopy and flow cytometry. EV-associated miRNAs were
quantified by traditional RT-qPCR and droplet digital PCR for absolute quantification.
The
actual EV-mediated passage of RNA molecules from cell to cell was followed through
RNA-specific fluorescent staining and Treg-derived EV effect on Tconv cell transcriptome
was
evaluated by RNAseq.
Results: In healthy conditions, the treatment of Tconv cells with Treg-derived
EVs was shown to cause the specific repression of genes involved in the proteasome-dependent
proteolytic process, known to be crucial for T cell activation. In MS, Treg-derived
EVs may
have lost this capability as a direct consequence of a significantly decreased expression
of
miR-142-3p, able to target key factors of the proteasome system.
Summary/Conclusion: Our results unveil a novel molecular mechanism for
Treg-mediated maintenance of self-tolerance based on EV-associated miR-142-3p and
its
potential alteration in human autoimmunity.
Funding: Fondazione Italiana Sclerosi Multipla, FISM, # 2016/R/10 and #
2018/R/4
OS29.3
Revealing the proteome of Brain Derived Extracellular Vesicles
isolated from human Amyotrophic Lateral Sclerosis post-mortem tissues.
Natasha Vassileff
a, Laura J.
Vellab, Harinda Rajapakshac, Mitch Shambrookd,
Amirmohammad Nasiri Kenarie, Jacky Chanf, Catriona McLeang,
Andrew F. Hillh and Lesley Chengh
aThe Department of Biochemistry and Genetics, La Trobe
Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.,
Melbourne, Australia; bThe Florey Institute of Neuroscience and Mental Health,
University of Melbourne, Melbourne, Australia; cLa Trobe Institute for Molecular
Science, Melbourne, Australia; dDepartment of Biochemistry and Genetics, La Trobe
Institute for Molecular Science, Australia, Melbourne, Australia; eDepartment of
Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University,
VIC, Australia, Melbourne, Australia; fThe Florey Institute of Neuroscience and
Mental Health, Melbourne, Australia; gThe Florey Institute of Neuroscience and
Mental Health, The University of Melbourne, Parkville, Victoria, Australia., Melbourne,
Australia; hDepartment of Biochemistry and Genetics, La Trobe Institute for
Molecular Science, La Trobe University, Melbourne, Australia
Introduction: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative
disease characterised by the deposition of misfolded proteins in the motor cortex
and motor
neurons. Although a multitude of ALS-associated proteins have been identified, few
have been
associated with extracellular vesicle (EV) trafficking, a form of inter-cellular
communication. Additionally, the role of EVs in ALS is undetermined, specifically
in
relation to pathogenic stress granule formation, a response to cellular stress involving
aggregation of non-coding RNAs and their RNA binding proteins. Therefore, this study
aimed
to determine the proteome of brain derived small extracellular vesicles (BDEVs) isolated
from ALS subjects and identify novel ALS-associated deregulated proteins and their
potential
contributions to pathogenic pathways in ALS.
Methods: BDEVs were isolated from human post-mortem ALS (n = 10) and control
(n = 5) motor cortex brain tissues through an ultracentrifugation protocol (Vella
et al.,
2017). Following thorough characterisation, BDEVs that successfully met the minimum
criteria
required by The International Society for Extracellular Vesicles were classified as
EVs. The
BDEVs subsequently underwent mass spectrometry analysis on the Thermo Scientific Q-Exactive
HF with Ultimate 3000 RSLCnano. Proteins identified to be statistically significant
differentially expressed then underwent validation by western blotting.
Results: A panel of 16 statistically significant differentially packaged
proteins were identified in the ALS BDEVs. This included several up-regulated RNA
binding
proteins and a down-regulated cell adhesion molecule; DHX30, STAU1 and VCAM1, respectively.
Pathway analysis revealed that the BDEVs were enriched in proteins associated with
stress
granule dynamics, exosomal and lysosomal pathways.
Summary/Conclusion: The identification of the RNA binding proteins in the ALS
BDEVs suggests there may be a relationship between ALS-associated stress granules
and ALS
BDEV packaging. The packaging of stress granule associated RNA binding proteins into
ALS
BDEVs may be an attempt by the cells to compensate for lysosomal dysfunction caused
by
stress granule accumulation, a feature of ALS. Thus, these results highlight a potentially
novel role for EVs in the pathogenesis of ALS.
Funding: Australian Government Research Training Program Scholarship
National Health and Medical Research Council Program Grant
Motor Neuron Disease Grant
OS29.4
Separation of microglial EVs from human brain-derived
EVs
Tanina Arab
a, Yiyao
Huangb, Vasiliki Machairakic, Juan Troncosoc, Olga
Pletnikovád and Kenneth Witwere
aDepartment of Molecular and Comparative Pathobiology, Johns
Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, USA, Baltimore,
USA;
bDepartment of Molecular and Comparative Pathobiology, Johns Hopkins University
School of Medicine, Baltimore, USA; cDepartment of Neurology, Johns Hopkins
University School of Medicine, Baltimore, USA; dDepartment of Pathology, Johns
Hopkins University School of Medicine, Baltimore, USA; eDepartment of Molecular
and Comparative Pathobiology and Department of Neurology, The Johns Hopkins University
School of Medicine, baltimore, USA
Introduction: Microglia cells are the resident macrophages of the central
nervous system (CNS) and maintain tissue homoeostasis under physiological conditions.
During
chronic inflammation and neurodegenerative diseases, this balance is lost. Studying
microglia in the CNS is difficult in part because microglial markers like Iba1, CD11b,
CD68
and CD45 are also found on macrophages. Recently, TMEM119 was reported be exclusively
expressed by microglia cells in both mouse and human CNS. We have sought to determine
whether brain-derived extracellular vesicles (bdEVs) display Tmem119 and betray the
state of
microglia of origin.
Methods: bdEVs were separated from human cortical samples and characterized
per MISEV2018 guidelines by Western blot, NanoView, transmission electron microscopy
and
nanoflow (NanoFCM). Tmem119 antibodies were tested by immunocytochemistry (ICC) of
microglia
SV40 cells and differentiated U937 macrophage-like cells. LPS treatment was used to
mimic
inflammatory conditions. A TMEM119 antibody was then chosen for microglial EVs capture
from
total bdEVs. Efficiency was cross-validated by Western blot.
Results: bdEVs from human cortex had typical EV morphology and size
distribution and expressed abundant CD9 and CD81 but lower CD63. Cellular marker GM130
was
depleted in EVs vs tissue. Particle counts number range from 10e7-10e8 particles/100 mg
tissue ICC revealed that TMEM119 was upregulated during inflammation by microglial
cells but
not by macrophage-like cells. A TMEM119-EV+ population was successfully separated
by
immunoaffinity.
Summary/Conclusion: An efficient and reproducible protocol was used for bdEV
isolation, and TMEM119 could be used to separate EVs of presumed microglial origin.
Further
optimization is underway, with the goal of using TMEM119 to identify microglial EVs
and
their informative cargo in the periphery.
Funding: Michael J. Fox foundation 00900821
Poster Presentations
PT01:
EVs in the Central and Peripheral Nervous Systems
Chair: Seena Ajit, PhD – Drexel University College of Medicine
PT01.01
miR-451a highly expressed in extracellular vesicles secreted
from Human foetal mesencephalic neural progenitor cells is one of key therapeutic
factors
for Parkinsonism multiple system atrophy
Chulwoo Lima, Jae Hyun Parka, Yuri
Choia, Ji-Min Parka and Jisook
Moonb
aCollege of Life Science, Department of Biotechnology, CHA
University, Seongnam-si, Republic of Korea; bCollege of Life Science, Department
of Biotechnology, CHA University, Seongnam, Republic of Korea
Introduction: We have successfully developed human foetal mesencephalic neural
progenitor cells (hmNPCs) for long-term cultivation . The whole cultivation process
of
tissue preparation, cultivation, and cryopreservation has been established using strict
serum-free conditions under a good manufacturing practice. Long-term-cultivated hmNPCs
retained stemness and hmNPCs have excellent differentiation efficiency into dopaminergic
neurons. hmNPCs reversed impaired motor function in a rodent model of parkinson’s
disease
(PD). Based on the promising results in animal experiments, the clinical trial is
under way
(NCT01860794).
Multiple-system atrophy (MSA) is one of fatal neurodegenerative diseases with a combination
of progressive autonomic nervous system disorders, Parkinson’s syndrome, and cerebellar
pyramid syndrome. There are three types of MSA such as MSA-A, MSA-C, and MSA-P. In
case of a
MSA-P type, it is difficult to diagnose due to the similarity of symptoms with Parkinson’s
disease (PD).
Methods: In Vitro and In Vivo animal MSA Model were established and Rotational
behavioural was performed.
NPC cells were isolated and cultured based on Moon et al.
miRNA sequencing (BGI) was performed and several bioinformatics analyses were done.
Results: Based on the finding that hmNPCs exhibited therapeutic effects on PD,
we hypothesize that hmNPCs will have a therapeutic effect on MSA-P, where sympotoms
are
largely common with PD. As expected, transplanted hmNPCs survived, integrated, and
differentiated in to dopamine neurons in the host brain, consequently leading to the
functional recovery in the MSA-P model. To further investigate the therapeutic key
factors
of hmNPCs in MSA-P, miRNA sequencing of the extracellular vesicles (EVs) secreted
from
hmNPCs was performed.
We found that miR-451a highly expressed in the NPC-derived EVs is one of key regulators
of
inflammatory response via NFkB pathway. We further experimentally demonstrated that
miR-451a
had anti-inflammatory effect on cells of MSA-P condition such that the level of CX3CL1
expression and its receptor, CX3CR1 were both decreased in the MSA-P modelled cells
and in
severe inflammatory environment in MSA brain.
Summary/Conclusion: Our study first showed that miR-451a in hmNPCs-EVs is one
of key therapeutic factors for the recovery of brain damage through immuno-modulation
in
MSA-P.
Funding: The Bio & Medical Technology Development Program of the NRF
funded by the Korean government, MSIP (NRF-2019M3A9H1103765) and the Ministry of Trade,
Industry, and Energy (MOTIE), Korea, under the “Regional Innovation Cluster Development
Program (OpenLab, P0004793)”
PT01.02
Patterns of apolipoproteins J, D and oxidative markers in
circulating extracellular vesicles from MCI and Alzheimer’s patients
Morgane Perrottea, Mohamed Raâfet Ben Khedhera,
Mohamed Haddada, Tamas Fulopb and Charles
Ramassamya
aINRS-Institut Armand-Frappier, Laval, Canada;
bUniversité de Sherbrooke, Sherbrooke, Canada
Introduction: Oxidative insults are known to be involved in the
pathophysiology of Alzheimer’s disease (AD). We have previously demonstrated that
some
blood-based redox-signature were associated to the cognitive scores in mild cognitive
impairment patients and in AD (Perrotte et al., 2019). The aim of this study was (1)
to
evidence the presence of some oxidative markers in circulating extracellular vesicles
(EVs),
and (2) to compare to their plasma levels.
Methods: Plasma samples from healthy, MCI and AD patients were from the Memory
Clinic of Sherbrooke (Québec, Canada). AD patients were stratified in three groups
(moderate, mild and severe) according to the MMSE and MoCA scores. Total plasma
extracellular vesicles (pEVs) were isolated from plasma with the Total Exosome Isolation
reagent (Invitrogen™ by Life Technologies Inc.). pEVs were then characterized by electronic
microscopy, NTA, DLS and Western Blot. Antioxidants apolipoprotein J, D (apo J, ApoD),
the
glyoxalase-1 and protein carbonyls were determined by Western blot.
Results: In pEVs, we found that apo D levels were higher in MCI patients but
not in AD patients. Protein carbonyls levels were higher later, in pEVs from moderate
and
severe AD while apo J levels were not different in pEVs from the five groups of
patients.
In plasma, the pattern of apo J and apo D was different. The levels of apo D was not
different in the five groups of patients while apo J levels were elevated in MCI and
in all
AD groups. Protein carbonyls were higher earlier from mild AD group, earlier than
in pEVs.
The levels of the detoxifying enzyme glyoxalase-1 were higher in pEVs than in plasma
and
were significantly decreased in early AD as compared to control subjects and MCI
Summary/Conclusion: These results demonstrate a differential regulation of
redox homoeostasis in plasma and in pEVs from AD patients.
Funding: Acknowledgements: This work was supported by the Chaire Louise &
André on Alzheimer’s disease, Foundation Armand-Frappier (CR) and CIHR grant (TF).
PT01.03
Extracellular vesicle biomarkers of complement activation and
synaptic loss in Multiple Sclerosis
Carlos J. Nogueras-Ortiza
, Pavan
Bhargavab, Sol Kimb, Francheska Delgado-Perazaa, Peter
Calabresib and Dimitrios Kapogiannisa
aLaboratory of Clinical Investigation, National Institutes of
Ageing, Baltimore, USA; bDepartment of Neurology, Johns Hopkins University School
of Medicine, Baltimore, USA
Introduction: Multiple Sclerosis (MS) is a neurological disorder characterized
by white matter demyelination and extensive synaptic pathology. Recent studies have
shown
synaptic loss in the grey matter of MS brains in the absence of demyelinating lesions
which
could account for disease progression independent of demyelinating episodes. Opsonization
of
synapses with complement components is a mechanism by which phagocytic cells normally
prune
synapses, but, when occurring in excess, it may underlie pathologic synapse loss.
We sought
to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss
in MS
using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles
(NEVs and
AEVs).
Methods: NEVs and AEVs were immunocaptured in parallel from the plasma of
60 MS patients (45 with Relapsing Remitting, 15 with Progressive MS) and 31 healthy
controls, targeting the neuronal-specific marker L1CAM and the astrocyte-specific
marker
GLAST, respectively. We measured the protein levels of pre- and post-synaptic proteins
synaptopodin and synaptophysin in NEVs using ELISAs and multiple complement cascade
components (C1q, C3, C3b/iC3b, C4, C5, C5a, C9, Factor B, Factor H) in AEVs using
a Luminex
array.
Results: Synaptopodin and synaptophysin protein levels in NEVs of MS patients
compared to controls were markedly reduced (2.5-fold; p < 0.0001 for both), whereas
multiple complement components in MS AEVs were markedly increased (C1q: 2.5-fold change;
C3:
1.25-fold change; C3b/iC3b: twofold change; C5: 1.4-fold change; C5a: 1.4-fold change;
Factor: 1.5-fold change; p < 0.0001); differences were not observed in total circulating
EVs or neat plasma. Strikingly, we found the NEV-associated synaptopodin/synaptophysin
and
the AEV-associated complement levels to be negatively correlated in people with MS
(synaptopodin vs: C1q, r = −0.7 and p < 0.0001; C5, r = −0.6 and p < 0.0001; Factor
H,
r = −0.46 and p < 0.0002/synaptophysin vs: C1q, r = −0.75 and p < 0.0001; C5,
r = −0.65 and p < 0.0001; Factor H, r = −0.52 and p < 0.0002), but not in
controls.
Summary/Conclusion: Circulating EVs provide markers of synaptic loss and
complement activation in MS and suggest a link between astrocytic complement production
and
synaptic decline.
Funding: This research was supported in part by the Intramural Research
Program of the National Institute on Ageing, National Institutes of Health.
PT01.04
Methylglyoxal and glyoxal affect the protein cargoes in
neuronal-derived extracellular vesicles
Mohamed Haddada, Morgane Perrottea, Mohamed
Raâfet Ben Khedhera, Tamas Fulopb and Charles
Ramassamya
aINRS-Institut Armand-Frappier, Laval, Canada;
bUniversité de sherbrooke, Sherbrooke, Canada
Introduction: Advanced glycation end-products (AGEs) and their receptor RAGEs
are known to be involved in the pathogenesis of Alzheimer’s disease (AD). Methylglyoxal
(MG)
or glyoxal (GO) are the precursors of AGEs and particularly N-(1-carboxymethyl)-L-lysine
(CML), the most abundant AGEs. MG induced tau hyperphosphorylation and causes hippocampal
damage and memory impairment in mice.
The aim of our study was to analyse the effects of MG and GO on the neuroprotective,
neurotrophic factors, inflammatory and neurodegenerative markers in the human cell
line
SK-N-SH and their release into the neuronal derived-EVs.
Methods: Briefly, SK-N-SH cells were incubated in FBS free media with MG and
GO (0.5 mM) for 24 hours. Neuronal derived-EVs (nEVs) from culture media were isolated
as
previously described (Haddad et al. 2019). nEVs were characterized by electronic microscopy,
NTA and by Western Blot.
Cellular and nEVs concentrations of BDNF, PRGN, NSE, APP, MMP9, ANGPTL-4, LCN2, PTX2,
S100B, RAGE, DJ-1 and alpha synuclein were determined by a Luminex assay from R&D
Systems, Inc. Aβ1-40, Aβ1-42, pTau T181 and total tau levels were measured also with
luminex
assay from EMD Millipore Corp.
Results: We found that both AGEs precursors, at non toxic concentration,
reduced the neuronal levels of NSE with no effect on BDNF, PTRX-2, LCN-2, DJ-1, on
neurodegenerative markers and on CML. GO decreased the levels of PRGN, APP, ANGPL-4
while
the expressions of MMP-9 and ANGPL-4 were, respectively lower and higher in the presence
of
MG.
MG and GO greatly reduced the release of LCN-2 by neuronal cells in nEVs. BDNF and
PRGN in
nEVs were reduced in the presence of GO. Both MG and GO did not modify the release
of NSE,
APP, MMP9, AGNTL-4, PTX-2, DJ-1, Aβ, pTau and CML in nEVs.
Summary/Conclusion: Our data demonstrated that MG and GO differently affect
the content of some protein cargoes in nEVs and suggest that targeting MG and GO may
be a
promising therapeutic strategy to prevent neurodegeneration.
Funding: Acknowledgements: This work was supported by the Chaire Louise &
André on Alzheimer’s disease, Fondation Armand-Frappier (CR) and CIHR grant (TF).:
PT01.05
Cigarette smoke extract alters extracellular vesicle release and
circular RNA expression
Ashley E. Russella
, Emily R.
Mallickb, Bonita H. Powellc, Zhaohao Liaob and Kenneth W.
Witwerc
aJohns Hopkins University School of Medicine, Laurel, USA;
bDepartment of Molecular and Comparative Pathobiology, Johns Hopkins University
School of Medicine, Baltimore, USA; cJohns Hopkins University School of Medicine,
Baltimore, USA
Introduction: Peripherally circulating brain-derived extracellular vesicles
(EVs) and their encapsulated RNAs may serve as biomarkers for HIV-associated neurocognitive
disorders (HAND). However, rates of cigarette smoking are significantly higher in
HIV+
individuals than the general population, and smoking can modulate the expression of
these
markers. To better understand how cigarette smoke might modulate RNA expression and
EV
release, we examined several CNS-derived cell lines, representing astrocytes (U87 MG),
microglia (SV40), and oligodendrocytes (HOG).
Methods: Cigarette smoke extract (CSE) was prepared by bubbling through
culture medium using a standardized and published method. All cell types were exposed
to
either 0% or 50% CSE for 24 hours. Cell viability was assessed by MuseTM Cell Analyser,
and
EVs were isolated from culture conditioned media (CCM) by size exclusion chromatography.
The
void (fractions 1–6), EV (7–10), and protein (11–14) enriched fractions were pooled
and
concentrated. EVs were characterized by transmission electron microscopy (TEM), microfluidic
resistive pulse sensing, and Western blotting. Total RNA was isolated from cells and
circular RNA (circRNA) expression was assessed with a circRNA Microarray.
Results: In response to CSE exposure, cell viability was only slightly reduced
for all cell types. TEM images validate the presence of vesicles in the EV fractions,
and
their absence in the void and protein fractions. Spectradyne particle counts indicated
CSE
exposure substantially increased the CCM particle count in the EV fraction when compared
with control. The presence of expected EV markers (CD63, CD81, and TSG101) in the
EV
fractions, and their absence in the void and protein fractions was observed via Western
blot. Intracellular circRNA expression was significantly altered in all three cell
lines.
Summary/Conclusion: CNS cells display physiologic responses to CSE that
include vesiculation pathways and significant alterations in circRNA expression. We
are now
studying the effects of CSE exposure on circRNA expression in released EVs.
Funding: This work is supported by DA040385, DA047807, and AI144997.
PT01.06
A method for exosomal RNA extraction from paired human brain and
blood specimens
Emily N. Moyaa
, Lillian
Wilkinsa, Esther Chenga, Lisa Linaresb, Brian
Kopellb, Navneet Dograc, Bojan Losica and Alexander
Charneya
aIcahn School of Medicine at Mount Sinai, New York, USA;
bMount Sinai Hospital, New York, USA; cDepartment of Genetics and
Genomic Sciences, Department of Pathology, Icahn School of Medicine, Mount Sinai,
New York,
USA
Introduction: Diagnosis and treatment of neuropsychiatric disorders has made
little progress in the last half-century likely in large part due to the absence of
a
scalable technique to profile the complex biological activity of the brain in a living
person. Exosomes are nanovesicles 30–150 nm in size that mediate intercellular communication
and contain proteins, lipids, and nucleic acids. It has been shown that brain derived
exosomes can be found in peripheral blood, but determining whether peripheral exosomes
truly
reflect ongoing brain processes has to date not been possible due to the absence of
paired
living brain and blood specimens. Here, we present a novel method for paired sampling
of the
dorsolateral prefrontal cortex (DLPFC) and peripheral blood from living human subjects
for
exosomal RNA profiling.
Methods: Informed consent, approved by the IRB at the Icahn School of Medicine
at Mount Sinai, was obtained for patients undergoing Deep Brain Stimulation (DBS).
Paired
brain and blood specimens were collected from 8 patients at two deep brain stimulation
(DBS)
electrode implantation procedures: left hemisphere followed by right hemisphere (total
of 30
samples). We developed protocols to profile RNA from exosomes of brain tissue extracellular
matrix (ECM) and peripheral blood. Exosomes were isolated via our in-house protocol
using
ultracentrifugation. RNA was then extracted from the exosomes using the Qiagen miRNeasy
Mini
Kit protocol. Quality control (QC) was performed to determine whether RNA obtained
was
sufficient for next-generation sequencing.
Results: We demonstrate the safety of a novel procedure to sample the brain in
living human subjects. Bioanalyzer traces and QC data show a mean total RNA of 14.83
ng
(range 1.72–137.17 ng) and no samples fell below the threshold required for library
preparation and sequencing (10 pg) determined by in-house optimization on the SMART-seq
v4
Ultra low input kit.
Summary/Conclusion: To our knowledge, we have performed the first study to
sample pairs of DLPFC and blood from living human subjects for exosomal RNA for subsequent
next-generation sequencing. Ongoing analyses by our group promise to establish peripheral
exosomal RNA transcripts reflective of brain activity. This non-invasive approach
to probing
neurobiology in the living human brain may facilitate the development of exosome-based
diagnostics for neuropsychiatric disorders.
Funding: The Friedman Brain Institute Pilot Grant
PT01.07
MicroRNAs from adipocyte-derived small extracellular vesicles
are associated with neurodegeneration
Rachael Batabyala
, Madeleine
Goldberga, Brennan Harmona, Clemma Jacobsen Mullerb, C.
Dirk Keenec, Elaine Peskindc, Ge Lic, Kristoffer
Rhoadsd, Thomas Montinee, Evan Nadlera, Dedra
Buchwaldf and Robert Freishtata
aChildren’s National Hospital, Washington, USA;
bWashington State University College of Medicine, Seattle, USA;
cUniversity of Washington, Seattle, USA; dUW Medicine Harborview,
Seattle, USA; eStanford University, Stanford, USA; fElson S Floyd
College of Medicine Washington State University, Seattle, USA
Introduction: The relationship between obesity and dementia is complex. While
obesity in middle age triples the risk of dementia 30 years later, many patients with
Alzheimer’s Disease (AD) are cachectic, and a decline in adiposity portends progression
of
dementia. This suggests adipose-derived factors are important to nervous system
homoeostasis. We previously showed that adipocyte-derived small extracellular vesicles
(ad-sEVs) induce pathologies critical to developing obesity-related diseases and may
provide
a mechanistic link between adiposity and dementia. We hypothesized that altered expression
of ad-sEV microRNAs involved in neurodegenerative pathways is associated with more
severe
cognitive impairment
Methods: We studied serum and cerebrospinal fluid (CSF) from 19 participants
with AD and 14 non-AD controls. Ad-sEVs were isolated from samples by precipitation
and
immunoselection. Ad-sEV microRNA expression was profiled in both biofluids and compared.
Results: Serum and CSF microRNA expression correlated strongly (r2 = 0.98). In
serum, 189 microRNAs were differentially expressed by a Fold Change ≥|1.1| in the
AD and
control groups (p ≤ 0.1) and 251 microRNAs were differentially expressed in CSF. Using
Ingenuity Pathways Analysis, we identified mRNAs expressed in nervous system tissue
that are
targeted by the differentially expressed microRNAs. The mRNAs map to 145 diseases
and
functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental
pathways are highly represented. Of the 189 differentially expressed microRNAs in
serum, 6
were moderately correlated with participants’ score on the Mini-Mental State Exam,
a test of
cognitive function (rs = |0.488–0.609|). As validation, RenCell Cx cortical derived
neuronal
stem cells had decreased doubling time when exposed to ad-sEVs from obese adipose
tissue in
vitro.
Summary/Conclusion: These findings support our hypothesis that altered
expression of circulating ad-sEV microRNAs are involved in neurodegenerative pathways
associated with cognitive impairment. These findings support using serum ad-sEVs as
a
surrogate for CSF ad-sEVs. Functional validation is underway to define the connection
between ad-SEVs and AD. Understanding the link between obesity and AD is crucial as
the
population ages and the global obesity epidemic grows.
Funding: Supported by UW ADRC (NIH:P50AG005136)
PT01.08
Expression of extracellular vesicles after acute traumatic brain
injury: an exploratory flow-cytometry study
Monisha A. Kumara
, Maggie
Schmierera, Erika Silvermana, Nimay Kulkarnib, Stefanie
Soergaard-Ballestara, Eva Silvestroa, Richard
Schretzenmairb, Ramon Diaz-Arrastiaa, Wade Rogersb and
Jonni Moorea
aUniversity of Pennsylvania, Philadelphia, USA;
bUniversity of Pennsylvania, Philadelphia, USA
Introduction: Coagulation derangements related to disseminated intravascular
coagulation (DIC) are common after TBI and contribute to secondary neural injury.
Extracellular vesicles (EVs) are released from all cell types, including platelets,
endothelium, and lymphocytes, which are responsible for DIC. We hypothesized that
specialized flow cytometry techniques could identify a unique EV signature of DIC
in acute
TBI.
Methods: Using a modified flow cytometry instrument for detection of small
particles, fluorescence panels were created to assess for EVs from endothelial cells
(CD144,
CD105), platelets (CD31, CD62p, CD41a, CD42b), and erythrocytes (CD235) as well as
brain
biomarkers (S100b, UCHL-1, GFAP, tau and NSE) and T-lymphocytes (CD3, CD4, CD8, CD31).
Samples were prepared in Trucount tubes to determine volume and treated with triton
to
confirm presence of EVs.
Results: 13/17 study patients and 16/20 controls were male. 76% of study
patients presented with a Glasgow Coma Scale of 15. In the hypercoagulability panel,
of the
10 subsets with statistically significant differential expression, 4 involved S100b+
and
were elevated in patients. Platelet-derived CD41a EVs and UCH-L1 EVs were significantly
elevated in controls in 7 EV subsets identified in the brain-specific panel. Finally,
CD3+/31+ EVs, derived from T-cells and identified in the endothelial/T cell panel,
are
significantly lower in patients suggesting CNS recruitment.
Summary/Conclusion: Endothelial and platelet/erythrocyte EVs may be elevated
early after TBI. S100B-carrying EVs are significantly elevated in circulation of TBI
patients; if reproducible, this signature profile may be informative for diagnosis
and risk
stratification. Further study is warranted to evaluate whether this expression correlates
with secondary microvascular brain injury.
Funding: Intramural Award from the University of Pennsylvania
PT01.09
Enrichment of miR-451a in CNS extracellular vesicles following
impairment of the blood brain barrier
Nasser Nassiri Koopaeia
,
Ekram-Ahmed Chowdhuryb, Lais da Silvaa, Jinmai Jianga,
Behnam Nooranib, Ulrich Bickelb and Thomas D.
Schmittgena
aUniversity of Florida, Gainesville, USA; bTexas
Tech University, Amarilo, USA
Introduction: Extracellular RNAs (exRNAs) are present in essentially all
biofluids and include all types of RNA including miRNA. To enhance their stability
outside
of the cell, exRNAs are bound within ribonucleoprotein complexes or packaged into
extracellular vesicles (EVs). The blood brain barrier (BBB) is a dynamic interface
between
the systemic circulation and the CNS and is responsible for maintaining a stable
extracellular environment for CNS cells. The intent of this study was to determine
if EVs
and their contents are transferred from the peripheral circulation to the CNS under
conditions of an impaired BBB.
Methods: The BBB of mice was disrupted by hyperosmolar mannitol injections. To
validate that the BBB has been disrupted with mannitol, intravenously-dosed [13 C]-sucrose
was increased in the forebrain by 14-fold with mannitol compared to sham treated mice.
EVs
were isolated from the forebrain, hindbrain and spinal cord following gentle tissue
lysis
and differential ultracentrifugation. EVs were validated by NTA, TEM and western blotting.
miR-451a, a miRNA that is highly abundant in erythrocytes, was measured in the EVs
by
qPCR.
Results: qPCR showed that miR-451a in CNS tissue EVs increased with mannitol
treatment in the forebrain, hindbrain and spinal cord by 15-, 1.6- and twofold respectively.
qPCR analysis of mRNA from reported miR-451a target genes showed reduced target gene
expression with mannitol.
Summary/Conclusion: We demonstrate that EVs containing miR-451a, a highly
abundant miRNA present within erythrocytes and erythrocyte EVs, is enhanced in the
CNS upon
BBB disruption.
PT01.10
Astrocyte-derived extracellular vesicles in morphine
tolerance
Guoku Hu, Rong Ma, Naseer Kutchy, Yuetong
Zhao, Susmita Sil and Shilpa Buch
University of Nebraska Medical Center, Omaha, USA
Introduction: Opiates, such as morphine are used extensively in the clinical
setting owing to their beneficial effects. Paradoxically, however, the prolonged use
of
morphine often results in the development of tolerance, drug addiction, and ultimately
leading to various comorbidities associated with drug abuse. Although great efforts
have
been made, at present there is no treatment. The sonic hedgehog (SHH) plays a key
role in
brain development, and brain cells fine-tuning processes such as their proliferation,
patterning, and fate specification Recent findings have demonstrated that inhibition
of the
SHH signalling prevents morphine tolerance in rodent models. We thus hypothesize that
extracellular vesicles (EVs) derived from morphine exposed astrocytes and their cargo
such
as SHH are critical for the development of morphine tolerance.
Methods: Mice were received either saline or chronic morphine injection with
escalating doses of morphine for 5 days (subcutaneously; 10 mg/kg, day 1, 20 mg/kg
days 2–3,
and 40 mg/kg days 4–5). The development of tolerance was assessed by measuring the
tail-flick latency using Tail Flick Analgesia Metre (LE7106, Harvard Apparatus). EVs
were
isolated using either differential ultracentrifugation from astrocyte conditioned
media or
gradient ultracentrifugation from brain tissues. Western blotting and qPCR were performed
to
determine the expression/activation of SHH signalling pathway components.
Results: Our data showed that the levels of SHH protein were upregulated in
morphine exposed astrocyte-derived extracellular vesicles (morphine-ADEVs). Furthermore,
SHH
containing morphine-ADEVs activated SHH signalling in astrocytes. Our in vivo study
further
demonstrated the upregulation of SHH, as well as the activation of SHH signalling,
in
astrocytes of morphine-administered mice.
Summary/Conclusion: These findings thus demonstrated an autocrine mechanism
for SHH pathway activation in astrocytes associated with morphine tolerance. These
findings
could pave the way for the development of SHH signalling pathway targeted strategies
in the
prevention and treatment for substance use disorders.
PT01.11
Biophotonics-based platforms for the evaluation of circulating
extracellular vesicles as biomarkers of neurodegeneration in Alzheimer’s
disease
Silvia Picciolini
a, Cristiano
Carlomagnoa, Alice Gualerzia, Monia Cabinioa, Francesca
Baglioa and Marzi Bedonib
aIRCCS Fondazione Don Carlo Gnocchi, Milan, Italy;
bIRCCS Fondazione Don Carlo Gnocchi, Milano, Italy
Introduction: In the search for novel and non-invasive biomarkers of
Alzheimer’s disease (AD), both circulating brain-derived Extracellular Vesicles (EVs)
and
whole serum represent a valuable integration of the currently used classification
system. To
face the technological challenge of EVs and serum analysis, we propose the use of
biophotonics techniques as reliable, sensitive, fast and label free methods, potentially
useful in tailoring pharmacological and rehabilitation treatments.
Methods: Circulating EVs, isolated by SEC, and serum samples were collected
from 10 healthy subjects (HC) and 10 AD patients. All subjects were asked to complete
Montreal Cognitive Assessment scale and MRI examination. Surface Plasmon Resonance
(SPR) was
performed in order to detect EVs coming from neurons, astrocytes, oligodendrocytes
and
microglia and to characterize each of them for the amount of ganglioside M1 (GM1),
Aβ and
TSPO expressed on their surface. Serum analysis was performed using a Raman microscope
through the Surface Enhanced Raman Spectroscopy (SERS) effect by mixing serum with
Ag
nanoparticles. The Pearson’s correlation index was used to assess the linear correlation
between SPRi data and clinical, MRI data and data obtained from multivariate analysis
(MVA)
of SERS spectra.
Results: The SPR analysis of EVs showed that the selected bioactive molecules
are differently loaded on neural EV populations and that their amount is increased
on total
EVs in AD patients compared to HC. We observed a significant correlation between MVA
data
from SERS and the presence of Aβ on neuronal and microglial EVs and of TSPO on neural
EVs,
measured with the SPR array.
Summary/Conclusion: Thanks to our methodological innovation we have verified
the potentiality of EVs as AD biomarkers, correlating biophotonics blood-based analysis
with
clinical data. This platform could provide a powerful tool for the evaluation of AD
neurodegeneration.
Funding: The study was supported by the Italian Ministry of Health (Ricerca
Corrente 2017–2018 to IRCCS Fondazione Don Carlo Gnocchi).
PT01.12
Raman profiling of extracellular vesicles as new blood-based
biomarker for brain disorders: focus on Parkinson’s disease
Alice Gualerzi
a, Silvia
Picciolinia, Cristiano Carlomagnoa, Federica Terenzib,
Silvia Ramatc, Sandro Sorbid and Marzi Bedonie
aIRCCS Fondazione Don Carlo Gnocchi, Milan, Italy;
bDipartimento di Neuroscienze, Psicologia, Area de Farmaco e Salute del
Bambino, Università di Firenze, Florence, Italy; cUniversità degli Studi di
Firenze, Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino,
Firenze, Italy; dIRCCS Don Carlo Gnocchi, Fondazione Don Carlo Gnocchi, Florence,
Italy; eIRCCS Fondazione Don Carlo Gnocchi, Milano, Italy
Introduction: Extracellular vesicles (EVs) play a pivotal role in brain
homoeostasis and intercellular communication in both physiological and pathological
conditions. In Parkinson’s disease (PD), EVs are key players in the transfer of α-synuclein,
with blood EVs reported to undergo proteomic modifications. Nonetheless, the detection
and
characterization of the EV cargo is technologically challenging, limiting the use
of EVs as
biomarkers so far. Herein, we propose Raman spectroscopy for the label-free, bulk
characterization of blood EVs in PD patients.
Methods: EVs were isolated by SEC and ultracentrifugation from the serum of 18
healthy subjects (HC) and 22 PD patients. In all patients, the severity of PD was
evaluated
with the Unified Parkinson’s Disease Rating Scale (UPDRS) part III and with Hoehn
and Yahr
scores (HY). After proper EV characterization following MISEV2018 guidelines, Raman
analysis
was performed. The Raman microspectroscope was used with a 532 nm laser in the spectral
ranges 600–1800 cm-1 and 2600–3200 cm-1. Data from HC and PD patients were compared
by
multivariate statistical analysis (PCA-LDA).
Results: The Raman analysis of EVs highlighted differences in the biochemical
profile of the two groups, with the main variations in the spectral regions related
to
proteins, lipids and saccharides. A preliminary estimate of the accuracy of Raman
profiling
of blood EVs for PD diagnosis was obtained, demonstrating an accuracy of 71%. Even
more
interestingly, we demonstrated the correlation between the Raman spectra and the clinical
scales (UPDRS and HY) used to stratify PD patients.
Summary/Conclusion: In conclusion, the biochemical signature of blood EVs can
be detected by Raman spectroscopy in PD patients and the EV spectral modifications
can be
related to their clinical status. These data suggest the possibility to use the Raman
profile of circulating EVs as a biomarker for brain disorders, complementary to other
specific molecular markers.
Funding: The study was supported by the Italian Ministry of Health (Ricerca
Corrente 2017 to IRCCS Fondazione Don Carlo Gnocchi)
PT01.13
Impact of circulating extracellular vesicles on brain functions
and behaviours
Eisuke Dohi
a, Norimichi
Itoa, Ken Matobaa, Indigo Roseb, Takashi
Imaib, Rei Mitanib, Eric Choib, Kenneth Witwerc
and Shin-ichi Kanod
aDepartment of Psychiatry and Behavioural Neurobiology, The
University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, USA,
Birmingham, USA; bDepartment of Psychiatry and Behavioural Sciences, The Johns
Hopkins University School of Medicine, Baltimore, Maryland, USA, Baltimore, USA;
cDepartment of Molecular and Comparative Pathobiology and Department of
Neurology, The Johns Hopkins University School of Medicine, baltimore, USA;
dDepartment of Psychiatry and Behavioural Neurobiology, The University of Alabama
at Birmingham School of Medicine, Birmingham, USA
Introduction: Peripheral immune alterations have been described in psychiatric
disorders such as schizophrenia, depression, and autistic spectrum disorders. In addition,
behavioural changes have been observed in various immunodeficient animal models. However,
the mechanisms by which peripheral immune system influences brain development and
function
are not well understood. In this study, we explored the mechanisms by which circulating
extracellular vesicles (EVs) mediate immune-brain communication and influence mouse
behaviours.
Methods: Mice deficient for Rag1 or Rag2 gene (Rag KO mice) were used as a
model to study the effects of loss of adaptive immune cells (T and B cells) on brain
cellular phenotypes and behaviours. Circulating EVs were collected from their sera
and
analysed by using electron microscopy, nanoparticle tracking assay, and Western blotting.
Brain cellular phenotypes were assessed by immunofluorescent staining and gene expression
analysis. Behavioural phenotypes of Rag KO and WT mice were examined in social interaction
test. In vivo transfer of EVs was performed to see its effects on behavioural alterations
of
Rag KO mice.
Results: Rag KO mice displayed social behavioural deficits, accompanying by
enhance c-Fos immunoreactivity and altered microglia morphology in the medial prefrontal
cortex (mPFC). Circulating EVs were also affected in these mice and lacked the expression
of
markers for T cells. A set of microRNAs (miRNAs) in circulating EVs were diminished
in Rag
KO mice. In vivo transfer of circulating EVs rescues the social behavioural deficits
of Rag
KO mice and ameliorate the c-Fos immunoreactivities in mPFC of Rag KO mice.
Summary/Conclusion: Our data showed that circulating EV profiles were altered
in mice lacking adaptive immune cells and, accordingly, showing social behavioural
deficits.
Notably, our in vivo experiments suggest that circulating EVs may contribute to social
behaviours. Further study will provide a novel biological insight into the mechanisms
underlying peripheral-to-brain immune communication via EVs.
Funding: RO1 MH113645, R21 MH118492
PT01.14
MicroRNA profile of circulating extracellular vesicles are
associated with upregulation of neuroinflammatiory signalling pathway in aged
animals
Catherina I. Moszkowicz
a, Laura
Cechinela, Rachael Batabyalb, Robert Freishtatb and
Ionara Rodrigues Siqueirac
aUniversidade Federal do Rio Grande do Sul, Porto Alegre,
Brazil; bChildren’s National Hospital, Washington, USA; cUniversidade
Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
Introduction: The involvement of neuroinflammation on ageing process is widely
recognized. Extracellular vesicles (EVs), such as exosomes, are able to cross the
blood-brain barrier and were related to neuroinflammation. In this context, EVs have
been
considered a potential mechanism of spreading molecules, including microRNAs (miRNAs)
that
can promote mRNA degradation or inhibit translation of their targets. Our aim was
to
investigate the miRNA profile of circulating total EVs during ageing process and their
impact on canonical pathways.
Methods: The Local Ethics Committee (Comissão de Ética no Uso de Animais –
UFRGS; n 29818) approved all animal procedures and experimental conditions. Plasma
was
obtained from Wistar rats (3 and 21 months-old) and total EVs were isolated. EV microRNA
isolation and microarray expression analysis was performed to determine the predicted
regulation of targeted mRNAs.
Results: The analysis of global microRNA expression revealed 48 differentially
expressed microRNAs (p < 0.05; fold change of ≥ |1.1|); 18 miRNAs were up-regulated
and
30 were down-regulated in circulating total EVs from aged animals compared to young-adult
ones. A conservative filter was applied on Ingenuity Pathway Analysis (IPA) and only
experimentally validated and highly conserved predicted mRNA targets were used. IPA
showed
that neuroinflammation signalling is ranked among the top canonical pathway impacted
by
differentially expressed microRNAs and is upregulated in aged animals (p < 0.0001;
z-score: 3.413). The differentially expressed miRNAs impacted 32 molecules in the
neuroinflammation pathway. Interestingly, the ion channel GRIN2B is predicted to be
up
regulated and is a target of many EVs miRNAs; in accordance with our results GRIN2B
was
previously related to neurodegenerative diseases. Moreover, let-7a-5p is predicted
to be
downregulated and target all the 32 molecules of the neuroinflammation signalling
pathway.
Previous studies have correlated let-7a-5p and neurodegenerative diseases.
Summary/Conclusion: Our data suggest that circulating total EVs cargo,
specifically miRNAs, are altered by ageing and impact neuroinflammation pathway, suggesting
the involvement EVs miRNA on ageing-induced susceptibility of neurodegenerative
diseases.
Funding: CNPq (307980/2018-9) and CAPES (88881.189257/2018-01).
PT02: EVs in Dermatology
Chair: My Mahoney – Thomas Jefferson University
Chair: Fabio Quaglia – Thomas Jefferson University
PT02.01
Keratinocyte-derived exosomal packaging drives conversion of
injury-site macrophage in granulation tissue of murine wound
Xiaoju Zhoua, Amanda Siegela, Brooke
Brownb, Mohamed El Masrya, Amitava Dasa, Sashwati
Roya, Yi Xuana, Maangilal Agarwala, Robert
Leec, David Clemmerb, Chandan Sena and
Subhadip Ghataka
aIndiana University, Indianapolis, USA; bIndiana
University, Bloomington, USA; cOhio State University, Columbus, USA
Introduction: Bidirectional cell-cell communication via paracrine mechanisms
is critical for wound healing. A new paradigm involving exosome-borne distinctive
repertoire
of cargo such as miRNAs has emerged as a predominant mechanism of cellular communication
at
the site of injury. Unlike other shedding vesicles of similar size, exosomes selectively
package miRNA by sumoylation of heterogeneous nuclear ribonucleoprotein (hnRNP).
Methods: Keratinocyte-derived exosomes (Exoκ) were genetically labelled with
fluorescent reporter (GFP) using tissue nanotransfection. Purified, GFP-labelled Exoκ
were
isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation
followed by affinity selection using magnetic beads. Distributions of intact exosome
were
analysed using a prototype Jarrold-geometry charge-detection mass spectrometer to
directly
measure differences in particle mass and charge distributions. Complementary MS and
ion
mobility spectrometry (IMS)-MS experiments have been used to characterize surface
glycans
and glycopeptides. To selectively inhibit miRNA packaging within the Exoκ in vivo,
pH-responsive targeted siRNA functionalized lipid nanocarriers (TLNκ) were designed
using
materials that have prior history of FDA approval for human use.
Results: An increase in mass/charge ratio with glycan binding sites on the
surface of wound-edge Exoκ were observed compared to dorsal skin Exoκ. Wound-edge
Exoκ were
selectively taken up by the macrophages in the granulation tissue (n = 6). Keratinocyte
targeting siRNAhnRNP functionalized lipid nanocarriers (TLNκ) were designed with
encapsulation efficiency of 94.3%.
Application of TLNκ encapsulating siRNA of hnRNP (TLNκ/si-hnRNP) to murine dorsal
wound-edge significantly inhibited the expression of hnRNP by 80% in epidermis compared
to
control (TLNκ/si-control)(n = 10). Moreover, mice treated with TLNκ/si-hnRNP showed
impaired
barrier function, with significant presence of macrophage in granulation tissue at
day 10,
suggesting impaired conversion of macrophage in the granulation tissue.
Summary/Conclusion: This work provides a novel insight wherein exosomes of
keratinocyte lineage are recognized as a major contributor that directs macrophage
conversion in granulation tissue for wound healing.
PT02.02
Multifaced effects of Milk-exosome (Mi-Exo) as modulator of
scar-free wound healing
Gna Ahn, Hyo-won Yoon, Yang-Hoon Kim and
Ji-Young Ahn
Chungbuk National University, Cheong-ju, Republic of Korea
Introduction: Recently, milk exosome (Mi-Exo) has been focused particularly on
the possibility of oral distribution for therapeutic agents. However, studies related
to the
cosmeceutical effects associated with Mi-Exo are fairly limited. The purpose of this
study
is to suggest the anti-oxidant and anti-inflammatory effect of Mi-Exo and possibility
that
can be induced by scar free healing by micro RNA in Mi-Exo.
Methods: The characteristics of the extracted Mi-Exo were verified by size
measurement, morphological characteristics through Cryo-EM and western blot. For antioxidant
experiments, an ABTS assay was performed. Next, mRNA expression through four major
cytokines
(TNFα, IL-6, COX-2, iNOS) was used to evaluate anti-inflammatory effects. Finally,
cell
migration assay was performed to confirm the effect of scar-free healing and the detection
of miR-30b in Mi-Exo and VEGF mRNA expression confirmed.
Results: Mi-Exo using 1% acetic acid extraction showed the highest yield. The
average size of the exosomes is approximately 110 nm, confirmed the typical double
membrane
vesicle. As a result of antioxidant experiments, it was confirmed that the treatment
of
exosomes of 10^10 particles showed about 65% antioxidant activity. When 10^10 particles
were
treated, RNA expression of cytokines showed about 2 times more inhibitory effect than
control. ELISA test results also confirmed that the concentration-dependent decrease.
The
activation of the raw cell less proceeded as the treated Mi-Exo increased. The cell
scratch
assay cells did not close the cells as the number of milk exosomes increased (wound
closing
% of 10^10 particle = 4.7%). and miR-30b in milk exosomes was detected at Ct value = 22.
Summary/Conclusion: The antioxidant and anti-inflammatory effects of Mi-Exo
showed the greatest efficacy when 10^10 particles were treated. In addition, it induced
to
scar free healing rather than wound healing. Mi-Exo has great potential as a superior
natural material in the future cosmeceutical field.
Funding: This work was also supported by the National Research Foundation of
Korea (NRF) grant funded by the Korea government (MEST) (No. NRF-2019R1A2C1010860).
PT02.03
Extracellular vesicles in human milk expose tissue factor and
promote coagulation
Yong Hua
, Johannes
Thalerb, Ruth Kendlbacherc, Najat Hajjid, Chi
Haue, Annemieke van Damf, René Berckmansd, Lukas
Wisgrillg, Ingrid Pabingerb, Alain R. Brissonh and Rienk
Nieuwlande
aLaboratory of Experimental Clinical Chemistry, Vesicle
Observation Centre, Biomedical Engineering & Physics, Amsterdam UMC, locatie AMC,
University of Amsterdam, Amsterdam, Netherlands; bClinical Division of
Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna,
Vienna, Austria; cMedical University of Vienna, Vienna, Austria;
dLaboratory of Experimental Clinical Chemistry, Vesicle Observation Center,
Amsterdam UMC, Locatie AMC, University of Amsterdam, Amsterdam, Netherlands;
eDepartment of Clinical Chemistry, Amsterdam UMC, University of Amsterdam,
Amsterdam, the Netherlands, Vesicle Observation Center, Amsterdam UMC, University
of
Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands; fBiomedical
Engineering & Physics, Amsterdam UMC, Locatie AMC, University of Amsterdam, Amsterdam,
Netherlands; gClinical Division of Neonatology, Paediatric Intensive Care &
Neuropaediatrics, Department of Paediatrics and Adolescent Medicine, Medical University
of
Vienna, Vienna, Austria; hUniversité de Bordeaux, Bordeaux, France
Introduction: Tissue factor (TF), a transmembrane protein, initiates
coagulation by binding and activating coagulation factor VII (FVII). TF is associated
with
extracellular vesicles (EVs) in saliva and urine, but it is unknown whether also human
milk
(HM) contains EVs exposing coagulant TF.
Methods: HM was collected from six healthy nursing women with informed
consent. EVs were isolated by ultracentrifugation and size exclusion chromatography
(SEC).
The presence of TF antigen exposing EVs was studied by Western blot, flow cytometry,
cryo-electron microscopy (cryo-EM), and surface plasmon resonance imaging (SPRi).
The
ability of TF exposing EVs to trigger coagulation was investigated with a plasma fibrin
generation test (FGT), performed in the absence or presence of antibodies against
TF or
FVII(a).
Results: Addition of HM to plasma shortened the plasma clotting time, even
when HM was highly diluted. After ultracentrifugation of HM, both TF antigen and TF
activity
were detected in the EV-containing pellet. After SEC, TF antigen and TF activity were
present in the EV-containing fractions 8 and 9. The presence of TF-exposing EVs in
these SEC
fractions was confirmed by Western blot (CD9, CD63 and TF), flow cytometry, SPRi,
and FGT.
In addition, the presence of EVs in HM was confirmed by cryo-EM.
Summary/Conclusion: We demonstrate the presence of highly procoagulant
TF-exposing EVs in HM.
Funding: Y.H. was supported by a scholarship from the China Scholarship
Council (CSC).
J. T. was supported by an unrestricted travel grant from the International Society
on
Thrombosis and Haemostasis.
PT02.04
Scalable isolation of EVs from different probiotic strains with
potential as cosmetic ingredients
Laura Soriano-Romaní, Joaquin Espí and Begoña
Ruiz
Ainia, Paterna, Spain
Introduction: Extracellular vesicles (EVs) are increasing their application in
a number of fields. Recently, it has been shown that skin health may be affected not
only by
commensal skin bacteria, but also by the EVs that they secrete. However, because most
of the
efforts have been directed to the characterization and evaluation of EVs, the scaling
up of
the production process remains a bottleneck at the industrial level. In this work,
the goal
was to evaluate the potential applications of EVs produced by different probiotic
strains
commonly used in the cosmetic field, considering the economic and technical viability
of the
process.
Methods: To meet our goal, a standardized workflow was defined to isolate EVs
from probiotic strains such as Lactobacillus and Bifidobacterium species, that have
demonstrated cutaneous immuno-regulatory effects. The different bacterial strains
were
produced under standard culture conditions. To isolate the secreted bacterial EVs,
different
chromatographic techniques were performed starting from clarified growth medium. Then,
EVs
were evaluated in vitro for a number of biological effects related with skin health.
Results: The EV yields obtained after downstream processing were calculated
for each strain and isolation technique by means of nanoparticle tracking analysis
(NTA) and
total protein content. Moreover, EVs were visualized by electron microscopy. The in
vitro
evaluation of isolated EVs was based on changes in the expression of five biomarkers
related
with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell
types to
identify possible cosmetic claims that could be associated to each probiotic source.
Summary/Conclusion: The potential of EVs obtained from probiotic strains as
cosmetic active cell-free ingredients was preliminarily assessed with this work, where
the
process yield and cosmetic function were evaluated. However, additional experiments
will be
needed in order to increase and optimize the productivity of each step of the EV
manufacturing process.
PT02.05
Acerola derived exosome-like nanovesicles enhances the repair of
ultraviolet B-induced DNA damage in cultured skin fibroblasts
Tomohiro Umezu, Masakatsu Takanashi, Yoshiki
Murakami and Masahiko Kuroda
Tokyo Medical University, Shinjyuku, Japan
Introduction: Acerola (Melpighia emarginata DC.) is a fruit is known to
contain not only high amounts of ascorbic acid but also various nutritional components
such
as carotenoids and polyphenols. Previous reports showed the acerola juices are able
to
confer protection against Ultraviolet radiation B (UVB), to improve barrier function
of
skin. UVB is the main cause of DNA damage in epidermal cells, generating several types
of
pro-mutagenic lesions, like cyclobutene prymidine dimers (CPDs) and prymidine (6–4)
prymidinone photoproducts (6–4PPs): if not repaired, this DNA damage leads to skin
cancer.
In this study, we investigated the biological property of the acerola derived exosome-like
nanovesicles (ADENs), aiming to clarify the involvement of ADENs in repair of UV-induced
DNA
damage.
Methods: Normal human dermal fibroblasts (NHDFs) were purchased from Lonza
Inc. The exosome-like nanovesicles were isolated from acerola juices using exoEasy
Maxi kit
(Qiagen). The morphology and size distribution of ADENs were checked by transmission
electron microscopy (TEM) and nanoparticle tracking analysis (NTA, NanoSight LM10,
Malvern).
NHDFs were exposed to UVB (1 mJ/cm2) with pre- or post-ADENs. Effect of UVB was assessed
by
examining cell viability, cell morphology, and DNA damage levels through biochemical
assays,
microscopy and protein expression studies.
Results: Purified ADENs were compatible with NTA or TEM for assessing the
nanovesicle size range and concentration (200–400 nm). When NHDFs were added with
ADENs and
incubated at 37°C for 48 h, there was no effect of ADENs on cell proliferation of
NHDFs. We
found that ADENs treatment to UVB exposed NHDFs significantly reduced CPDs and 6–4PPs
DNA
adduct formation. Present results showed that ADEN treatment prevented UVB induced
DNA
damage in NHDFs.
Summary/Conclusion: We confirm that ADENs have the effect of repairing DNA
damage caused by UVB. These results provide that ADENs can be a new source to protect
human
skin from UV-induced skin cancer.
PT03: Engineering and Loading EVs
Chair: André Görgens – Department of Laboratory Medicine, Clinical Research Centre,
Karolinska Institutet
PT03.01
EV-mediated HOTAIR delivery for increased
angiogenesis
Louis J. Born
a and Steven M.
Jayb
aUniversity of Maryland, College Park, USA;
bUniversity of Maryland, College Park, College Park, USA
Introduction: Introduction: Despite the development of a variety of therapies,
complex wounds resulting from disease, surgical intervention, or trauma remain a major
source of morbidity. Extracellular vesicles (EVs) derived from mesenchymal stem/stromal
cells (MSCs) have been shown to improve wound healing, especially via enhanced wound
angiogenesis. However, despite their clearly established potential, EVs have limitations
that may limit clinical relevancy, such as low potency.
Hypothesis: Increased expression of pro-angiogenic lncRNA HOTAIR within MSC EVs enhances
their pro-angiogenic effects and thus their wound healing properties.
Methods: Methods: HOTAIR was overexpressed in human dermal microvascular
endothelial cells (HDMECs) to determine any molecular or functional pro-angiogenic
effects.
Anti-angiogenic miRNAs and angiogenic mRNA levels were quantified by RT-qPCR. Effects
of
HOTAIR on proliferation of HDMECs was also determined. HOTAIR was then loaded into
MSC EVs
by delivering a CMV-based HOTAIR plasmid to MSCs for endogenous loading via a concentration
gradient. EVs were collected by differential centrifugation. HOTAIR content within
EVs was
confirmed by gel electrophoresis and RT-qPCR. Effects on migration of HDMECs by
HOTAIR-loaded MSC EVs were determined using a scratch assay.
Results: Results: Overexpression of HOTAIR decreased miR-29 c and miR-107,
while increasing VEGF and HIF-1a. HDMEC proliferation was also increased in HDMECs
overexpressing HOTAIR (p < 0.01). HOTAIR was visually confirmed in HOTAIR-loaded MSC
EVs
by gel electrophoresis, but was undetectable in unmodified MSC EVs. RT-qPCR confirmed
a
900-fold increase of HOTAIR compared to control MSC EVs. HDMECs showed a more statistically
significant rate of gap closure when treated with HOTAIR-loaded EVs (p < 0.01) than
compared to control MSC EVs (p < 0.05).
Summary/Conclusion: Summary: Loading lncRNA HOTAIR into MSC EVs is achievable
by a concentration gradient-dependent method and offers potential to enhance the angiogenic
properties of MSC EVs.
PT03.02
Nanomaterial labelling of exosomes for cell biology
Rabab Hamzah
a, Walter
Harringtonb, Alexandru Birisc and Robert Griffinb
aUniversity of Arkansas at Little Rock, LR, USA;
bUniversity of Arkansas for Medical Sciences, Little Rock, USA;
cUniversity of Arkansas at Little Rock, Little Rock, USA
Introduction: Exosomes are vesicles secreted by many, if not all, cell types
and have been known about for decades. Among larger micro vesicles that are produced
directly from the cell membrane, the small (30–150 nm), exosomes are similar in size
to a
virus surrounded by a lipid bilayer. We and others have demonstrated that exosomes
contain
proteins, lipids, RNA, and DNA, making them promising materials for diagnosing and
treating
diseases, including many cancers such as brain cancer. In addition, exosomes from
neurons
and glial cells represent a novel type of intercellular communication. However, their
size
makes them hard to track with traditional fluorescence microscopy. To address this,
we
developed photothermal microscopy (PTM), which uses gold nanomaterial labelling to
track
exosomes’ interaction with and effect on cells/tissue.
Methods: Exosomes secreted by tumour cells and general exosomes found in the
blood were isolated using differential ultracentrifugation or a commercially available
kit
(Invitrogen). Next, the exosomes were characterized by (TEM), (NTA), and western blotting
to
determine shape, size, morphology and the protein profile in the exosomal membrane.
After
characterization, the exosomes were labelled with gold nanoparticles via sonication.
Next,
the samples were washed, and the exosomes were labelled with fluorescence dye to stain
the
membrane. After staining and labelling, the exosomes were added to U87 cells in culture
and
incubated for 3 h. They were then fixed by 4% paraformldehyde and imaged by PTM.
Results: PTM found that exosome-cell interactions are exosome-type dependent,
as U87 cells took up exosomes from other U87 cells but not human serum exosomes. This
suggests that exosome uptake is a selective process and depends on the source of the
donor
cells.
Summary/Conclusion: Exosomes can be labelled with gold nanoparticles via
sonication then successfully tracked by PTM to study the effect of exosome source
on
exosome-cell interactions and communication. Cells incubated with U87 exosomes took
the
vesicles up rapidly, while cells incubated with serum exosomes had little uptake.
PTM will
help us design selective exosome-based strategies to treat different conditions, including
brain cancer and CNS damage.
Funding: NSF EPSCoR RIII Award 1457888.
PT03.03
Loading of goat´s whey extracellular vesicles with spiked
microRNA and curcumin as an strategy for developing new nanocarriers for acellular
therapies
Pedro Pablo Silvaa, Ana Mançanaresb, Jose
Manriqueza, José Leóna, Yat Wonga, Lleretny
Rodriguez-Alvarez
c and Fidel Ovidio Castroc
aDepartment of Animal Science. Faculty of Veterinary
Sciences. Universidad de Concepción, Chillan, Chile; bDepartment of Animal
Science. Faculty of Veterinary Sciences. Universidad de Concepción, Chillán, Chile;
cUniversidad de Concepcion, Chillan, Chile
Introduction: Extracellular vesicles (EV) are involved in cell signalling and
are present in a variety of cell secretions such as milk, from which enormous amount
of EV
can be purified, thus milk is an attractive raw material for scaling up EV production
for
therapeutic, cosmetic or other uses. Here we isolated EVs from the whey fraction of
goat´s
milk and demonstrated that such EVs can be loaded with molecules like polyphenols
and
miRNA.
Methods: To achieve this, milk was collected from lactating goats and
fractionated by acidification and centrifugation into whey and caseins. EVs were purified
from the former fraction by serial centrifugation and precipitation with commercial
kit
(Total Isolation/Thermo Fisher) and characterized by Electron Transmission Microscopy
(TEM),
Western Blot to identify surface markers and measurement of size through Nanotracking
Analysis. Once isolated, EVs were loaded with different concentration of a spiked
synthetic
miR39 or with the polyphenol curcumin. miRNA or curcumin were co-incubated over night
with
EVs at 4oC, precipitated and purified as described above, with an additional washing
and
precipitation for curcumin. Concentration of miRNA uploaded by EVs was quantified
using
miR39 specific qPCR. Curcumin was measured using a spectrophotometer at 420 nm.
Results: EVs isolated from whey had an average size of 120 nm, were positive
for HSP70, CD9 and Alix. In TEM, EVs were identified with their natural conformation
and
corresponding size to exosomes. qPCR showed a significant difference of expression
of miR39
in relation to control (loaded with shame) and the negative control (p < 0.05). Curcumin
presence was also confirmed after washing and precipitacion.
Summary/Conclusion: In conclusion, milk EVs and exosomes can be loaded with
miRNA and a polyphenol and can be used as alternative nanocarrier for acellular
therapies.
Funding: Supported by CONICYT grant 21161052 to PS.
PT03.04
Development of novel tool for purification and characterization
of extracellular vesicles in ageing and disease
Madhusudhan Bobbili
a, Stefan
Vogtb, Markus Schossererb, Wolfgang Holnthonera, Heinz
Redla, André Görgensc, Samir El-Andaloussic and Johannes
Grillarid
aLudwig Boltzmann Institute for Experimental and Clinical
Traumatology, Vienna, Austria; bUniversity of Natural Resources and Life
Sciences, Vienna, Vienna, Austria; cDepartment of Laboratory Medicine, Clinical
Research Centre, Karolinska Institutet, Huddinge, Sweden; dChristian Doppler
Laboratory for Biotechnology of Skin Ageing, Department of Biotechnology, BOKU – University
of Natural Resources and Life Sciences, Vienna, Austria Evercyte GmbH Austrian Cluster
for
Tissue Regeneration Ludwig Boltzmann Institute for Experimental and Clinical Traumatology,
Vienna, Austria, Vienna, Austria
Introduction: Extracellular vesicles (EVs) are cell- derived lipid membrane
nanoparticles that serve as messengers of intercellular communication, transferring
bioactive molecules to recipient cells. EVs have a natural therapeutic potential with
high
flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic
delivery vehicles. Considering the importance of EVs, their isolation methods are
still a
bottleneck. To get insights into the tissue-specific cargo in vivo for complete exploitation
of EVs as therapeutic, biomarker and diagnostic tools, EV purification methods are
critical.
The aim of the study was brought about to develop an efficient EV purification method
both
in vitro and in vivo and to further investigate function of EVs in cellular senescence.
Methods: To isolate tissue- specific EVs in vivo we developed recombinant EVs
by genetically fusing snorkel-tag to the CD81. The snorkel-tag enables on-column protease
treatment for purifying EVs which does not rely on traditional immunoaffinity purification
protocols using low pH or high salts solutions.
Results: We systematically evaluated the purification of EVs harbouring
snorkel-tag by employing different methodologies. Our findings suggest that EVs harbouring
snorkel-tag indeed can be purified at high purity without altering EV biophysical
properties. Furthermore, we expressed CD81-snorkel-tag under p16ink4a promoter and
were able
to purify EVs derived from senescent cells.
Summary/Conclusion: Finally, we are developing an in vivo model with
CD81-snorkel-tag under p16ink4a promoter. This will provide us detail insights into
the EV
cargo secreted from senescent derived cells, by purifying EVs harbouring snorkel-tag
under
pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools.
Summarized, we have here developed novel tool for studying content and function of
EVs in
the context of ageing and disease. This tool will now pave the way for studying the
molecular mechanisms underlying these EV functions in vivo.
Funding: This work was funded by the Austrian Science Fund PhD Program
BioToPeBiomolecular Technolgy of Proteins (W1224).
PT03.05
Engineering exosomes with GATA-4
Jie Xu, Christian Paul, Yi-gang Wang and
Meifeng Xu
University of Cincinnati, Cincinnati, USA
Introduction: Exosomes, are small vesicles (30–150 nm) secreted from cells
that can transport and deliver of their components such as lipids, proteins, DNA,
mRNA, and
miRNA to target cells. GATA-4, a cardiac transcription factor, has been shown to regulate
differentiation, proliferation, and survival of a wide range of cell types. Delivering
GATA-4 protein into ischaemic tissues may be one of the most straightforward approaches
to
improve cardiac function following myocardial infarction. Here, exosomes were engineered
with GATA-4 by infusing GATA-4 with exosome targeting peptide.
Methods: The open reading frame of mouse GATA-4 cDNA was ligated to XPACK
lentivirus vector (XPACK-GATA-4) and pLVX-EF1-IRES-Pouro lentivirus vector (pLVX-GATA-4),
respectively. HEK 293 cells were transduced by lentivirus, then exosomes were isolated
from
conditioned medium of HEK293 cells using ultracentrifugation. Exosomes were identified
using
transmission electronic microscope (TEM), and the expression of GATA-4 was semi-quantified
using western blot. The internalization of exosomes was tracked via treating bEnd3
cells
with exosomes pre-labelled with PKH26.
Results:
DNA sequencing confirmed the open reading frame of GATA-4 cDNA in frame with exosome
target peptide.
Isolated exosomes from HEK293 cells transduced with XPACK-GATA-4 and pLVX-GATA-4
appeared as diverse round-shaped entities and sized about 25–160 nm under TEM.
GATA-4 was expressed in both cell lysis of HEK293 cells which were transduced with
XPACK-GATA-4 and pLVX-GATA-4. However, GATA-4 was only expressed in exosomes isolated
from HEK293 cells transduced with XPACK-GATA-4, and not in exosomes from HEK293 cells
transduced with pLVX-GATA-4.
Exosomes expressing GATA-4 can be internalized by bEnd3 cells.
Summary/Conclusion: Exosomes can be directly engineered with GATA-4 and
internalized by bEnd3 endothelial cells, which may be a potential effective approach
for
delivery of GATA-4 to target cells.
Funding: NIH: HL140962
Pathology Pilot Grant of University of Cincinnati: F102150
PT03.06
Chinese hamster ovary cells engineered for production of
GFP-loaded extracellular vesicles
Braulio Carrillo Sanchez
a, Matthew
Hinchliffeb and Daniel Bracewella
aUniversity College London, London, UK; bUCB,
Slough, UK
Introduction: Chinese hamster ovary (CHO) cells have dominated as the
mammalian cell host for the manufacture of humanized biologics, in part owing to their
genomic plasticity and robust growth in suspension culture. There is great interest
surrounding the use of extracellular vesicles (EVs) as novel therapeutics owing to
their
capacity to deliver bioactive molecules. However, much remains unknown about the mechanisms
involved in EV cargo loading, limiting their development as novel biologics. To this
end, we
have engineered CHO cells to stably express constructs enabling loading of GFP into
EVs.
Methods: Tetraspanins are established markers of EV identity. Accordingly,
CD81 was selected as a tethering point to generate EVs with GFP cargo and constructs
were
generated via golden gate assembly. CHO cells were stably transfected by electroporation
and
expression was verified with fluorescence microscopy and western blotting. Growth
in batch
culture was monitored to establish maximum viable cell densities for EV harvest and
recovered EVs were characterized by nanoparticle tracking analysis (NTA). Finally,
uptake of
GFP-EVs was studied using time-lapse fluorescence imaging in co-culture experiments.
Results: Strong localization of CD81-GFP was observed at the cell membrane and
blotting confirmed intact tetraspanin fusion present at the expected molecular weight.
Additionally, cells were confirmed to retain high GFP expression post-cryopreservation.
Stable cell pools were able to reach viable densities greater than 7 million cells/mL
in
batch culture and NTA allowed for detection of GFP cargo even prior to EV isolation.
EV-mediated transfer of functional GFP to recipient cells was found to occur over
a period
of hours.
Summary/Conclusion: Collectively, our findings indicate that tetraspanins can
be used as targets to package recombinant protein cargo into mammalian derived EVs.
Moreover, CHO cells overexpressing cargo destined for EVs can reach high cell densities
and
produce functional EVs to facilitate yield challenges often associated with EV recovery.
Funding: Funded by the UK Engineering & Physical Sciences Research Council
(EPSRC) Centre for Doctoral Training in Emergent Macromolecular Therapies hosted at
University College London (UCL).
This research is sponsored and supported by UCB S.A. working in collaborating with
UCL.
PT03.07
Manufacturing extracellular vesicles derived from human
mesenchymal stromal cells (MSC) in bioreactors for cancer therapy
Miguel Almeida Fuzetaa
, Filipa
Oliveirab, Ana Catarina Costac, Ana
Fernandes-Platzgummerc, Sunghoon Jungd, Rong-Jeng Tsenge,
William Milligane, Brian Leed, Nuno Bernardesc, Diana
Gasparb, Joaquim Cabralc and Cláudia Silvac
aDepartment of Bioengineering and iBB – Institute for
Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa,
Lisboa,
Portugal; Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de
Lisboa,
Lisboa, Portugal, Lisboa, Portugal; bInstituto de Medicina Molecular, Faculdade
de Medicina, Universidade de Lisboa, Lisboa, Portugal, Lisboa, Portugal;
cDepartment of Bioengineering and iBB – Institute for Bioengineering and
Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal,
Lisboa,
Portugal; dPBS Biotech Inc., Camarillo, CA, USA, Camarillo, USA;
eAventaCell Biomedical Corp., Atlanta, GA, USA, Atlanta, USA
Introduction: Extracellular vesicles (EVs) are considered promising for
therapeutic applications. EVs resemble the cell membrane, allowing high biocompatibility
to
target cells, while their small size makes them ideal candidates to cross biological
barriers. Despite the promising potential of EVs for therapeutic applications, robust
manufacturing processes that would increase the scalability and consistency of EV
production
are still lacking.
Methods: In this work, EVs were produced by mesenchymal stromal cells (MSC),
isolated from different human tissue sources (bone marrow, umbilical cord matrix and
adipose
tissue). MSC were selected as these cells allow for a scalable production of EVs,
while
displaying low immunogenicity. A Vertical-Wheel™ bioreactor system was implemented
for the
production of MSC-derived EVs and compared with traditional static systems. The obtained
EV
products were characterized by nanoparticle tracking analysis, atomic force microscopy,
zeta
potential and Western blot.
Results: The bioreactor system allowed to obtain EVs at higher concentration
and productivity, as well as more homogeneous size distribution profiles, when compared
to
traditional static culture systems.
Functional studies were performed using breast cancer and lung cancer cell lines.
Proliferation assays allowed to determine the dose-response profiles of these cell
lines
when exposed to MSC-derived EVs. A bell-shaped profile was observed for most cases,
since
raising the EV concentration lead to increased cell proliferation until a certain
point
(25–50 µg/mL), after which cell proliferation was attenuated with increasing EV
concentrations.
Summary/Conclusion: The bioreactor culture system allowed a substantial
improvement in the production of MSC-derived EVs, while the obtained dose-response
profiles
will be valuable to determine the most appropriate EV concentrations for anticancer
drug
delivery. Overall, we demonstrate that this culture system is able to robustly manufacture
human MSC-derived EVs in a scalable manner towards the development of novel therapeutic
products such as anticancer drug delivery systems.
Funding: Fundação para a Ciência e Tecnologia (SFRH/PD/BD/128328/2017,
SFRH/PD/BD/135046/2017, PTDC/EQU-EQU/31651/2017, UID/BIO/04565/2019).
PT03.08
Biodistribution and cellular location of inhaled exosomes and
liposomes in the lung
Kristen Popowskia
, Phuong-Uyen
Dinha, Halle Lutzb, Arianna Georgea, Mallory
Flanaganb, Devlyn Andersonb and Ke Chengb
aNC State University, Raleigh, USA; bNorth
Carolina State University, Raleigh, USA
Introduction: Increasing evidence reveals the potential role of extracellular
vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment
of
lung diseases. Encapsulation and delivery of potential RNA and microRNA targets into
liposomes and exosomes are attractive drug delivery methods, but remain difficult
to deliver
to the pulmonary parenchyma to reach target lung cell types. Here, we demonstrate
effective
delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma
via
inhalation treatment in a murine model of idiopathic pulmonary fibrosis.
Methods: Human lung stem cells (LSCs) were generated and expanded from healthy
whole lung donors. LSC-exosomes were purified via ultrafiltration and DiI-labelled
using
Vybrant☐ labelling solution according to the manufacturer’s instructions. DsRed-labelled
liposomes were generated using Lipofectamine™ RNAiMAX Transfection Reagent and BLOCK-iT™
Alexa Fluor™ Red Fluorescent Control according to the manufacturer’s instructions.
LSC-exosomes and liposomes were delivered via nebulization to CD1 mice with
bleomycin-induced pulmonary fibrosis. Exosome and liposome delivery and biodistribution
were
visualized 4- and 24-hours post-treatment through histological analysis. The study
was
approved by the Institutional Animal Care and Use Committee of North Carolina State
University and complied with all national and state ethical standards.
Results: Exosome and liposome delivery to the pulmonary parenchyma was
confirmed by the presence of DiI and DsRed fluorescence in lung histological sections
that
penetrated the mucus-lined respiratory epithelium. More exosomes and liposomes surpass
mucus-lined surfaces 24-hours post-treatment compared to 4-hours post-treatment. Fluorescent
colocalization of exosomes and liposomes with alveolar type I cells, alveolar type
II cells,
basal lung cells, and CD68+ macrophages was observed through immunohistochemistry
analysis.
More exosomes and liposomes colocalize with these cell types 24-hours post-treatment
compared to 4-hours post-treatment.
Summary/Conclusion: LSC-exosomes and liposomes penetrate the mucus-lined
respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment.
LSC-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells,
and
interstitial macrophages with improved biodistribution 24-hours post-treatment.
Funding: This study was supported by the NC State Chancellor’s Innovation
Fund.
PT03.09
Transfection reagent artefact accounts for some reports of
extracellular vesicle function
Russell McConnell, Madeleine Youniss and
Jonathan Finn
Codiak BioSciences, Cambridge, USA
Introduction: Extracellular vesicle (EV) functions are frequently investigated
by transiently transfecting cells with plasmid DNA to produce EVs modified with protein(s)
or nucleic acid(s) of interest. However, EVs and the DNA-complexes used to transduce
cells
are physically similar, raising the possibility that they may co-purify during differential
ultracentrifugation, the most common EV isolation procedure. Activities attributed
to EVs
may therefore be due to contaminating DNA -transfection reagent complex.
Methods: EV producing cells were transiently transfected with plasmid DNA
encoding gene-editing or split enzymes fused to EV-targeting protein sequences. Differential
and density gradient ultracentrifugation were used to purify EVs from cell culture
supernatant or DNA lipoplexes from cell-free culture media. Protein expression and
localization to EVs was confirmed by Western blot. Cell lines stably expressing fluorescent
or luminescent reporters were used to assess functional enzyme delivery in recipient
reporter cells.
Results: Reporter cells treated with ultracentrifuge pellet material (UCP)
from media of transiently transfected cells showed robust and reproducible signal,
however
fractionating the UCP with an iodixanol density gradient revealed that reporter activity
was
associated with high-density fractions that were depleted in EVs. UCP isolated from
identical transfection conditions, but lacking cells (and exosomes), showed identical
biological activity levels and distribution in iodixanol gradients, suggesting that
the
activity was due to contaminating transfection reagent complexes and not EVs. Serial
media
changes on EV producing cells post-transfection did not significantly reduce UCP activity
on
reporter cells. Treatment with nucleases did not digest complexed DNA, did not significantly
reduce DNA levels in the UCP as measured by qPCR, and did not decrease activity in
reporter
cells treated with UCP from either transfected cells or no-cell controls.
Summary/Conclusion: We find that DNA-transfection reagent complexes are not
separated from EVs using differential ultracentrifugation and that common approaches
to
remove such complexes, including media exchanges and nuclease treatment, are ineffective.
Due to the pernicious nature of the DNA-complex in these cellular assays, it is likely
that
some reports of EV function are likely artefacts produced by contaminating DNA-complexes.
We
find that density gradient centrifugation can effectively separate EVs and DNA-complexes,
highlighting the importance of validating elimination of contaminating transfection
reagent
complexes when using transient transfection to interrogate EV function.
PT04: EV Protein Biomarkers
Chair: Suresh Mathivanan – La Trobe University
PT04.01
Cancer stem cell-derived exosomes: potential biomarkers for
early diagnosis and prognosis in pancreatic cancer
Haobin Wanga
, Shijing
Yueb, Yingshu Luoc and Zoller Margotd
aAffiliated Hospital of Southwest Jiaotong University/The
Third People’s Hospital of Chengdu, Chengdu, China (People’s Republic); bNankai
University, Tianjin, China (People’s Republic); cUniversity Hospital of
Electronic Science and Technology, Chengdu, China (People’s Republic);
dHeidelberg University, Heidelberg, Germany
Introduction: Pancreatic cancer (PaCa) is the most deadly manlignancy, due to
late daignosis and early metastatic spread, which prohibits surgery. it is urgently
for
relaible, early detection. Research shows that tumour-derived exosomes, which had
been
present in the blood in the early stage of tumour formation and before metastasis,
is the
vanguard forces of tumour formation and metastasis; Cancer stem cell-derived exosomes
(CSC-Exos) has stronger migration ability, so the detection of blood CSC-Exos for
early
diagnosis and monitoring of progress for PaCa has great research potential and the
value of
application.
Methods: Protein markers were selected according to expression in exosomes of
PaCa cell line culture supernatants, but not healthy donors’ serum-exosomes. According
to
these preselections, serum-exosomes were tested by flow cytometry for the pancreatic
cancer
stem cell marker CD44v6 and Tspan8.
Results: The majority (95%) of patients with PaCa and patients with
nonPa-malignancies reacted with anti-CD44v6 and anti-Tspan8. Serum-exosomes of healthy
donors’ and patients with non-malignant diseases were not reactive. Recovery was tumour
grading and staging independent including early stages.
Summary/Conclusion: Thus, the evaluation of pancreatic CSC-derived exosomes
awaits retrospective analyses of larger cohorts, as it should allow for a highly sensitive,
minimally-invasive PaCa diagnostics.
Funding: Supported by the National Natural Science Foundation of China (No.
81702963)
PT04.02
Characterization of Extracellular vesicles separated from
biospecimens of former national football League players at a risk for chronic traumatic
encephalopathy
Satoshi Muraokaa
, Mark
Jedrychowskib, Zijian Yangc, Harutsugu Tatebed, Annina
Deleoa, Kayo Yukawaa, Jina Koe, Yuzhi Wanga,
Seiko Ikezua, Steven Gygib, David Issadoref, Takahiko
Tokudad, Robert Sterna and Tsuneya Ikezua
aBoston University, Boston, USA; bHarvard Medical
School, Boston, USA; cUniversity of Pennsylvania, Philadelphia, USA;
dKyoto Prefectural University of medicine, Kyoto, Japan;
eMassachusetts General Hospital, Wyss Institute at Harvard University, Cambridge,
USA; fDepartment of Bioengineering, University of Pennsylvania, Philadelphia,
USA
Introduction: Chronic Traumatic Encephalopathy (CTE) is a tauopathy that
affects individuals with a history of mild repetitive brain injury frequently seen
in
contact sports. Initial neuropathologic change of CTE include perivascular deposition
of
phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation
of
neurofibrillary tangles in neurons throughout the brain. Extracellular vesicles (EV)
are
known to carry neuropathogenic molecules in neurodegenerative disease and able to
cross the
blood brain barrier. We therefore examined the protein composition of EV separated
from
cerebrospinal fluid (CSF) and plasma in former national football League (NFL) players
with
cognitive dysfunction, and an age-matched control group with no history of contact
sports.
Methods: EVs were separated from CSF and plasma from former NFL players
(n = 4, 14) and controls (n = 5, 12) by affinity separation method or size exclusion
chromatography, respectively. The EV protein profiling was characterized by SIMOA
for tau
and p-tau and mass spectrometry. The protein data was analysed for EV enrichment,
differentially expressed proteins, pathway analysis and correlation with cognitive
function,
head impact and tau/p-tau levels by biostatistics and bioinformatics.
Results: The level of total tau and p-tau in CSF EVs was not significantly
changed, but significantly elevated in plasma EVs from former NFL players. The 95
proteins
were commonly identified between the paired plasma-CSF from the same patients, but
there was
no significant correlation with disease status. Collagen alpha-3(VI) chain (COL6A3),
−1(VI)
chain (COL6A1) and Reelin (RELN) were differentially expressed in former NFL players’
plasma
EVs. A combination of these 3 proteins in plasma EV can distinguish former NFL players
from
controls with 85% accuracy by machine learning.
Summary/Conclusion: The interacting plasma-CSF EV proteomes provide an
original resource to EV biomarker development for neurodegenerative disease, and COL6A3,
RELN and COL6A1 in plasma EVs can be potential biomarker for monitoring the CTE
development.
PT04.03
Density-based fractionation of urine to unravel the proteome
landscape of extracellular vesicles in prostate cancer
Bert Dhondt, Olivier de Wever and An
Hendrix
Laboratory of Experimental Cancer Research, Department of Human
Structure and Repair, Ghent University, Belgium; Cancer Research Institute Ghent,
Belgium,
Ghent, Belgium
Introduction: Current diagnostic tests are unable to discriminate indolent
from aggressive prostate cancer (PCa), leading to overdiagnosis and overtreatment,
and an
intense interest in biomarkers to improve clinical decision making. Urine is considered
an
ideal proximal fluid for biomarker identification in PCa due to its direct contact
with the
urogenital system. The discovery and translation of extracellular vesicle (EV) content
into
PCa biomarkers remains challenging due to the difficulty of obtaining urinary EV (uEV)
with
high specificity.
Methods: We developed a step-by-step protocol to separate uEV by orthogonal
implementation of ultrafiltration and bottom-up density gradient centrifugation (BU-ODG).
We
implemented complementary particle and protein measurements to identify uEV (lower
density)
and protein rich fractions (higher density) and assess the performance of BU-ODG
(specificity, efficiency and reproducibility). Using mass spectrometry-based proteomics
we
interrogated uEV and protein rich fractions from matched urine and radical prostatectomy
tissue samples from PCa patients (n = 4), and urine from men with PCa prior to (n = 12)
and
after local treatment (n = 10), benign prostatic hyperplasia (n = 12) and other urological
cancers (n = 11).
Results: BU-ODG separated uEV from soluble proteins and Tamm-Horsfall Protein
(THP) complexes with high specificity and reproducibility, outperforming differential
ultracentrifugation, ExoQuick and size-exclusion chromatography. Comparison of the
uEV
proteome from men with benign or malignant prostate disease, allowed us to expand
the known
human uEV proteome and identify a PCa specific uEV proteome not uncovered by the analysis
of
the protein rich fraction. Proteomic analysis of EV separated from prostate tumour
interstitial fluid and matched uEV confirmed PCa specificity of the uEV proteome.
Analysis
of the uEV proteome from patients with bladder and renal cancer provided additional
evidence
of the selective enrichment of protein signatures in uEV reflecting their respective
cancer
tissues of origin.
Summary/Conclusion: We identified hundreds of previously undetected proteins
in uEV of PCa patients and developed a powerful toolbox to map uEV and protein rich
fractions, ultimately supporting biomarker discovery for urological cancers.
PT04.04
Immunoglobulin A coating of faeces-derived bacterial vesicles as
a marker of inflammatory bowel disease in humans
Nader Kamelia
, Frank
Stassenb, Heike Beckerc, John Pendersc, Daisy
Jonkersd and Paul Savelkoulb
aDepartment of Medical Microbiology, School of Nutrition and
Translational Research in Metabolism (NUTRIM), Maastricht University Medical Centre+,
Maastricht, The Netherlands., Maastricht, Netherlands; bDepartment of Medical
Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM),
Maastricht University Medical Centre+, Maastricht, The Netherlands, Maastricht, Netherlands;
cSchool of Nutrition and Translational Research in Metabolism (NUTRIM),
Department of Medical Microbiology, Maastricht University Medical Centre+, Maastricht,
The
Netherlands., Maastricht, Netherlands; dDepartment of
Gastroenterology/Hepatology, NUTRIM school of Nutrition and Translational Research
in
Metabolism, Maastricht University Medical Centre, Maastricht, The Netherlands, Maastricht,
Netherlands
Introduction: IgA is the most abundant antibody in mucosal secretions and
plays a crucial role in maintaining the balance between the host and the gastrointestinal
microbiome. Recent studies suggested that pronounced IgA coating is especially prominent
among inflammatory commensals which drive intestinal disease. Membrane vesicles (MVs,
nano-sized particles released by bacteria) have also been found to interact with the
host
and modulate development and function of the immune system. However, their interaction
with
IgA has not been studied yet. Here we developed a method to isolate and characterize
the MVs
from faecal samples and checked for possible differences in IgA coating patterns of
MVs in
health and disease.
Methods: MVs were isolated by using a combination of ultrafiltration and size
exclusion chromatography from faecal samples of 6 healthy controls (HC), 6 patients
with
active Crohn disease (CD) and 6 CD patients in a remissive state. Quantification and
verification have been done with tunable resistive pulse sensing (TRPS-based analysis)
bead-based flow-cytometer (BBFC) and transmission electron microscope (TEM). MVs were
selected with specific antibodies for capturing (Gram+: LTA, Gram-: OmpA) followed
by
PE-conjugated anti-human IgA antibodies as detection.
Results: We could successfully isolate 1*109-5*109 particles/ml from 500 mg of
faeces. BBFC in combination with TRPS provide a valuable method for (semi-)quantitative
measurements of mixed populations. Intriguingly, remarkable differences were found
between
IgA coating MVs derived from healthy controls and active and remissive CD patients
as MVs
derived from healthy controls were significantly more coated compare to both CD patient
groups. In details, for selected G-ve derived MVs: 60% of the total population of
MVs
derived from HC were coated, 20% from remissive CD patients, and <5% of active CD
patients; and for selected G+ ve derived MVs: 55% of the total population of MVs derived
from HC were coated, 34% from remissive CD patients, and 25% of active CD patients.
(Data
are represented as the mean).
Summary/Conclusion: Here we demonstrate for the first time that MV isolated
from the faecal samples are also coated with IgA, and surprisingly MVs from healthy
volunteers were more densely coated than MVs from diseased patients. The possible
consequence of this difference remains to be determined in future studies.
PT04.05
Monitoring altered tetraspanin and PSMA expression in prostate
cancer derived extracellular vesicles via Advanced Image Flow Cytometry (ISX)
Lukas W. Prausea
, Christopher
Millanb, Natalie Henskyc, Tullio Sulserc and Daniel
Eberlic
aUniversityHospital Zurich, Zurich, Switzerland;
bUniversity of Zurich Hospital, Schlieren, Switzerland; cUniversity of
Zurich Hospital, Zurich, Switzerland
Introduction: New diagnostic and therapeutic options for patients with
prostate cancer are urgently needed. Prostate-specific membrane antigen (PSMA)-based
imaging
and therapy are increasingly used for prostate cancer management. Unfortunately, as
a
membrane protein, PSMA is not found as a soluble protein in the blood and therefore
has
limited utility as a diagnostic biomarker. However, PSMA has reportedly been observed
as a
cargo protein of prostate cancer-derived extracellular vesicles (EVs).
Methods: We demonstrate altered PSMA expression on EVs derived from prostate
cancer cell cultures (C4-2, LNCaP) in response to novel next-generation androgen receptor
inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental
nonsteroidal antiandrogen (Epi-001) that binds covalently to the N-terminal domain
of the
androgen receptor and dihydrotestosterone (DHT). Additionally, EVs were isolated from
the
plasma of prostate cancer patients who participated in the proCOC biobank campaign
at the
USZ. Plasma was taken and stored from patients both pre- and post- prostatectomy.
Results: Transmission electron microscopy, nanoparticle tracking analysis and
simple Western (WES) analysis show stable size distribution and amount of EVs produced
by
treated and non-treated cells. Using advanced image-based flow cytometry, altered
tatraspanin and PSMA expression could be detected in EVs isolated from cell culture
supernatants of LNCaP and C4-2 prostate cancer cells following their treatment.
Summary/Conclusion: Measuring PSMA expression on extracellular vesicles might
pave the way to use image flow cytometry of EVs to develop a blood based diagnostic
test for
prostate cancer patients with a wide range of possible applications including: 1)
monitoring
response to therapy and, 2) early indications of potential relapse.
Funding: Vontobel Fondation.
PT04.06
Proteomic profiling of human neural cells derived extracellular
vesicles to identify human brain cell-type specific markers
Tsuneya Ikezua
, Yang
Youa, Venkata Viswanadh Edarab, Satomi Stacyb, Amanda
McQuadec, Samuel Hersha, Mathew Blurton-Jonesc and Anuja
Ghorpaded
aBoston University, Boston, USA; bUniversity of
North Texas Health Science Center, Fort Worth, USA; cUniversity of California,
Irvine, Irvine, USA; dMedical Innovation Collaborative of North Texas, Fort
Worth, USA
Introduction: Alzheimer’s Disease (AD) is a common neurodegenerative brain
disease which affects appropriately 30 million patients worldwide. One of the major
challenges in AD is to develop reliable biomarkers for early diagnosis and disease-modifying
therapies, especially before the clinical symptoms. Extracellular vesicles (EVs) carry
cargos of proteins, lipids and nucleic acids. There was no comprehensive characterization
of
EVs isolated from specific brain cell types, which may be useful for cell type-specific
biomarkers. The purpose of this study is to isolate EVs from human induced pluripotent
stem
cell (iPSC)-derived brain cells for proteomic profiling and characterization of cell
type-specific molecules.
Methods: Human iPSCs-derived neurons, microglia and primary cultured
astrocytes were differentiated in EV-depleted media. The EVs were isolated by differential
centrifugation combined with size exclusion chromatography, followed by characterization
using nanoparticle tracking analysis and mass spectrometry. The proteomic data were
subjected to bioinformatics analysis
Results: We identified 153 proteins from neuron-derived EV (NDE), 215 proteins
from microglia-derived EV (MDE) and 380 proteins from astrocyte-derived EV (ADE) by
proteomics. Gene ontology analysis indicated that most of these proteins are associated
with
EVs. Furthermore, 15, 48 and 251 proteins are present individually in NDEs, MDEs and
ADEs.
Among them, high levels of ATP1A3 and SYT1 in NDEs, ITGAM and CD300A in MDEs, and
EAAT1 and
GFAP in ADEs were found, all of which are typically and highly expressed in the original
cells.
Summary/Conclusion: Our results provide us the potential candidates for
cell-type specific EV markers, which will be helpful to develop non-invasive tools
to enrich
EV originating from specific brain cells and may lead to the development of new biomarkers
for neurodegenerative disorders.
Funding: NIH RF1AG054199, NIH R56AG057469, Abbvie, Cure Alzheimer’s Fund
PT04.07
Quality control for bacterial EVs
Simon Swifta
, Jiwon
Honga, Vanessa Changb, Priscila Dauros-Singorenkob,
cherie blenkironb and Anthony Phillipsa
aUniversity of Auckland, Auckland, New Zealand;
bThe University of Auckland, Auckland, New Zealand
Introduction: The MISEV guidelines of 2018 (DOI:
10.1080/20013078.2018.1535750) are a tremendous resource for extracellular vesicle
(EV)
research, but they are heavily focussed on mammalian EVs, i.e. EVs from humans and
laboratory animals, where protein cargoes are well characterised, and a wide selection
of
antibodies are commercially available. Protein markers can be used to identify and
define
the types of mammalian EV and to determine the presence of any contaminants that might
confound functional studies. Similar resources are not as readily available for bacterial
EVs as these are not as well characterised, commercially available antibodies are
much less
abundant and immunological variation between different bacterial species (and there
are 1
trillion bacterial species on planet Earth!) means that each species, strain, or group
of
related species may require different antibodies.
Methods: To identify quality markers for bacterial EVs, we have characterised
the proteome of cells, crude EVs (ultracentrifuge pellet from cell free culture supernatant)
and size exclusion chromatography or density gradient centrifugation purified EVs
from two
different (pathogenic vs probiotic) strains of Escherichia coli grown under two different
environmental conditions, and one strain of Mycobacterium marinum grown in one medium.
Results: Our results identify a selection of proteins enriched in purified EV
preparations, and proteins that are depleted after purification steps.
Summary/Conclusion: Our results allow the identification of potential markers
for EV purity and non-EV contaminants, but also highlight the variability in bacterial
EV
preparations and suggest potential targets that can be used to investigate the heterogeneity
of bacterial EV populations.
Funding: This work was supported by the School of Medicine Performance-Based
Research Fund from the University of Auckland; The Hugo Charitable Trust; Maurice
and
Phyllis Paykel Trust Project Grant [8.1.17]; Lottery Health Research Grant [326702];
Health
Research Council of New Zealand Explorer Grant [14/805]; Ministry of Business, Innovation
and Enterprise of New Zealand, Smart Ideas Grant [UOAX1507], New Zealand.
PT04.08
Relationship of extracellular vesicle cargo with clinical
markers of mortality and race
Nicole Noren Hootena
, Minna
McFarlandb, David Freemanc, Nicolle Moded, Ngozi
Eziked, Alan Zondermand and Michele K. Evansd
aNational Institute on Aging, National Insitutes of Health,
Baltimore, USA; bNational Institute on Aging, National Institutes of Health,
Chapel Hill, USA; cNational Institute on Aging, National Institutes of Health,
Salt Lake City, USA; dNational Institute on Aging, National Institutes of Health,
Baltimore, USA
Introduction: Recent findings indicate an increase in mid-life mortality rates
in the USA and persistent, significant race-related health disparities exemplified
by
differential mortality rates. This suggests that exploring new molecular markers that
may be
linked to mortality could provide novel insights into factors that are driving mortality
rates. Accumulating data suggests that extracellular vesicles (EVs) circulating in
blood may
be potential biomarkers of age-related disease. EVs are nano-sized membranous vesicles
that
bear molecular cargo and mediate intercellular communication between different cells
and
tissues. Little is known about whether EV characteristics differ by race or whether
EVs are
associated with clinically relevant mortality risk factors.
Methods: In this cross-sectional study, plasma EVs were isolated from
middle-aged African American (AA) and white males and females.
Results: We report no significant differences in EV size or concentration with
race or sex. There were significantly higher EV levels of phospho-p53, total p53,
cleaved
caspase 3, ERK1/2 and phospho-AKT in whites compared to AAs. Higher EV levels of
phospho-IGF-1R were found in females compared to males. We examined EV characteristics
and
protein cargo in the context of well-established clinical mortality risk factors.
EV
concentration was significantly, and positively, associated with several mortality
markers
including, high-sensitivity C-reactive protein (hsCRP), homoeostatic model assessment
of
insulin resistance (HOMA-IR), alkaline phosphatase, pulse pressure, body mass index,
and
waist circumference. The relationship of EV concentration and cargo with mortality
markers
differs by race.
Summary/Conclusion: Our data show that EV-associated proteins can differ by
race and sex and are associated with mortality risk factors. This study provides insight
into the characterization of EVs in middle-aged AAs and whites, which may aid in the
development of EV-based diagnostics.
Funding: This study was supported by the National Institute on Ageing
Intramural Research Program of the National Institutes of Health.
PT04.09
Repurposing specialised cell-free DNA blood collection tubes for
extracellular vesicle isolation
Jessica Heatliea, Vanessa Changb, Sandra
Fitzgeraldb, Yohanes Nursalimb, Kate Parkerb, Benjamin
Lawrenceb, Cristin Printb and Cherie
Blenkironb
aThe University of South Australia, Adelaide, Australia;
bThe University of Auckland, Auckland, New Zealand
Introduction: Liquid biopsies offer a minimally invasive approach to patient
disease diagnosis and monitoring. However, many plasma processing protocols have been
designed with a single biomarker in mind. Here we investigate whether specialised
DNA blood
stabiliser tubes could be repurposed for the analysis of extracellular vesicles (EVs).
Methods: Peripheral blood (n = 3) was collected into K3-EDTA, Roche or Streck
cell-free DNA (cfDNA) blood collection tubes and processed using sequential centrifugation
immediately or after storage for 3 days. MicroEV were collected from platelet poor
plasma by
10,000 g centrifugation and NanoEVs isolated using size exclusion chromatography.
Particle
size and counts were assessed by Nanoparticle Tracking Analysis, protein by BCA assay
and
dot blotting for blood cell surface proteins.
Results: Major variations in Micro and NanoEVs were seen with delayed time to
processing. NanoEV counts did not change with processing delay or tube collection
type but
the associated protein amount increased, indicative of cell lysis or activation. The
protein
was predominantly derived from from platelets (CD61) and red blood cells (CD235a).
The
increase in associated protein was seen more in the K3-EDTA and Streck tubes indicating
that
the Roche tubes may offer improved cell stability. Conversely, MicroEVs increased
in both
quantity and protein content with delay to processing indicative of both lysis and
cell
activation, irrespective of tube type. Epithelial cell surface marker EpCAM abundance
remained the same across conditions in both Micro and NanoEVs demonstrating that EpCAM+
EVs
were stable.
Summary/Conclusion: Specialised cfDNA collection tubes can be repurposed for
Micro and NanoEV analysis, however simple counting or using protein quantity as a
surrogate
of EV number may be confounded by pre-analytical processing. The EVs would be suitable
for
disease selective EV subtype analysis if the molecular target of interest is not present
in
blood cells.
Funding: Translational Medicine Trust, University of Auckland.
PT04.10
Role of exosomes in the management of cellular disease in the
genetic susceptible participants in the Chilean population
Maria Paz Weisshaara
, Jamal
Ghanamb, Rafael Barrac, Miguel Riosc and Jorge
Escobard
aUniversity of Applied Sciences Bonn-Rhein-Sieg, Bonn,
Germany; bUniversity of Applied Sciences, Bonn- Rhein-Sieg, Dept. of Natural
Sciences, 53359, Rheinbach, Bonn, Germany; cUniversity of Santiago of Chile,
Santiago, Chile; dPontifical Catholic University of Valparaíso, Chile,
Valparaíso, Chile
Introduction: Nutrigenomics and nutrigenetics have been defined as the effect
of nutrients on gene expression and genetic variation on dietary response, respectively.
Here, we propose the isolation and characterization of exosomes from donors carrying
different alleles of HLA-DQA1 and HLA-DQB1, to investigate their involvement in coeliac
disease (CD) management.
Methods: A Chilean population (n = 30) was investigated for SNPs mutations in
HLA Class II alleles associated to CD predisposition (as well as other mutations related
to
other food intolerances), using the GenoChip Food Technology. Exosomes have been isolated
from donors’ serum by ultracentrifugation and characterized by SDS-PAGE, Western Blotting
(CD63 and CD9), and transmission electron microscopy. Exosomes were also studied for
their
interleukins (IL-6 and IL-1ra) content.
Results: Among the studied population, 86% present at least one of the alleles
leading to CD development and 60% carry alleles encoding for α- and β-chains heterodimers
associated with very high risk to develop CD. In parallel, isolated exosomes from
donors
with low to extremely high risk for CD showed high IL-1ra content (108.8 ± 15.91 to
148.8 ± 12.37), as the persons were not following any treatment. However, values of
IL-1ra
decrease in exosomes isolated form persons receiving treatment for CD. A relationship
between exosomes’ content and genetic susceptibility for CD has been observed, which
may
suggest their possible use as biomarkers for CD as the diagnostic of this disease
is still a
big issue.
Summary/Conclusion: Until this point of this underway project, we demonstrate
the existence of a relationship between the exosomes’ content in IL-1ra and genetic
susceptibility for CD. Furthermore, the genetic predisposition to CD could also modulate
the
gut colonization process, another important player in intestinal homoeostasis. In
the next
step, extracellular vesicles from gut microbiota will be isolated and analysed to
determine
their role in CD management.
PT04.11
Subpopulations of EVs in serum and plasma
Nasibeh Karimia
, Razieh Dalir
Fardoueia, Jan Lötvalla and Cecilia Lässerb
aKrefting Research Centre, Institute of Medicine, Sahlgrenska
Academy at University of Gothenburg, Göteborg, Sweden; bKrefting Research Centre,
Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg,
Sweden
Introduction: The ability to isolate extracellular vesicles (EVs) from blood
is vital in the development of EVs as disease biomarkers. Both serum and plasma can
be used
but few studies have compared them in terms of amount and type of EVs. We have previously
developed a method to isolate EVs from plasma with minimal contamination of lipoprotein
particles (Karimi et al 2018). The aim of this study was to compare the presence of
different subpopulations of EVs in plasma and serum.
Methods: Blood was collected from healthy subjects, from which plasma and
serum were isolated. EVs were isolated using a combination of density cushion and
size
exclusion chromatography (SEC) (protocol 1) or a combination of density cushion and
density
gradient (protocol 2) or immune-capturing (anti-CD63, anti-CD9 and anti-CD81 beads)
(protocol 3). Purity and yield of EVs were determined by nanoparticle tracking analysis
(NTA), Western blot, electron microscopy (EM), ExoView, flow cytometry and mass spectrometry
(LC-MS/MS).
Results: As determined by NTA and protein measurement more EVs could be
isolated from plasma with protocol 1 and the majority of the vesicles were CD9/CD41a
positive as determined with ExoView and Western blot. Additionally, flow cytometry
and
Western blot showed that more CD9/CD41a positive EVs where also identified with protocol
3.
Furthermore, Western blot showed increased amount of CD41a in plasma samples in protocol
2.
When labelled EVs were spiked in freshly collected blood, no difference in recovery
was seen
for plasma and serum.
Summary/Conclusion: This study shows that a larger amount of EVs could be
isolated from plasma compared to serum when three different isolation methods were
used.
Firstly, this suggests that more EVs are present in plasma. Secondly, it suggests
that these
vesicles are probably released by platelets and that EVs are not trapped in the clot
during
serum formation. Future studies are needed to answer how this affects the use of
blood-derived EVs as biomarkers from serum and plasma.
PT04.12
Tumour
-derived extracellular vesicles contain
distinct integrin proteins
Stephanie N. Hurwitza
and David G.
Meckesb
aUniversity of Pennsylvania, Philadelphia, USA;
bFlorida State University, Tallahassee, USA
Introduction: Cargo profiling, including proteomic analyses, of tumour
cell-derived extracellular vesicles (EVs) may provide ripe opportunities for further
understanding cancer growth, drug resistance, and metastatic behaviour. Accumulating
data
suggest that cancer-derived EVs contain membrane-bound integrin proteins which may
aid in
cell detachment, migration, and homing to future metastatic niches. We have previously
published an extensive proteomic profile of secreted vesicles from the NCI-60 panel
of human
cancer cells.
Methods: Here, we further examine the distinct integrin components in these
cancer-derived EVs, and additionally profile EVs released from benign epithelial cells
by
liquid chromatography and tandem mass spectrometry for comparison.
Results: We demonstrate the enrichment of integrin receptors in cancer EVs
compared to vesicles secreted from benign epithelial cells. Total EV integrin levels,
including the quantity of integrins α6, αv, and β1 correlate with tumour stage across
a
variety of epithelial cancer cells. In particular, integrin α6 also largely reflects
breast
and ovarian progenitor cell expression, highlighting the utility of this integrin
protein as
a potential circulating biomarker of certain primary tumours. Other integrins including
α4,
αL, and β2 are enriched in vesicles derived from leukaemia cells, and may provide
a means to
distinguish haematopoietic cell-derived EVs.
Summary/Conclusion: This study provides preliminary evidence of the value of
vesicle-associated integrin proteins in detecting the presence of cancer cells and
prediction of tumour stage. Differential expression and selective packaging of integrins
into EVs may contribute to further understanding the development and progression of
tumour
growth and metastasis across a variety of cancer types.
PT04.13
Effect of nicotine and menthol on cytochrome P450 and
antioxidant enzymes in rat plasma-derived extracellular vesicles
Asit Kumar
a, Namita
Sinhab, Sanjana Haquec, Tengfei Wanga, Angel G.
Martinezc, Hao Chenc and Santosh Kumarb
aUniversity of Tennessee Health Science Center, Memphis, USA;
bUTHSC, Memphis, USA; cThe University of Tennessee Health Science
Center, Memphis, USA
Introduction: Tobacco products such as e-cigarettes pose potential adverse
health effects caused by direct exposure to aerosolized nicotine, flavorants such
as
menthol, and other particulates. Here, we aimed to study the hypothesis that whether
nicotine and menthol modulate nicotine-metabolizing cytochrome P450 2A6 (CYP2A6),
antioxidant enzymes (AOEs), SOD1 and catalase in plasma extracellular vesicles (EVs).
Modulation of these enzymes would eventually lead to nicotine-induced toxicity and
HIV-1
pathogenesis via EVs-based cell-cell interactions.
Methods: We isolated and characterized EVs from rat plasma before and after
nicotine self-administration (NIC) with audiovisual cue (AV) and menthol and characterized
using EV markers according to the ISEV guidelines. Protein associated with CYP2A6,
SOD1, and
catalase were quantified by western blot.
Results: We measured size, total protein, and acetylcholine esterase activity
of EVs and found no significance difference in these characteristics before and after
NIC.
To investigate the effect AV, menthol alone or in combination in the absence and presence
of
NIC, first we evaluated the expression of EV markers CD9 and CD63. The results showed
menthol and AV together increased the levels of CD9 (p ≤ 0.05), the marker of small
vesicles, in the presence of NIC. The NIC with menthol and AV showed a pattern of
increased
levels of small vesicle but could not reach to significance. Next, we demonstrated
that the
NIC with AV increased the level of SOD1 (p ≤ 0.05), which showed a pattern of increased
levels of catalase and CYPA6, though statistically non-significant. The expression
of
nicotine receptor did not change under any conditions used. The results showed an
increased
level of CYP2A6 (p ≤ 0.01), SOD1 (p ≤ 0.05), and catalase (p ≤ 0.05) in plasma EVs
in the
menthol-NIC group compared to menthol group only. NIC group with a combined AV and
menthol,
showed further increase in the levels of CYP2A6 (p ≤ 0.01), and catalase (p ≤ 0.05).
Further
analysis of plasma EVs on inflammatory cytokines/chemokines in these groups, and the
effect
of plasma EVs on nicotine-induced toxicity and HIV pathogenesis are underway.
Summary/Conclusion: Nicotine administration increased, though not
statistically significant, the levels of circulatory EVs. Moreover, the study provided
evidence that nicotine in the presence of menthol, AV, and/or menthol+AV increased
nicotine-metabolizing CYP2A6 in all the groups and AOEs in specific groups.
Funding: We thank the National Institute on Drug Abuse (Grant #DA047178,
DA-047638) for supporting our work.
PT04.14
Proteomic signatures in breast cancer exosomes and cell
lines
Zoraida Andreu
a, Francisco
García-Garcíab, Marta Hidalgoc, Esther Masiád, Maria
Jesús Vicente and Zoraida Andreu Martínezf
aPolymer Therapeutics Lab 1. Centro de Investigación
Principie Felipe, Valencia, Spain; bBioinformatics Biostatics Unit 3. Principe
Felipe Research Centre, Valencia, Spain; cBioinformatics and Biostatistics Unit
3. Principe Felipe Research Centre, Valencia, Spain; dPolymer Therapeutic Lab1.
Screening Platform2 Principe Felipe Research Centre, Valencia, Spain; ePolymer
Therapeutic Lab. 1 Screening Platform 2. Principe Felipe Research Centre, Valencia,
Spain;
fCentro de Investigación Principie Felipe, Valencia, Spain
Introduction: Biomarker discovery in breast cancer (BC) is a clinical need for
therapeutics and non-invasive diagnostics. Tumour exosomes are involved in pre-metastatic
niche formation and drug resistance and represent a source of non-invasive biomarkers.
The
identification of tumour exosomal biomarkers provides, not only, the possibility to
discriminate patient groups also potential targets to control cancer progression that
could
be exploited to develop innovate BC therapeutic strategies.
Methods: We have performed a comparative differenti.al proteomic profile of
four BC cell lines and their derived- exosomes, representative of the most relevant
BC
subtypes in clinic to search non-invasive biomarker candidates. Then, we have carried
on two
bioinformatics approaches: 1) protein association network analysis interaction (STRING)
and
2) pathway inference analysis (Hipathia), to characterize the functional profiling
for each
BC subtype.
Results: We have found differentially-expressed proteins, in both cells and
exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance.
Exosome proteome profile reflects their different BC cell origin suggesting potential
indicators of BC subtype. Further, bioinformatics analysis reveals a differential
role of
exosomes in BC signalling pathways in recipient cells, according to their protein
cargo and
cell origin.
Summary/Conclusion: Our results show a set of cells and exosome proteins that
highly discriminate BC subtypes and may significantly contribute to further studies
for the
design of BC biomarker predictor to stratify BC patients and the development of novel
therapeutic strategies.
Funding: A set of potential biomarkers to discriminate breast cancer
subtypes.
PT04.15
Circulatory EVs as potential biomarkers of HIV-drug abuse
interactions and neurological dysfunction in HIV-Infected subjects and alcohol/tobacco
Users
Sunitha Kodidela
a, Kelli
Gertha, Namita Sinhaa, Asit Kumarb, Prashant
Kumara and Santosh Kumara
aUTHSC, Memphis, USA; bUniversity of Tennessee
Health Science Center, Memphis, USA
Introduction: Abuse of alcohol and tobacco can exacerbate HIV pathogenesis and
its associated complications. Further, the diagnosis of neurocognitive disorders associated
with HIV infection and drug abuse using CSF or neuroimaging are invasive or expensive
methods, respectively. Therefore, extracellular vesicles (EVs) can serve as reliable
non-invasive markers due to their bidirectional transport of cargo from the brain
to the
systemic circulation. Hence, we aimed to study the specific EVs proteins, which are
altered
in both HIV and drug abusers to identify a physiological marker to indicate the immune
status and neuronal dysfunction of HIV-positive drug abusers.
Methods: EVs were isolated from plasma of the following subjects: a) Healthy
b) HIV c) Alcohol drinkers d) cigarette Smokers e) HIV+alcohol drinkers f) HIV+cigarette
smokers. Quantitative proteomic profiling of EVs was performed by mass spectrometry
and
potential EV proteins associated with neuronal dysfunction were quantified by
westernblot.
Results: The EVs were characterized according to the ISEV guidelines. A total
of 343 proteins were detected in EVs of all the study groups. Comparison of proteins
among
all the study groups revealed that hemopexin was significantly altered in HIV+drinkers
compared to drinkers and HIV subjects. Further, our study is the first to show properdin
expression in plasma EVs, which was decreased in HIV+smokers and HIV+drinkers compared
to
HIV patients. Though we couldn’t identify the few other CNS-specific proteins, G-FAP
and
L1-CAM, associated with neuronal dysfunction in plasma EVs by Mass spectrometry, we
could
detect those by westernblot. The protein expression of GFAP (p < 0.01) was significantly
enhanced in plasma EVs obtained from HIV-positive subjects and drinkers compared to
healthy
subjects, suggesting enhanced activation of astrocytes in those subjects. The L1CAM
expression was found to be significantly elevated in smokers (p < 0.05). Both GFAP
and
L1CAM levels were not further elevated in HIV+smokers compared to HIV+nonsubstance
users.
Summary/Conclusion: The present findings suggest that hemopexin, and properdin
show potential as markers for HIV-drug abuse interactions. Further, astrocytic and
neuronal-specific markers (GFAP and L1CAM) can be packaged in EVs and circulate in
plasma,
which is further elevated in the presence of HIV infection, alcohol, and/or tobacco
and thus
may represent as potential biomarkers for neurological dysfunction in those subjects.
Funding: We thank the National Institute on Drug Abuse (DA047178) for
supporting our work.
.
PT04.16
Electrochemical detection of miRNA-21-5p
Verónica Serrano
a, Ana Rita
Cardosob and Maria G. Salesc
aUCoimbra/Biomark, Coimbra, Portugal;
bISEP/Biomark, Porto, Portugal; cBioMark Sensor Research/UC, Faculty
of Sciences and Technology, Coimbra University, Coimbra, Portugal, Porto, Portugal
Introduction: MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA
species that regulate gene expression post-transcriptionally, and are transported
by
extracellular vesicles (EVs). They play an essential role in biological processes,
such as
development, cell proliferation, apoptosis, stress response and tumorigenesis. Thus,
miRNAs
are considered relevant biomarkers in health. More particularly, miRNA-21-5p is expressed
in
neurons after traumatic brain injury, being expectably transported to peripheral fluids
by
brain EVs that cross the blood-brain barrier. The main goal of this work is to develop
an
electrochemical biosensor for the detection of miRNA-21-5p in serum.
Methods: Overall, the experimental assembly of the biosensor was made in three
stages. The first one consisted in the electrodeposition of AuNPs, the second one
in the
incubation of anti-miRNA21-5p on the carbon screen-s printed electrodes and the final
stage
in the incubation of mercaptosuccinic acid for blocking unspecific bindings. The probe
was
hybridized with the target miRNA21-5p by a consecutive incubation of several standard
solutions. Each modification was evaluated with cyclic voltammetry (CV), electrochemical
impedance spectroscopy (EIS) and square wave voltammetry (SWV). The electrochemical
behaviour of the biosensor was followed in all steps by monitoring the electron transfer
features of a standard redox system. The redox probe selected for this purpose was
[Fe(CN)6]4-/[Fe(CN)6]3-.
Results: The results indicated that the electrodeposition of gold was more
effective for −1.5 V for 600 s and could lead to better signals upon anti-miRNA-21-5p
hybridization.
Summary/Conclusion: In general, the experiments showed increasing charged
transfer resistance upon the incubation of higher concentrations of miRNA-21-5p.
Funding: The authors acknowledge the financial support European
Commission/H2020, through the project MindGAP/FET-Open/GA829040. ARC also acknowledges
funding to National Foundation for Science and Technology, I.P., FCT, through the
PhD Grant
SFRH/BD/130107/2017.
PT05: Advance in EV Quantitation
Chair: Malene M. Møller – Department of Clinical Immunology, Aalborg University
Hospital
Chair: Rienk Nieuwland, Ph.D. – University of Amsterdam
PT05.01
Accurate EV concentration is critical for experimental rigour
and reproducibility
Jean-Luc Fraikin, Lew Brown, Ngoc Do, Mark
Ruane and J. B. Vasquez
Spectradyne, Torrance, USA
Introduction: Extracellular vesicles (EVs) are studied for their potential as
powerful therapeutics and biomarkers of health and disease. Key to success in this
research
are rigorous experiments to quantify the cargo and biological activity of different
vesicle
preparations. In these experiments, EV concentration is a critical variable that must
be
carefully controlled to ensure scientific rigour and reproducibility: Without controlling
for concentration (dose), experimental outcomes will exhibit excess variability that
could
mask important biological discoveries.
In this study, three orthogonal methods are compared for accuracy in EV quantification:
Microfluidic Resistive Pulse Sensing (MRPS) and Nanoparticle Tracking Analysis (NTA)
were
compared to each other and relative to the gold standard method, Transmission Electron
Microscopy (TEM). The ability of NTA to accurately measure particle concentration
is shown
to depend on the polydispersity of the sample itself. Results validate the accuracy
of MRPS
and emphasize the importance of using orthogonal techniques to quantify EVs.
Methods: Reference urinary vesicles were prepared and analysed with the three
methods and the relative concentration accuracy of NTA and MRPS were compared as a
function
of particle size. The hypothesis that NTA concentration accuracy was impeded by sample
polydispersity was tested using polystyrene bead mixtures having a range of polydispersity.
A theoretical argument based on fundamental physics explains the experimental
observations.
Results: TEM and MRPS measurements of the EVs were in excellent agreement and
showed a broad, polydisperse particle size distribution with no peak on the measured
size
range (50 nm – 400 nm diameter). NTA differed significantly from TEM and MRPS by reporting
a
steep decrease in measured concentration below about 150 nm that resulted in a peak
in the
reported particle size distribution. Bead measurements confirmed the hypothesis to
be
tested: Sample polydispersity significantly affects the ability of the NTA method
to
accurately measure concentration, even for particles as large as 150 nm diameter.
Summary/Conclusion: These experiments validate MRPS as an accurate method for
quantifying EVs and highlight the importance of using orthogonal measurement methods
in
accordance with MISEV2018 guidelines.
PT05.02
Clinically relevant synthetic reference materials to standardize
concentration measurements of extracellular vesicles: state-of-the-art and future
prospects
Zoltan Vargaa
, Edwin van der
Polb, Daniel Geißlerc, Ute Resch-Gengerc and Rienk
Nieuwlandd
aBiological Nanochemistry Research Group, Institute of
Materials and Environmental Chemistry, Research Centre for Natural Sciences, Budapest,
Hungary, Budapest, Hungary; bDepartment of Clinical Chemistry, Amsterdam UMC,
University of Amsterdam, Amsterdam, the Netherlands; Vesicle Observation Center, Amsterdam
UMC, University of Amsterdam, Amsterdam, the Netherlands; Department of Biomedical
Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands,
Amsterdam, Netherlands; cBundesanstalt für Materialforschung und – Prüfung,
Berlin, Germany; dDepartment of Clinical Chemistry, Amsterdam UMC, University of
Amsterdam, Amsterdam, the Netherlands, Vesicle Observation Center, Amsterdam UMC,
University
of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands
Introduction: There is an unmet need to standardize concentration measurements
of extracellular vesicles (EVs). Flow cytometry is the clinically most applicable
method,
but the currently available reference materials for calibration are insufficient.
For
example, the refractive index (RI) between standard particles and EVs substantially
differs,
whereas concentration and fluorescence calibration particles are too bright. The goal
of
this study is to ascertain the most desired properties of reference materials to standardise
EV measurements.
Methods: An online survey was prepared within the MEVES II project to measure
the desired size, concentration range, optical properties, choice of fluorochromes,
and
stability of synthetic EV reference materials for flow cytometry (FCM) measurements.
Besides
the desired properties of EV reference particles, also the available instrumentation
was
assessed in the survey, which was sent to the members of the Stakeholder Committee
of METVES
II project and members of the EV Flow Cytometry Working Group.
Results: The most desired size, concentration, and RI range for EV reference
particles is 50 nm to 500 nm, 10E7 to 10E10 1/mL, and 1.35–1.45, respectively. Based
on
Mie-theory evaluation of the sensitivity of the available instruments, none of the
respondents would be able to detect 50 nm particles with RI = 1.35 with their current
instruments. Regarding fluorescence intensity, the most desired range according to
the
responses is from 10 molecules of equivalent soluble fluorochromes (MESF) to 10 000
MESF.
Considering the sizes of EVs and fluorescent labels, the maximal MESF that can be
obtained
for EV reference particles with 50 nm diameter and high molecular mass fluorescent
dyes is
in the range of several hundreds. Typical antigen densities on EVs fall below 100
copies per
EV with 50 nm diameter, i.e. MESF values above 100 are probably not physiologically
relevant
in this size range.
Summary/Conclusion: A part of the desired properties of EV reference materials
precludes either their physical feasibility of production or their detection at most
currently available FCMs, meaning that the intended reference materials will be
future-proofed.
Funding: This work was supported under 18HLT01 METVES II project by the
European Metrology Programme for Innovation and Research (EMPIR). The EMPIR initiative
is
co-funded by the European Union’s Horizon 2020 research and innovation programme and
the
EMPIR Participating States.
PT05.03
Comparison of production and activity of amniotic fluid stem
cell extracellular vesicles from 3D hollow fibre bioreactor and 2D culture.
Charmi Dedhiaa, Paolo Neviania, John
Cadwellb, Sargis Sedrakyanc and Laura
Perinc
aChildren’s Hospital Los Angeles, Los Angeles, USA;
bFiberCell Systems, New Market, USA; cChildren’s Hospital Los
Angeles-University of Southern California, Los Angeles, USA
Introduction: EV clinical translation is limited by scale-up of EVs
production. Hollow fibre bioreactors (HFBR) support culture of large numbers of cells
at
high densities. Culture conditions may affect EV composition and potency. Here we
compare
production, potency, identity and therapeutic potential of EVs collected from cells
grown in
culture dish (2D) versus HFBR (3D).
Methods: Human clonal AFSC were derived from patient-consented amniotic
fluids. 1x10e6 hAFSC were seeded in 2D (145cm2), and 1.6X10e7 hAFSC on a small 20kd
MWCO
HFBR (FiberCell-C2025D, 450cm2) with fibronectin coating; both cultured in Chang medium
with
20% of ES-FBS, starved for 24 hr and then EVs collected. The effect of harvest frequency
was
tested (8hrs, 24 hr, 72hrs,1 wk). 2D-EVs and 3D-EVs were compared by Nanosight, potency
assay (by WB), identity (by Exoview analysis) and therapeutic effect (in vivo in an
animal
model of kidney disease, Alport Syndrome).
Results: 2D production was ~5.5X10e9 EV/ml/24 hrs while 3D was
~2.8X10e10 EV/ml (first four 24 hrs) and ~4.4X10e10 EV/ml (two days of hourly harvests).
Very little difference in EV concentration and very similar size distribution (~130 nm)
were
observed during harvest intervals; possibly indicating either significant EV re-uptake
or
inhibition of EV secretion dependent upon free EV in the supernatant. 3D-EVs trapped
VEGF
(an in vitro established potency assay) as efficiently as 2D-EVs, and expressed CD9,
CD81,
CD63, CD80, CD86 and VEGFR1 as 2D-EVs.
Summary/Conclusion: 3D-EVs had comparable properties and bio-activity to
2D-EVs, but the HFBR produced 10x more EVs. HFBR cell culture conditions for hAFSC
still
need optimization, however an available 1.2 m2 cartridge provides a 50X scale up potential.
The HFBR, a cGMP closed system, can produce sufficient numbers of EV to support pre-clinical
and clinical applications with at least similar properties to EVs produced by conventional
2D methods.
Funding: – Intramural funding
- intramural EV Core Pilot funding
PT05.04
Demonstration of High Gain mode in combination with Imaging Flow
Cytometry for improved EV analysis
Haley R. Pugsley and Raymond Kong
Luminex Corporation, Seattle, USA
Introduction: Extracellular vesicles (EVs) are membrane-derived structures
that include exosomes, microvesicles, and apoptotic bodies. In recent years, the importance
of EVs has become apparent, as they are key mediators of intercellular communication.
However, quantifying and characterizing EVs in a reproducible and reliable manner
is
challenging due to their small size – exosomes range from 30 to 100 nm in diameter.
It is
well-known that flow cytometers were originally designed to measure and detect cells,
and
due to the quantitative power flow cytometry offers, there has been a push to quantify
and
characterize EVs using flow cytometric methods. However, these systems have not been
designed to measure objects smaller than a cell.
Methods: Here, we describe the use of High Gain mode on the Amnis®
ImageStream® Imaging Flow Cytometer to address the challenges of measuring small particles.
In this new High Gain mode, the charge-coupled device (CCD)-camera is manually adjusted
to
higher gain settings, increasing the signal obtained from the EV. Object thresholds
and
masking have also been adjusted to better identify and detect small particles.
Results: Preliminary results using murine leukaemia virus-sfGFP reference
particles have shown up to a fivefold increase in the number of GFP-positive objects
collected in High Gain mode, when compared to standard gain on the ImageStream System.
Summary/Conclusion: In this study, we demonstrate improved small particle
detection, including EVs, using this new High Gain mode on the ImageStream Imaging
Flow
Cytometer.
PT05.05
Distance-controlled accelerated catalysed hairpin DNA circuit
for multiple and sensitive detection of exosomes-associated miRNAs
Ye Zhanga
, Shihua Luob
and Lei Zhengc
aDepartment of Laboratory Medicine/Guangdong Engineering and
Technology Research Centre for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern
Medical University, Guangzhou, 510515, Guangdong Province, PR China, guangzhzou, China
(People’s Republic); bDepartment ofLaboratory Medicine, Nanfang Hospital,
Southern Medical University, Guangzhou, 510515, China, guangzhou, China (People’s
Republic);
cDepartment of Laboratory Medicine, Nanfang Hospital, Southern Medical
University, Guangzhou, 510515, China, guangzhou, China (People’s Republic)
Introduction: Sensitive and simultaneous monitoring of multiplexed
exosome-associated RNAs is of great value for early cancer diagnosis remains a
challenge.
Methods: Here, we report a simple, multiple and sensitive exosomes-associated
multiplex miRNAs detection method that uses distance-controlled accelerated catalysed
hairpin DNA circuit (CHDC) system without any complex operation or enzymatic amplification.
The distance-controlled accelerated CHDC can directly enter the plasma exosomes to
generate
fluorescent signal quantitatively by specifically targeting miRNAs without any transfection
means.
Results: We show that distance-controlled accelerated CHDC strategy with
signal amplification capability could selectively and sensitively identify low level
RNAs in
serum EVs, distinguishing patients with early- and late-stage breast cancer from healthy
donors and patients with benign breast disease.
Summary/Conclusion: This simple, accurate, sensitive, and cost-effective
liquid biopsy by the distance-controlled accelerated CHDC method is potent to be developed
as a non-invasive breast cancer diagnostic assay for clinical applications.
PT05.06
Impact of isolation methods on biophysical heterogeneity of
single extracellular vesicles
Shivani Sharma
University of California Los Angeles, CA, Los Angeles, USA
Introduction: Current biophysical analysis of extracellular vesicles (EVs)
typically encompasses particle density and size distribution determinations using
various
techniques. However, variabilities in EV isolation methods and the structural complexity
of
these biological-nanoparticles (sub-100 nm) necessitate more rigorous nanoscale biophysical
characterization of single EVs to facilitate more reliable and comparable EV-based
assays.
Methods: Combining atomic force microscopy (AFM), super-resolution optical and
conventional particle sizing light scatter and microfluidic techniques, we compared
the
unique sub-nanometre scale biophysical properties of breast cancer cell-derived EV
isolates
obtained using different isolation methods.
Results: AFM and dSTORM particle size distributions showed coherent unimodal
and bimodal particle size populations in centrifugation and immune-affinity isolates
respectively. More importantly, AFM imaging revealed striking differences in nanoscale
morphology, surface undulations, and vesicle-to-non-vesicle ratios among EV isolates
from
different isolation methods. Our findings demonstrate the effectiveness of orthogonal
high-resolution biophysical characteristics of single EVs, not discernable via particle
size
distributions and counts alone.
Summary/Conclusion: The identified nanoscale biophysical characteristics of EV
isolates represent a strategic and complementary framework to resolve differences
in the
heterogeneity and purity of EVs from different cell types and isolation techniques.
Funding: We acknowledge funding (1R21CA218386-01A1) from the National Cancer
Institute/NIH Program to Assess the Rigour and Reproducibility of Exosome-Derived
Analytes
for Cancer Detection, and the UCLA SPORE in Brain Cancer – Career Enhancement Program
Award.
PT05.07
Ready-to-use, pre-labelled and stable human EVs for validating
new standardization procedures of EV concentration measurements
Britta A. Bettina
, Kathariina
Maaninkab, Chi Hauc, Rienk nieuwlandc, Pia
Siljanderd and Edwin van der Pole
aDepartment of Clinical Chemistry, Amsterdam UMC, University
of Amsterdam, Vesicle Observation Center, Amsterdam UMC, University of Amsterdam,
Amsterdam,
the Netherlands, Amsterdam, Netherlands; bEV group, Molecular and Integrative
Biosciences Research Programme, Faculty of Biological and Environmental Sciences,
and CURED,
Drug Research Program, Faculty of Pharmacy, UnIversity of Helsinki, Helsinki, Finland;
cDepartment of Clinical Chemistry, Amsterdam UMC, University of Amsterdam,
Amsterdam, the Netherlands, Vesicle Observation Center, Amsterdam UMC, University
of
Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands; dFaculty of
Biological and Environmental Sciences, University of Helsinki, Finland, Helsinki,
Finland;
eDepartment of Clinical Chemistry, Amsterdam UMC, University of Amsterdam,
Amsterdam, the Netherlands; Vesicle Observation Center, Amsterdam UMC, University
of
Amsterdam, Amsterdam, the Netherlands; Department of Biomedical Engineering and Physics,
Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam,
Netherlands
Introduction: Extracellular vesicle (EV) concentrations measured by flow
cytometry are incomparable. To improve comparability, the METVES II consortium is
developing
traceable reference materials and procedures, which require validation by test samples.
In
previous interlaboratory comparison studies, however, a main source of variation was
introduced by pre-analytical variables and measurement artefacts introduced by test
samples.
To minimize variation introduced by test samples, our aim is to develop off-the-shelf
biological test samples containing pre-labelled EVs.
Methods: Human urine and plasma were collected from healthy donors. EVs were
labelled with lactadherin-FITC, isolated by size-exclusion chromatography to remove
free dye
and minimize swarm detection, and mixed with dimethyl sulphoxide (DMSO), ExoCap or
trehalose, frozen in liquid nitrogen and stored at −80°C. After thawing, EV concentrations
were measured by a calibrated flow cytometer (Apogee A60-Micro).
Results: Compared to the EV concentrations measured in fresh plasma and urine,
the concentrations decreased 27% in plasma (p = 0.04; mean of the 3 cryopreservation
agents)
and 35% in urine (p = 0.05) after one day of storage. After 5 months of cryopreservation,
the concentration of plasma EVs decreased 2% (DMSO and Exocap) and 8.5% (trehalose)
compared
to one day of storage, whereas the concentration of urine EVs decreased 6% (Exocap)
and 18%
(DMSO and trehalose).
Summary/Conclusion: We have developed ready-to-use, pre-labelled human EVs
that are stable up to 5 months and dedicated for use in interlaboratory comparison
studies.
To further increase stability, other cryopreservation agents will be tested. Our biological
test samples will be key to validate the new reference materials and procedures developed
by
METVES II in 2021.
Funding: This project has received funding from the EMPIR program co-financed
by the Participating States and from the European Union’s Horizon 2020 research and
innovation program.
PT05.08
Understanding intracellular fate of EV-delivered
content
Killian O’Briena
, Stefano
Ughettob and Xandra O Breakefieldc
aMassachusetts General Hospital, Boston, USA;
bMGH-Harvard Medical School, Charlestown, USA; cMGH-Harvard Medical
School, Charlestown, USA
Introduction: Despite much work performed on evaluating the potential effects
of extracellular vesicles (EVs), the functional uptake of their cargo is still
controversial. This project aimed to demonstrate that EV content (protein and mRNA)
is
protected and can be subsequently transferred with functional activity into recipient
cells,
while also developing a tool to assess and quantify functional EV uptake.
Methods: Fusion proteins used were mitochondrial localized
coxVIII-CFP-nanoluc(Cox) and nuclear localized H2B-RFP-nanoluc(H2B).
Results: HEK293 T cell-derived EVs protected Cox proteins from proteinase K
digestion while demonstrating significantly improved efficiency of uptake when compared
to
free protein, as measured by bioluminescence that was still detectable in recipient
cells 96
hrs post-EV-exposure. To confirm functional uptake, recipient cells exposed to EVs
containing H2B for 72 hrs were imaged and some recipient cells manifested fluorescent
red
nuclei. To demonstrate the presence of functional mRNA within EVs, producer cells
were
transfected for such a duration as not to have detectable levels of protein in the
EVs while
still containing detectable levels of mRNA (qPCR) even after RNaseA treatment. Transfer
of
these EVs to HeLa cells showed an increase in expression of H2B which was blocked
by
cyclohexamide, confirming translation of the mRNA (2.2kb). To determine if recycling
of EV
delivered proteins occurs, recipient HeLa cells were exposed to EVs containing Cox
for
72 hrs. All extracellular EVs were removed and cells were trypsinized (0.25% for 30 min)
to
remove any non-internalized Cox protein. 48 hrs later, EVs (CD63+ and CD9+) released
from
cells contained Cox suggesting recycling of protein or possibly recycling of entire
EVs.
Lastly, an assay was developed to measure functional EV uptake. Nanoluc protein was
split in
two and fused to mTurquoise2(N65) or mScarlet-I(66 C). Expression of each fragment
alone
exhibited non-detectable levels of luminescence while expressing both together had
a
significantly increased signal. Delivery of either fragment within an EV to a cell
expressing the corresponding fragment worked as confirmation and quantification of
EV uptake
(HEK293, U87, HeLa cells).
Summary/Conclusion: This study robustly demonstrates EV delivery of functional
mRNA and protein to cells, while also establishing a simple assay to quantify and
validate
functional EV uptake.
PT05.09
Theoretical model of EV losses due to adsorption on the tube
walls. Application for immunomagnetic detection of the vesicles
Mikhail A. Livshitsa, Irina Yu. Petrushankoa,
Victoria N. Lavrenovab, Eduard V. Generozovb and Elena
Khomyakova
c
aEngelhardt Institute of Molecular Biology, Russian Academy
of Sciences, 32, Vavilova str., Moscow, 119991, Russian Federation, Moscow, Russia;
bFederal Research and Clinical Center of Physical-Chemical Medicine of Federal
Medical Biological Agency, 1a, Malaya Pirogovskaya str., Moscow, 119435, Russian Federation,
Moscow, Russia; cFederal Research and Clinical Centre of Physical-Chemical
Medicine of Federal Medical Biological Agency, Paris, France
Introduction: Short-term storage of unfrozen samples of vesicles, mainly at
4°C, overnight or during a couple of days is rather common laboratory practice. However,
it
was found to lead to significant losses of vesicle concentration supposedly due to
adsorption on the walls of the tube. The present work develops a theoretical model
intended
to describe the vesicle adsorption process. The experimental validation of the model
was
made using method of immunomagnetic precipitation.
Methods: The theoretical model considers the “diffusion-limited” case of
vesicles storage. The maximal adsorption capacity of the surface of contact between
the tube
and the solution is given as the number of vesicles in hexagonally packed monolayer.
For experiment, the vesicles were purified from HT29 cell culture supernatant by
differential centrifugation, aliquoted and kept at −80 C. Further the aliquots were
consequently unfrozen, and placed into the tubes with different surface treatment
and kept
at +4 C. The kinetics of vesicles loss was measured by anti CD9 immunomagnetic capturing
followed by CD81, EpCam and CD166 staining and flow cytometry.
Results: The model allows the estimation of the adsorption-associated losses
as dependent on initial vesicles concentration, volume of the solution, tube geometry,
the
storage temperature and duration case of quiet vesicles storage (without mixing) and
also
accounts an expected effect of active agitation of the solution (EV-beads complexes
formation).
Theoretical calculations were illustrated by analysis of EV at different storage conditions
and during reaction of immunomagnetic precipitation of the vesicles.
Summary/Conclusion: It was demonstrated that application of tubes surface
treatment allows increasing sensitivity of immunomagnetic precipitation method to
2x10^5 for
CD81+, 5x10^5 for EpCam+ and 2x10^8 for CD166+ vesicles.
PT05.10
Videodrop by Myriade: the use of interferometric microscopy to
quantify vesicles in complex samples such as faecal filtrates
Romain Sausset
a, Marie-Agnès
Petitb and Marianne Depaepeb
aINRA, UMR1319, Micalis, Paris, France; bINRA,
UMR1319, Micalis, Jouy en Josas, France
Introduction: It is now largely accepted that the intestinal microbiota plays
a key role in Intestinal Bowel Diseases (IBD). An imbalance in the composition and
diversity
of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed
out by
several teams. There are also indications that extracellular vesicles produced by
bacteria
and exosomes produced by epithelial cells might be increased in this family of diseases.
Methods: In order to differentiate healthy and IBD faecal samples on the basis
of their vesicle profiles, we want to develop a means to enumerate rapidly particles
in
faecal samples, based on interferometric microscopy. The Videodrop technology, developed
by
Myriade, relies on the creation of single beam interferences between two signals from
the
same light path by nanoparticles such as small vesicles. It will permit to compare
on large
scales the viral load of healthy subjects and IBD patients.
Results: This fast and easy-to-use device was compared to the NTA on several
types of eukaryotic and prokaryotic vesicles and our preliminary results are
encouraging.
Summary/Conclusion: If IBD patients indeed have an increased number of
vesicles in their stools, the Videodrop could be a new diagnosis tool for such
conditions.
PT06: EVs in Stem Cells (including Cancer Stem Cells)
Chair: Sai Kiang Lim, MDPhD – Institute of Medical Biology, Agency for Science,
Technology and Research, Singapore. Department of Surgery, Yong Loo Lin School of
Medicine, National University of Singapore, Singapore
Chair: Gareth R. Willis – Harvard University & Boston Children’s
Hospital
PT06.01
Characterization of small extracellular vesicles produced by
human mesenchymal stromal cells in an improved extracellular vesicle-free
medium
Arthur V. Sampaio, Jennifer Christie, Isaac
Jonker, Connor Johnson, Mandy Chan, Adil Kassam, Ravenska Wagey, Allen Eaves, Stephen
Szilvassy and Sharon A. Louis
STEMCELL Technologies Inc., Vancouver, Canada
Introduction: Small extracellular vesicles (sEVs) produced by mesenchymal
stromal cells (MSC-sEVs) may be useful in cell-free therapies for immunomodulation
and
tissue regeneration.
Methods: To characterize MSC-sEVs produced ex vivo, human bone marrow MSCs
were cultured in MesenCult-ACF Plus (MACFP), an EV-free and animal component-free
culture
medium for 3 days and spent medium collected to isolate sEVs by ultracentrifugation
(UC).
Analyses of sEVs were performed by Nanoparticle-tracking analysis (NTA), western blot
(WB),
and human umbilical vein endothelial cell (HUVEC) tube formation assay.
Results: Analysis of fresh uncultured MACFP by UC, NTA and WB for CD63, CD81,
and CD9 confirmed the absence of sEVs. MSC-sEVs isolated from spent MACFP by UC ranged
from
80–150 nm in size and were positive for CD63, CD9, and CD81 proteins. These sEVs could
be
stored at −80°C for >4 months in solution or lyophilized with minimal loss based on
NTA
and WB analysis. The MSC-sEVs contained the MSC-associated microRNAs let7a, miR21,
and
miR26a as per qPCR analysis. The biological function of ex vivo isolated MSC-sEVs
was
assessed using a human umbilical vein endothelial cell (HUVEC) tube formation assay.
HUVECs
treated with MSC-sEVs generated tubes as early as 6 h after seeding, which were not
observed
in control HUVEC cultures until 15 h. Moreover, the number of branch points present
in such
tube structures was >fourfold higher in HUVEC cultures (n = 5) supplemented with MSC-sEVs
versus control, with the former lasting >60 h and the latter lasting <50 h in culture.
Direct comparison of the performance of MACFP medium to media containing non-depleted
or
EV-depleted foetal bovine serum demonstrated that only MSCs cultured in MACFP (n = 3)
were
able to expand robustly with a doubling time of 1.1, 2.1 and 8.9 days in these media,
respectively. Lastly, methods for isolating sEVs using newly developed EasySep-EV™
magnetic
separation kits and size exclusion columns will be presented.
Summary/Conclusion: Taken together, these data demonstrate that MSC-sEVs can
be produced in high yield in MACFP medium and that these possess similar physical,
phenotypic and functional characteristics as sEVs in vivo.
Funding: This work was privately funded by STEMCELL Technologies Inc.
PT06.02
Comparison of classical and imaging flow cytometry platforms for
the characterization of stem cell-derived extracellular vesicles
Elżbieta Karnasa
, Katarzyna
Kmiotek-Wasylewskaa, Zbigniew Madejab and Ewa K.
Zuba-Surmab
aMalopolska Centre of Biotechnology, Jagiellonian University,
Krakow, Poland, Kraków, Poland; bDepartment of Cell Biology, Faculty of
Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland,
Kraków,
Poland
Introduction: Extracellular vesicles (EVs) are heterogeneous group of small
vesicular structures released by different types of cells, including stem cells (SCs).
As
recent studies demonstrate that they may enclose bioactive content and transfer it
into the
target cells, growing interest is placed on the utilization of EVs in the field of
biomedical research. However, there is still lack of standardized methods of EVs
characterization. As an example, typical flow cytometry-based protocols, commonly
used for
cells phenotyping, may be inadequate for the characterization of EVs as particles
with size
close to the detection limit of conventional cytometers. Thus, the aim of this study
was to
optimize and compare the use of different flow cytometry platforms for the multiparameter
analysis of EVs isolated from different types of SCs populations.
Methods: EV samples were obtained by ultracentrifugation of conditioned media
collected from selected SCs types, including human induced pluripotent SCs (iPS) and
mesenchymal SCs (MSCs). Next, several high resolution flow cytometry systems: Cytoflex,
Apogee (A50 and A60 Micro-Plus) and Image Stream Mk II were employed to compare their
sensitivity and resolution, as well as influence of “swarm” effect. Furthermore, we
examined
EVs phenotype, including expression of tetraspanins and other surface markers.
Results: Our results have revealed that tested flow cytometry systems may be
utilized for the phenotypic characterization of EVs secreted by SCs populations. However,
the conventional staining and gating strategy protocols have to be thoroughly optimized.
Additionally, depending on a type of tested cytometer, we have demonstrated the difference
in a “swarm” effect and its influence on obtained results regarding EVs phenotype.
Finally,
imaging flow cytometry platform was also employed to visualize EVs on the single particle
level.
Summary/Conclusion: In conclusion, we have demonstrated that tested
high-resolution flow cytometry platforms are convenient methods for the multiparameter
characterization of EVs produced by different types of SCs populations. However, careful
selection of particular measurement parameters should be performed depending on a
type of
employed system.
Funding: This study was funded by NCBR grant STRATEGMED III
(STRATEGMED3/303570/7/NCBR/2017) to EZS.
PT06.03
Evaluation of ATCC’s exosomes from cell culture supernatant as
reference standards in research and development.
Siddhartha Paula
, Heather
Branscomeb, Nanda Mahashettyc, Dezhong Yina, Lili Lili
Wangd, David A. Routenberge, Elzafir Elsheikhd, Wyatt
Vreelandd and Thomas Cleveland Clevelandd
aAmerican Type Culture Collection, Gaithersburg, USA;
bAmerican Type Culture Collection, Manassas, USA; cAmerican Type
Culture Collection, Gaithersburg, USA; dNational Institute of Standards and
Technology, Gaithersburg, USA; eMeso Scale Diagnostics, LLC., Rockville, USA
Introduction: Exosomes are subcellular particles 30–150 nm in size released
from cells through a fusion of multicellular bodies with the plasma membrane. Exosomes
are
stable carriers of cell-free cargo in the form of DNA, RNA, and proteins, thereby
making
them an attractive candidate for diagnostic and therapeutic applications. However,
isolating
a consistent population of exosomes can be challenging and there is an unmet need
for highly
characterized exosomes for use as reference standards in extracellular vesicle research
(EV).
Methods: Exosomes were isolated from cell culture supernatants of different
ATCC cell lines including stem cells and cancer cell lines representing the most prevalent
cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using
Tangential flow filtration (TFF). These exosomes underwent sterility and mycoplasma
tests as
a part of their quality control. The morphology and size distribution of these exosomes
were
evaluated through multiple strategies including Nanoparticle tracking analysis (NTA),
Asymmetrical flow field-flow fractionation (AF4), Cryo-electron microscopy (Cryo-EM)
and
Spectra DyneTM particle analyser. Exosome surface markers were also analysed through
multiple strategies such as electro chemiluminescent ELISA, flow cytometry and western
blotting. Also, stem cell exosomes and cancer exosomes were further evaluated for
functionality through in vitro functional assays including migration assay, angiogenesis
and
anchorage independent growth assay.
Results: Our optimized TFF method resulted in high yields of > 1 × 1010
exosomes/mL and average protein equivalent of more than 1 mg/mL. More than 90% of
the
exosomes population had an average size distribution of 50–200 nm and median size
of 110 nm
confirmed through a number of different size distribution instruments. Although cell
line
dependent, we were able to obtain similar expression levels of different cell surface
markers including tetraspanins (CD63, CD81, CD9) when evaluated through different
methods.
Our functional data demonstrated stem cell exosomes were functionally active in promoting
cell migration and tubule formation. Additionally, cancer cell exosomes were found
to
promote a malignant phenotype in an anchorage independent growth assay.
Summary/Conclusion: Collectively, we demonstrated our ability to reproducibly
manufacture production-scale batches of exosomes from multiple different cell types.
Our
purified exosomes are of high yield, meet well-established quality control specifications,
and are robust in maintaining size distribution, surface marker expression, and
functionality in vitro. Therefore, they can serve as ideal reference materials that
can
support different EV-based research applications.
PT06.04
Exo-cise: Extracellular vesicles enriched from plasma
post-exercise promotes myogenesis and neurogenesis
Bianca Parisa, Yaomeng Liua, Vicente
Pagalday-Vergaraa, Julie Daviesb, Priya Samuela, Ayman
Abu Seera, Johnny Colletta, Laura Gathercolea, Ken
Howellsa, Karl J. Mortenb, Zhidao Xiaa, Daniel
Anthonyb, David R F. Cartera, Helen Dawesa and
Ryan C. Pinka
aOxford Brookes University, Oxford, UK;
bUniversity of Oxford, Oxford, UK
Introduction: Physical activity brings about a widespread physiological
response and elicits the beneficial adaptation of several tissues and organs. Furthermore,
regular participation in physical activity reduces the risk of developing major
non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis,
and dementia. Two important processes known to occur following physical activity are
myogenesis and neurogenesis; both of which involve the activation and proliferation
of
specialised tissue-resident stem cells. The molecular mechanisms regulating these
processes
following exercise are poorly understood to date. Here, we investigated the contribution
of
extracellular vesicles, which are released into the circulation after exercise, to
benefit
adult myogenesis and neurogenesis.
Methods: Small extracellular vesicles were enriched from the blood of healthy
participants before and following maximum and moderate intensity exercise. Differentiation
and proliferation using a range of methods was measured following vesicle treatment
onto
primary myoblasts and neuronal primary ex-vivo stem cells. Activation of key cellular
pathways were measured.
Results: We show significant proliferation and differentiation changes of both
stem cell types. This is independent of extraction method, extracellular vesicle depleted
fractions and is interestingly conserved across mammalian species. Remarkably, we
see an
age-related effect.
Summary/Conclusion: This advocates that short single bouts of exercise may
promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles
which
opens an interesting field in endogenous EV therapies.
Funding: The Royal Society and Oxford Brookes University Nigel Groome
Studentship.
PT06.05
Extracellular vesicle characterization in human induced
pluripotent stem cell-derived retinal pigment epithelium
Takerra K. Johnsona
, Ameera
Mungaleb, Rafael Villasmilb, Peter Backlundc, Temesgen
Fufab and Robert Hufnagelb
aNational Eye Institute, Laurel, USA; bNational
Eye Institute, Bethesda, USA; cNational Institute of Child Health and Human
Development, Bethesda, USA
Introduction: Clinical-grade induced pluripotent stem cell-derived retinal
pigment epithelial cells (hiPSC-RPE) show promise as a cell-based therapy for retinal
degeneration. While clinical trials are ongoing, the potential of extracellular vesicles
(EVs) as biomarkers for monitoring eye health and disease is not well studied. This
study
characterized the EV surface profile and cargo of hiPSC-RPE to offer a baseline assessment
in normal and disease conditions. Moreover, we evaluated the importance of PNPLA6,
a gene
involved in membrane integrity and when mutated causes retinal degeneration, in EV
biogenesis and secretion.
Methods: EVs were isolated from serum-free culture medium of hiPS-RPE and
identified with nanoparticle tracking analysis, transmission electron microscopy,
and
immunoblot analysis of exosomal markers, including Alix, TSG101, and CD63. Surface
marker
detection and proteomic profiling were completed using an EV surface marker kit and
mass
spectrometry, respectively. Small interfering RNA targeting PNPLA6 was used to knockdown
the
expression in hiPSC-RPE and EVs were characterized.
Results: Nanoparticle tracking analysis confirmed the presence of both
microvesicles (>150 nm) and exosomes (<150 nm) by size distribution and the
concentration of EVs (1x108 particles/ml) from RPE. TEM displayed typical morphological
characteristics of EVs. The presence of known EV markers, Alix, TSG101, and CD63 was
confirmed via immunoblot and flow cytometry. Surveillance of EV surface markers revealed
enrichment of epithelial markers (CD326) and stem cell markers (CD133/1) that depict
donor
cell origin and functional proteins including integrin-binding (CD29) and TGF-beta
receptors
(CD105). In addition, proteomic analysis revealed regulators of inflammation and RPE
function, including hemopexin, clusterin, complement factor I, and pigment
epithelium-derived factor. Furthermore, reduction in PNPLA6 expression reduced vesicle
secretion and vesicle size compared to non-targeting controls.
Summary/Conclusion: HiPSC-RPE expresses a population of EVs reflective of
normal RPE function. The knockdown of PNPLA6 negatively impacts vesicle secretion
and
suggests a role for EVs in retinal health. Future studies will elucidate the role
of EVs in
retinal maintenance and disease states.
Funding: NEI Intramural Funding and Knights Templar Eye Foundation, Inc
PT06.06
Increased VEGF secretion from MSC cell culture in normoxic
condition
Angliana Chouw, Geofanny Facicilia, Annisa Nur
Arofah and Cynthia Retna Sartika
PT. Prodia StemCell Indonesia, Jakarta, Indonesia
Introduction: Vascular endothelial growth factor (VEGF) is a potent angiogenic
factor and was first described as an essential growth factor for vascular endothelial
cells.
VEGF plays a role in normal physiological functions such as bone formation, haematopoiesis,
wound healing, and development. Mesenchymal stem cell (MSC) was found to secretes
potential
growth factors such as VEGF when cultured in vitro. However there are some beliefs
that
Foetal Bovine Serum (FBS) which usually used as serum in cell culture content VEGF.
Methods: MSC seeded in in 96-well plate in with concentration of 5,000
cell/well. Cells were incubated for 24 hours and fasted for another 16 hours using
only
DMEM. Cells were treated with complete medium consist of DMEM and 10% FBS. Culture
medium
were collected after 5, 12, and 24 hours after treatment. Cell were culture in 37ºC
dan 5%
CO2. VEGF concentration was detected using ELISA technique.
Results: VEGF concentration was not found in FBS which do not contact with
MSC. An increasing of VEGF concentration in time-dependent manner was shown when culture
medium was used in MSC cell culture in normoxic condition. The result of VEGF concentration
when culture 5, 12, and 24 hours were 74.022 pg/mL, 76.67 pg/mL, and 93.58 pg/mL,
respectively. The mechanism of MSC release growth factor is still under investigated.
However, the Classic growth factors and cytokines serves paracrine control molecules
which
were important in regenerative medicine. VEGF was found to be an important molecules
in
angiogenesis process and determine the fate of cells.
Summary/Conclusion: MSC secreted VEGF and concentration increased in
time-dependent manner.
PT06.07
Isolation and characterization of exosomes from canine stem
cells
David L. Simpsona
, Anthony
Diominob and Dori Borjessonc
aWentworth Institute of Technology, Boston, USA;
bUniversity of California, San Diego School of Medicine, San Diego, USA;
cUniversity of California, Davis, Davis, USA
Introduction: Unlike induced disease models using laboratory animals,
naturally occurring disease models display pathophysiologic attributes that are more
similar
to human diseases. Unfortunately these models are underutilized in translational
regenerative medicine research. This is partly due to the slow development of
species-specific experimental therapeutics to investigate comparative efficacy. Thus,
we set
out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem
cells
(cAD-MSC) to use as a comparative therapeutic in dogs. To accomplish this, we optimized
an
isolation and purification strategy and characterized their molecular properties.
Methods: Exosomes were isolated by sequential centrifugation and subsequent
ultrafiltration. The proteome was characterized by tandem mass tag (TMT) mass spectrometry
and the miRNA cargo was identified using a canine specific PCR array with subsequent
target
and enrichment analysis using TargetScan and the Panther platform, respectively. Also,
nanoparticle tracking analysis and transmission electron microscopy were used to determine
exosome size and structure. To investigate bioactivity, we measured the ability of
exosomes
to inhibit collagen production in an in vitro model of fibrosis.
Results: Exosomes were purified by ultrafiltration using a 100 KDa cut-off.
Proteomic analysis by TMT mass spectrometry identified 1262 unique proteins. 90% of
the
ExoCarta top 100 were identified from this list. Additionally, we identified the miRNA
cargo
within exosomes and found 27 highly expressed miRNAs. Enrichment analysis identified
multiple pathways of probable regulation including angiogenesis (Fold Enrichment = 3.1;
p < 0.0001) and transforming growth factor-beta (TGFb) signalling (Fold Enrichment = 4.1;
p < 0.0001). Exosome size was quantified to be 119.7 ± 3.4 nm with a modal average
of
98 nm. Lastly, in the presence of exosomes, TGFb stimulated fibroblasts deposited
32.2% less
collagen than vehicle controls (p = 0.036).
Summary/Conclusion: In summary, cAD-MSCs exosomes display structural and
functional features comparable to stem cell derived exosomes from other species. Use
of
these exosomes in naturally occurring disease canine models may provide superior predictive
value for human clinical trials.
Funding: Support provided by the CCAH, School of Veterinary Medicine, UC
Davis.
PT06.08
Mesenchymal stem cells-derived exosomes promote in vitro the
progression of triple negative breast cancer cells
Maria A. Jurja
, Olga
Soritaub, Cristian Moldovanc, Laura Popc, Cornelia
Braicuc and Ioana Berindan-Neagoec
aUniversity of Medicine and Pharmacy Iuliu Hatieganu,
Cluj-Napoca, Romania; bThe Oncology Institute “Prof. Dr. Ion Chiricuţă”,
Cluj-Napoca, Romania; cUniversity of Medicine and Pharmacy, Cluj-Napoca,
Romania
Introduction: Mesenchymal stem cells (MSCs) are multipotent stromal cells and
have been described as key regulators of different aspects of tumour physiology. In
tumour
pathogenesis, MSCs can integrate the tumour microenvironment after recruitment and
are able
to interact with cancer cells to promote tumour modifications by affecting
epithelial-to-mesenchymal transition (EMT). It was revealed that exosomes derived
from MSCs
are critical players in the tumour niche. Exosomes are a novel way of cell-to-cell
communication and play crucial roles in the majority of pathways that contribute and
affect
response to therapy, cell-adhesion molecules and the progression of tumour cells.
Because of
the known importance of this communication we decided to investigate the implication
of MSCs
with triple negative breast cancer (TNBC) cell lines as well as exosomal profiles
between
the experimental conditions.
Methods: The interactions of MSCs with triple negative breast cancer cell
lines (MDA-MB-231 and Hs578T) was performed by coculturing MSCs (or TNBC cell lines)
with
exosomes derived from TNBC cell lines (or MSCs). Physical characterization of isolated
exosomes was performed followed by their molecular investigations. Cell proliferation
was
detected by MTT assay and migration was analysed by wound healing assay using 2D cultures.
Moreover, we also used 3D culture to assess the exosomes uptake and to observe their
capability of internalization into a 3D structure. The alterations in expression level
of
some transcripts (mRNAs and miRNAs) and protein profile were investigated by qRT-PCR,
western blot and immunofluorescence staining.
Results: We found that MSCs-derived exosomes are actively incorporated by
triple negative breast cancer cell lines (2D culture). In coculture, in TNBC cells
the
expression level of mesenchymal markers and EMT markers (E-cadherin, vimentin) at
mRNA and
at protein levels, as well as miRNA-derived exosomes targeting mesenchymal genes were
significantly affected. Using bioinformatics tools, we highlighted the important biological
processes which were activated by promoting tumour modifications. In addition, using
3D
culture we provided a comprehensive understanding regarding exosomes internalization
in 3D
structures, which closely mimics in vivo conditions, compared to 2D culture.
Summary/Conclusion: In this work, we focus on the investigation of
MSCs-derived exosomes in order to highlight their implication in several biological
processes, including tumour proliferation and progression of triple negative breast
cancer
cells. All these alterations affect the response to therapy and should be considered
for
developing efficient therapeutic strategies.
PT06.09
Natural killer cell-derived extracellular vesicles have a potent
anti-leukaemic effect and selectively target the cancer stem cell subpopulation
Allyson M. Cochrana
and Jacki
Kornbluthb
aSaint Louis University, St. Louis, USA;
bDepartment of Pathology, Saint Louis University School of Medicine, St. Louis,
USA
Introduction: Natural killer (NK) cells of the immune system recognize and
kill tumour cells. Extracellular vesicles (EVs) secreted from NK cells are capable
of
killing tumour cells independent of the cell to cell contact required for NK cell
activation. Cancer is a leading cause of death, primarily due to metastasis and recurrence.
Cancer stem cells (CSC) within tumours are resistant to chemotherapy and immune attack,
and
cause metastasis and relapse. Identification of the cancer types killed by NK EVs
is
limited, and the effect of NK EVs on CSCs has not been described. Here we determine
whether
NK-derived EVs kill a myeloid leukaemia cell line and its CSC subpopulation.
Methods: NK EVs were isolated from our NK cell line, NK3.3, derived from
normal human lymphocytes. NK3.3 EVs were characterized by immunoblotting, proteomics,
and
next generation RNA sequencing. Human K562 leukaemia cells were treated with NK3.3
EVs in
vitro and analysed for proliferation and markers of cell death.
Results: NK3.3 EVs contain EV-associated proteins Alix, CD63, HSP70, and
Tsg101, NK effector molecules perforin, granzymes A and B, granulysin and NKLAM/RNF19b,
an
E3 ubiquitin ligase required for maximal NK cytotoxicity, and tumour suppressor miR-186.
NK3.3 EV treatment of K562 significantly decreased its expression of proliferation
markers
CD71 and Ki67, and increased the frequency of apoptotic and necrotic cells, paralleled
by
elevated levels of active caspases −3 and −7. Non-tumorigenic cells were unaffected
by NK EV
treatment. Most notably, NK3.3 EV treatment significantly reduced the frequency of
K562
cells highly expressing ALDH, a CSC marker.
Summary/Conclusion: NK3.3-derived EVs have a robust anti-tumour effect on K562
myeloid leukaemia cells and selectively target the CSC population, suggesting they
may
circumvent the evasion and resistance mechanisms used by CSCs. NK3.3 EVs therefore
have the
potential to be a safe alternative, or synergistic partner, to current cancer
therapeutics.
Funding: This study was supported by a VA Merit Award (1 IOBX000705) to J.K.
from the U.S. Department of Veterans Affairs, Biomedical Laboratory Research and Development
Service. Additional funding was provided by the Lottie Caroline Hardy Charitable Trust
and
the NIH/National Center for Advancing Translational Sciences (NCATS) grant UL1TR002345.
PT06.10
Scalable manufacturing system for MSC-EV generation
Katrina Adlerz, Josephine Lembong, Divya
Patel, Jon Rowley and Taby Ahsan
RoosterBio Inc, Frederick, USA
Introduction: Due to their potential as a key bioactive agent in regenerative
medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly
being
investigated as a clinical therapy. Manufacturing that generates enough EVs for product
development and clinical doses is currently a limitation in the field and clearly
a scalable
manufacturing solution will be necessary for successful translation. Moreover, a
complementary approach that increases the EV productivity, i.e. the number of EVs
produced
per cell, could further help to accelerate the development of MSC-EVs as a therapy.
Methods: We developed a process that leverages a series of new cell culture
reagents to couple to our established cell-media system for scalable manufacturing
of
MSC-EVs. Briefly, human bone marrow- or umbilical cord-derived MSCs were rapidly expanded
under xeno-free conditions (i.e. >150X expansion within 10 days). Cultures were then
switched to our proprietary EV collection medium and EVs were harvested for up to
three
additional days. At the end of culture, the EVs in the conditioned media were concentrated
using a tangential flow filtration (TFF) system. To increase the productivity of MSCs,
two
medium supplements were developed that increased EV yield by either increasing the
number of
EVs generated per cell in a shortened culture process or increasing the number of
collected
EVs by lengthening the EV collection culture period.
Results: This scalable MSC-EV manufacturing method was implemented in both 2D
flask and 3D bioreactor culture and generated over 2,000 particles per cell in 2D
and over
4,000 particles per cell in 3D. With the addition of a medium supplement to increase
EVs
produced per cell, the EV productivity was increased >2x after 24 hrs. Alternatively,
EV
productivity was also increased >2x by addition of the medium supplement that extended
EV
collection culture period.
Summary/Conclusion: MSC-EV success in clinical translation will be reliant on
a manufacturing method that can scalably and reliably generate large amounts of EVs.
These
results present one such solution. Furthermore, increasing EV productivity, for instance
by
medium supplements that increase EVs per cell or lengthen culture times could further
address the limitation of generating the EVs required for development and translation
of
clinical therapies.
PT06.11
Simplifying scalable MSC EV production in a microcarrier-based
bioreactor system
Divya Patel, Josephine Lembong, Katrina
Adlerz, Jon Rowley and Taby Ahsan
RoosterBio Inc, Frederick, USA
Introduction: The growpt0ing numbers of MSC-EV clinical applications drives
the need for a scalable MSC-EV production platform. While most MSC- EVs are generated
while
cells are attached to tissue culture plastic, such 2D cultures cannot be scaled up
to meet
the yields necessary for commercialization of EV-based therapeutics. We have shown
that 3D
bioreactors can be used to generate MSC-EVs and that paradigm can be scaled directly
in
terms of yield from the 3 to 15 L scales. The technical expertise of seeding cells
onto
microcarriers for expansion in bioreactors, however, requires technical expertise
not
available to all those in the EV field. Therefore, our goal here is to simplify and
expedite
the EV collection process in bioreactors by cryopreserving cells on microcarriers,
such that
end users can merely thaw and then collect MSC-EVs.
Methods: MSCs were expanded in 2D and then seeded on three different
microcarriers and cultured in a bioreactor for 5 days. When confluent, cells on
microcarriers were cryopreserved. To evaluate the microcarriers and the cryopreservation
protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth
media for
24 hours, then in EV collection media for 3 additional days. Cell recovery and EV
production
upon thaw was evaluated and compared to EV collection from fresh, non-cryopreserved
cells.
Results: Total cell counts 24 hrs post thaw were comparable to those before
cryopreservation and to fresh samples prior to EV collection. Following 3-day EV collection,
concentration of particles collected from cryopreserved cells on microcarriers were
similar
to those collected from the fresh cells (2E9 particles/ml). This process was validated
for
two different microcarriers using two separate cryopreservation solutions.
Summary/Conclusion: Our results show that cryopreserved hMSCs on microcarriers
can support EV collection in a 3D bioreactor process with a particle yield that is
comparable to those collected from fresh cells. This cryopreserved product can simplify
EV
production, reducing cost and time by removing process steps associated with the hMSC
expansion, with in a paradigm suitable for scale-up.
PT06.12
The whitening, anti-wrinkle, and wound-healing effects of
Extracellular vesicles from Orbicularis oculi muscle-derived stem cells.
Kyung Min Lima
, Ahmed Abdal
Dayema, Minchan Gila, Soo Bin Leea, Sehee
Kimb, Yu Jin Choic, Yoon Joo Leea, Hyun-Jin
Shind and Ssang-Goo Choc
aKonkuk University, Seoul, Republic of Korea;
bKonkuk University, Seoul, Republic of Korea; cKonkuk University,
seoul, Republic of Korea; dKonkuk University Hospital, Seoul, Republic of
Korea
Introduction: Skeletal muscle-derived stem cells possess potent therapeutic
activities in the treatment of muscle-related disorders. In our study, we tried to
isolate
and characterize orbicularis oculi muscle (ORM)-derived stem cells (ORM-SCs) from
the
discarded human tissues which were obtained from the ocular surgery-subjected patients.
We
also prepared the natural extracellular vesicles (EVs) from the cultured ORM-SCs and
assessed the their therapeutic actitities including the skin whitening, anti-wrinkle,
and
wound healing effects.
Methods: We isolated the ORM-SCs from the patients subjected to ocular surgery
and characterized the ORM-SCs by analysing cell morphology, proliferation, expression
levels
of the cell surface and stemness-associated markers, and tri-lineage differentiation
and
colony-forming capacities, confirming the stemness properties of the ORM-SCs. Then,
we
prepared the natural EVs from the ORM-SCs via the centrifugation and filtration of
the media
supernatants and their therapeutic activity was investigated.
Results: The isolated ORM-SCs showed spindle-like morphology and positive
expression of CD105, CD 90, and CD73, but they were negative in expression of CD45
and CD34.
The ORM-SCs showed the capacity of osteogenic, adipogenic, and chondrogenic
differentiations. The EVs from ORM-SCs (ORM-SC-EVs) possessed the apparent inhibitory
effect
on the melanin synthesis in B16F10 cells by blocking the tyrosinase activity, although
ORM-SC-EVs treatment did not dramatically change the expression level of
melanogenesis-related genes, such as microphthalmia-associated transcription factors
(MITF),
tyrosinase (TYR), tyrosinase-related protein1(TYRP-1), and TYRP-2. In addition, we
confirmed
that ORM-SC-EVs could stimulate skin cell migration and increase the expression level
of
anti-wrinkle related genes and wound-healing properties.
Summary/Conclusion: This study revealed the stem cell property of ORM-SCs and
the whitening, anti-wrinkle, and wound healing effects of ORM-SC-EVs, suggesting that
ORM-SCs and ORM-SC-EVs can be successfully used for stem cell-based EV therapy and
cosmetics, by regulation the melanogenesis, wrinkle, and wound.
Funding: This work was supported by grants from the National Research
Foundation (NRF) funded by the Korean government (2019M3A9H1030682).
PT06.13
Use of stem cell extracellular vesicles as a holistic approach
towards CNS repair
Heather Branscomea
, Dezhong
Yinb, Weidong Zhouc, Lance Liottac, Fatah
Kashanchid and Siddhartha Paulb
aAmerican type culture collection, Manassas, USA;
bAmerican Type Culture Collection, Gaithersburg, USA; cCenter for
Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA,
Manassas,
USA; dLaboratory of Molecular Virology, School of Systems Biology, George Mason
University, Manassas, VA, Manassas, USA
Introduction: Neurological diseases and disorders are leading causes of death
and disability worldwide. Many of these pathologies are associated with high levels
of
neuroinflammation and irreparable tissue damage. We have previously shown that extracellular
vesicles (EVs) from infected cells contain viral by products (non-coding RNAs and
proteins)
and that these EVs can exert deleterious effects on recipient cells1-3. Therefore,
in the
context of neurotrophic viruses EVs may contribute to or perpetuate processes relating
to
neuroinflammation and neurodegeneration. Due to their multipotent properties, stem
cells
have broad applications for tissue repair; additionally, stem cells have been shown
to
possess both immunomodulatory and neuroprotective properties. In recent years it has
been
well-established that stem cell EVs play a critical role in the functionality associated
with stem cells. The diverse biological cargo contained within these vesicles are
proposed
to mediate their effects and, to date, the reparative and regenerative effects of
stem cell
EVs have been demonstrated in a wide range of cell types. While a high potential for
their
therapeutic use exists, there is a gap of knowledge surrounding their characterization,
mechanisms of action, and how they may regulate cells of the central nervous system
(CNS).
Methods: We have isolated and recovered high yields of EVs from large scale
cultures of both induced pluripotent stem cells (iPSCs) and mesenchymal stem cells
(MSCs)
using tangential flow filtration. Our EV characterization includes both phenotypic
(size,
tetraspanin expression) and biochemical assays. EV functionality has also been assessed
in
vitro utilizing several cell-based assays related to cellular viability, migration,
angiogenesis, and immunomodulation in both healthy and damaged recipient cells with
relevance to the CNS.
Results: Our data suggests that EVs from different sources of stem cells
display unique phenotypes, exhibit differential association with various cytokines,
proteins, and long non-coding RNAs, and have the ability to significantly enhance
processes
that are critical for cellular repair4. Lastly, utilizing an iPSC-derived neurosphere
model,
we have observed a robust uptake of stem cell EVs and have found that these EVs are
able to
effectively penetrate these 3D structures.
Summary/Conclusion: Collectively, these results highlight the “holistic”
properties of stem cell EVs by demonstrating their ability to partially reverse or
reduce
damage in various cell types.
Funding: This work was supported by National Institutes of Health (NIH) Grants
AI078859, AI074410, AI127351-01, AI043894, and NS099029 to FK and R33 CA206937 and
R01AR068436 to LAL.
PT06.14
The effect of cell culture media on extracellular vesicle
secretion from mesenchymal stem cells and human pluripotent stem cell-derived
neurons
Mette Heiskanen
a, Jenni
Karttunenb, Tiina Jokic, Anu Hyysalod, Vicente
Navarro-Ferrandisa, Susanna Narkilahtic and Asla
Pitkänena
aAIV Institute for Molecular Sciences, University of Eastern
Finland, Kuopio, Finland; bDepartment of Veterinary Medicine, University of
Cambridge, Cambridge, UK; cNeuroGroup, Faculty of Medicine and Health Technology,
Tampere University, Tampere, Finland; dRegenerative Neuroscience, Institute of
Biotechnology, HiLIFE unit, University of Helsinki, Helsinki, Finland
Introduction: Cell culture media and its supplements are known to affect the
secretion and isolation of extracellular vesicles (EVs) from cell cultures. Identification
of these effects is crucial especially when planning to use EVs as therapeutic agents.
Here,
we investigated the effect of cell culture media on EV yield from human mesenchymal
stem
cells (MSCs) and human pluripotent stem cell (hPSC)-derived neurons.
Methods: EVs were collected from cell-conditioned media (CCM) and no cell
control (NCC) media using size-exclusion chromatography (SEC). MSCs were cultured
in
DMEM/F12:neurobasal medium or in Opti-MEM reduced serum medium, both supplemented
with
exosome-depleted foetal bovine serum (FBS). The EV yield from hPSC-derived neurons
was
compared at two maturation time points (day 46 and 60), in DMEM/F12:neurobasal or
in
Opti-MEM, with and without 3-hour KCl stimulation. SEC fractions were analysed by
nanoparticle tracking analysis (NTA), protein concentration assay and blinded transmission
electron microscopy (TEM).
Results: CCM samples had a clear peak of EVs in SEC fractions 7–10, which was
not detected with NCC. Interestingly, a second population of EVs eluted in SEC fractions
13–17 in both CCM and NCC, indicating presence of EVs in exosome-depleted FBS. Moreover,
this second population differed largely between used media batches. Culture medium
had no
significant effect on MSC EV yield (DMEM: 8.30E+08 particles/ml, Opti-MEM: 9.61E+08
particles/ml). With neuronal cultures, no significant differences in EV yield were
found
between culture media or cell maturation time points. In contrast to earlier findings,
3-hour stimulation of neurons by KCl resulted in significantly smaller EV yield compared
to
non-stimulated controls (stimulated: 5.28E+10 particles/ml, non-stimulated: 1.43E+11
particles/ml, p < 0.02).
Summary/Conclusion: Our results indicate that exosome depleted-media are not
entirely devoid of vesicles, which can cause bias in downstream analyses. However,
SEC is a
good method to separate cell-secreted EVs from the contaminating medium-derived EVs.
Culture
medium did not affect the number of EVs secreted by MSCs or neurons; instead, we observed
larger differences between media batches. This data emphasizes the importance of analysing
the NCC as negative control in all cell culture experiments.
PT06.15
Mouse mesoangioblast stem cell extracellular vesicles are able
to influence macrophage cell activity
Maria Magdalena Barrecaa and Fabiana
Geraci
b
aDept STEBICEF University of Palermo, Palermo, Italy,
Palermo, Italy; bDept STEBICEF, University of Palermo, Italy, Palermo, Italy
Introduction: It is largely demonstrated that stem cells release extracellular
vesicles (EVs) that are able to modify target cell behaviour. Interestingly, there
is a
bidirectional signalling exchange between stem cell EVs and damaged cells. Moreover,
it is
well known that macrophages, could also play a role in wound repair and tissue regeneration.
It was also demonstrated that stem cell EVs are involved in immune cell regulation.
For this
reason, today takes hold the idea that EVs could replace stem cells in regenerative
medicine. The aim of our work was to evaluate if EVs released by mouse mesoangioblast
stem
cells (A6) could have a role in immune cell regulation. Specifically, we have investigated
the possible A6 EV effect on murine macrophages (RAW264.7) in terms of cell proliferation,
migration and phagocytic ability, and cytokines/chemokine release.
Methods: A6 EVs were collected from conditioned milieu by ultracentrifugation.
Raw 264.7 cell proliferation with or without A6 EVs was evaluated via CFSE assay.
Scratch
test was performed to assay their migration ability. To study RAW264.7 cell phagocytosis
they were treated with 2 μm beads. Finally, cytokine array was used to monitor their
secretion after EV treatment.
Results: We have found that A6 EVs inhibited macrophage proliferation as
proved by a proliferation index significantly reduced after EV treatment. Simultaneously,
we
have noticed that EVs increases RAW264.7 migration ability. Furthermore, A6 EVs are
able to
increase macrophage phagocytic activity. As it is known that Hsp70 is involved in
for
macrophagic activity increase and A6 EVs express Hsp70 on their surface, we performed
phagocytosis assays assay by blocking the protein or its receptor TLR2, TLR4 and CD91.
Our
data demonstrated that A6 EVs increase phagocytosis through Hsp70 and its receptors.
We have also proved that A6 EVs modify the expression pattern of cytokines/chemokines
released in the extracellular milieu by RAW264.7 cells. In particular, we observed
an
increase in anti inflammatory cytokines, and a decrease in some inflammatory ones,
suggesting that EVs could polarize macrophages towards an anti inflammatory M2
phenotype.
Summary/Conclusion: In conclusions, our data show that A6 EVs influence
macrophage activity and additional studies could provide a new insight into understanding
the underlying potential of EVs in tissue regeneration.
PT07: EV Biogenesis (from Prokaryotes to Euraryotes), Component
Loading, and Release
Chair: Pascale Zimmermann – INSERM/KU Leuven
Chair: Caitlin McAtee – Vanderbilt University
PT07.01
Analytical ultracentrifugation identifies distinct populations
of extracellular vesicles (EVs) carrying the evolutionarily-conserved polycystin-2
protein
in C. elegans
Juan Wanga, Inna
Nikonorovab
and Maureen Barra
aRutgers University, Human Genetics Institute of NJ,
Piscataway, USA; bRutgers University, New Brunswick, USA
Introduction: Autosomal dominant polycystic kidney disease (ADPKD) affects
~1/800. ADPKD is one of the most common, life threatening human genetic diseases and
leading
cause of end-stage renal failure in adults and children. 95% of ADPKD cases are caused
by
mutations affecting the ciliary PKD1 and PKD2 complex (Polycystic Kidney Disease 1
and 2).
In a healthy kidney, the polycystins localize to renal cilia. Mutations that abrogate
ciliary localization of PKD2 (yet preserve its channel function) also cause cysts.
Besides
cilia, PKD2 is also found in other subcellular locations including extracellular vesicles
(EVs) of human urine. How dysfunction of PKD2 trafficking and localization leads to
the
kidney pathology remains unknown.
PKD2 is evolutionarily conserved across all members of Eumetazoa. In C. elegans, PKD-2
is
exclusively expressed in ciliated male‐specific neurons, where it is trafficked to
cilia and
EVs. GFP-tagged PKD-2-containing EVs play a signalling role in inter‐organismal
communication between animals. Conservation of polycystin-2 cellular localization
between
worm and human suggests that their network of molecular interactions may also be conserved.
We propose that PKD-2 plays distinct roles in cilia versus ciliary EVs.
Methods: To understand the role of EVs in C. elegans inter‐organismal
signalling, we aim to identify the PKD-2‐associated EV proteome, transcriptome, and
metabolome. We established a pipeline for fluorescent labelling and tracking specific
EV
cargoes in a living animal using super‐resolution microscopy. We used fluorescence
of the
PKD-2 carrying EVs to optimize biochemical procedures for their enrichment.
Results: Our initial analysis revealed two populations of PKD-2‐carrying EVs
that differ in their densities: 1.11–1.12 versus 1.14 g/mL. We are currently characterizing
these two distinct populations using transmission electron microscopy and refining
our
enrichment procedure for protein identification by mass spectrometry, sequencing of
their
RNA cargoes and metabolome analysis.
Summary/Conclusion: What function human PKD2 plays within the cilia and within
the urinary EVs is not well understood. Identification of molecular mediators of C.
elegans
PKD-2 EV signalling will inform on the interactome of human PKD2 and its function
in cilia
versus EVs.
Funding: NIH DK059418 and DK116606 (MB), KUMC PKD Center (JW).
PT07.02
C. elegans ciliated sensory neurons release distinct
subpopulations of extracellular vesicles
Michael Clupper, Rachael Gill, Denis
Touroutine and Jessica Tanis
University of Delaware, Newark, USA
Introduction: Ectosomes play roles in many physiological and
pathophysiological processes, and their precise is dependent on molecular cargo and
parent
cell type. A single cell can release distinct subpopulations of EVs enriched with
different
molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis
mechanisms. In the nematode C. elegans, ectosomes bud from sensory neuron cilia and
are
released into the environment to modulate animal behaviour.
Methods: C. elegans is genetically tractable and optically transparent,
allowing for live imaging of fluorescently tagged EV cargo. We express all tagged
cargo at
endogenous levels, adding physiological relevancy.
Results: We discovered that the calcium homoeostasis modulator ion channel
CLHM-1 localizes to cilia of EV-releasing neurons and observed GFP-tagged CLHM-1 in
ciliary
EVs. Using super resolution microscopy, we imaged EVs released from animals co-expressing
tdTomato-tagged CLHM-1 and GFP-tagged PKD-2 (another vesicle cargo) in the same neurons.
While the two proteins colocalize in the cilia, CLHM-1::tdTomato and PKD-2::GFP rarely
colocalize in EVs. This indicates that separate subpopulations of EVs are being released
from the same neurons. To determine how the CLHM-1 subpopulation is formed, we are
investigating candidate genes. ANOH-1, a homolog of the Ca2+ scramblase TMEM16F, localizes
to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet.
In
anoh-1 mutants, the number of CLHM-1::GFP EVs released is significantly decreased
but the
number of PKD-2::GFP EVs does not significantly change. In addition, I am using FACS
to
isolate CLHM-1 and PKD-2 containing EVs and analysing the respective proteomes with
LC-MS/MS.
Summary/Conclusion: We are elucidating mechanisms that give rise to distinct
subpopulations of ciliary EVs in C. elegans and defining cargoes being enriched in
these EV
subpopulations to gain insight into EV cargo sorting and biogenesis mechanisms in
ciliated
neurons.
PT07.03
Ceramide accumulation induces exosome secretion through
lysosomal protein LAPTM4B
Kohei Yuyama, Hui Sun and Yasuyuki
Igarashi
Hokkaido University, Sapporo, Japan
Introduction: Exosomes, a type of extracellular vesicles originated from
multivesicular bodies (MVB), are important carriers of cellular molecules and have
critical
roles in intracellular communication in both health and disease. Ceramides (Cer) are
implicated in biogenesis of exosome, however the molecular machinery that mediates
exosome
secretion remains obscure. Lysosome-associated protein transmembrane-4B (LAPTM4B)
is a
lysosome/late endosome-resident transmembrane protein, which has been reported to
bind Cer.
We demonstrate here that LAPTM4B is involved in the exosome secretion, which are induced
by
exogenous Cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells.
Methods: Neuroblastoma SH-SY5Y cells were treated with Cer (porcine
brain-derived Cer or synthetic d18:1/C2:0~ C24:0 Cer) for 24 h. Exosomes were isolated
from
the culture supernatants by sequential centrifugation and their amounts were measured
using
PS Capture exosome ELISA kit. To analyse MVB transport, MVB and recycling endosomes
are
visualized with GFP-CD63 and Rab11 immunostaining, respectively.
Results: We found that exogenous treatment of Cer, especially those with C16
and C18 fatty acids, resulted in a marked increase in exosome secretion. In addition,
lysosomal Cer accumulation induced by acid ceramidase inhibition also accelerated
exosome
production. Knockdown of LAPTM4B significantly prevented the ceramide-dependent exosome
release. In addition, we showed that these Cer loading promoted colocalization of
CD63-positive MVB with Rab11-positive recycling endosomes, further demonstrated that
LAPTM4B
knockdown cancelled the Cer-dependent increase of the colocalization.
Summary/Conclusion: These data suggest that lysosomal Cer binds to LAPTM4B and
promote the transport of MVB to plasma membrane, resulting in an increase of exosome
secretion in neuronal cells.
PT07.04
Chloroquine-mediated lysosomal inhibition alters composition and
function of cancer-derived extracellular vesicles
Jing Xua
, Kevin Yanga,
Shane Colbornea, Elham Hosseini-Beheshtib, Gregg Morina,
Emma Gunsb and Sharon M. Gorskia
aBC Cancer, Vancouver, Canada; bThe Vancouver
Prostate Centre, Vancouver, Canada
Introduction: Small extracellular vesicles (sEV) are signalling entities
released by many types of eukaryotic cells. sEV are of special interest in cancer
due to
their reported roles in modulating the cancer microenvironment and facilitating cancer
cell
invasion. Macroautophagy (hereafter autophagy) is a catabolic process well-known for
the
recycling of cytosolic cargos through lysosome-mediated degradation. In this study,
we
profiled the changes in sEV content and function under lysosome inhibition and investigated
the involvement of autophagy machinery in sEV content.
Methods: Chloroquine (CQ) was used to inhibit lysosomal degradation and
autophagy turnover in triple-negative breast cancer (TNBC) cell lines. sEV were collected
via precipitation after pre-clearing and concentration of conditioned media. Western
blotting, NanoSight and transmission electron microscopy were used to profile sEV.
Quantitative mass spectrometry was used to characterize CQ-induced changes in the
sEV
proteome. Antibody-conjugated magnetic beads were used in immunoprecipitation of sEV.
Results: CQ treatment did not substantially alter the physical properties of
TNBC-derived sEV. However, CQ treatment altered the sEV proteome and growth effects
of sEV
on normal and endothelial recipient cells. CQ treatment induced co-localization of
mammalian
ATG8 proteins with endolysosomal markers in the cytoplasm, which coincided with an
enrichment of ATG8 s and their adaptor proteins in sEV. CQ-induced enrichment of ATG8 s
in
sEV required lipidation, and occured preferentially in one subset of sEV.
Summary/Conclusion: Our study reveals changes in the content and function of
cancer cell-derived sEV in response to perturbation of intracellular trafficking pathways,
demonstrates the flexibility and heterogeneity of sEV composition, and has implications
for
CQ efficacy in therapeutic settings.
Funding: CIHR with Avon Foundation for Women-Canada grant OBC127216 and BC
Cancer-Pfizer Research Innovation Fund. JX was supported by a CIHR Doctoral Award.
PT07.05
Evaluation of culture conditions on the presence of Ago2 in
extracellular vesicles
Lizandra Jimeneza
, Bahnisikha
Barmanb, Roxanne Pelletierb and Alissa Weaverc
aVanderbilt University, Nashville, USA;
bVanderbilt University, Nashville, USA; cDepartment of Cell and
Developmental Biology, Vanderbilt University School of Medicine, Nashville, USA
Introduction: Introduction: Argonaute 2 (Ago2) is the essential component of
the RNA-Induced Silencing Complex (RISC) that binds miRNAs and promotes mRNA degradation.
Extracellular vesicle (EV)-carried miRNAs have been shown to influence gene expression
and
functional phenotypes in recipient cells. Many investigators have found Ago2 in EVs
and it
is postulated that Ago2 is a major transporter of miRNAs into small EVs (SEVs), such
as
exosomes. Others have reported extracellular Ago2 that is non-vesicular. We set out
to
evaluate the effect of growth factor signalling and serum contamination on the detection
of
Ago2 in SEVs.
Methods: Methods: Wildtype KRAS colorectal cancer cells, DKs8, were
conditioned with 3 different culture media (serum-free DMEM, DMEM supplemented with
EV-depleted FBS, and Opti-MEM). EVs were purified from conditioned media by cushion-density
gradient ultracentrifugation. Western blot analysis of DKs8 total cell lysates, large
EVs
and density gradient fractions was performed, probing for Ago2 and EV marker proteins.
The
size and concentration of the EVs were determined by Particle Metrix analysis.
Results: Results: In all conditions, we found the highest abundance of SEVs in
fractions 6 and 7, as assessed by Western blot analysis. Ago2 was detected in the
same
fractions as SEVs in both the serum-free DMEM and Opti-MEM conditions, although the
levels
of Ago2 was higher in the serum-free DMEM fractions compared to that of Opti-MEM.
In
contrast, Ago2 was present in both vesicular and non-vesicular fractions in the DMEM
supplemented with EV-depleted FBS condition. No significant differences were observed
in the
size and number of EVs collected in the three conditioning methods.
Summary/Conclusion: Summary/conclusion: The presence or absence of Ago2 in EVs
has been controversial. Multiple factors may affect the ability to detect vesicular
Ago2,
including serum and growth factors in the conditioned media that may provide sources
of
extravesicular Ago2 and also regulate the trafficking of Ago2 into vesicles.
PT07.06
Extracellular vesicles secreted from melanoma cell lines contain
similar gangliosides and associated molecules with lipid rafts on the cellular
membrane
Koichi Furukawaa
, Satoko
Yamamotoa, Mariko Kambeb, Akiko Tsuchidac, Yuhsuke
Ohmia, Yuki Ohkawaa and Keiko Furukawaa
aChubu University College of Life and Health Sciences,
Kasugai, Japan; bChubu University, Kasugai, Japan; cNoguchi Institute,
Tokyo, Japan
Introduction: Cancer-associated glycosphingolipids have been utilized as
tumour markers and targets of cancer therapy. We have investigated roles of gangliosides
in
cancers, and clarified that cancer-associated gangliosides enhance malignant properties
of
cells by forming complexes with membrane molecules in lipid rafts. In this study,
we
analysed contents of gangliosides and membrane molecules on extracellular vesicles
(ECVs)
secreted from melanoma cell lines.
Methods: Melanoma cell lines with various ganglioside patterns were used for
isolation of ECVs. Ganglioside-modified melanomas with genetic engineering were also
used.
Genetic modification was done by cDNAs of ganglioside synthase genes. ECVs were collected
by
ultra-centrifugation, or by Tim4-beads. Contents in ECVs were analysed by immunoblotting
or
flow cytometry. Roles of lipid rafts in the generation and secretion of ECVs were
analysed
by treating cells with 1 mM methyl β-cyclodextrin.
Results: Using 4 melanoma cell lines, ECVs were isolated by
ultra-centrifugation, and their sizes were analysed by NanoSight. All samples showed
uniform
sizes between 100 and 200 nm. Protein amounts in ECVs were measured, showing heterogeneous
levels at 10 ~ 80 μg/100 mL. Then, gangliosides expressed on ECVs from these cell
lines were
analysed using Tim4-beads and flow cytometry. GD3 and GD2 were detected on ECVs almost
proportionally with expression levels of those gangliosides on the cell surface. Then,
immunoblotting was performed to analyse integrin levels in ECVs from transfectant
cells
expressing high levels of GD3, showing increased levels of integrins in ECVs from
GD3+ cells
compared with those from GD3- cell lines. Integrin levels in cell lysates from these
cells
(GD3+ and GD3 – cells) were almost equivalent. Treatment of a GD3-expressing melanoma
cell
line by 1 mM methyl β-cyclodextrin resulted in marked reduction of secreted ECVs and
amounts
of TSG101 in them.
Summary/Conclusion: Ganglioside expression patterns on melanoma cells were
well reflected in the expression of gangliosides on ECVs. These results as well as
increased
levels of integrins in ECVs from GD3+ cells suggest that gangliosides and lipid rafts
are
involved in the generation and secretion of ECVs.
Funding: This study was supported by a Grant of JST Basic Research Program
(CREST) of Japan.
PT07.07
Hypoxia alters the metabolic and miRNA content of breast
cancer-derived exosomes
Kathleen M. McAndrewsa
, Margo
Caina, Noritoshi Katob, Sonia A. Meloa, Sara
Lovisaa, Pedro Correa de Sampaioa, Laura Snowdena, Hikaru
Sugimotoa, Valerie LeBleua and Raghu Kalluric
aUT MD Anderson Cancer Center, Houston, USA;
bHarvard Medical School, Boston, USA; cDepartment of Cancer Biology,
Metastasis Research Center, University of Texas MD Anderson Cancer Center, Houston,
USA
Introduction: Hypoxia, or low oxygen tension, is a common feature associated
with tumour growth and is known to regulate tumour cell function, especially through
rewiring of cell metabolism. However, how hypoxia influences tumour cell interactions
with
surrounding cells is not fully elucidated. We sought to evaluate how hypoxia alters
metabolite and metabolism-associated miRNA packaging in exosomes.
Methods: Exosomes were isolated from 4T1 breast cancer cells cultured in
normoxia (21% O2) and hypoxia (5% O2) via ultracentrifugation, Optiprep gradients,
and size
exclusion chromatography. Exosomes were further characterized by Nanosight, Qubit
protein
quantification, and flow cytometry analysis of exosome markers. Metabolite and miRNA
profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia.
Results: Secretion of exosomes was increased under hypoxic conditions.
Metabolite profiling revealed alterations in metabolites specific to exosomes derived
from
hypoxic cells. Profiling of exosomal miRNA showed packaging of metabolism-related
miRNA into
exosomes derived from hypoxic cells.
Summary/Conclusion: Hypoxia alters the metabolite and miRNA profiles of cancer
cells, with selective packaging of these molecules into exosomes. We identified metabolites
and miRNA that are depleted and enriched in exosomes compared to cells. These studies
identify hypoxia-associated shifts in exosome cargo, providing insight into exosome
cargo
packaging with potential implications for understanding how cancer cell-derived exosomes
regulate recipient cell function.
PT07.08
Lysosomotropic agents prompts the release of extracellular
vesicles carrying autophagy-associated markers: evidence of a general mechanism of
secretion driven by lysosomal impairment
Lorena Urbanellia
, Krizia
Saginib, Sandra Burattaa, Federica Deloa and Carla
Emiliania
aUniversità di Perugia, Perugia, Italy; bDept. of
Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo,
Norway
Introduction: Drug-induced lysosomal storage disorders (LSDs) are due to the
transient intracellular accumulation, mostly of phospholipids, into multilamellar
inclusion
bodies within late endosomal/lysosomal compartment. They represent a major side-effect
for
many drugs of several pharmacological categories. Most LSDs inducers are cationic
amphiphilic drug (CAD), but the molecular mechanisms leading to accumulation of undigested
substrates are unknown. Extracellular vesicles (EVs) have been implicated in cell
waste
disposal, but it is unclear whether they might be involved in extracellular release
of
undigested substrates.
Methods: To investigate this aspect, we developed Hek cells stably expressing
the fluorescent fusion proteins EGFP-CD63 and mCherry-CD63, separated EVs by differential
ultracentrifugation and quantified by EV-associated fluorescence and NTA particle
count.
Results: EVs released by these models upon treatment with drugs inducing the
accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed
the
release of fluorescent medium/large EVs (10k fraction) and small EVs (100k fraction),
whose
size and distribution were similar to the same vesicles released by control cells,
but
enhanced the recovery of medium/large EVs and to a lower extent of small EVs, Analysis
of
EVs associated markers revealed a dose-dependent increase of autophagy-associated
markers in
medium/large and small EVs. Similar results were obtained when autophagic flux was
impaired
by drugs raising lysosomal pH by different mechanisms, such as chloroquine and bafilomycin,
but not when autophagic flux was stimulated by drugs such as curcumin or overexpression
of
the endosomal/lysosomal regulator TFEB.
Summary/Conclusion: Overall results show that impairment of autophagic flux,
either by indigested substrates or higher lysosomal pH, is associated with an increased
release EVs enriched in autophagy markers, compatible with autophagomes and/or amphisomes,
unravelling a connection with secretory autophagy.
PT07.09
Roles of extracellular vesicles for lenalidomide-resistance in
multiple myeloma
Tomofumi Yamamotoa
, Yusuke
Yamawakib, Yutaka Hattoric and Takahiro Ochiyaa
aTokyo Medical University, Shinjuku-ku, Japan;
bNational Cancer Center Research Institute, chuo-ku, Japan; cKeio
University Faculty of Pharmacy, Minato-ku, Japan
Introduction: Multiple myeloma (MM) is a haematological tumour. Last decade,
the prognosis of MM has improved by the development of therapeutic drugs; however,
MM cells
acquire drug resistance by long-term exposure of these therapeutic drugs. One of the
possible explanations of drug resistance is that cells with drug resistance transmit
information to other MM cells and their microenvironmental cells. Although the elucidation
of the mechanism of drug resistance in MM have been desired, it remains poorly
understood.
Methods: In order to understand the mechanism of drug resistance in MM,
lenalidomide resistant cell lines were established by long-term exposure of low
concentration of lenalidomide. Drug resistance was assessed by MTS assay and caspase
assay.
The amount of EV was measured by ExoScreen, which is ultra-sensitive detection method
of EVs
by measuring surface protein of EVs, such as, CD9 and CD63 (Yoshioka et al., Nat Commun.,
2015). To identify the genes which involved in drug resistance, RNA sequence among
the
drug-resistant cell lines and their parental cell lines was performed.
Results: Firstly, characterization of these cells was confirmed. We found that
all of the lenalidomide resistant cell lines secreted more EVs than their parental
cell
lines. In addition to this, the size of EV derived from resistant cells are smaller
than
those of parental cells. Next, we collected EVs from resistant cells and parental
cells by
using ultracentrifugation, and added them to parental cells in the presence of lethal
dose
of lenalidomide. Compared with EV derived from parental cell lines, the EVs derived
from
lenalidomide resistant cell lines increased a number of living parental cells. These
results
suggested that the EVs derived from lenalidomide resistant cells can affect the lenalidomide
sensitive cells.
As a result of RNA sequence, several genes highly expressed in resistant cell line
we
found, which associated with lysosome pathway. Among them, attenuating the SORT1 and
LAMP2
genes could significantly reduce the EV secretion in MM cells, leading to enhance
the
lenalidomide sensitivity.
Summary/Conclusion: Our results showed that EV secretion via SORT1 or LAMP2
could induce the drug resistance in MM.
PT07.10
Study on biological stimulate mechanism of stem cell-derived
exosome generation by nanoparticles
Dong Jun Parka
, Wan Su
Yunb, Jeong-Eun Parka, Su Hoon Leea, Sunmok
Hac, Jin Sil Choia and Young Joon Seoa
aDepartment of Otorhinolaryngology, Yonsei University Wonju
College of Medicine, Wonju, Republic of Korea; bDepartment of Biomedical
Engineering, Yonsei University, Wonju, Republic of Korea; cDepartment of
Biomedical Laboratory Science, Yonsei University Wonju College of Medicine, Wonju,
Republic
of Korea
Introduction: Mesenchymal stem cells (MSCs) are pluripotent stromal cells
known to release extracellular vesicles (EVs) containing various growth factors and
antioxidants that can positively affect surrounding cells. Nanoscale MSC-derived EVs,
such
as exosomes, have been developed as bio-stable nano-type materials, but had low yield
and
were difficult to quantify. We hypothesized that the mechanism of nanoparticle-enhanced
exosome production would stimulate intracellular molecules. The aim of this study
was to
elucidate the molecular mechanisms of exosome generation by comparing the internalization
of
surface-modified positively charged nanoparticles and exosome generation from MSCs.
Methods: Mesenchymal stem cells (MSCs) were cultured in MEM-alpha with 10% FBS
and 1× antibiotics. The positively charged nanoparticles were synthesized by
Poly-lactide-co-glicolide (PLGA) and polyethylenimine (PEI) with cy5.5 for tracking
nanoparticles. All of the exosome image were identified using an electron microscope.
Additionally, it was confirmed the internalization of the nanoparticles by IF. The
primary
antibodies used were anti-EEA1, anti-Rab7 and anti-GM130. In order to prove the development
of exosomes, RT-PCR using autophagy-related mRNA was performed. Real-time RT-PCR was
performed using the Applied Biosystems sequence detection system 7900. Lastly, miRNA
from
MSC-derived exosome analysed automatically in the Affymetrix data extraction protocol
using
the provided Affymetrix GeneChip® Command Console® Software (AGCC). All statistical
testing
and visualization of differentially expressed genes was conducted using R statistical
language 3.3.3
Results: We determined that Rab7, located in the MVB and autolysosomal
membrane, was increased upon exosome expression and was associated with autophagosome
formation. These results suggested that nanoparticles migrated to lysosomes during
treatment; however, intracellular exosome-forming factors were stimulated during endosomal
maturation simultaneously.
Summary/Conclusion: Therefore, MSC-derived exosome research using
nanoparticles is useful for increasing exosome yield and the discovery of
nanoparticle-induced genetic factors.
Funding: This research was supported by a grant of the Korea Health Technology
R&D Project through the Korea Health Industry Development Institute(KHIDI), funded
by
the Ministry of Health & Welfare, Republic of Korea (grant number: HI19C1334).
PT07.11
Theoretical description of formation of extracellular vesicles
by budding of membrane
Veronika Kralj-Iglič
a, Gabriella
Pocsfalvib, Luka Mesarecc, Vid Sustard, Henry
Hagerstrande, Ales Igličf and Veronika Kralj-Iglicg
aLaboratory of Clinical Biophysics, Faculty of Health
Sciences, University of Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia;
bInstitute of Biosciences and BioResources (IBBR) – National Research Council
(CNR), Naples, Italy; cFaculty of Electrical Engineering, Ljubljana, Slovenia;
dLymphocyte Cytoskeleton Group, Medical University of Turku, Turku, Finland,
Turku, Finland; eCell Biology, Faculty of Science and Engineering, Åbo Akademi
University, Åbo/Turku, Finland Novia University of Applied Sciences, Ekenäs, Finland,
Abo/Turku, Finland; fUniversity of Ljubljana, Ljubljana, Slovenia;
gLaboratory of Clinical Biophysics, Faculty of Health Sciences, University of
Ljubljana, Slovenia, Ljubljana, Slovenia
Introduction: Understanding mechanisms of extracellular vesicles (EVs)
formation is of utmost importance for their effective use in science, medicine and
technology. In particular, the discovery of universal mechanisms explaining the phenomena
taking place in vesiculation appears to be crucial and highly warranted. Mammalian
erythrocytes and giant phospholipid vesicles have been largely used as model systems
to
study principles of membrane budding and vesiculation. The mechanisms conveniently
studied
in these simple systems are then generalized to other types of biological membranes.
We
present a theoretical description of membrane budding and compare the theoretically
obtained
shapes with the observed ones.
Methods: In accordance with the fluid crystal mosaic model, membrane is
considered as composed of constituents (inclusions) subjected to the local curvature
field
created by surrounding constituents. Constituents can attain different in-plane orientations
in the membrane which correspond to different energies. The thermal motion oposes
the
complete orientational ordering. The single-constituent energy expresses a mismatch
of the
curvature of the membrane at the position of the constituent and the intrinsic principal
curvatures of the constituent and in – plane orientation of their principal axes.
The free
energy of the whole membrane is obtained by summing up (integration) the contributions
of
the constituents and using methods of statistical physics, and minimized by using
numerical
methods.
Results: To outline the principle of (outward and inward) budding, respective
sequences of shapes corresponding to a formation of one (outward and inward) spherical
bud
were calculated by minimization of the free energy. Also the corresponding shapes
observed
in EVs (imaged by electron microscopy) and in erythrocytes and giant phospholipid
vesicles
(imaged by optical microscopy) are shown. It can be seen that theoretically calculated
shapes and experimentally observed ones agree well over up to 3 orders of magnitude
(the
order of the size of giant phospholipid vesicles is between 1 and 100 micro metres,
in
erythrocytes it is about 5 micro metres and in EVs it is about 100 nanometres).
Summary/Conclusion: Budding of the membrane is an universal mechanism in
formation of external and internal vesicles.
Funding: This work was supported by the Slovenian Research Agency under grants
P3-0388, J1-9162, J2-8166 and J2-8169 and EU Commission under grant Ves4us. Authors
are
indebted to Anna Romolo for technical assistance in preparing the manuscript.
PT07.12
Role of Arrdc4-mediated ubiquitination in extracellular vesicle
biogenesis and protein trafficking
Ammara Usman Farooq
a, Kelly
Gembusb, Natalie J. Footc and Sharad Kumard
aUniversity of South Australia, Adelaide, Australia;
bResearch Asistant, Adelaide, Australia; cUniSA, Adelaide,
Australia; dUniversity of South Australia, Adelaide, Australia
Introduction: The release of extracellular vesicles (EVs) from cells is
important for many cellular mechanisms both in normal physiology and in disease. Arrdc4
(arrestin domain containing protein 4) is an adaptor protein known to facilitate the
ubiquitination of target substrates by Nedd4 family ubiquitin ligases. It also traffics
cargo to extracellular vesicles. Previous studies show the involvement of Arrdc4 in
the
trafficking of the divalent metal ion transporter DMT1 to EVs in a ubiquitin-dependent
manner, and we aimed to further understand this mechanism.
Methods: We performed mass spectrometry to identify ubiquitinated lysine
residues in Arrdc4. We then generated Arrdc4 WT and lysine mutant clones and expressed
these
in cells to determine the effect on EV biogenesis and protein trafficking.
Results: Mass spectrometry data identified 5 potential ubiquitinated lysine
residues. Out of these, lysine 270 appeared to be the most important for Arrdc4 function.
Arrdc4K270 R mutation caused a decrease in the number of EV released by the cell compared
to
Arrdc4 WT, and a reduction in trafficking of DMT1 to EVs. Furthermore, we also observed
a
decrease in DMT1 activity and an increase in its intracellular degradation in the
presence
of Arrdc4K270 R. K270 also appeared to be ubiquitinated with K29 polyubiquitin chains
by the
ubiquitin ligase Smurf1.
Summary/Conclusion: Our data suggests that K29 polyubiquitin chains are the
signal for Arrdc4-mediated EV biogenesis and protein trafficking, and loss of this
signal
causes cargo to be rerouted to intracellular degradation mechanisms.
PT08: Scientific Outreach and Collaboration
Chair: Tanina Arab – Department of Molecular and Comparative Pathobiology, Johns
Hopkins University School of Medicine
PT08.01
A 3D-printed model to represent the structure and nature of
extracellular vesicles, for public engagement and education events.
Christian Burtona, Sara
Veigaa
, Jason Webbera, Kate Milwarda,
Muireann Ni Bhaoighilla, Lauren Evansa, Andreia De
Almeidab, Rachel J. Erringtona and Aled Claytona
aCardiff University, Cardiff, UK; bCardiff
University, Research Associate, UK
Introduction: Explaining the field of extracellular vesicles to the lay public
and young audiences can often be challenging. Whilst diagrams and images of EVs may
be
helpful, conveying clearly the shape and composition of an EV by these means is not
always a
success. Whilst many members of the audience may be familiar with concepts of cells
and
related structures, others will find such discussions very abstract and challenging.
In
order to aid interactions with lay audiences we embarked on the design of a physical
hand-held plastic model, representing a typical EV. Incorporating flexibility in the
design
allowing the community to adapt it to showcase their own research. The second goal
was to
ensure manufacturability using widely available 3D-printing technologies.
Methods: The basic model design was conceived by Dr C. Burton, and iteratively
developed using Solidworks, 2019, then exported for use in any CAD environment (STL
format).
A model showing a halved EV hemisphere, with a visible lipid-bilayer was developed.
Attachable rings allow trans-membrane-molecules to be represented, current designs
include
MHC Class-I, HSPGs, integrins, tetraspanins and supported by handouts accompanying
the
models. Intraluminal cargo is included via removeable “pegs”, and examples representing
RNA
or simple globular proteins, and a template has been created.
Results: The design is free and open source, and available to the community
at: https://www.thingiverse.com/thing:3986565. Instructions for 3D printing are available
from the UK Extracellular Vesicle Society website;
https://www.ukev.org.uk/public-engagement-materials/. Models have been produced using
entry-level 3D printers and trialled at engagement events with good early responses.
Summary/Conclusion: The authors hope the community will use and develop this
3D-model design and that the approach provides an additional and helpful tool for
educating
audiences about the complexities and roles of EVs in biology and disease.
PT08.02
Centrifugal filtration-SEC is promising for extracellular
vesicle isolation from D492 and D492HER2+ breast epithelial cell lines
Berglind E. Benediktsdottira
, Helga
Snorradottira and Sarah Steinhäuserb
aUniversity of Iceland, Reykjavik, Iceland;
bUniversity of Iceland, Reykjavík, Iceland
Introduction: Despite recent developments in breast cancer therapy, there is
still need for a more targeted approach. Extracellular vesicles (EVs), endogenous
nanovesicles released from human cells, are an attractive choice as nanodrug carriers
due to
their size, stability and their unique targeting specificity. The aim of this study
was to
determine if centrifugal filtration (CF) combined with size exclusion chromatography
(CF-SEC) would be useful for EV isolation from two epithelial breast cell lines D492
and
D492HER2+, representing the tissue of interest, and the amount of cell culture needed
to get
measurable EV concentrations.
Methods: Cell culture media (without serum) from the immortalized breast
epithelial cell lines D492 and D492HER2+ was concentrated with centrifugal filtration
(CF)
followed by isolation with size-exclusion chromatography (SEC) using HiPrep 16/60
Sephacryl
S-400 column run with ÄKTA Start (280 nm), 240 min runs. Each fraction (4–5 ml) was
collected with fraction collector. Dulbecco’s particle free PBS was used as mobile
phase.
The resulting particles were analysed with nanoparticle tracking analysis (NTA, NanoSight
NS300, camera gain 10, static mode, capture time 90 sec), western blotting (WB), microBCA
and transmission electron microscopy (TEM, samples fixed with 2% formaldehyse and
stained
with 2% uranyl acetate, run at 80kV).
Results: Although SEC did not show any prevalent peaks from early eluting
regions previously shown to contain extracellular vesicles, these fractions (F1-F3,
40–130 min) were collected from D492HER2+ cell culture medium. Interestingly, both
NTA and
TEM suggest that F2 and F3 contained EVs as the isolated particles measured 65 and
58 nm,
respectively and TEM revealed spherical particles 20–50 nm in diameter. WB was unable
to
detect the EV associated protein Alix (but was present in the whole cell lysate).
Soluble
proteins and protein aggregates eluted late in the SEC chromatogram (180 min), with
protein
analysis (microBCA), TEM and WB confirming their presence.
Summary/Conclusion: CF-SEC is a promising method for EV isolation for
pharmaceutical applications, but further work is needed to optimize the isolation
process
using ÄKTA Start for these cell lines.
Funding: University of Iceland Research Fund and “Göngum saman” Research Fund
(Research fund supporting fundamental research of breast cancer in Iceland) is gratefully
acknowledged.
PT08.03
Customer stories from the EV Core of University of
Helsinki
Karina A. Barreiroa
, Mari
Palviainenb, Pia Siljanderb and Maija Puhkaa
aInstitute for Molecular Medicine Finland FIMM, University of
Helsinki, Finland, Helsinki, Finland; bFaculty of Biological and Environmental
Sciences, University of Helsinki, Finland, Helsinki, Finland
Introduction: The EV core, world’s first EV-dedicated technology platform
established in 2016, is a joint venture of two extracellular vesicle (EV) research
laboratories at University of Helsinki. As an academic research/service facility,
the EV
core provides infrastructure, state-of-the-art and emerging EV-technologies for research
groups, hospitals, companies and authorities in the EV-field. The EV core provides
EV
isolation, purification and characterization services and offers contacts to downstream
analyses in other core facilities based on optimized EV protocols. Here, we present
and
discuss the customer experiences and prospects with the aim to further develop EV
core
services.
Methods: Our most wanted services are nanoparticle tracking analysis, electron
microscopy, EV isolation and RNA isolation and consultation. Currently, the key down-stream
analysis methods are (mi)RNA sequencing, metabolomics, flow cytometry and functional
assays.
Results: We present the stories from our customers starting with their
research questions and need for the EV expertise/consultation and equipment. Next,
we show
how the projects advanced and what types of EV core -derived or other downstream services
helped them to achieve their aims. In the end, we will acknowledge the customers experience
and current status of their research.
Summary/Conclusion: Narratives of customer stories are an effective starting
point for fruitful discussions about the current status and next developments in the
young
EV service field.
PT08.04
Recent ISEV Workshops: Open, reproducible and standardized EV
research (Ghent, 2019) and EVs in immunology (Buenos Aires, 2020)
Kenneth W. Witwer
a, Edit
Buzásb, Albertina Romaniukc, Florencia Sabbioned,
Clotilde Thérye, Analia Trevaniad, Marca H.M. Waubenf,
Olivier de Weverg, An Hendrixg and Matias Ostrowskih
aJohns Hopkins University School of Medicine, Baltimore, USA;
bSemmelweis University, Department of Genetics, Cell and Immunobiology, MTA-SE
Immune-Proteogenomics Extracellular Vesicle Research Group, Budapest, Hungary and
HCEMM_SE
Extracellular Vesicle Research Group, Budapest, Hungary; cInstituto INBIRS,
Universidad de Buenos Aires, Buenos Aires, Argentina; dAcademia Nacional de
Medicina, Buenos Aires, Argentina; eINSERM U932, Institut Curie, PSL Research
university, Paris, France; fDepartment of Biomolecular Health Sciences, Faculty
of Veterinary Medicine, Utrecht University, Utrecht, Netherlands, Utrecht, Netherlands;
gLaboratory of Experimental Cancer Research, Department of Human Structure and
Repair, Ghent University, Belgium; Cancer Research Institute Ghent, Belgium, Ghent,
Belgium;
hInstituto INBIRS, Universidad de Buenos Aires-CONICET, Buenos Aires,
Argentina
Introduction: Since its founding in 2012, ISEV has sought to further
extracellular vesicle research in various ways including scientific meetings. These
events
encompass annual meetings as well as smaller, topically focused workshops, with the
first
ISEV Workshop (on RNA and EVs) organized in New York City in October, 2012. In December,
2019, the Workshop “Open, Reproducible, and Standardized EV Research” was held in
Ghent,
Belgium. In March, 2020, the Workshop, “EVs in Immunology” was held in Buenos Aires,
Argentina, with a preceding Education Day.
Methods: The international organizing committees of the 2019 and 2020 ISEV
Workshops prepared scientific programs around key themes of EV rigour and standardization
(Ghent, Belgium, 2019 workshop) and EVs in immunology (Buenos Aires, Argentina, 2020
workshop). Abstract and application submissions were invited. Applications were reviewed
and
ranked by panels of EV experts for each event, and participants were invited.
Results: The 2019 and 2020 Workshops assembled a total of more than 100
individuals for talks and discussions around the themes of Rigour and Standardization
and
EVs in Immunology. The Buenos Aires Workshop was preceded by an Education Day, coordinated
by the ISEV Executive Committees for Education and Science and Meetings. During these
two
workshops, poster presentations were permitted for the first time, affording additional
presentation and interaction opportunities. The Rigour and Standardization Workshop
also
featured real-time discussant polling to facilitate discussion.
Summary/Conclusion: ISEV workshops such as those addressing Rigour and
Standardization (Ghent, 2019) and EVs in Immunology (Buenos Aires, 2020) continue
to provide
opportunities for focused discussion of small groups of experts on key topics in the
field.
Often followed by published products, ISEV workshops help to lead and coordinate progress
in
EV science. For future ISEV workshops, educational activities may again expand the
reach of
each event, while poster sessions and app-driven real-time responses should be considered
for enhanced interactions and participant canvassing.
PT08.05
EV Journal Club: exchanging pizza for a worldwide audience
during COVID-19
Kenneth W. Witwer
Johns Hopkins University School of Medicine, Baltimore, USA
Introduction: A monthly journal club focused on extracellular vesicle science
was established at Johns Hopkins University in 2016, featuring lunch and presentations
by
academic and industry participants. When COVID-19 prevented in-person meetings beginning
in
March, 2020, the journal club was converted to a virtual, weekly format on the popular
online meeting app Zoom. The journal club has persisted despite initial problems with
online
vandalism. Most sessions are also made public on a YouTube channel,
https://www.youtube.com/c/ExtracellularVesicleClub.
Methods: Weekly EV Club sessions are arranged by the host. Most focus on a
specific manuscript related to EVs, but some weeks feature presentations of published
or
soon-to-be-published research by the presenting authors. Sessions are advertised one
week to
several days in advance on social media platforms such as LinkedIn, Twitter, and Facebook,
asking interested parties to sign up to join a mailing list via SurveyMonkey. The
log-in
information is then sent to the mailing list. Upon clicking the link, participants
are
placed in a virtual waiting room for vetting by the host and volunteers. After admission,
all parties but the host and presenter are muted to avoid distractions. Questions
and
comments may be placed in a chat box. Contributions are monitored and compiled by
the host
and volunteers to build a question-and-answer session at the end of the presentation.
Recorded sessions–with or without editing as needed–are placed on the YouTube channel
for
additional access.
Results: Despite initial problems with online vandalism known as
“Zoombombing,” the journal club has continued weekly during the COVID-19 shutdown
in the
host country (US). An audience of between 80 and 150 individuals is typical. Participants
typically ask more questions than can be answered in a one-hour time frame. The online
format also allows for debate-style events and polling of the audience.
Summary/Conclusion: This EV Journal Club is an example of how online tools can
be used to facilitate international scientific interactions. Further development of
such
formats could provide alternative approaches for ISEV activities in the Science, Education,
and Communication areas.
PT09: EV Immunology, Autoimmunity, and Inflammation
Chair: Heather H. Pua, MD, PhD – Department of Pathology, Microbiology and Immunology,
Vanderbilt University Medical Centre
Chair: Evan Keller – University of Michigan
PT09.01
Air pollution exposure and allergic rhinitis exacerbation: the
role of nasal microbiota and extracellular vesicle communication
Jacopo Mariania
, Simona
Iodiceb, Laura Cantoneb, Paolo Marracinic, Emanuele
Confortid, Raymond Ignare, Pallav Bulsarae, Maria Stella
Lombardif, Robert Howling, Luca Ferrarib and Valentina
Bollatib
aEPIGET LAB, Department of Clinical Sciences and Community
Health, Università degli Studi di Milano, Milan, Italy; bEPIGET LAB, Department
of Clinical Sciences and Community Health, Università degli Studi di Milano, Milano,
Italy;
cFondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Milano,
Italy; dSchool of Occupational Medicine, Università degli Studi di Milano,
Milano, Italy; eGlaxoSmithKline Consumer Healthcare, Warren, New Jersey, USA,
Warren, USA; fGlaxoSmithKline Consumer Healthcare, Nyon, Switzerland, Nyon,
Switzerland; gGlaxoSmithKline Consumer Healthcare, Weybridge, Surrey, UK,
Weybridge, UK
Introduction: Allergic Rhinitis (AR) is a systemic airway disease involving
the respiratory tract. Lifestyles and environmental factors (e.g. particulate matter,
PM)
have a role in disease pathogenesis and recurrence.
The study aim is to assess whether the exposure to PM10 and PM2,5, chosen as paradigmatic
environmental stressors, could modify the composition of nasal microbiota (NM) and
extracellular vesicle (EV9 signalling network, showing a role in allergic AR
exacerbation).
Methods: NM analysis were performed on V3-V4 16 S rRNA gene regions amplified
from upper-airway tracts of 25 AR cases and 25 healthy individual controls to perform
NM
analyses. EV size, concentration and cellular origin for each subject were assessed
by
nanoparticle tracking analysis (NTA) and flow-cytometry (FC). Information on daily
PM10 and
PM2,5 concentrations at the municipality of residence in the 7 days preceding nasal
sampling
(i.e. Day −1 to Day −7) was assigned to each subject by ArcGIS software. Multivariable
and
logistic analyses were applied on NM, NTA and FC outcomes.
Results: When taxonomy composition was considered, in controls Actinobacteria
(50.8%) was the most represented, followed by Firmicutes (34.7%) and Proteobacteria
(12.8%)
while in cases Proteobacteria were 38.8%, Actinobacteria were 37.1% and Firmicutes
were
23.4%.
Cases showed a higher concentration of all the investigated EV types, derived from
platelets (CD61+), activated endothelium (CD62e+), monocytes (CD14+), eosinophils
(CD294+),
neutrophils (CD177+), mastocytes (CD203 c+), epithelial cells (EPCAM+), GRAM+ bacteria
(Lipoteichoic Acid+), GRAM- bacteria (LPS+). The effect was greatest in the case of
mastocytes EVs which were increased 2.5-fold in cases versus controls (p < 0.001).
EVs
were modified by PM exposure at several time lags. In particular, a negative association
between PM10 and eosinophil EVs was observed (beta = −0,016; p-value = 0,017).
As we clustered subjects according to their NM, we observed this variable was a strong
effect modifier of the association between PM exposure and EV release.
Summary/Conclusion: Our findings start to provide an insight on the effect of
air pollution on EVs, taking into account the effect of NM, in patients with AR. Further
research is necessary to disentangle the mechanism exerted by inhaled pollutants in
modulating EVs and NM, and therefore AR exacerbation.
Funding: GSK Investigator Sponsered Study
PT09.02
Aryl hydrocarbon receptor activation induces the expression of
specific MicrorRNAs in Th17 cells that are release into extracellular vesicules and
associated with arthritis
Paula Barbim Donatea
, Kalil Alves
de Limab, Raphael Sanches Peresc, Fausto Almeidaa, Tatiana
Nerry Cecilioa, Sandra Alves Fukadaa, Daniele Bernardo
Nascimentoa, Jhimmy Talbotd, Rêne Almeidaa, José Carlos
Alves-Filhoa, Thiago Mattar Cunhaa, Paulo Louzadaa and
Fernando Queiroz Cunhae
aUniversity of São Paulo, Ribeirão Preto, Brazil;
bUniversity School of Medicine in Saint Louis, St. Louis, USA;
cKennedy Institute of Rheumatology, University of Oxford, Oxford, UK;
dNew York University School of Medicine, New York, USA; eUniversity
of São Paulo, Ribeirão Preto, Brazil
Introduction: In Rheumatoid Arthritis (RA), an autoimmune disorder
characterized by a chronic sinovial inflammation, smoking is a major risk factor
contributing to disease progression, and poor response to therapy. Th17 cell is actively
involved in worsening smooking-associates inflammation mediated by Aryl hydrocarbon
receptor
(AhR), a cytoplasmic transcription factor involved in xenobiotic metabolism. Both,
AhR and
Th17 cells, has important implications during RA development. Considering that cigarette
smoke is a potent epigenetic modifier, we hypothesized that AhR activation, by cigarette
components, would transcribe specific microRNAs in Th17 cells as a molecular mechanism
to
exacerbate inflammation in arthritis.
Methods: MicroRNA expression was evaluated by large-scale approach or
real-time PCR. C57/BL6 and AhR null mice were submitted to arthritis experimental
models and
exposed or not to cigarrete smoke (ethical committee approved 048/2012). Extracellular
vesicles (EVs) were isolated by ultracentrifugation, and characterized by western
blot and
nanosight. RANKL-induced osteoclasts (OCs) differentiation in vitro was stained for
TRAP.
Inhibition of miRNAs were performed using anti-miRs transfection.
Results: We identified a specific group of miRNAs induced in Th17 cells after
AhR activation. During arthritis progression, the microRNAs are expressed and increases
after exposure to cigarette smoke. In the absence of AhR their levels were drastically
reduced. Interestingly, we found that these microRNAs are released by Th17 cells into
EVs,
and are able to promote osteoclastogenesis. OCs differentiation in vitro increases
in the
presence of Th17-derived EVs, and this process is reduced in the absence of microRNAs.
Summary/Conclusion: MicroRNA-mediated gene regulation plays crucial roles in
the immune system functions, and their abnormal expression is highly correlated with
the
pathogenesis of RA. EVs are known to function in cell-to-cell communication and are
able to
transmit their contents and cause changes in the target cell. Our findings demonstrate
a new
molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis;
through the activation of AhR receptor in Th17 cells, inducing the transcription of
specific
microRNAs that are released into EVs, and act as pro-inflammatory mediators.
Funding: Capes, FAPESP
PT09.03
Extracellular Vesicles released by Trypanosoma cruzi
trypomastigotes under different stress conditions
Camilla Ioshida Vasconcelosa, Normanda Souza
Meloa, Juliana Terzi Maricatoa, Patricia Xanderb, Wagner
Luiz Batistaa, Rodrigo Pedro Soaresc, Sergio Schenkmana and
Ana Claudia Torrecilhas
a
aUNIFESP, Sao Paulo, Brazil; bUniversidade Federal
de São Paulo campus Diadema, Sao Paulo, Brazil; cFIOCRUZ Rene Rachou, Belo
Horizonte, Brazil
Introduction: Chagas disease (CD) is caused by the flagellated protozoan T.
cruzi. Trypomastigote forms are capable of releasing extracellular vesicles (EVs)
that
contain the major surface molecules of the parasite. The parasite has a complex life
cycle
that leads to it a rapid adaptation in the environmental changes in the hosts. However,
the
effects of stress on on EVs release are not completely understood. Objetive: we evaluated
the release of EVs by trypomastigotes incubated under different stress conditions
and the
immunomodulatory role of these EVs in pre-activated bone marrow-derived macrophages
(BMDM).
Methods: Nanoparticle tracking analysis (NTA) and scanning electron microscopy
(SEM) showed an increase in EVs releasing by trypomastigotes at 4°C under acidic conditions,
EVs released was affected and triggered amastigogenesis process.
Results: Treatment with sodium azide (NaN3) also caused changes in the release
of EVs regarding size and concentration. Nitrosative stress caused by sodium nitrite
(in
culture medium mildly acidic, pH 5.5; in this condition NaNO2 releases Nitric Oxide)
stimulated an increase in production of EVs by T. cruzi. When the parasites were treated
with 100 nM S-nitrosoglutathione (SNOG), we observed a reduction in size and concentration
of vesiculate material by trypomastigotes. At a higher SNOG concentration (100 µM),
the
concentration of the vesiculate material increased. T. cruzi-derived EVs exposed to
stress
conditions increased the expression of iNOS, Arg 1, IL-12 and IL-23 genes in IFN-γ
and LPS
pre-activated BMMs.
Summary/Conclusion: Results suggest that the viability and/or integrity of the
parasite are necessary for the EVs releasing. In those in vitro conditions they triggered
a
proinflammatory response in host cells. This may be a strategy developed by the parasite
to
favour its establishment in the host.
Funding: FAPESP, CNPq, CAPES and FAPEMIG PPM-X 00102/6.
PT09.04
Immuno-toxicological evaluation of human mesenchymal stem
cell-derived extracellular vesicles
Dong-oh Kima
, Min-Hui
Yooa, Sang-Jin Parka, Aram Leea, Ji-Young
Kima, Geon Yooa, Hyunju Kima, YongWoo Chob and
Kyoung-Sik Moona
aKorea Institute of Toxicology, Deajeon, Republic of Korea;
bDepartment of Materials Science and Chemical Engineering, Hanyang University
ERICA, Ansan, Gyeonggi-do 15588, Republic of Korea, Ansan, Republic of Korea
Introduction: Mesenchymal stem cells (MSCs) have been widely used to the field
of autoimmune diseases or tissue regeneration therapy. Recently, many research groups
have
reported that MSCs showed their ability via secreted paracrine mediators including
extracellular vesicles (EVs) rather than cell-to-cell contact. MSCs mainly exist on
bone
marrow, peripheral blood, umbilical cord and adipose and can mostly secrete EVs. It
has
emerged that EVs alone are responsible for the therapeutic effect of MSCs in plenty
of
animal diseases models. Hence, MSC-derived EVs may be used as an alternative MSC-based
therapy in regenerative medicine.
Methods: As part of safety programme for human therapeutics, we performed
immunotoxicological assessment of EVs obtained from human MSCs (hEVs) in mice and
human
peripheral blood mononuclear cells (hPBMCs). Firstly, mice were treated intravenously
with a
negative control, a positive control (LPS; 0.4 mg/kg), or low-dose (1x10e8 paticles/head)
and high-dose (1x10e9 paticles/head) of hEVs every other day for 10 days and then
analysed
lymphocyte subsets from collected spleen by FACS.
Next, we treated the EVs on hPBMCs for 3 days with low conc. (1x10e8 particles/ml),
high
conc. (1x10e9 particles/ml), PMA/Ionomycin as a cell activator or CPT (10 μM) as an
apoptotic inducer. Annexin V/PI and CSFE were analysed by FACS.
Results: As a result, splenic NK cells and B cells were slightly increased
about 2 ~ 7% in hEVs- treated mice, without biological significance, compared with
a
positive control (LPS) as an immunogenicity inducer. And there were no effects on
serum
levels of inflammatory cytokines in mice. In addition, hEVs had no cytotoxic effect
on
hPBMCs at both low and high conc. Under the culture medium with EVs-depleted FBS,
the hEVs
appeared minimal anti-apoptotic effect on hPBMCs. For the CFSE assay, the hEVs showed
slight
proliferation on hPBMCs and PBMC activation induced by PMA/Ionomycin.
Summary/Conclusion: In conclusion, the hEVs have little immuno-toxicological
effects in mice and hPBMCs. Further detailed studies to elucidate immunological response
of
hEVs for development of human therapeutics are needed.
Funding: This research was supported by a grant (18172MFDS173) from Ministry
of Food and Drug Safety.
PT09.05
Investigation of immune response to mesenchymal stem
cell-derived extracellular vesicles in the cancer setting
Katie E. Gilligana
, Oliver
Treacyb, Clodagh O’Neillc, Elan McCarthyd, Emma
McDermotte, Adriele Prina-Mellof, Peter Dockerye, Aideen
Ryang and Róisín Dwyerh
aDiscipline of Surgery, National University of Ireland
Galway, Galway, Ireland; bRegenerative Medicine Institute, National University
Ireland Galway, Galway, Ireland; cDiscipline of Surgery, National University
Ireland Galway, Galway, Ireland; dPrecision Oncology Ireland & Discipline of
Surgery, National University Ireland Galway, Galway, Ireland; eDiscipline of
Anatomy, National University Ireland Galway, Galway, Ireland; fSchool of
Medicine, Trinity College Dublin, Ireland, Dublin, Ireland; gRegenerative
Medicine Institute and CÚRAM, SFI Research Centre for Medical Devices, National University
Ireland Galway, Galway, Ireland; hDiscipline of Surgery, CÚRAM, SFI Research
Centre for Medical Devices and Precision Oncology Ireland, National University Ireland
Galway, Galway, Ireland
Introduction: Mesenchymal Stem Cell-derived extracellular vesicles (MSC-EVs)
are thought to be a fingerprint of the secreting cell and therefore may retain the
cancer
targeting and immune privilege of MSCs. Thus MSC-EVs hold immense potential as
tumour-targeted therapeutics for breast cancer. The aim of this study was to determine
whether MSC-EV administration in tumour bearing immunocompetent animals would initiate
an
immune response.
Methods: EVs were isolated from conditioned media of both human and murine
bone marrow-derived MSCs through sequential differential centrifugation, micro-filtration
and ultracentrifugation. EVs were characterized by Nanoparticle Tracking Analysis
(NTA),
Western Blot and Transmission Electron Microscopy (TEM). 1 x 10(8) human or murine
MSC-EVs
were administered intravenously into 4T1 breast tumour bearing Balb/c mice (n = 6)
and
healthy controls (n = 6). Tumour tissue, draining lymph node and spleen were then
harvested,
dissociated and flow cytometry performed targeting markers associated with a range
of immune
cells including T-cells, macrophages and natural killer (NK) cells.
Results: EVs were successfully isolated from murine and human MSCs with the
appropriate size of small EVs (sEVs: 30–150 nm) and morphology including a lipid bilayer
observed by TEM. EVs expressed tetraspanins CD63, CD81, CD82; Cytosolic protein TSG101
and
were negative for Calnexin. EV concentrations ranged from 4.08 x 10(9) – 6.6 x 10(9)/ml.
In
order to study a range of immune cell populations two antibody panels were created
using
complimentary fluorescent dyes. The proportion of T-cells (CD4+, CD8+, CD25+), Neutrophils
(GR-1+, LY-6 C+), Dendritic cells (CD11 c+), Macrophages (CD11b+, MHCI+, MHCII+),
NK cells
(CD27+) and B cells (CD19+) remained stable in the tumour, draining lymph node and
spleen of
all tumour-bearing animals that received either human or murine MSC-EVs, with no significant
change observed in any category.
Summary/Conclusion: The data presented supports the hypothesis that MSC-EVs
retain the immune privilege of the secretory cell, with human cell-derived EVs illiciting
no
immune response in mice. This is encouraging and reinforces the potential for use
of MSC-EVs
in the therapeutic setting.
Funding: Irish Research Council Government of Ireland Postgraduate Scholar
2016 GOIPG/2016/978
PT09.06
Outer membrane vesicles of Mycobacterium avium modulate
inflammatory response in monocytes
Jingyu Wanga
, Lei Zhengb
and XiuMei Huc
aDepartment of Laboratory Medicine, Nanfang Hospital,
Southern Medical University, Guangzhou, China, Guangzhou, China (People’s Republic);
bDepartment of Laboratory Medicine, Nanfang Hospital, Southern Medical
University, Guangzhou, 510515, China, guangzhou, China (People’s Republic);
cSouthern medical university, guangzhou, China (People’s Republic)
Introduction: Mycobacterium avium (M. avium) is a slow growth rate
non-tuberculous mycobacterium (NTM). M. avium infection is a severe global health
problem.
But the mechanisms of pathogenicity of M. avium are poorly understood. Outer membrane
vesicles (OMVs) that traverse the cell wall and contain a varied bioactive components
inculding DNA, RNA, protein and toxins. Previous studies have suggested that these
OMVs are
produced in vitro and during animal infection, but the role of OMVs secretion during
the
interaction of M. avium with host cells remains unknown.
Methods: In this study, M. avium were grown in Middlebrook 7H9 medium (M7H9)
supplemented with 10% (v/v) OADC enrichment and 0.5% (v/v) glycero. M. avium OMVs
were
isolated by ultracentrifugation method. Characterization of OMVs by transmission electron
microscopy (TEM) and nanoparticle tracking analysis (NTA). The RAW 264.7 murine macrophages
were incubated with the M. avium OMVs to analyse inflammatory response and production
of
nitric oxide (NO) and reactive oxygen species (ROS) of macrophage.
Results: In this study, we demonstrate by fluorescence microscopy that murine
macrophages can phagocytosis OMVs produced by M. avium. Incubation of M. avium OMVs
with
murine macrophages resulted in increased levels of extracellular tumour necrosis factor
alpha (TNF-α), interleukin-1β (IL-1β), terleukin-6 (IL-6) and interleukin-12 (IL-12).
Meanwhile OMVs stimulated macrophages produce NO and ROS.
Summary/Conclusion: The above results indicate that the outer membrane
vesicles of M. avium could stimulate macrophage function and induce inflammatory immune
responses comparable to M. avium infection but do not cause cell apoptosis. Our findings
suggest that M. avium secretory outer membrane vesicles have the potential to influence
the
interaction of M. avium with host cells.
Funding: supported by China Postdoctoral Science Foundation under Grant No
2019M662990
PT09.07
Sepsis alters the differential expression of microrna in
platelet-derived small extracellular vesicles of paediatric patients
Elizabeth W. J. Kerrisa
, Madeleine
Goldbergb, Claire Hoptayc and Robert Freishtatb
aChildren’s National Hospital, Silver Spring, USA;
bChildren’s National Hospital, Washington, USA; cChildrens National
Hospital, Washington, USA
Introduction: Hospital associated venous thromboembolism (HA-VTE) in
paediatric patients is the second most common contributor to harm in hospitalized
children.
Platelet-endothelial interactions are integral to the formation of VTE, especially
in
inflammatory conditions such as sepsis. Small extracellular vesicles (sEVs) have the
ability
to reprogramme target cell phenotypes via their microRNA contents and are known to
contribute to VTE formation. We hypothesize that sepsis alters platelet-derived sEV
microRNAs capable of net upregulation of vascular endothelial procoagulant and
downregulation of anticoagulant pathways.
Methods: Using a precipitation solution and size exclusion chromatography, we
isolated sEVs from platelet poor plasma of children admitted to the paediatric intensive
care unit for sepsis and from healthy controls. We positively selected platelet-derived
sEVs
using immunomagnetic isolation for CD42B platelet antigen and confirmed selection
using flow
cytometry. MicroRNA was profiled using Affymetrix GeneChip miRNA 4.0 array.
Results: MicroRNA from 9 sepsis patients (median age 7.0 years; IQR:1.2–13 and
77% female) with a median pSOFA score of 6 (IQR:2.5–10) and from 5 healthy controls
(median
age 9 years; IQR:6.5–13.5 and 20% female) was isolated and compared. In septic vs.
healthy
patients 30 miRNAs were differentially expressed (false discovery rate (FDR)<0.05;
fold
change ≥|1.5|) affecting 921 mRNA pathways. In septic children, pathways affecting
chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores
≥2.
Summary/Conclusion: We developed a method to successfully isolate
platelet-derived sEVs. Sepsis alters the platelet-derived sEV microRNA profile in
paediatric
patients with sepsis. These microRNAs are predicted to target chemotaxis and cell
movement
pathways, important contributors in the formation of HA-VTE. Further analysis into
specifically targeted pathways should be conducted as a potential target for the prevention
of HA-VTE in sepsis.
Funding: Dr. Kerris’ institution received funding from a Ruth L. Kirschstein
National Research Service Award (NRSA) Institutional Research Training Grant awarded
to the
Children’s Research Institute Hematology Training Program by the National Heart, Lung
and
Blood Institute (NHLBI) of the National Institutes of Health (NIH) (5T32HL110841-07).
PT09.08
T cell-exosome-derived miR-142-3p impairs glandular cell
function in Sjögren´s syndrome
Juan Cortesa
, Niki
Moutsopoulosb and Luigi Alevizosc
aNIDCR, NIH, bethesda, USA; bNational Institute of
Dental and Craniofacial Research (NIDCR),Oral Immunity and Inflammation Unit, NIH,
Bethesda,
USA; c1Sjögren’s Syndrome and Salivary Gland Dysfunction Unit, National Institute
of Dental and Craniofacial Research, National Institutes of Health., Bethesda, USA
Introduction: Sjögren´s syndrome (SS) is a systemic autoimmune disease that
mainly affects salivary and lacrimal glands. Mechanisms of SS pathogenesis are poorly
understood. It is thought that inflammation leads to destruction of exocrine glands,
however
the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology
are not characterized. Our work identifies T cell-exosome-derived miR-142-3p as a
pathogenic
driver of immunopathology in SS. MicroRNAs (miRNAs) are endogenous small noncoding
RNA
molecules that regulate the expression of target genes through translational repression
of
mRNAs. Through transcriptomic profiling studies our group had previously documented
a
significant upregulation of miR-142-3p in patient SS tissues and in serum exosomes.
Methods: Structured search for target genes of miR-142-3p involved in salivary
gland (SG) physiology was performed with mirDIP 4. SERCA2b, RyR2 and AC9 were selected
for
further validation and functional analysis. Binding of the miRNA was confirmed by
luciferase
reporter assays in HSG cell lines and human-derived primary epithelial cells. The
mRNA and
protein levels of SERCA2b, RyR2 and AC9 were determined by qPCR and Western blot,
respectively. To investigate the cell-specific distribution of miR-142-3p in relation
to the
expression levels of SERCA2b, RYR2, and AC9, a double fluorescent in situ hybridization
was
performed. Ca2+ signalling and cAMP levels were measured using fluorescent sensor.
To
isolate exosomes, the T cell medium and serum of SS-patients and healthy volunteers
(HV)
were collected.
Results: We show that miR-142-3p is over-expression in the SGs of SS-patients.
Next, we demonstrated that miR-142-3p is contained in exosomes in serum of SS-patients
significantly more than serum of HV. We also show that activated T cells secrete exosomes
containing miR-142-3p which transfer into glandular cells and affecting intracellular
Ca2+ signalling, cAMP production and protein production by miR-142-3p targets (SERCA2B,
RyR2
and AC9).
Summary/Conclusion: This study provides evidence for a functional role of the
miR-142-3p in SS pathogenesis and promotes the concept that T cell-activation directly
may
impair epithelial cell function through secretion of mi-RNA containing exosomes.
PT09.09
Treg-Derived IL35-Coated Extracellular Vesicles Promote
Infectious Tolerance
William J. Burlinghama
, Jeremy
Sullivanb, Tomita Yusukec, Ewa Jankowska-Gand, Diego
Lemad, Matthew Arvedsone, William Bracamonte-Baranf,
Seungpyo Hongd and Dario Vignalig
aUniversity of Wisconsin, Madison, USA;
bUniversity of Wisconsin-Madison, Madison, USA; cTokyo Women’s
Hospital, Tokyo, Japan; dUW-Madison, Madison, USA; eStanford
University, Palo Alto, USA; fUT-Odessa, Odessa, USA; gUniversity of
Pittsburgh, Pittsburgh, USA
Introduction: Interleukin-35 (IL35) is an immunosuppressive cytokine composed
of Epstein-Barr-virus-induced protein 3 (Ebi3) and IL12-alpha chain (p35) subunits,
yet the
forms that IL35 assumes and its role in peripheral tolerance, remain elusive.
Methods: We induce CBA-specific, IL35-producing T regulatory (Treg) cells in
TregEbi3 WT C57BL/6 reporter mice, and identify IL35 producers by expression of Ebi3TdTom
gene reporter, plus Ebi3 and p35 proteins.
Results: Curiously, both subunits of IL35 were displayed on the surface of
tolerogen-specific Foxp3+ and Foxp3neg (iTr35) T cells. Furthermore, IL35 producers,
although rare, secrete Ebi3 and p35 on extracellular vesicles (EV) targeting a 25-
to
100-fold higher number of T and B lymphocytes, causing them to acquire surface IL35.
This
surface IL35 is absent when EV/exosome production was inhibited, or if Ebi3 is genetically
deleted in Treg cells.
Summary/Conclusion: The unique ability of EV to coat bystander lymphocytes
with IL35, promoting exhaustion in, and secondary suppression by, non-Treg cells,
identifies
a novel mechanism of infectious tolerance.
Funding: NIH Grants R01-AI119140-03 (to W.J.B.), R01 CA203689 and P30 CA047904
(to D.A.A.V.) and the University of Wisconsin Carbone Cancer Center Support Grant
P30
CA014520.
PT09.10
Unique formulated dual targeting antigen specific and delivered
miRNA-150 gene regulating exosomes acting at the immune synapse to induce APC-derived
secondary suppressive exosomes
Phil W. Askenasea
, Krzysztof
Bryniarskib, Katarzyna Nazimekb and Francisco
Sánchez-Madridc
aYale University, Hamden, USA; bDepartment of
Immunology, Jagiellonian University Medical College, 31–121 Kracow, Poland;
cUniversity of Madrid, Madrid, Spain
Introduction: An exosome-APC circuit we uncovered may be applicable beyond
skin immunity we study in mice.
Methods: High antigen dose tolerized CD8 + T cells make suppressive
antigen-specific exosomes due to chosen surface antibody light chains that enable
targeting
antigen presenting cells (APC) antigen-specifically for delivery of also chosen inhibitory
miRNA-150 to mediate specific functional gene alterations.
Results: Both antigen and gene specificity aspects are lent to naïve but
activated exosomes by simple in vitro incubations alone. For mechanism, these primary
exosomes bind antigen peptides in MHC on APC that in turn make secondary suppressive
exosomes that act peptide/MHC-specifically on the effector T cells at the immune synapse.
They transfer another miRNA for strong prolonged inhibition of active delayed-type
hypersensitivity (DTH) for days even, when the primary miRNA-150-pos exosomes are
administered orally at the height of the in vivo response, in a physiological dose.
Summary/Conclusion: It is shown possible to induce therapeutic exosomes with
Ag targeting of choice due to placed Ab on the surface and that also target specific
gene
functions of acceptor cells due to carriage of a selected miRNA. This dual Ag and
gene-specific therapy has applications in treatment of Cancer, Autoimmunity and
Allergies.
Funding: This research was supported by the grant of Polish Ministry of
Science and Higher Education No 507 K/ZDS/006148 to K.B. and partly by grant No AI-1053786
from the National Institutes of Health to P.W.A.
PT09.11
The role of Mac-1 and calcium signalling in the formation of
neutrophil granulocyte derived extracellular vesicles
Viktória Szeifert
a, Ákos M.
Lőrinczb and Erzsébet Ligetia
aDepartment of Physiology, Semmelweis University, Budapest,
Hungary; bSemmelweis University, Department of Physiology, Budapest, Hungary
Introduction: Previously, our group characterized distinct populations of
extracellular vesicle (EV) released from neutrophilic granulocytes: EV formed spontaneously
(sEV) and upon activation with opsonized particles (aEV). The aEV differs in protein
cargo
and its ability to inhibit bacterial growth. We described that Mac-1 integrin (CR3
receptor)
plays key role in the aEV production and extracellular calcium supply is crucial in
this
signalization. In the present work, our aim was to investigate whether Mac-1 activation
or
Ca-signalling on their own are sufficient for the initiation of the aEV biogeneis.
Methods: We isolated neutrophil derived EVs from peripheral human blood and
murine bone marrow by two-step centrifugation and filtration. We tested the effect
of
Ca-ionophore and examined the EV production on C3bi coated surface and in soluble
form. We
quantified the vesicles by flow cytometry and determined their protein content by
Bradford
assay. We examined their antibacterial effect in parallel with optical density-based
measurement and our flow cytometry based method.
Results: On C3bi coated surface, we observed an increased EV production, and
these EVs possessed antibacterial capacity. However, in soluble condition, C3bi did
not
induce further EV production, and these EVs did not show any antibacterial property.
We
found that Ca-ionophore initiated EV formation, but these EV did not show antibacterial
effect. We observed EV production increase after Ca-ionofore treatment both in the
presence
and in the absence of extracellular Ca. The Ca-ionophore slightly increased the opsonized
particle induced EV production, but did not potentiate their antibacterial capacity.
Summary/Conclusion: Mac-1 activation is not just crucial, but sufficient in
initiation of the aEV biogenesis. Clustering of this receptor is required. While the
Ca-signal is crucial, it is not sufficient in the generation of aEVs.
Funding: ÚNKP-19-19 NEW NATIONAL EXCELLENCE PROGRAM OF THE MINISTRY FOR
INNOVATION AND TECHNOLOGY (Hungary).
PT09.12
Extracellular vesicles and their microRNA cargo in retinal
health and degeneration: mediators of homoeostasis, and immune modulation
Yvette S. M. Wooff, Adrian Cioanca, Riemke
Aggio-Bruce, Joshua Chu-Tan, Ulrike Schumann and Riccardo Natoli
The Australian National University, Canberra, Australia
Introduction: Photoreceptor cell death and inflammation are known to occur
progressively in retinal degenerative diseases such as age-related macular degeneration
(AMD). However, the molecular mechanisms regulating these biological processes are
largely
unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication
with emerging roles in the modulation of immune responses. EVs, including exosomes,
encapsulate and transfer microRNA (miRNA) to recipient cells and in this way can modulate
the environment of recipient cells. Dysregulation of EVs however is correlated to
a loss of
cellular homoeostasis and increased inflammation. In this work we investigated the
role of
isolated retinal small-medium sized EV (s-mEV) in the regulation of homoeostasis and
immune
modulation in both the healthy and degenerating retina.
Methods: Isolated s-mEV from healthy and degenerative (photo-oxidative
damaged) mouse retinas were characterized using dynamic light scattering, transmission
electron microscopy and western blot, and quantified using nanotracking analysis.
Small
RNA-seq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy
and
degenerating retinas. Finally, the effect of exosome inhibition on s-mEV-mediated
immune
modulation was investigated using systemic daily administration of exosome inhibitor
GW4869
and analysed by in situ hybridization of s-mEV-abundant miRNA. Electroretinography
and
immunohistochemistry were performed to assess functional and morphological changes
to the
retina as a result of exosome depletion.
Results: Our results demonstrated an inverse correlation between s-mEV
concentration and photoreceptor survival, with decreased s-mEV numbers following retinal
degeneration. Small RNA-seq revealed that s-mEVs contained uniquely enriched miRNAs,
however
no differential composition in s-mEV miRNA cargo following photo-oxidative damage
was
observed. Exosome inhibition using GW4869 exacerbated photoreceptor degeneration,
with
reduced retinal function and increased levels of inflammation and cell death seen
following
photo-oxidative damage. Further, reduced translocation of the photoreceptor-derived
s-mEV
was demonstrated following exosome-inhibition in photo-oxidative damaged mice.
Summary/Conclusion: Taken together, we propose that retinal s-mEV and their
miRNA cargo play an essential role in maintaining retinal homoeostasis through
immune-modulation, and have the potential to be targeted using gene therapy for retinal
degenerative diseases.
PT09.13
Impacts of agricultural dust exposure on human lung-resident
mesenchymal stromal/stem cells and their extracellular vesicles
Stefanie N. Sveiven, Gary B. Adkins, Wenwan
Zhong and Tara M. Nordgren
University of California, Riverside, Riverside, USA
Introduction: Agricultural dust is considered a high-risk occupational hazard
by the CDC, with impacts reaching throughout the communities surrounding these industries,
leading to increased incidence of respiratory illness and disease among individuals
within
this occupation and these communities. Lung-resident mesenchymal stromal/stem cells
(lr-MSC)
have an important role in maintaining homoeostasis in the lung, and mediating pro-
and
anti-inflammatory effects, particularly during exposure to inhaled irritants, like
agricultural dust. One way in which these lr-MSC promote lung homoeostasis is through
the
release of extracellular vesicles (EV), with a variety of cargo that elicit changes
among
target cells. We hypothesize that exposure to agricultural dust modifies the quantity
and
cargo of EV released by lr-MSC to promote lung tissue homoeostasis.
Methods: Primary human lung-resident mesenchymal stromal cells were exposed to
extracts of dusts collected from swine confinement facilities (DE) for 8 or 48hrs
and the
media from these exposures were collected and enriched for EV by opti-prep density
gradient
ultracentrifugation. The quantity of these EV were assessed by nanoparticle tracking
analysis. Additionally, cytokine and chemokine release by lr-MSC were analysed by
enzyme-linked immunoassays.
Results: As assessed at 48 hr following treatment, DE-exposed lr-MSC released
pro-inflammatory cytokines, IL-6 and IL-8, with IL-8 release reaching statistical
significance at 0.1%, 0.5%, and 1% DE concentrations (p = 0.0367, <0.0001, and <0.0001
respectively) and IL-6 trending a similar dose response but only statistically significant
at 1% DE (p = <0.0001). DE exposure of lr-MSC also induced changes in the lr-MSC-derived
EV populations when compared to vehicle control, where lr-MSC released significantly
more EV
in the 5 and 10% iodixanol fractions (p = <0.0001 and 0.0219, respectively) at 8 hr
following DE treatment. Alternatively, there were significantly less EV in the 20
and 40%
density fractions in the media of DE-exposed lr-MSC versus vehicle control.
Summary/Conclusion: Following exposure to agricultural dusts, lr-MSC-derived
EV populations more likely consist of exosomes and ectosomes, which play an important
role
in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation.
PT10: Single-EV Analysis
Chair: Patricia M. Ozawa –
PhD student, Department of Genetics, Universidade Federal do Paraná
PT10.01
Clinically applicable method to avoid swarm detection during
flow cytometry analyses of single extracellular vesicles in human plasma
Bo Lia
, Edwin van der
Polb, lei Zhengc and Rienk Nieuwlandd
aDepartment of Laboratory Medicine, Nanfang Hospital,
Southern Medical University, Guangzhou, China (People’s Republic); bDepartment of
Clinical Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands;
Vesicle Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the
Netherlands; Department of Biomedical Engineering and Physics, Amsterdam UMC, University
of
Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands; cDepartment of
Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515,
China, guangzhou, China (People’s Republic); dDepartment of Clinical Chemistry,
Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Vesicle Observation
Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam,
Netherlands
Introduction: During analyses of single extracellular vesicles (EVs) by flow
cytometry (FCM), particles below the detection limit may exceed the trigger threshold,
which
is called swarm detection and generates false-positive counts. Serial dilutions are
recommended to find the minimal dilution for which swarm detection is absent. However,
because particle concentrations in plasma vary, the optimal dilution differs >100-fold
between donors, but it is unfeasible to do serial dilutions for each clinical sample.
Therefore, our aims are to (1) develop a faster method to avoid swarm detection, and
(2)
increase the number of detected EVs per second.
Methods: We measured serial dilutions of CD61-stained EVs in platelet free
plasma (PFP), with and without spiking of FITC beads, by FCM (Apogee A60-Micro). We
triggered either on side scatter or fluorescence.
Results: For scatter triggering with our FCM, swarm detection consistently
occurred for plasma samples exceeding a (total particle) count rate of 4,100–4,200
events/s.
The CD61+ EVs concentration scaled linearly over 1.5 orders of magnitude of the dilution
and
most donors required >1000-fold dilution to avoid swarm detection, thereby reducing
CD61+ EV counts. For fluorescence triggering, the CD61+ EVs concentration scaled linearly
over >3 orders of magnitude of the dilution. For all donors, swarm detection was absent
after 32-fold dilution (relative to pure plasma). The count rates of CD61+ EVs were
30–100-fold higher compared to scatter triggering. The spiked FITC beads confirmed
that the
median signals remained constant.
Summary/Conclusion: We have developed two clinically applicable ways to avoid
swarm detection. For scatter triggering, the count rate provides direct feedback on
the
presence of swarm detection in plasma samples. For fluorescence triggering, swarm
detection
was absent for all plasma samples diluted ≥32-fold and compared to scatter triggering,
count
rates of CD61+ EVs were 30–100 fold higher, thereby improving statistical significance.
Funding: Edwin Van der Pol is supported by the Netherlands Organisation for
Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research
programmes
VENI 15924.
PT10.02
Benchmarking flow cytometric analysis of nanoparticles: a
cross-platform study for single extracellular vesicle detection
Estefanía Lozano-Andrésa
, Ye
Tianb, Ger Arkesteijna, Sten F. W. M. Libregtsc, An
Hendrixd, Xiaomei Yanb and Marca H. M. Waubene
aUniversity of Utrecht, Utrecht, Netherlands;
bXiamen University, Xiamen, China (People’s Republic); cUtrecht
University, Utrecht, Netherlands; dLaboratory of Experimental Cancer Research,
Department of Human Structure and Repair, Ghent University, Belgium; Cancer Research
Institute Ghent, Belgium, Ghent, Belgium; eDepartment of Biomolecular Health
Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands,
Utrecht,
Netherlands
Introduction: Despite flow cytometry being widely used to analyse cells in
suspension, most commercial instruments lack sensitivity when measuring nanoparticles
(NPs)
and extracellular vesicles (EVs). Furthermore, the use of appropriate reference materials
(RMs) for calibration and quality control are essential to compare results acquired
with
different instruments. To work towards successful clinical applications for EV biomarker
profiling, benchmarking studies including state-of-the-art flow cytometers are required.
We
here investigated the ability of three different flow cytometers to detect NPs and
EVs.
Methods: The instrument sensitivity of light scattering detection was
evaluated by using synthetic NPs of different sizes and refractive indices. Fluorescent
calibration was investigated by using molecules of equivalent soluble fluorophores
(MESF)
beads. Biological recombinant EVs (rEVs) were used to validate the detection and
quantification of fluorescent EVs in a side-by-side cross-platform study using an
N30
Nanoflow Analyser (NanoFCM), an optimized BD Influx and a CytoFLEX LX.
Results: We found that when light scatter based detection was used, the
NanoFCM detected the smallest non-fluorescent NPs, the BD Influx was able to provide
reliable FSC information from the smallest detected NPs and the CytoFLEX performance
was
greatly improved by the use of Violet-SSC. Biological rEVs showed that the NanoFCM
could
clearly resolve fluorescent EVs while the BD Influx and CytoFLEX were unable to fully
resolve rEVs from background, although fluorescence threshold improved detection.
In
addition, our findings revealed that different concentrations are required to ensure
single
EV detection in these platforms.
Summary/Conclusion: We identified several strengths and limitations for each
platform with respect to single EV analysis. Furthermore, our results showed that
proper
calibration and RMs are of utmost importance to ensure reliable interpretation of
EV flow
cytometric data.
Funding: European Union’s Horizon 2020 research and innovation program under
the Marie Skłodowska-Curie (grant agreement No 722148) and the National Natural Science
Foundation of China (grants 21934004 and 21627811).
PT10.03
Caution when using membrane dyes for sequential extracellular
vesicle analysis
Diana Pham, Michael Wong, Desmond Pink and
John lewis
Nanostics, Edmonton, Canada
Introduction: Confirmation that particles detected by microflow cytometry are
actually extracellular vesicles (EVs), or at least membranous in composition, can
be
achieved through a variety of methods. Positively staining particles with a membrane
dye
strongly suggest that the particle contains a membrane; loss of stain (or detection)
after
detergent solubilization of the membrane-dyed particles provides even stronger evidence
that
the particles were EVs. It is important to recognize that the labelling protocol provided
by
the membrane dye manufacturer may not be ideal for all types of EV-containing biological
samples, such as blood, urine, semen etc.). Removal of excess dye from stained EVs
is very
difficult and can be impractical depending on the nature of the experiment. However,
this
means that the potential for excess dye to contaminate subsequent sampling is high.
Therefore, it is important to determine optimal working concentrations and labelling
conditions when using membrane dyes for EV detection to understand properties that
may
impact your analyses.
Methods: To assess the utility of membrane dyes, titration curves were
generated to determine the optimal working concentrations of membrane dyes for EV
detection
in conditioned media and human serum samples. Once the optimal concentration was determined
the potential of dye carry-over from sample to sample during microflow cytometry detection
was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline
(PBS)
blanks and matched, unlabelled, sample replicates.
Results: We found that optimal concentration of any membrane dye is dependent
on sample type. Even with the inclusion of system washes to prevent sample carryover,
there
was carryover of low amounts of dye+ particles into sequentially analysed PBS blanks.
If
unstained samples were analysed following a stained sample, excess dye (or at least
dye+
events) appeared in the data. A sample concentration effect was also seen; samples
of lower
concentrations were more susceptible to dye carryover.
Summary/Conclusion: When using membrane dyes to stain EVs in biological
samples, especially if an autosampler is employed to run a series of tests, it is
critical
to determine the optimal concentration of dye for each type of sample, as excess dye
can
carry over to the next sample in the queue. In addition, determining the necessary
steps to
clean any excess dye following each sample run will improve the accuracy of EV detection
and
analyses.
Funding: Nanostics
Alberta Innovates
Alberta Cancer Foundation
PT10.04
Correlation between size and protein expression of single
exosomes by combined atomic force and fluorescence microscopy
Federico Peverea, Sara
Cavallarob
, Carolina Pabac, André Görgensd,
Samir El-Andaloussid, Jan Linnrosb and Apurba Deva
aUppsala University, Uppsala, Sweden; bKTH Royal
Institute of Technology, Stockholm, Sweden; cPolitecnico di Torino, Turin, Italy;
dDepartment of Laboratory Medicine, Clinical Research Centre, Karolinska
Institutet, Huddinge, Sweden
Introduction: Exosomes are biological nanoparticles released by all types of
cells, healthy as well as cancer cells. By travelling in body fluids (blood, saliva,
etc.)
exosomes carry proteins and genetic cargo between cells in distant organs. Therefore,
they
are involved in intercellular communication and play an important role in the development
of
several diseases, such as cancer. For diagnostic and therapeutic purposes, it is fundamental
to measure the size and the molecular content of exosomes. Nevertheless, a direct
correlation between these two quantities at the single-exosome level has remained
elusive
mainly due to their small size (~30-200 nm) and their biological/soft and heterogeneous
nature.
Methods: In this work, we overcome the challenges in correlative measurements
by combining two powerful techniques: atomic-force (AFM) and fluorescence microscopy.
AFM
allows accurate size-measurement of exosomes in liquid, thus preventing their damage.
On the
other hand, fluorescence microscopy is used to estimate the amount of surface
proteins/markers of exosomes attached on a glass substrate.
Results: Exosomes from HEK293 cell line were first bound onto patterned glass
coverslips in a cell containing 1x phosphate buffer saline (PBS). Surface proteins
of such
exosomes could be labelled with antibodies or alternatively by bioengineering. The
AFM,
mounted on top of the fluorescence microscope and aligned with the optical axis, allowed
combined size-fluorescence measurements on individual exosomes. In particular, to
prevent
size/shape alteration of the soft nanoparticles, force curve-based (or quantitative)
imaging
was performed in liquid environment. The correlation data was acquired for different
samples/surface proteins/markers.
Summary/Conclusion: In conclusion, we have measured the size and the protein
expression level of single exosomes by combining AFM and fluorescence microscopy.
This
approach, first of its kind for exosomes, allows to accurately estimate both properties
at
the single-exosome level, thus giving valuable information for diagnostic and therapeutic
use of these nanoparticles, as well as for their fundamental knowledge.
PT10.05
Development of a panel of antibodies for capture and labelling
of EVs from neural cell types
Kristin Luther and George Daaboul
NanoView Biosciences, Boston, USA
Introduction: There are no universal markers of extracellular vesicles, but
often they are identified by the presence of tetraspanins in their membrane. Based
on this,
products have been developed to precipitate or quantify EVs by acting upon CD9, CD81,
and
CD63. However, EVs also carry proteins from their parent cells, and capturing EVs
based
their presence allows for a more complete understanding of vesicle heterogeneity from
a
single cell type, and for EVs derived from specific tissues to be enriched from other
biofluids in support of biomarker assessment. For example, EVs derived from the brain
could
be captured from the general population of serum EVs for better assessment of cargo
associated with proteinopathy. The goal of this study was to identify specific antibodies
to
capture and label EVs bearing the neural markers CD171, SNAP25, α-Synuclein, Tau,
and
NCAM.
Methods: The targets were overexpressed in HEK293 T cells through transient
transfection of plasmids (Origene). Media was conditioned for 24–48 hours, and then
centrifuged to remove cell debris. Cell lysates and concentrated conditioned media
(CM) were
analysed by Western blot. Unpurified CM, or CM after performing size exclusion
chromatography (SEC, Izon), were analysed in the ExoView R100 system. Diluted CM was
incubated on custom antibody microarray chips overnight. Then the chips were labelled
with a
cocktail of 3 labelled antibodies, washed and imaged. Vesicles were counted, sized,
and
phenotyped. Next, commercially available pooled human CSF was analysed in a similar
fashion
to determine their abundance in a relevant biofluid.
Results: Multiple antibody clones were tested in different combinations for
capture and labelling for the five different neuronal enriched proteins of interest,
and
optimal combinations were identified. Some markers were identified on particles >
50 nm
in size that were negative for tetraspanins, while others colocalized with tetraspanins.
Through comparing permeabilized and intact EVs with and without SEC to remove non-vesicular
proteins, we found that tau could be on the vesicle surface, within the vesicle, and
free in
solution.
Summary/Conclusion: The ExoView platform can be customized to enable the
detection of proteins of interest and to determine whether they are on the EV surface,
intravesicular, or non-EV associated.
PT11: EVs in Cardiovascular Diseases and Vascular
Disorders
Chair: Saumya Das – Massachusetts General Hospital, Harvard Medical School
Chair: J. Brian Byrd – University of Michigan
PT11.01
Association between circulating extracellular vesicles and
thrombogenic risk markers for cardiovascular disease
Esra Bozbasa
, Ruihan
Zhoua, Plinio Ferreirab, Keith Allen-Redpatha and Parveen
Yaqooba
aUniversity of Reading, Reading, UK; bImperial
College London, London, UK
Introduction: Elevated numbers of circulating extracellular vesicles (cEVs)
have been observed in different diseases, most notably cardiovascular diseases (CVDs).
EVs
are reported to have thrombogenic properties, but there is very little information
about the
association between circulating EV numbers and thrombogenic risk markers for CVD.
The aim of
this research was to investigate whether thrombogenic risk markers for CVD are associated
with increased numbers of circulating EVs.
Methods: Forty non-smoking male and female subjects (40–70y) at moderate risk
for CVD were recruited for the study. EVs from platelet-free plasma (PFP) were isolated
using size exclusion chromatography (SEC). The concentration and size distribution
of EVs
were measured by Nanoparticle Tracking Analysis (NTA) and flow cytometry (FCM). Three
EV
markers, including Annexin V for the circulating phosphatidylserine-positive (PS+)
EVs, CD41
for platelet-derived EVs and CD105 for endothelial-derived EVs were used for phenotyping.
In
addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser
(Hemacore). Platelet aggregation to determine platelet function was assessed by a
high-throughput platelet function assay with a wide range of concentrations of agonists,
including adenine diphosphate (ADP), collagen-related peptides (CRP-XL), epinephrine,
thrombin receptor activating peptide (TRAP-6) and U46619. The association between
thrombogenic risk markers for CVD and EV numbers was tested by Pearson’s correlation
coefficient and linear regression model using the statistical program, SPSS.
Results: Circulating EV concentration with threshold of 71 nm, measured by
NTA, were positively associated with coagulation-related risk markers, including rate
of
clot growth (r = 0.568; p = 0.01) and clot size at 30 min (r = 0.480; p = 0.002).
PS+ EVs
derived from endothelial cells, determined by FCM, were negatively associated with
lysis
onset time (r = −0.410; p = 0.009), whereas they were found positively correlated
with lysis
progression (r = 0.417; p = 0.007). Both mean and mode size of cEVs, detected by NTA,
were
significantly correlated with U46619-induced platelet aggregation (r = −0.338; p = 0.033,
r = −0.382; p = 0.015, respectively).
Summary/Conclusion: In subjects at moderate risk for CVD, cEV numbers were
positively related to rate of clot growth and clot size and size of cEVs was negatively
related to platelet activity. Higher numbers of endothelial cell-derived PS+ cEVs
were
associated with lower rates of fibrinolysis. This suggests that cEVs promote clot
growth and
reduce fibrinolysis, and may therefore be an indicator for greater risk of CVD.
Funding: This project is founded by Biotechnology and Biological Sciences
Research Council (BBSRC)-Diet and Health Research Industry Club (DRINC) in the UK
and the
Ministry of National Education (Turkey).
PT11.02
Beyond stem cells: extracellular vesicles from human induced
pluripotent stem cells (hiPSC) and hiPSC-cardiomyocytes as therapeutic approaches
for
heart failure
Ana F. Louro, Henrique Vazão Almeida, Patrícia
Gomes-Alves, Paula Alves and Margarida Serra
iBET, ITQB-NOVA, Oeiras, Portugal
Introduction: Heart failure is caused by a variety of underlying diseases, the
most common being myocardial infarction. Initially regarded as an alternative to
pharmacological approaches, stem cell transplantation has failed to demonstrate clinically
meaningful results. Instead, it has become increasingly apparent that the therapeutic
effects of transplanted cells are largely mediated by their secretome, while mounting
evidence suggests Extracellular Vesicles (EVs) play a major role in cardiac repair.
Within
this framework, EVs from human induced pluripotent stem cells (hiPSC) and hiPSC-derived
cardiomyocytes (hiPSC-CM), hold a tremendous potential to treat cardiovascular disease.
We
isolated EVs from conditioned culture media at key stages of the hiPSC-CM differentiation
and maturation processes, i.e. from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV),
immature
(CMi-EV) and mature (CMm-EV) cardiomyocytes, with the aim of studying their potential
role
as therapeutics, and whether their effectiveness was influenced by the state of their
parent
cell.
Methods: hiPSC were differentiated into cardiomyocytes in a 3D culture
approach, using the protocols developed by our group. EV isolation was performed on
an
iodixanol density gradient, and the EVs were characterized in terms of particle size
and
particle size distribution, presence of EV-specific markers, and imaging through
transmission electron microscopy. Functional studies were performed using human umbilical
vein endothelial cells (HUVECs) to evaluate EV-uptake, cell migration and angiogenesis.
Results: EVs from all hiPSC and cardiac derivatives presented a typical
cup-shaped morphology and expressed CD63 and CD81. EV yield varied along differentiation,
with a minimum for CPC and a maximum for CMi. PKH26-labelled EVs were uptake by HUVECs,
and
colocalized with calnexin, a protein from the endoplasmic reticulum. Wound healing
assays
showed an increased cell migration in HUVECs treated with cardiomyocyte-derived EVs,
in
comparison with control EVs isolated from foetal bovine serum.
Summary/Conclusion: Our findings suggest a different EV secretion profile
along CM differentiation and maturation, with preliminary assays showing EV functionality.
Ongoing work aims at elucidating the possible differences in function and cargo amongst
these types of EVs.
Funding: FCT PhD fellowship PD/BD/139078/2018; IC&TD Project “MetaCardio“
(PTDC/BTM-SAL/32566/2017), Project NETDIAMOND (SAICTPAC/0047/2015), and iNOVA4Health
Research Unit (LISBOA-01-0145- FEDER-007344).
PT11.03
Endothelial cells differentially load and secrete extracellular
vesicle-derived microRNAs into apical and basolateral compartments
Sneha Rajua, Jamie Starkb, Shawn C.
Veitchb, Dakota D. Gustafsonb, Rathnakumar
Kumaragurubaranb, Emilie Boudreauc, Jason Fishb and
Kathryn L. Howeb
aUniversity of Toronto, Toronto, Canada;
bUniversity of Toronto, Toronto, Canada; cUniversity Health Network,
TGHRI, Toronto, Canada
Introduction: Endothelial cells (EC) are a major secretory organ that
selectively release biologically active molecules. Given that extracellular vesicle
(EV)-derived miRNAs mediate cell-cell communication and serve as biomarkers, we hypothesized
that they are secreted in a polarized manner in steady and activated states.
Methods: Primary human aortic endothelial cells and human umbilical vein
endothelial cells (HUVECs) were cultured in transwells (± apical IL-1β, 10 ng/ml,
4 h).
Apical and basolateral EVs were isolated from supernatants by polyethylene glycol
precipitation (ExoQuick-TC, System Biosciences, Palo Alto, CA, USA). EV-miRNA profiles
were
determined by next generation sequencing (HTG Molecular Diagnostics Inc., Tucson,
AZ, USA)
and differential expression evaluated using Partek Genomics Suite software (version
8.0). EC
barrier function was assessed by FITC-dextran flux (40 kDA) and localization of VE-cadherin
expression (MAB9381, R&D Systems). Directional miRNA transfer was assessed in
miR-39-transfected HUVECs by seeding onto transwells with monocytes (THP-1 cells,
apical and
basolateral compartment, 24 h).
Results: EV isolation was confirmed with nanoparticle tracking analysis
(130 nm ±19.57, n = 12) and protein content-based characterization (CD63 positive,
Calnexin
negative). EV size and concentration was unaffected by IL-1β. IL-1β-stimulated ECs
demonstrated a 1.89 ± 0.098 fold change in FITC permeability (p < 0.0001, n = 3),
but
VE-Cadherin expression was maintained at adherens junctions (n = 2). Sequencing performed
on
EC-EVs isolated from each compartment showed differential miRNA expression (p < 0.05,
fold change >1.5; n = 7). Polarized EV-miRNA secretion was maintained after IL-1β
stimulation but demonstrated altered miRNA profiles. Selective and directional release
of
EV-miRNA was demonstrated in a model of cellular communication, whereby ECs transfected
with
exogenous miRNA-39 preferentially deliver this to monocytes in the apical compartment
(274 ± 163 fold increase, n = 2).
Summary/Conclusion: ECs selectively secrete EV-miRNA in a polarized fashion
and may therefore participate in selective and directional cell-cell communication.
These
findings have important implications for discovery of EV-miRNA based biomarkers and
therapeutics.
Funding: University Health Network, Division of Vascular Surgery, Peter Munk
Cardiac Centre.
PT11.04
Insights into EV subpopulations positive and negative for
Annexin A1 from smooth muscle cells and valvular interstitial cells
Kristin Luthera
, Maximillian A.
Rogersb, Samantha Atkinsb, George Daaboula and Elena
Aikawab
aNanoView Biosciences, Boston, USA; bCenter for
Interdisciplinary Cardiovascular Sciences, Division of Cardiovascular Medicine, Brigham
and
Women’s Hospital, Harvard Medical School, Boston, MA, 02115, Boston, USA
Introduction: Annexin A1 associates with extracellular vesicles (EVs) and can
mediate vesicle aggregation. This may play a role in microcalcification in calcific
aortic
valve disease (CAVD), but this is poorly understood. Annexin A1 is thought to be a
marker of
membrane-derived EVs, but because it can be found on the cytoplasmic or extracellular
side
of the plasma membrane, its localization within or on the surface of EVs is unclear.
The
goal of this study was to determine whether annexin A1 is found on the surface of
EVs in two
cell lines relevant to CAVD, and develop an assay that can be used to determine whether
this
changes under pathogenic conditions.
Methods: EVs were isolated by differential ultracentrifugation from the
conditioned medium (CM) of smooth muscle cells (SMC) and valvular interstitial cells
(VIC).
Total protein in the cell lysates and EV pellets was analysed by western blot. EVs
from
cells treated with control siRNA or Anxa1-siRNA were enumerated and phenotyped using
the
ExoView R100 platform. EVs with surface expression of CD9, CD81, CD63, and annexin
A1 were
captured using a customized antibody microarray chip. Then EVs were labelled with
fluorescent antibodies to assess EV number, size, and colocalization of EV proteins.
The
knockdown of annexin A1 allowed us to assess the specificity of the selected annexin
A1
antibody.
Results: The EV fraction was positive for CD9, and lacked markers of other
vesicle types. Western blot on the EV pellet and supernatant in ± EDTA indicated that
there
is annexin A1 both on the surface of and within the EVs. Using the antibody microarray
chips, numerous annexin A1+ EVs were captured on the annexin A1 spots from the control
CM,
and there was a marked decrease in capture and labelling from Anxa1-siRNA treated
cells.
Under both conditions, vesicles were also captured on tetraspanin probes, with the
greatest
number captured on CD9, then CD63 and CD81. There was a significant population of
annexin
A1+ EVs that was negative for tetraspanins.
Summary/Conclusion: Annexin A1 is found on the surface of EVs. The assay
developed in collaboration with NanoView Biosciences is well suited for assessing
the number
and phenotype of annexin A1+ EVs derived from SMC and VIC cell lines, which could
provide a
useful method for understanding EV populations in CAVD patient cell lines.
Funding: This work was supported by HL136432 and HL147095.
PT11.05
Possibility of exosomal microRNAs associated with chronic
limb-threatening ischaemia, the end stage of atherosclerosis, as a promising
biomarker
Shinsuke Kikuchia
, Yusuke
Yoshiokab, Kurataka Otsukac, Naoya Kuriyamaa, Yusuke
Yamawakid, Yuri Yoshidaa, Nobuyoshi Azumaa and Takahiro
Ochiyae
aDepartment of Vascular Surgery, Asahikawa Medical
University, Asahikawa, Japan; bDivision of Molecular and Cellular Medicine,
Institute of Medical Science Tokyo Medical University, chuo-ku, Japan; cDivision
of Cellular Signaling, National Cancer Center Research Institute, chuo-ku, Japan;
dNational Cancer Center Research Institute, chuo-ku, Japan; eTokyo
Medical University, Shinjuku-ku, Japan
Introduction: Chronic limb-threatening ischaemia (CLTI), the end stage of
peripheral artery disease (PAD), has poor prognosis and is attributed to life-style
disease.
With increasing of atherosclerotic disease all over the world, establishment of biomarker
for should play a pivotal role for early detection and preventing aggravation of the
disease. The aim of this study is to explore the possibility of liquid biopsy for
atherosclerotic disease by analysis of CLTI-associated exosomal microRNAs.
Methods: CLTI due to PAD was diagnosed by ankle-brachial blood pressure index,
skin perfusion pressure (<40 mmHg) and angiography. Ten preoperative CLTI patients
and 10
control patients without PAD were analysed (All patients with diabetes and 50% of
patients
had end-stage renal failure [ESRD]). To identify biomarkers associated with CLTI,
exosomes
were extracted from patient’s serum after ultracentrifugation and total RNA including
small
RNA was isolated from the exosomes. The expression profile of exosomal microRNAs associated
with CLTI were evaluated using a next generation sequencing.
Results: Forty-three exosomal miRNAs associated with CLTI were identified.
Intriguingly, these miRNAs were clearly categorized with ESRD, which was well known
as
end-stage of life-style disease: these were stratified into 20 microRNAs for ESRD
patients
and 23 microRNAs for non-ESRD patients. Since ESRD is the most important factor
significantly related to patient’s prognosis in CLTI, exosomal microRNAs reflected
patient’s
comorbidity onto the expression profile.
Summary/Conclusion: A portion of the expression profile of exosomal microRNAs
associated with CLTI was identified. Exosomal microRNA could be a biomarker to stratify
patient’s condition along with their comorbidities and is very promising for individualized
diagnosis in atherosclerotic diseases with risk diversity.
PT11.06
Postoperative plasma exosomal miR-21 and miR-133a signature in
patients with left ventricular reverse remodelling after surgical mitral valve
repair
Fausto Pizzinoa, Giulia
Furinib
, Valentina Casieria, Massimiliano
Marianic, Marco Solinasc, Stefano Maffeic, Dante
Chiappinoc, Giovanni Donato Aquaroc and Vincenzo
Lionettia
aIstituto di Scienze della Vita, Scuola Superiore Sant’Anna,
Pisa, Italy; bIstituto di Scienze della Vita, Scuola Superiore Sant Anna, Pisa,
Italy; cFondazione Torscana G. Monasterio, Massa, Italy
Introduction: Patients with mitral valve regurgitation (MR) show volume
overload and progressive heart remodelling with increased left ventricular (LV)
end-diastolic volume (EDV). Surgery is recommended for patients with substantial decay
of
global cardiac function (LV ejection fraction, LVEF) in order to reverse LV remodelling.
However, approximately 10% patients show poor recovery. Identification of patients
at high
risk of post-operative LV remodelling may help preventive strategies. In this scenario,
microRNAs delivered by plasma exosomes (pEXOs) might have a predictive value as well
as
complement the routine clinical measures of surgical outcome.
Methods: Primary MR patients (N = 19; 45–71 y.o.) underwent implantation of a
prosthetic mitral ring. LV remodelling was assessed by cardiac magnetic resonance
imaging
and pEXOs were isolated by optimized ultracentrifugation before surgery (T0) and six
months
after surgery (T1). Isolated pEXOs were quantified by nanoparticle tracking analysis
and
miR-1, miR-21, miR-133a, and miR-208a were measured by RT-qPCR. The same analysis
was
performed on healthy subjects with normal cardiac function (N = 8). Local ethical
committee
approved the study (EMIGRATE study, approval n°1529) and informed consent was obtained
from
all patients.
Results: pEXOs levels at T0 were lower (−32%, p = 0.02) in patients with worst
postoperative LV function, while they were higher at T1 (+31%, p = 0.03) in patients
with
reversed LV remodelling after surgery. At T1, the increase in pEXOs levels was associated
to
decreased heart mass index (−13%, p = 0.02) and higher levels of exosomal miR-21 (+78%,
p = 0.02) and miR-133a (+69%, p = 0.05) were detected in patients with improved LV
function.
Summary/Conclusion: Higher postoperative levels of pEXOs delivering miR-21 and
133a depict LV reverse remodelling after surgical mitral valve repair. Monitoring
of
exosomal microRNAs cargo might predict postoperative outcome in patients with MR.
PT11.07
Expression of lipocalin-2 (LCN2) in circulating extracellular
vesicles (EVs) and femoral plaque-derived EVs of peripheral arterial disease
patients.
Goren Saenz-Pipaon
a, Josune
Orbeb, Esther Martinez-Aguilarc, José Antonio Paramod,
José Antonio Rodriguezb and Carmen Roncalb
aLaboratory of Atherothrombosis, Program of Cardiovascular
Diseases, Cima Universidad de Navarra, Pamplona, Spain; IdiSNA, Instituto de Investigación
Sanitaria de Navarra, Pamplona, Spain., Pamplona, Spain; bLaboratory of
Atherothrombosis, Program of Cardiovascular Diseases, Cima Universidad de Navarra,
Pamplona,
Spain; IdiSNA, Instituto de Investigación Sanitaria de Navarra, Pamplona, Spain; CIBERCV,
Instituto de Salud Carlos III, Madrid, Spain., Pamplona, Spain; cIdiSNA,
Instituto de Investigación Sanitaria de Navarra, Pamplona, Spain; Departamento de
Angiología
y Cirugía Vascular, Complejo Hospitalario de Navarra, Pamplona, Spain., Pamplona,
Spain;
dLaboratory of Atherothrombosis, Program of Cardiovascular Diseases, Cima
Universidad de Navarra, Pamplona, Spain; IdiSNA, Instituto de Investigación Sanitaria
de
Navarra, Pamplona, Spain; CIBERCV, Instituto de Salud Carlos III, Madrid, Spain; Hematology
Service, Clínica Universidad de Navarra, Pamplona, Spain
Introduction: Peripheral arterial disease (PAD) is the most prevalent
cardiovascular condition globally and it is estimated to increase greatly with the
ageing of
the population. Extracellular vesicles (EVs) have emerged as potential components
of liquid
biopsy related to their protein and nucleic acid cargo. The transcriptomic analysis
of
circulating EVs has revealed the expression of genes related to the immune response
in PAD,
including lipocalin-2 (LCN2) or NGAL, which has been involved in atherogenesis in
preclinical studies.
Methods: To study if the content of circulating EVs might reflect molecular
changes locally, LCN2 levels were determined by RT-qPCR in femoral atherosclerotic
plaques
(n = 5) and in medium/large EVs released from femoral plaques into cell culture medium
ex
vivo (n = 5). EVs were isolated by centrifugation (2x20000xg) and characterized by
NTA, TEM,
and western blot. The study was approved by the Institutional Review Board of Complejo
Hospitalario de Navarra (ref. 30/10), according to the Declaration of Helsinki on
medical
research. Written informed consent was obtained from all patients.
Results: LCN2 mRNA was detected in femoral plaques and in tissue derived EVs
by RT-qPCR. Additional PCR experiments in plaque derived EVs showed the presence of
the LCN2
full length transcript within EVs.
Summary/Conclusion: The transcriptional content of plasma EVs (liquid biopsy)
might reflect in part the molecular changes within atherosclerotic tissue, and femoral
plaque derived EVs.
Funding: European Fund for Economic and Regional Development (FEDER) funds
[PI18/01195], Ministry of Economy and Competitiveness, Institute of Health Carlos
III
(CB16/11/00371).
PT11.08
Cardioprotective effects of extracellular vesicles from
immortalized parental stromal cells: a translational strategy
Rafael Sáncheza, Akaitz Dorronsoroa, Marc
Buiguesb, Ignacio Reinalb, Marta Gómezc, Elena
Amarob, Estela Villanuevaa and Pilar
Sepulveda
a
aAssociated Unit for Cardiovascular Repair La Fe-Centro de
Investigación Príncipe Felipe (IISLAFE-CIPF) Health Research Institute, Valencia,
Spain,
Valencia, Spain; bInstituto de Investigación Sanitaria La Fe, Valencia, Spain;
cInstituto de Investigación Sanitaria La Fe., Valencia, Spain
Introduction: Myocardial is a significant cause of mortality and morbidity in
developed countries and since current therapies are only palliative this pathology
has
arisen in the last decade. Mesenchymal stromal cell (MSC) derived extracellular vesicles
(EVs) emerged as potential therapeutic element to reduce tissue damage after the ischaemic
insult. EVs are known to recapitulate healing benefits exert by MSC transplantation
in
preclinical ischaemic models but EV based therapies face relevant difficulties such
as high
effective dose and manufacturing standardization. In this piece of work, we immortalized
EV
secreting MSCs to get high amount of EVs from a standizable and scalable source.
Methods: MSC were immortalized and secreted EVs (EVMSC-I) isolated by
sequential ultracentrifugation. In order to evaluate the therapeutic potential of
EVMSC-I we
used both human and rat cardiomyocytes as well as cardiac microvascular cultures subjected
to isquemia/reperfusion injury. We evaluated the capacity of EVMSC-I to reduce the
consequences of the ischaemic insult measuring cell viability and functional parameters
like
contractile capacity in the cardiomyocytes and angiogenic properties in the cardiac
microvascular cultures. EVMSC-I therapeutic effect was assessed in vivo in a rat model
of
cardiac infarction followed by histopathological studies.
Results: EVMSC-I significantly reduced cardiomyocyte death (22%±7.219%) and
revert the beating rate reduction in a 45%±17.31% trigger by the ischaemic event.
In this
regard, EVMSC-I inhibit caspase 3 cleavage and reduce the appearance of ROS. In terms
of
post-ischaemic neoangiogenesis, we observed that EVMSC-I increased the capacity of
vascular
cells to form tubular structures. In addition, we observed that EVMSC-I reduce the
consequences of cardiac infarction in vivo. Finally, intramuscular injection of EVMSC-I
after permanent ligation of the descendent coronary reduced the infarct area in rats
from
46%±10.98% of the left ventricular area in control group to a 31.27%±8.619% in the
group
treated with EVMSC-I
Summary/Conclusion: Taking all together, our results show that EVMSC-I
mitigate the consequences of the cardiac infarction by reducing tissue damage. More
work
needs to be done to understand the underlying biological processes.
Funding: ACIF/2017/318, RD160011/0004, PI19/00245, Co-funder by FEDER “Una
manera de hacer Europa”
PT12: Biodistributio, Signalling, and ECM
Chair: Bong Hwan Sung – Department of Cell and Developmental Biology, Vanderbilt
University
PT12.01
Acinetobacter baumannii transfers the efflux pump related
substances via outer membrane vesicles
Hongye Jianga
and Lei
Zhengb
aShunde Hospital, Southern Medical University (The First
people’s hospital of Shunde), Guangzhou, China (People’s Republic); bDepartment
of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou,
510515,
China, guangzhou, China (People’s Republic)
Introduction: Clinically, the drug resistance situation of Acinetobacter
baumannii is becoming increasingly serious, and its drug resistance has become a difficult
problem for nosocomial infection and clinical treatment. In view of the relatively
slow
development of antibacterial drugs, exploring the resistance mechanism of Acinetobacter
baumannii is of great significance to improve bacterial resistance and help clinical
treatment. Studies have shown that outer membrane vesicles (OMVs) can transmit resistance
genes to mediate the spread of drug resistance, and recent studies have confirmed
that high
expression of efflux pumps play an important role in the multidrug resistance of A.
baumannii. In this study, we want to explore whether the outer membrane vesicles of
Acinetobacter baumannii can transfer the efflux pump related substances.
Methods: First, ultracentrifugation and density gradient centrifugation were
used to extract the OMVs of Acinetobacter baumannii antimicrobial-sensitive strains
(ATCC19606) and antimicrobial-resistant strains. Then, nanoparticle tracking analysis
(NTA)
technology was used to analyse the particle size and distribution range of OMVs.
Transmission electron microscopy (TEM) was used to identify their morphology and structure.
Bradford method was used to determine the protein concentration of OMVs. Next, the
OMVs of
antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains
and
then the drug susceptibility test was done to determine whether OMVs of
antimicrobial-resistant strains could transmit antimicrobial-resistance information
to the
antimicrobial-sensitive strains. Finally, PCR, qPCR and mass spectrometry were used
to
determine whether the efflux pump related genes were higher expression in OMVs of
antimicrobial-resistant strains than those in antimicrobial-sensitive strains.
Results: Nanoparticle tracking analysis (NTA) detected the concentration and
size distribution of OMVs of Acinetobacter baumannii strains. It showed that the extracted
OMVs have a relatively uniform particle size and a size between 100–250 nm. TEM showed
that
OMVs had a typical vesicle structure. OMVs co-culture experiments showed that OMVs
of the
antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive
strains. And the efflux pump related genes were higher expression in OMVs of
antimicrobial-resistant strains than those in antimicrobial-sensitive strains.
Summary/Conclusion: OMVs of the antimicrobial-resistant strains can indeed
pass resistance to the antimicrobial-sensitive strains. The cause of acquiring antimicrobial
resistance in sensitive strains may be caused by resistant strains passing efflux
pump-related genes or proteins to sensitive strains.
PT12.02
Characterization of melanocytic extracellular vesicles during
ageing of the choroid
Kelly Coutanta
, Léo
Piqueta, Nathan Schoonjansb, Philippe Gros-Louisa, Julie
Bérubéc, Stéphanie Proulxa, Alain R. Brissond and Solange
Landrevillea
aUniversité Laval, Quebec City, Canada;
bUniversité de Lille, Lille, France; cCentre de recherche du CHU de
Québec-Université Laval, Quebec City, Canada; dUniversité de Bordeaux, Bordeaux,
France
Introduction: The choroid is located at the backside of the light-sensitive
retina and is highly vascularized. It contains pigmented melanocytes, and their melanin
protects them against oxidative stress. Since ageing reduces the number of melanosomes
in
melanocytes and generates a stiffer extracellular environment, our hypothesis is that
surrounding choroidal cells and the retinal pigment epithelium (RPE) are subject to
more
oxidative stress-related damages. This study aimed to characterize EVs released by
human
choroidal melanocytes in the context of intercellular cooperation during ocular ageing.
Methods: Melanocytic EVs were recovered from the conditioned culture medium of
young/old melanocytes grown on hydrogels of varying stiffness (0.5–20 kPa) by differential
centrifugation. The concentration and size distribution of melanocytic EVs were determined
by high-sensitivity flow cytometry. Cryo-transmission electron microscopy combined
with
receptor-specific gold labelling were used to reveal their morphology, size and phenotype.
The relative abundance of 8 surface markers was evaluated with the Exo-Check Exosome
Antibody Array. The uptake of fluorescent melanocytic EVs by the RPE and choroidal
endothelial cells was assessed by confocal microscopy.
Results: Choroidal melanocytes released EVs positive for Annexin-5 and the
tetraspanin CD63. Young melanocytes produced more Annexin-5 positive EVs and EVs larger
than
500 nm compared to older donors. The stromal stiffness impacted the concentration
and size
of melanocytic EVs. We confirmed the uptake of melanocytic EVs by endothelial and
RPE
cells.
Summary/Conclusion: EVs from choroidal melanocytes are internalized by
surrounding endothelial cells and RPE. Age-related stressors modify the phenotype
of
melanocytic EVs. The identification of melanocytic factors that can protect retina/choroid
cells from oxidative stress-induced cell death could lead to more efficient therapy
for
patients suffering from dry age-related macular degeneration.
Funding: Vision Health Research Network; Fonds de recherche du Québec – Santé
(FRQS); Canada Foundation for Innovation
PT12.03
Extracellular vesicle heterogeneity and the impact of cellular
phenotypic drift on the vesicular proteome
Scott Bonnera
, Hakimi
Kassimb, André Görgensc, Mariana Conceicaod, Eduard
Willmse, Matthew Wooda, David R F. Carterf, Imre
Mägerd and Samir El-Andaloussic
aUniversity of Oxford, Oxford, UK; bUniversity of
Oxford, Permatang Pauh, Malaysia; cDepartment of Laboratory Medicine, Clinical
Research Centre, Karolinska Institutet, Huddinge, Sweden; dDepartment of
Paediatrics, University of Oxford, Oxford, UK; eLa Trobe University, Oxford, UK;
fOxford Brookes University, Oxford, UK
Introduction: Owing to their proposed biocompatibility and ability to cross
biological barriers, EVs represent an attractive therapeutic delivery platform. However,
EVs
are eminently heterogeneous. A better understanding of EV heterogeneity and its origins
will
allow for improved design of EV-based therapeutics. EV heterogeneity is mainly studied
by
focusing on distinct EV subpopulations. Other sources of heterogeneity, such as
heterogeneity within EV secreting cells themselves, have been investigated in lesser
detail.
In this study, we assessed the phenotypic drift of cell derived EVs to explore the
origins
of EV heterogeneity and its potential impact.
Methods: Three independent samples of two MDA-MB-231 breast cancer cell
sub-clones were cultured for six weeks. EVs were harvested weekly and analysed using
the
MACSplex Exosome Flow Cytometry kit. At two time points the proteome of EVs was analysed
by
LC-MS/MS mass spectrometry with subsequent Gene Ontology and REACTOME Pathway Analysis.
Results: The expression of over 600 proteins was de-regulated in EVs derived
from the two different cell clones. Many de-regulated proteins were associated with
biological processes predicted to affect potential EV toxicity (platelet activation,
neutrophil degranulation, blood coagulation) and EV biological activity (antigen
presentation, inflammation, TGF-beta/mTOR/WNT signalling). More surprisingly, within
only
two weeks, over 400 EV proteins, many associated with immune modulation, apoptosis,
interleukins, cytokines and cell signalling pathways (including those affecting
T-cell/B-cell receptors) were de-regulated between the two EV isolation time points.
Summary/Conclusion: Results suggest that temporal changes can be observed in
the EV proteome (potentially by clonal drift, epigenetic changes or cellular genomic
instability) over short time periods. These changes could cause significant differences
in
biological effects and delivery capabilities between EVs harvested from the same cells
at
different time points and conditions.
PT12.04
In vivo tracking and biodistribution analysis of mesenchymal
stem cell-derived extracellular vesicles in a radiation injury murine model
Sicheng Wena
, Elaine
Papab, Mark Doonerb, Michael Del Tattob, Mandy
Pereirab, Yan Chengb, Laura Goldbergb, Giovanni
Camussic and Peter Quesenberryb
aRhode Island Hospital/Brown University, providence, USA;
bRhode Island Hospital, Providence, USA; cDepartment of Medical
Sciences, University of Torino, Turin, Italy
Introduction: Recent studies indicated that Extracellular vesicles (EVs) play
key roles in intercellular communication and have great potential for clinical application.
Understanding the biodistribution of EVs is therefore essential. Our previous works
have
shown the ability of mesenchymal stem cell (MSC)-derived EVs to protect haematopoietic
cells
from radiation damage. In this study, we evaluated the biodistribution of MSC-EVs
in a
radiated mouse model.
Methods: Human MSC-EVs were harvested by ultracentrifugation and labelled with
DiD lipid dye. The reliability of the labelling EVs was confirmed by sucrose gradient
fractionation analysis. The distribution of EVs in radiation-exposed mice after EV
intravenous administration were evaluated by Fluorescence Molecular Tomography and
further
confirmed by flow cytometry and confocal microscopy analysis.
Results: We observed that DiD labelled MSC-EVs appeared highest in liver and
spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in
heart,
kidney and lung. We found the significantly increased MSC-EV accumulation in spleen
and bone
marrow post-radiation appeared with an increase of uptake of MSC-EV by CD11b+ and
F4/80+ cells, but not B220+ cells, compared to those organs from non-irradiated mice.
However, there was a predominant EV accumulation in lung and less accumulation in
spleen and
liver; in mice infused with human lung fibroblast cell derived EVs (LFC-EVs) and there
was
no significant LFC-EVs accumulation change in the spleen or liver after radiation.
We
further found that increasing levels of irradiation caused a selective increase in
vesicle
homing to marrow and spleen. This accumulation of MSC-EVs at the site of injured bone
marrow
could be detected as early as 1 hour after MSC- EV injection and was not significantly
different between 2 and 24 hrs. post-MSC-EV injection.
Summary/Conclusion: This study indicated the specific accumulation of MS-EVs
at the site of injury of haematopoietic tissue in radiation injury mice.
Funding: This work was supported by the NIH grants 5UH2TR000880,
3UH3TR000880-03 S1, 5P20GM119943, and 5T32HL116249.
PT12.05
Linking fat to colorectal cancer: extracellular vesicle
crosstalk
Robert Tempest, Daniel Kelly, Caroline Dalton
and Nicholas Peake
Sheffield Hallam University, Sheffield, UK
Introduction: Colorectal cancer is the third most common cancer worldwide, and
fourth leading cause of malignancy related mortality. Understanding the mechanisms
of its
growth and metastasis is key to elucidating new therapeutic targets and developing
treatments in the clinical setting. Epidemiological evidence indicates an increased
risk of
cancer in obese patients, pointing to bidirectional communication between colon and
adipose
cells. Extracellular vesicles (EVs) are small membrane enclosed packages released
by cells,
capable of transporting bioactive cargo from donor to recipient cells and inducing
phenotypic changes. Adipocytes are a key component of the tumour microenvironment
and
interactions between adipose tissue and tumour cells may be important in the growth
and
metastasis of cancer. In this study, we investigate the effects of colorectal cancer
EVs on
adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic,
pro-inflammatory phenotype.
Methods: EVs were isolated from SW480 and HT29 human colorectal cancer cell
lines by differential ultracentrifugation and mature adipocytes generated by differentiation
of the SGBS human pre-adipocyte cell line. Adipocytes were treated with EVs and their
lipid
content measured by oil red O to determine loss of lipids. Inflammatory cytokine profile
was
measured by ELISA to assess any increase in pro-inflammatory behaviour, and expression
of
late adipogenesis markers were determined by western blot.
Results: EV treatment was shown to reduce lipid accumulation in adipocytes,
with up to 80% reduction in lipids observed at the 50 µg/mL dose. Treatment was also
shown
to reduce the expression of late adipogenesis markers, and increase secreted levels
of
pro-inflammatory cytokines IL-6 and IL-8 by over 3 fold and 10 fold respectively.
These
results provide evidence for colorectal cancer derived EV involvement in the
dedifferentiation observed in cancer associated adipocytes in vivo, displaying an
altered
phenotype, releasing lipid energy stores to fuel tumour growth and increasing
pro-inflammatory signalling.
Summary/Conclusion: Studies have shown colorectal cancer EVs may be involved
in signalling which induces functional changes in cells within the tumour microenvironment.
Our work indicates that EV mediated dedifferentiation of resident adipocytes may potentially
contribute to a microenvironment favouring cancer cell growth and metastasis. Further
work
aims to elucidate the specific EV cargo which mediates these effects.
PT12.06
Regulation of cellular senescence by Interferon Induced
Transmembrane Protein 3 contained in small Extracellular Vesicles
Juan Fafián-Labora, Michela Borghesan, Olga
Eleftheriadou, Paula Carpintero-Fernaández and Ana O’Loghlen
Epigenetics & Cellular Senescence Group, Blizard Institute, Barts
and The London School of Medicine and Dentistry, Queen Mary University of London,
4 Newark
Street, London E1 2AT, UK
Introduction: Ageing is a major risk factor for many human diseases. It is a
complex process that progressively compromises most of the biological functions of
the
organisms, resulting in an increased susceptibility to disease and death. Senescence
is a
cellular phenotype characterized by a stable cell cycle arrest. Senescent cells are
accumulated in the body during ageing. It contributes to develop age-related diseases
and
cancer. The alteration in intercellular communication with age has been demonstrated
to be
due to senescent cells developing a phenomenon denominated senescence-associated secretory
phenotype (SASP). Exosomes are small extracellular vesicles (sEV) (30–120 nm) of endocytic
origin whereas microvesicles are formed by shedding of the plasma membrane. They contain
nucleic acids, proteins and lipid that generally reflect the status of the parental
cell and
can influence the behaviour of neighbouring cells.
Methods: In this study, we demonstrated that the small extracellular vesicles
(sEV) contribute for transmitting paracrine senescence to proliferative cells Firstly,
we
evaluated the presence of exosome-like particles in the sEV from senescent cells by
detection of exosome markers (Alix, Tsg101 and CD63), Transmission Electronic Microscopy
(TEM) and Nanoparticle Tracking Analysis (NTA). To determine that sEV from senescent
cells
are mediators of the paracrine senescence, we performed functional assays using Cre-LoxP
reporter system and high-throughput
Results: Besides, we confirmed at a single-cell level that the proliferative
cells internalizing sEV from senescent cells activate senescence process using the
Cre-reporter system. sEV protein analysis from senescent cells by mass spectrometry
(MS) and
validation of top candidates using a functional siRNA screen identify Interferon Induced
Transmembrane Protein 3 (IFITM3), a component of non-canonical interferon (IFN) pathway,
as
partially responsible for transmitting senescence to proliferative cells.
Summary/Conclusion: In conclusion, we found that sEV are regulators of
paracrine senescence and IFITM3 contained in senescent sEV has an important role in
the
intercellular communication mediated through sEV during cellular senescence1.
Funding: A.O.’s lab is supported by the BBSRC (BB/P000223/1) and The Royal
Society (RG170399). M.B. is funded by the MRC (MR/K501372/1) and the Centre for Genomics
and
Child Health. P.C.-F. (IN606B 2017/014) and J.F.-L. (ED481B 2017/117) are funded by
the
Xunta de Galicia.
PT12.07
Stiff matrix induces exosome secretion and promotes tumour
progression
Bin Wua
, Lei Guana, Ye
Xua, LiKang China, Ting Lia, Youhai Chena,
Gordon Millsb, Jinqi Rena, Ravi Radhakrishnana, Rebecca
Wellsa and Wei Guoa
aUniversity of Pennsylvania, Philadelphia, USA;
bOregon Health & Science University, Portland, USA
Introduction: Extracellular matrix (ECM) remodelling and stiffening are
associated with solid tumour progression. Stiff ECM promotes cell proliferation,
epithelial-to-mesenchymal transition (EMT), metastasis and chemoresistance. Hepatocellular
carcinoma (HCC) appears frequently in patients with liver cirrhosis or fibrosis while
the
mechanism remains unclear. Exosomes have been determined to serve as messengers to
mediate
intercellular communication and influence the extracellular. Tumour-derived exosomes
have
been shown to influence tumour progression, metastasis, drug resistance, angiogenesis
and
immune regulation. Thus, determining whether exosomes provide a mechanism by which
stiff
matrix modulates tumour microenvironment for tumour progression opens a new way to
understand cirrhosis and oncogenesis. Here we identified the molecular mechanism of
matrix
stiffening induced exosome secretion and showed the different effect of exosomes induced
by
soft or stiff matrix on tumorigenesis.
Methods: Huh7 cells were cultured on acrylamide gels with the stiffness was
modulated to 500 Pa (soft) or 10 k Pa (stiff). The exosomes in conditioned media were
collected and analysed by nanoparticle trafficking analysis (NTA) and immunoblotting.
Protein expression level in cells was screened by Reverse Phase Protein Array (RPPA).
Inhibitor or shRNA were used to inhibit target proteins function. In vitro phosphorylation
and GEF assay were used to verify Rabin8 phosphorylation and activation. Exosomes
from cells
on soft or stiff matrix were injected into mice to study their effect on tumour growth.
Results:
Stiff matrix promoted exosomes secretion.
Akt was activated by stiff matrix and was required for exosome secretion.
Rab8 was activated by Akt and regulated exosome secretion.
Rabin8 was a direct target of Akt.
Stiff exosomes promote tumour growth.
Summary/Conclusion: Matrix stiffening promotes exosome secretion via
Akt-Rabin8-Rab8 pathway, contributing to tumorigenesis.
PT12.08
Tridimensional fibroblast culture revealed a novel
exososome-dependent extracellular matrix secretion mechanism
Vincent Clémenta
, Bastien
Paréb, Cassandra Gouleta, Thiéry De Serres-Bérarda,
Stéphane Bolduca, François Berthoda and François
Gros-Louisa
aUniversité Laval, Québec, Canada; bNorgen Biotek
Corp., Thorold, Canada
Introduction: The extracellular matrix (ECM) is constituted of a variety of
proteins and polysaccharides that are secreted locally and assembled into a thick
3D
meshwork to provide biophysical and biochemical support to the surrounding cells,
and
regulate numerous cellular functions such as adhesion, migration and proliferation.
Dysregulation of ECM components or aberrant ECM remodelling can lead to various pathologies,
as well as to play important roles in wound healing. Although ECM secretion pathways
are
still largely unknown, the current paradigm is that ECM-associated proteins are synthesized
in the endoplasmic reticulum and transported via the endosomes to the Golgi apparatus
en
route to the cell surface and released by exocytosis.
Methods: To study ECM secretion pathway, we used 3-dimensional (3D) cultured
fibroblasts. This culture method technique has been used widely to generate
tissue-engineered self-assembled stromal tissues, free of exogenous materials, and
rely on
long-term supplementation of sodium ascorbate into the culture medium. Non-cancerous
fibroblasts, grown in conventional two-dimensional (2D) cellular cultures, are known
to be a
poor source of secreted exosomes when compared to cancerous fibroblasts.
Results: Here, we provide evidence that non-cancerous dermal fibroblasts can
secrete high amounts of exosomes, containing different ECM proteins, when cultivated
in a 3D
fashion. We also demonstrated that dermal fibroblast-derived exosomes had the capacity
to
travel from one cell to another, induce cellular migration and promote wound healing.
Summary/Conclusion: Altogether, these findings reveal a novel
exosome-dependent ECM deposition mechanism and suggest that the use of 3D-fibroblast
cellular culture may emerge as an innovative approach in precision medicine to better
study
the role of patient-derived exosomes and ECM proteins in the establishment of cellular
microenvironment in health and disease.
PT12.09
Redirected tropisms of extracellular vesicles and exomeres yield
distinct biodistribution profiles
Anthony Yan-Tang. Wu
a, Charles Lai,
Yun-Chieh Sungb, Steven T. Chouc, Vanessa Guoc, Jasper C.
Chienc, John J. Koc, Alan L. Yangc, Ju-Chen
Chuangc, Hsi-Chien Huangb, Syuan Wuc, Meng-Ru
Hod, Maria Ericssone, Wan-Wan Linf, Koji
Uedag, Yunching Chenh, Chantal Hoi Yin Cheungi and
Hsueh-Fen Juanj
aDepartment and Graduate Institute of Pharmacology, National
Taiwan University, Taipei, Taiwan (Republic of China); bInstitute of Biomedical
Engineering and Frontier Research Centre on Fundamental and Applied Sciences of Matters,
National Tsing Hua University, Hsinchu, Taiwan (Republic of China); cInstitute of
Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan (Republic of China);
dInstitute of Biological Chemistry, Academia Sinica, Taipei, Taiwan (Republic
of China); eDepartment of Cell Biology, Harvard Medical School, Boston, USA;
fInstitute of Pharmacology,College of Medicine, National Taiwan University,
Taipei, Taiwan (Republic of China); gJapanese Foundation For Cancer Research,
Koto-ku, Japan; hNational Tsing-Hua University, Hsinchu, Taiwan (Republic of
China); iDepartment of Life Science, National Taiwan University, Taipei, Taiwan
(Republic of China); jNational Taiwan University, Taipei, Taiwan (Republic of
China)
Introduction: Bionanoparticles including extracellular vesicles and exomeres
(collectively termed EVs), have been shown to play significant roles in diseases and
therapeutic applications. However, their spatiotemporal dynamics in vivo have remained
largely unresolved in detail due to the lack of a limited suitable method.
Methods: We developed a bioluminescence resonance energy transfer (BRET)-based
reporter, PalmGRET, to enable pan-bionanoparticle labelling ranging from exomeres
(<
50 nm) to small (< 200 nm) and medium and large (> 200 nm) EVs and larger EVs (>
50 nm).
Results: PalmGRET emits robust, sustained signals and allows the
visualization, tracking and quantification of bionanoparticles from whole-animal to
nanoscopic resolutions under different imaging modalities, including bioluminescence,
BRET,
and fluorescence. Using PalmGRET, we show that EVs released by lung metastatic
hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to
other
major organs in immunocompetent mice. EV proteomics identified HCC-EV lung tropic
protein
candidates associated with cancer progression, in which SLCO2A1 and CLIC1 expression
on
non-tropic EVs conferred lung-tropism, while CD13 gave spleen tropism. Our results
further
demonstrate that redirected lung tropism decreases EV distribution to the liver, whereas
the
spleen tropism significantly reduces over time delivery to most major organs distribution
including the liver and kidney.
Summary/Conclusion: We established a multimodal and multi-resolution PalmBRET
method to enable pan-bionanoparticle labelling and imaging and therefore quantification
in
live cells, whole animals, and preserved tissues. The method can resolve the intricate
spatiotemporal dynamics of EVs. PalmGRET revealed that EVs derived from lung metastatic
HCC
are lung tropic, and the tropism can be conferred to non-lung-tropic EV-293 T by decorating
EVs with identified HCC-EV membrane proteins. Importantly, the enhanced EV delivery
to
tropic organs also significantly alters its distribution to other major organs. Our
findings
suggest that the dynamics of EV biodistribution and targeted design should be investigated
at the organ systems level in EV biology and therapeutic developments, respectively.
Funding: Ministry of Science and Technology (MOST) grants
104–2320-B-007-005-MY2 (C.P.L.), 106–2320-B-007-004-MY3 (C.P.L.), Academia Sinica
Innovative
Materials and Analysis Technology Exploration (i-MATE) Program AS-iMATE-107-33 (C.P.L.),
and
Academia Sinica Career Development Award AS-CDA-109-M04 (C.P.L.).
PT12.10
Tracking mesenchymal stem cell-derived extracellular vesicles
(EVs) in a in vivo cancer model
Clodagh O’Neill
a, Christy
Barberb, Katie E. Gilliganc, Emma McDermottd, Adriele
Prina-Melloe, Peter Dockeryd, Zhonglin Liub and Róisín
Dwyerf
aDiscipline of Surgery, National University Ireland Galway,
Galway, Ireland; bDepartment of Medical Imaging, University of Arizona, Tucson,
AZ 85724 USA, Tuscon, USA; cDiscipline of Surgery, National University of Ireland
Galway, Galway, Ireland; dDiscipline of Anatomy, National University Ireland
Galway, Galway, Ireland; eSchool of Medicine, Trinity College Dublin, Ireland,
Dublin, Ireland; fDiscipline of Surgery, CÚRAM, SFI Research Centre for Medical
Devices and Precision Oncology Ireland, National University Ireland Galway, Galway,
Ireland
Introduction: Small Extracellular vesicles (sEVs) are nanoparticles (30–120mn)
encircled by a phospholipid bilayer, derived from the endocytic pathway and released
by all
cells. sEVs have an inherent role in cell communication and deliver cargo to target
cells.
Mesenchymal stem cells (MSCs) and have a natural ability to home to tumours and metastases
while avoiding the host immune response. It is hypothesised that MSC derived sEVs
(MSC-sEVs)
also possess tumour-homing and immune-evading capacities therefore could provide a
novel
targeted delivery vehicle for treatment of cancer. It is imperative to elucidate MSC-sEVs
migratory itinerary in vivo to support translation to the clinical setting.
Methods: This study aimed to image the interaction of labelled MSC-sEVs with
cancer cells in real time in vivo.
sEVs were isolated from wildtype MSCs and MSCs with stably expressing red fluorescent
protein (RFP) (via lentivirus) by the combined techniques of differential centrifugation,
microfiltration and ultracentrifugation. Isolated sEVs were extensively characterised
by
Transmission electron Microscopy (TEM), Nanoparticle Tracking Analysis and Western
Blot. NOD
SCID Gamma (NSG) mice with dorsal skinfold window chamber (DSFWC) were injected with
either
MDA-MB-231 luciferase (Luc) expressing cells or HT-29-Luc cells. Bioluminescence imaging
was
performed to confirm tumour formation. A dose of 1x10^7 MSC-RFP-sEVs was directly
added to
the window chamber and RFP expression detected using a microscope with RFP filter
attachments. 8x10^7 EVs were incubated with the radionuclide, Technetium-99m tagged
Duramycin (99 mTc-Dur) for 30 minutes at room temperature. Excess radiolabel was removed
using exosome spin column (Invitrogen™). The 99 mTc-Dur-sEVs were then added directly
to the
window chamber and charged particle imaging carried out.
Results: 18 hours post-administration; the RFP signal was localised at the
tumour site. Radiolabelled sEV signal could be detected 10 minutes and 4 hours after
administration. MSC-sEVs were successfully detected at the tumour site following direct
administration using two different tagging and imaging approaches.
Summary/Conclusion: This promising preliminary data supports the potential of
this approach for tracking MSC-sEV migration In Vivo. Future studies will investigate
systemic tracking of MSC-sEV migration.
Funding: This work was funded by the Irish Association of Cancer Research
(IACR) AOIFA mobility award. C.O.N. is supported by funding from the National Breast
Cancer
Research Institute (NBCRI).
.
PT12.11
Response To Irradiation Of Small Extracellular Vesicles Derived
From Prostate Tumors
Vaughn Garcia1; Aejez Sayeed2; Rachel
DeRita1; Shiv Ram Krishn3; Peter A. McCue1; Adam
Dicker1; Lucia R. Languino1
1Thomas Jefferson University, Philadelphia, USA;
2Thomas Jefferson.edu, Philadelphia, USA; 3Thomas Jefferson
University, Philadelphia, USA, Philadelphia, USA
Introduction: Tumor-derived small extracellular vesicles (sEVs) have emerged
recently as mediators of tumorigenesis. However, the role of sEVs in response to
irradiation, a widely used therapy in prostate cancer, is not fully understood.
Methods: Our study involved the TRAMP mouse model of prostate cancer. We used
plasma sEVs isolated using differential ultra-centrifugation and further isolated
using
iodixanol gradient fractionation. We also used Nanoparticle Tracking Analysis (NTA)
to
analyze sEVs. Mouse pelvises were irradiated using 10 Gy, for 5 consecutive days.
Results: We first observed that upon pelvic irradiation of TRAMP mice, the
levels of the signaling oncogene c-Src are reduced in plasma-derived sEVs, while the
average
size of sEVs is increased from 50-100nms to 70-250nms. Furthermore, we show that the
sEVs
from irradiated cells lose the ability to stimulate anchorage independent growth and
migration of recipient cancer cells. Additionally, sEVs from irradiated mice increase
the
amount of DNA damage in recipient cancer cells.
Summary/Conclusion: Overall, our data show that irradiation of TRAMP mice (and
prostate cancer cells) significantly reduces the pro-metastatic and
pro-anchorage-independent growth potential of sEVs when tested on human cells. Changes
to
the composition and behavior of a cancer cell sEV population via radiation therapy
offers
promise for future therapeutic approaches for prostate cancer.
Funding: This study was supported by NIH, P01 CA-140043 (to LRL). This project
is also funded, in part, under a Commonwealth University Research Enhancement Program
grant
with the Pennsylvania Department of Health (H.R.); the Department specifically disclaims
responsibility for any analyses, interpretations or conclusions.
Please provide any keywords if applicable.: Small extracellular vesicles,
TRAMP, prostate cancer, irradiation
PT13: Advances in EV Separation and Concentration
Chair: Navneet Dogra – Department of Genetics and Genomic Sciences, Department of
Pathology, Icahn School of Medicine, Mount Sinai
PT13.01
Development and characterization of extracellular vesicles
separation methods from human brain tissues and its application to Alzheimer’s
disease
Satoshi Muraokaa
, Annina
Deleoa, Manveen Sethia, Weiwei Lina, Zijian
Yangb, Mei Chenc, Kayo Yukawaa, Jina Kod, John
Hogana, Seiko Ikezua, Weiming Xiaa, Santhi
Gorantlae, Howard Gendelmane, Andrew Emilia, David
Issadoref, Joseph Zaiaa and Tsuneya Ikezua
aBoston University, Boston, USA; bUniversity of
Pennsylvania, Philadelphia, USA; cEdith Nourse Rogers Memorial Veterans Hospital,
Bedford, USA; dMassachusetts General Hospital, Wyss Institute at Harvard
University, Cambridge, USA; eUniversity of Nebraska Medical Center, Omaha, USA;
fDepartment of Bioengineering, University of Pennsylvania, Philadelphia,
USA
Introduction: There are emerging physiological and pathological functions of
extracellular vesicles (EVs) in neurodegenerative diseases including Alzheimer’s disease
(AD). Brain derived-EVs contain pathogenic proteins, such as tau, amyloid beta (Aβ),
which
have been reported to contribute to cell-to-cell propagation in those diseases.
Investigation of the brain-derived EV cargo, therefore, is important to further understand
the mechanisms of progression in neurodegenerative diseases. We developed the EV separation
method from unfixed frozen mouse and human brain tissues and assessed the protein
composition.
Methods: To establish the EV separation method, we separated EVs from frozen
mouse brain tissue using sucrose density gradient ultracentrifugation (SG-UC) or size
exclusion chromatography to compare the results from the particle number, morphology
and
protein profiling by NTA, TEM and Mass spectrometry. EVs were then separated from
cortical
grey matter of AD (n = 20) and Control (n = 18) by SG-UC. Tau and Aβ in the EVs were
measured by immunoassay. Differentially expressed EV proteins were observed by quantitative
proteomics employing machine learning.
Results: The separated EVs were enriched in EV molecules and devoid of
contaminant proteins by SG-UC, showing our method was successful. The levels of pS396
tau
and Aβ1-42 were significantly increased in AD EVs. Annexin A5 (ANXA5), neurosecretory
protein VGF, neuronal membrane glycoprotein M6-a (GPM6A), and Alpha-centractin (ACTZ)
were
differentially expressed in AD EVs. A combination of these 4 proteins were confirmed
to
predict AD with the 88% accuracy by machine learning.
Summary/Conclusion: These data suggest our method were suitable for the
separation of brain-derived EVs and EV ANXA5, VGF, GPM6A and ACTZ can be potential
biomarkers for monitoring the progression of AD.
PT13.02
EDTA stabilizes the concentrations of extracellular vesicles
during blood collection
Naomi C. Buntsmaa
, Aleksandra
Gąseckab, Yvo Roosa, Ton G. van Leeuwenc, Edwin van der
Pold and Rienk Nieuwlande
aDepartment of Neurology, Amsterdam UMC, University of
Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands; b1st Chair and
Department of Cardiology, Medical University of Warsaw, Warsaw, Poland; cVesicle
Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands;
Department of Biomedical Engineering and Physics, Amsterdam UMC, University of Amsterdam,
Amsterdam, the Netherlands, Amsterdam, Netherlands; dDepartment of Clinical
Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands; Vesicle
Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands;
Department of Biomedical Engineering and Physics, Amsterdam UMC, University of Amsterdam,
Amsterdam, the Netherlands, Amsterdam, Netherlands; eDepartment of Clinical
Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Vesicle
Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands,
Amsterdam, Netherlands
Introduction: To establish reliable biorepositories for research on
extracellular vesicles (EVs) as disease biomarkers, the release of EVs during blood
collection and handling must be avoided. Currently, citrate is recommended as the
anticoagulant for blood EV research, but citrate does not inhibit the release of EVs
from
activated platelets. The release of platelet-derived EVs excludes pneumatic tube transport
and makes assays time dependent, thereby limiting clinical compatibility. Therefore,
we aim
to stabilize the release of platelet EV concentrations.
Methods: Blood samples were collected from healthy individuals and subjected
to common circumstances known to induce platelet activation. Blood was (i) incubated
with or
without thrombin receptor-activating peptide 6 (TRAP; n = 7), a potent platelet activator,
(ii) send to the lab by a routine blood transport (pneumatic tube system; n = 3),
and (iii)
stored at room temperature or at 4°C for 6 hours (n = 3). The concentrations of EVs
from
platelets (CD61+), activated platelets (P-selectin+), erythrocytes (CD235a+), and
leukocytes
(CD45+) were determined by flow cytometry (Apogee A60-Micro).
Results: Following activation by TRAP, concentrations of platelet-derived and
activated platelet-derived EVs increased 5.8-fold and 10.4-fold in citrate-anticoagulated
blood, compared to 1.4-fold and 2.0-fold in EDTA-anticoagulated blood (EDTA vs citrate:
p = 0.018 and p = 0.043, respectively). Preliminary data show that during pneumatic
tube
transport and routine sample handling, both platelet- and activated platelet-derived
EVs
were more stable in EDTA compared to citrate. The concentrations of EVs from erythrocytes
and leukocytes were unaffected under all studied conditions.
Summary/Conclusion: To conclude, EDTA stabilizes platelet EV concentrations
during and after blood collection, which would facilitate pneumatic tube transport,
enhance
reliability and thereby improves the establishment of reliable biorepositories for
EV
research.
Funding: N. Buntsma acknowledges the Dutch Heart Foundation, research grant
CINTICS 2018B031. A. Gasecka acknowledges funding from the Polish National Science
Centre,
research grant PRELUDIUM 2018/31/N/NZ7/02260. E. van der Pol acknowledges funding
from the
Netherlands Organisation for Scientific Research – Domain Applied and Engineering
Sciences
(NWO-TTW), research program VENI 15924.
PT14: Microfluidic and Other Devices
Chair: Colin L. Hisey – Research Fellow, The University of Auckland
PT14.01
Circular Dichroism-based Exosome (CDEXO) detection microfluidic
platform using their assemblies with chiral gold nanoparticles
Yoon-Tae Kanga
, Hee Jeong
Jangb, Ji-Young Kimc, Colin Palacios-Rolstonb, Sunitha
Nagratha and Nicholas A. Kotovb
aUniversity of Michigan, Ann Arbor, USA; bUMICH,
Ann Arbor, USA; cKorea Institute of Toxicology, Deajeon, Republic of Korea
Introduction: Cancer-cell secreted extracellular vesicles, called exosomes,
are an emerging biomarker for cancer liquid biopsy. Profiling of cancer-associated
exosomes
usually required lengthy, and multi-step procedures; therefore simple and easy-setup
sensing
methods are urgently needed for diagnosing cancer in a timely manner. Chirality, the
foundational property of all biomolecules, including exosomal proteins, can be utilized
for
exosome detection and differentiation using recent advances in chiral nanostructures.
We
found that microfluidic sensors can be successfully implemented for successful detection
of
cancer-associated exosomes taking advantage of unusually high circular dichroism (CD)
of
chiral gold nanoparticles (AuNPs).
Methods: Circular Dichroism-based Exosome (CDEXO) detection utilizes
chiroplasmonic enhancement of CD signatures of cancer-associated exosomes. We first
synthesized donut-shaped AuNPs conjugated with L-cysteine and immobilized the AuNPs
on a
glass slide using a layer-by-layer assembly. The AuNPs on slide glass were surface
functionalized by the standard biotin-avidin reaction after MUA treatment. Biotinylated
annexin V marker, targeting phosphatidylserine (PS) expression on cancer-associated
exosomes, was conjugated to the AuNP surface. 5 μl of exosome samples from cancer
cells
(A549 and H3255) or normal cells (MRC5) were injected into the PDMS microfluidic device
and
incubated for 30 minutes. The CD signal before and after exosome exposure was monitored,
compared, and systematically analysed as a rapid technique for the detection of exosomes
with high sensitivity.
Results: We showed that the CDEXO signals from cancer exosomes showed 26.6
folds absolute CD peak value change and 3.41 folds shift, respectively, compared to
that of
healthy exosomes. Importantly, the CDEXO sensing method takes less than 10 mins in
terms of
total scanning time and requires minimal sample volumes. From the preclinical studies
using
7 blood samples from cancer patients and healthy donors, we found that cancer patients
show
stronger band shift and signal change comparing to that of healthy donors, implying
our
platform could be used for cancer diagnosis.
Summary/Conclusion: This new versatile and sensitive method based on
chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes
in a
timely manner for minimal volumes of liquid biopsies.
PT14.02
EV classification and fractionation strategy using surface
charge labelling
Takanori Ichikia
, Hiroaki
Takeharaa, Hirofumi Shionob and Hiromi Kuramochia
aThe University of Tokyo, Bunkyo, Japan;
bInnovation Center of NanoMedicine, Bunkyo, Japan
Introduction: The development of new classification technology is required
based on the evaluation of physicochemical properties of exosome surfaces and the
diversity
of constituent molecules. In this presentation, we present the electric charge activated
exosome sorting platform comprising microfluidic device technology and electric charge
labelling technique.
Methods: The single nanoparticle analysis platform, which has been developed
by our research group, images Rayleigh scattered light (elastically scattered light)
obtained by irradiating nanoparticles with convergent laser light and provides information
of individual particles by image processing. The method that utilizes electrokinetic
phenomena, unlike the method using fluorescent labels, measures the properties of
the
particle surface without serious difficulty in principle even if the particle size
is on the
order of tens nanometres, and further enables to perform fractionation. Since the
number of
particles usually handled in exosome research or its envisioned application is enormous,
it
is not realistic to take an approach such as a cell sorter in which particles are
sequentially manipulated one by one following the measurement results of individual
particles.
Results: Particles receive attraction or repulsion by an external field
according to the charge density on the surface, so there is no need to control the
external
force, and it is possible to design a device that can autonomously fractionate particles
according to the difference in zeta potential.
Summary/Conclusion: In conclusion, we have proposed and demonstrated the new
concept of electric charge activated EV sorter.
Funding: This research was partially supported by the Center of Innovation
Program (COI STREAM) from the Japan Science and Technology Agency.
PT14.03
High throughput exosome analysis by using reversible
microfluidic electrochemical sensor system
Hyo-Il Junga
, Junmoo
Kima, Seung-Eun Kimb, Hogeyong Gwaka and Kyung-A
Hyuna
aYonsei University, Seoul, Republic of Korea;
bKorea Electronics Technology Institute (KETI), Seoul, Republic of Korea
Introduction: Exosome is one of the important extracellular vesicles (EVs)
released from parental cells and it contains various types of molecular cargos from
its
original cell including proteins, messenger RNA (mRNA), and micro RNA (miRNAs) [1].
The
exosomes have recently emerged as biomarkers for early stage cancer detection because
the
number of exosomes originated from cancerous cells are significantly higher than those
from
normal cells [2]. Since many different types of exosomes exist in the whole blood,
it is
necessary to isolate and detect disease-specific exosomes. For this reason, the isolation
and the detection of exosomes is an important research issue and has been studied
by many
groups. However, limitations such as low throughput and low recovery still make it
difficult
to use exosomes in diagnostics and therapeutics.
Methods: In this study, we developed an integrated microfluidic
electrochemical biosensor to extract plasma from whole blood and subsequently detect
cancer
related exosomes in a continuous manner. This consists of two parts. The first part
is a
channel for extracting plasma containing exosomes from whole blood, and the second
part is a
channel combined with an electrochemical sensor for multiple detection of various
exosomes
in the extracted plasma. Previously, a Multi-Orifice Flow Fractionation (MOFF) channel
that
consists of a series of expansion and contraction structures has been developed in
our
group. In this channel, the blood cells are moved to sides of channels by hydrodynamic
forces and then are eliminated to outlets. At this time, the plasma is moved to the
electrochemical sensor part, the exosomes in the plasma are captured to the electrodes
immobilized with the specific antibodies and are quantified the amount of cancer-related
exosomes.
Results: Using this chip, blood cells were eliminated from the whole blood
with over 90% of separation efficiency at 225 µl/min flow rate and exosomes were collected
continuously with high recovery (~70%). In order to quantify various types of exosomes,
a
label-free electrochemical biosensor with Electrochemical Impedance Spectroscopy (EIS)
was
used for the continuous detection of exosomes. The limit of detection was 1x10^6
exosomes/ml.
Summary/Conclusion: The developed device is an integrated device capable of
separating exosomes from whole blood with high purity and quantitating exosomes through
the
electrochemical sensor in a continuous manner.
Funding: The authors acknowledge the Bio-Medical Technology Development
Program of the National Research Foundation of Korea (NRF) for providing the following
grants: MSIP (No. 2015M3A9D7067364), (No. NRF-2018R1A2A2A15019814), and (No.
NRF-2081R1C1B6002499) to carry out this research.
PT14.04
High-throughput multiparametric characterization and
quantitation of extracellular vesicles by fluorescence-based microfluidic diffusion
sizing
Carolina Paganinia
, Britta
Hetticha, Marie R.G. Koppa, Adam Eördöghb, Umberto
Capasso Palmieroa, Mauro Mannoc, Antonella bongiovannid,
Pablo Rivera Fuentesb, Jean-Christophe Lerouxa and Paolo
Arosioa
aETH Zurich, Zurich, Switzerland; bEPFL, Lausanne,
Switzerland; cNational Research Council of Italy, Palermo, Italy;
dInstitute for Research and Biomedical Innovation (IRIB) – National Research
Council (CNR), Palermo, Italy
Introduction: Progress in the field of extracellular vesicles (EVs) is
hampered by the challenge to produce large amounts of EVs in a reproducible fashion
(Paganini et al, Biotech. J., 1800528, 2019). The development of high-throughput techniques
capable of simultaneously monitoring physical and biochemical properties of EVs would
significantly simplify and accelerate the characterization process. In this context,
microfluidic technology is emerging as an attractive platform. Here, we present a
microfluidic device based on the combination of diffusion sizing and multi-wavelength
fluorescence detection to simultaneously provide information on EV size, concentration
and
composition.
Methods: The diffusion of EVs in the microfluidic channel provides information
on their size distribution, and four different staining protocols with high signal-to-noise
ratios track different EV native molecules. EVs are separated from unbound fluorophores
directly during the microfluidic analysis, therefore avoiding the need for sample
pretreatments and allowing to operate the device as a single-step immunoassay.
Results: The microfluidic device coupled with complementary staining
techniques allows to individually detect and size particle populations with different
EV
components such as lipids, primary amines and the EV marker CD63. We demonstrate that
this
approach can probe the abundance of EV-specific markers and impurities such as lipoproteins
with high throughput and low sample consumption.
Summary/Conclusion: We present a microfluidic technique capable of
characterizing and quantifying EVs at low costs, in a time-scale of minutes and requiring
only up to 2 µL of non-pretreated sample. This method is an important complementary
tool to
the current array of biophysical methods for EV characterization, in particular for
high-throughput screening applications.
Funding: H2020‐EU.1.2.1‐FET Open programme via the Grant agreement 801338.
PT14.05
Immunomagnetic isolation of specific subpopulations of exosomes
for liquid biopsy via nano-architected porous materials
Andrew A. Lina
, Zhimin
Jiangb, James Pikulb and David Issadorec
aUniversity of Pennsylvania, Philadelphia, USA;
bDepartment of Mechanical Engineering and Applied Mechanics, University of
Pennsylvania, Philadelphia, USA; cDepartment of Bioengineering, University of
Pennsylvania, Philadelphia, USA
Introduction: Exosomes offer the potential to reveal significant biological
information in many areas of clinical importance by virtue of their RNA contents and
protein
surface markers. This abstract reports the fabrication of a device for high throughput
targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected
porous metal lattice materials.
Methods: We have invented a fabrication technique to precisely make millions
of nanoscale exosome sorting devices that can operate on unprocessed plasma. Each
nanoscale
device can precisely sort targeted exosomes from background vesicles but is too slow
for
practical use individually. However, the operation of millions of these devices in
parallel
preserves the precision of nanoscale sorting while also enabling high throughput and
robust
use on raw plasma samples. The metal lattice within which these devices are contained
is
assembled via metal electroplating onto a self-assembled polystyrene bead lattice
with
face-centred cubic (FCC) symmetry with 600 nanometre pores. The devices feature a
conformally-coated layer of nickel-iron with gold passivation atop a base layer of
nickel,
resulting in a lattice of millions of nanoscale pores capable of magnetic sorting
of
exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic
nanoparticles.
Results: Compared to our previous work on immunomagnetic exosome capture via
commercial track-etched membranes (TEMPO), this device offers superior capture due
to
increased surface pore density (>25x) and three-dimensional pore density (>1000x)
alongside lower required sample volume due to decreased non-capturing volume in the
device.
Finite-element analysis simulations show that strong magnetophoretic traps emerge
at the
pore boundaries in this structure between higher-permeability metals such as nickel-iron
permalloy and the lower-permeability sample fluid in the device. Preliminary experimental
data shows that this device can isolate iron nanoparticles in solution with >100x
enrichment from input and 49x capture efficacy versus TEMPO.
Summary/Conclusion: Current methods of exosome isolation such as
ultracentrifugation and column chromatography all suffer from low throughput and limited
yield. The application of inverse opal materials towards exosome capture offers the
potential for isolation of specific exosome populations from very low clinical sample
volumes or sparse biological signals.
PT14.06
Micropatterned growth surface topography affects extracellular
vesicle production
Colin L. Hiseya
, James
Hearnb, Yohanes Nursalima, Vanessa Changa, Cherie
Blenkirona and Lawrence W. Chamleya
aThe University of Auckland, Auckland, New Zealand;
bUniversity of Auckland, Grafton, New Zealand
Introduction: Extracellular vesicles are micro and nanoscale packages released
by all cells and play an important role in cell-to-cell communication by shuttling
biomolecules to nearby and distant cells. However, producing enough EVs for many in
vitro
studies using conventional tissue culture techniques can be challenging, and despite
the
success of some bioreactors in increasing EV-production, it is still unknown how many
independent culture conditions like growth surface topography can alter the production
and
content of EVs.
Methods: Standard 150 mm petri dishes were patterned with 2 µm tall
polystyrene microtracks spaced by 5, 10 and 20 µm across a 100 mm area using standard
microfabrication techniques including photolithography, soft lithography and microtransfer
printing. The micropatterns were characterized with SEM and profilometry, then activated
with oxygen plasma and UV sterilized. MDAMB231 cells were seeded onto patterned and
smooth
(control) dishes and grown in serum-free media for the final 48 hours of culture.
EVs were
isolated using sequential ultracentrifugation of conditioned media and characterized
using
NTA, TEM and western blot. Cell morphology was imaged using immunocytochemistry and
single
cell migration was characterized using time-lapse microscopy and manual single cell
tracking
in FIJI.
Results: We demonstrate the simple and repeatable fabrication of microtracks
across a large surface area in order to culture cells on topographically patterned
growth
surfaces. Furthermore, we show that the 5 µm spacing produced significantly more EVs
than
other patterns as well as the highest cell aspect ratio and average single cell migration
speed (p < .05).
Summary/Conclusion: These findings have implications in both biomanufacturing
of EVs and potentially in enhancing the biomimicry of EVs produced in vitro. However,
further experimentation to assess the differences in cargo on patterned growth surface
topographies compared to conventional methods is still required.
Funding: This project was funded by the Maurice and Phyllis Paykel Trust.
PT14.07
Using miscroscale thermophoresis and surface plasmon resonance
to measure the interactions of extracellular vesicles
Apolonija Bedina Zavec
a, Neža
Omersab, Ario DeMarcoc, Maja Jamnika, Aleksandra
Šakanovićb, Gregor Anderluha and Marjetka Podobnika
aNational Institute of Chemistry, Ljubljana, Slovenia;
bDepartment of Molecular Biology and Nanobiotechnology, National Institute of
Chemistry, Ljubljana, Slovenia, Ljubljana, Slovenia; cLaboratory for
Environmental and Life Sciences, University of Nova Gorica, Nova Gorica, Slovenia,
Nova
Gorica, Slovenia
Introduction: Microscale Thermophoresis (MST) is a recently developed
biophysical technology for the analysis of molecular interactions. The motion of molecules
in temperature gradients allows the quantification of biomolecule interactions by
changes in
conformation, charge and size of a molecule induced by a binding event. MST is a quick
method, easy to handle, has a low sample consumption, has no limitation on molecule
size,
and enables measurements in solution, either in various buffers or complex biological
liquids. These properties make MST an interesting tool for research of extracellular
vesicles (EVs); therefore, our aim is to apply this method to EVs.
Methods: EVs were isolated from Jurkat cell line by differential
centrifugation. Microscale Thermophoresis (MST) and Surface Plasmon Resonance (SPR)
were
used to analyse the interaction between antibody and EVs.
Results: We have demonstrated that interactions of EVs with antibodies could
be analysed by MST. However, the tiny glass capillaries for sample mounting represent
a
challenge due to adhesion of EVs to their surface. We have tested commercial capillaries
as
well as prepared capillaries in house coated by liposomes or bovine serum albumin.
The
interactions between EVs and antibodies were confirmed by Surface Plasmon Resonance
(SPR),
which is an established method for studying the interactions of EVs.
Summary/Conclusion: Microscale Thermophoresis (MST) is a promising method for
the analysis of EVs.
Funding: Financed by Slovenian research agency, grant No. P1-0391 and
J4-9322.
PT14.08
Acoustofluidic separation of complex suspensions
Melanie Colditz
a, Cynthia
Richardb, Armaghan Fakhfouria, Romy Kronstein-Wiedemannc,
Torsten Tonnc and Andreas Winklera
aLeibniz Institute for Solid State and Materials Research
Dresden, Dresden, Germany; bMonash University, Melbourne, Australia;
cExperimentelle Transfusionsmedizin, Medizinische Fakultät Carl Gustav Carus der
Technischen Universität Dresden/DRK-Blutspendedienst Nord-Ost gGmbH, Dresden, Germany
Introduction: The isolation of extracellular vesicles (EVs) from cell culture
supernatants and complex body fluids, such as blood and urine, is of high importance
for EV
research as well as for future medical applications in diagnostics and therapy.
Nevertheless, it is still challenging to reach the desired recovery, purity and specificity
due to many manual and time intensive sample preparation steps. Conventional centrifugation
for EV isolation or sample preparation prior to affinity-based separation methods
can damage
EVs and cells, leading to misinterpretation of results or inactive EVs. Alternative
field
flow fractionation methods employing acoustic fields are highly promising, but so
far
limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly
reproducible. Here, we present an innovative surface acoustic wave (SAW)-based
acoustofluidic device for gentle sorting of cells and particles.
Methods: Our device consists of interdigital transducers patterned on a
piezoelectric substrate generating SAW propagating on the substrate surface. Upon
interaction of SAW with our on-chip structured, fluid-loaden microchannels, an acoustic
pressure field is developed across the fluid wherein particles are suspended. This
pressure
field can be employed to simply manipulate cells and particles based on their intrinsic
properties, such as size, density and compressibility in continuous flow. The device
is
manufactured using precise and low-cost microtechnological methods and is suitable
for
reproducible mass fabrication.
Results: We demonstrated the separation of blood components, i.e. the sorting
of erythrocytes and thrombocytes. Furthermore, we could also show results on thrombocyte
activation indicating a gentle separation without damaging these shear-sensitive cells,
as
well as first results on plasma separation from whole blood samples and nanoparticle
sorting.
Summary/Conclusion: Our unique acoustofluidic sorting technology for complex
suspensions has the potential to overcome the need for time-effective, cheap and gentle
separation of EVs.
Funding: This work was supported by EFRE InfraPro project “ChAMP: Chip-based
acoustofluidic Medtech Platform”.
PT14.09
Nanophotonic platform for cancer-associated exosomal microRNA
detection
Raquel S. Vaz, Manuela F. Frasco and Maria G.
Sales
BioMark Sensor Research/UC, Faculty of Sciences and Technology, Coimbra
University, Coimbra, Portugal, Porto, Portugal
Introduction: Exosomes have an important role in intercellular communication
at physiological and pathological processes. Their cargo includes microRNAs (miRs),
single-stranded non-coding RNAs, involved in alterations on recipient cells, such
as
development of tumourous phenotype and metastasis. More particularly, miR-21 excels
due to
its association with several cancers. Determining exosomal miRs as cancer indicators
demands
selective and accurate methods, which are not currently available or entail high costs.
Colorimetric photonic-based assays are a promising label-free alternative, which dismisses
complex apparatus for signal reading since biorecognition is detected by colour change.
Moreover, the clinical and economic systems have also been demanding a decrease on
the green
footprint of biosensors, requirement fulfilled with naturally derived biomaterials.
Methods: Herein, the biosensor is constructed on a biopolymer matrix to meet
the requirements of an eco-friendly disposable device, and it is based on a photonic
structure obtained by imprinting a nanopattern on the polymer surface. Then, the surface
is
functionalized with the complementary oligonucleotide sequence of miR-21 as sensing
probe. A
label-free detection is thus envisioned and the sensor performance is evaluated by
changes
in the optical properties when the target is present.
Results: The combination of biological materials conducted to a biosensor
support with great flexibility and low water permeability, allowing easy surface
functionalization. The self-reporting ability of the photonic-based sensor enables
high
intensity colours detected by naked eye.
Summary/Conclusion: The alliance with the high selectivity of oligonucleotide
hybridization is expected to offer great exosomal miR-21 recognition ability and an
optimistic perspective for utilization in clinical setups.
Funding: The authors acknowledge the financial support from the European
Commission/H2020, through MindGAP/FET-Open/GA829040 project.
PT15 = OP1 Oral with Poster Session 1: Lungs
Chair: Uta Erdbrügger – University of Virginia
Chair: Peter Kurre, MD – Comprehensive Bone Marrow Failure Center, Children’s Hospital
of Philadelphia; Perelman School of Medicine, University of Pennsylvania
PT15.01 = OP1.01
Human urinary extracellular vesicles carry surface markers that
are indicative of haematopoietic origin
Veronika Mussacka
and Michael
Pfafflb
aTUM School of Life Sciences, Dept. Animal Physiology and
Immunology, Freising, Germany; bDivision of Animal Physiology and Immunology,
Technical University of Munich, Freising, Germany, Freising, Germany
Introduction: Urinary extracellular vesicles (uEVs) are important
intercellular communicators. By systems biology integration, uEVs prove to be relevant
in
genitourinary disease detection. However, it has recently been shown that labelled
EVs
administered to the circulation can be detected in the urinary system, as well. Thus,
this
pilot study aimed at phenotyping haematopoietic surface markers on uEVs to create
enough
plausibility for future non-invasive biomarker studies of circulation and immune disorders
that may translate into urine but are not yet timely recognized.
Methods: Urine was obtained from healthy men signing a written informed
consent (n = 31). Sampling was approved by the local ethics committee and in compliance
with
the Declaration of Helsinki. Cell-free urine was obtained by serial centrifugation
and
10 ml, each, were utilized for the MACSPlex Exosome Kit, human (Miltenyi Biotec).
The
manufacturer’s recommendations were followed to examine 37 distinct uEV surface markers
of
CD9+/CD63+/CD81+ vesicles in a multiplexed bead-based manner including respective
controls.
The Accuri C6 (BD) was utilized for data acquisition.
For further MISEV2018-compliant characterization, CD9+/CD63+/CD81+ uEVs were isolated
by
immunoaffinity and analysed by fluorescence nanoparticle tracking (f-NTA), transmission
electron microscopy (TEM) and western blotting (WB).
Urinary creatinine (Ucrea) was determined to control for variances in urinary dilutions
and
used for data normalization.
Results: Except CD209, all other 36 surface markers could be identified. The
most abundant markers were CD9 and CD63, which were detected in 93% of samples, followed
by
CD133/1 (84%), CD326 (81%), CD81 and CD24 (77%, each). CD3 (42%), CD9, CD45 (39%),
CD49e
(32%) and CD81 showed similar relative median fluorescent intensities (rMFI), while
CD63
yielded significantly higher (p = 0.009) and all other markers significantly lower
rMFI
(p < 0.011).
TEM and f-NTA revealed cup-shaped vesicles (137 ± 22 nm) with 8.8 ± 7.0 E + 10 particles/g
Ucrea. WB indicated uEV isolates that were positive for Alix, Syntenin, TSG101, CD9,
CD63
and CD81 without any uromodulin or calnexin contamination.
Summary/Conclusion: Our results imply that considerable quantities of
circulatory EVs are, indeed, filtered into urine and could serve as valuable non-invasive
biomarkers for systemic dysfunctions.
PT15.02 = OP1.02
Cardiovascular risk markers are strongly related to numbers of
circulating extracellular vesicles
Ruihan Zhoua
, Esra
Bozbasa, Plinio Ferreirab and Parveen Yaqooba
aUniversity of Reading, Reading, UK; bImperial
College London, London, UK
Introduction: Extracellular vesicles (EVs) are small plasma membrane-derived
vesicles released from various cells, which potentially affect many physiological
and
pathophysiological processes, and are emerging as a potential novel biomarker in
cardiovascular diseases (CVDs). However, there is little information about the association
of circulating EV levels with traditional cardiovascular risk markers and CVD risk
score.
Methods: • Subjects (n = 40) aged 40–70 yrs with moderate risk of CVDs were
recruited and assessed for body mass index (BMI), blood pressure (BP) and plasma lipid
profile (triacylglycerol, total cholesterol and high-density lipoprotein).
• EVs were isolated from platelet-free plasma by size exclusion chromatography and
analysed
by both Nanoparticle Tracking Analysis (NTA) and flow cytometry (FCM). NTA was used
to
measure the concentration and size distribution of EVs population, and EVs were phenotyped
by FCM via a 3-colour panel, which included Annexin V (for the majority of circulating
EVs),
CD41 (for platelet-derived EVs) and CD105 (for endothelial-derived EVs).
• The association between risk markers and EV numbers was examined by Pearson’s correlation
coefficient and stepwise multivariate regression model. Analysis of covariance (ANCOVA)
was
performed after adjustment for various variables to determine the correlation between
the
quartile range of EV numbers and 10-yr CVD risk detected by QRISK2.
Results: EV numbers, as determined by NTA, were strongly associated with BMI
(r = 0.602, p < 0.001), blood pressure (systolic BP: r = 0.359, p = 0.023; diastolic
BP:
r = 0.550, p < 0.001) and plasma triacylglycerol levels (r = 0.703, p < 0.001). Plasma
total cholesterol level was positively associated with platelet-derived EVs, determined
by
FCM (r = 0.330, p = 0.038). A multivariate regression model demonstrated that plasma
triacylglycerol and diastolic BP independently predicted total EV numbers, with plasma
triacylglycerol concentrations explaining 49.4% of the variance for total EV numbers.
An
additional 9.3% of the variance in total EV numbers was predicted by diastolic BP.
ANCOVA of
the 10-yr CVD risk score in the quartile range of total EV numbers were positively
and
independently associated.
Summary/Conclusion: BMI, blood pressure, plasma triacylglycerol and total
cholesterol levels are strongly associated with EV numbers. Plasma triacylglycerol
and
diastolic BP independently predict circulating EV numbers. Elevated numbers of EVs
are
independently associated with 10-yr CVD risk.
Funding: This project is supported by Biotechnology and Biological Sciences
Research Council (BBSRC)-Diet and Health Research Industry Club (DRINC) in the UK.
PT15.03 = OP1.03
Extracellular vesicles from cardiosphere-derived cells
potentiate regulatory T cells
Akbarshakh Akhmerov, Geoffrey de Couto,
Russell G. Rogers, Ahmed Ibrahim, Weixin Liu, Lizbeth Sanchez and Eduardo Marbán
Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Introduction: Extracellular vesicles from cardiosphere-derived cells (CDC-EVs)
are known to be anti-inflammatory in various disease models. To further dissect the
mechanism, we examined the effects of CDC-EVs on T lymphocytes.
Methods: Naïve CD4 + T cells were isolated from secondary lymphoid organs of
Foxp3-RFP reporter mice, using magnetic-activated and fluorescence-activated cell
sorting.
Cells were subsequently polarized into effector subtypes (Th1, Th2, and Th17), as
well as
regulatory T cells (Tregs), and the effects of exposure to human-derived CDC-EVs on
proliferation and cytokine production were assessed. CDC-EVs were isolated from serum-free,
15-day conditioned medium, using ultrafiltration by centrifugation.
Results: After polarization and culture for 5 days, CDC-EVs resulted in
dose-dependent and cell-specific proliferative responses. Effector T cells (Th1, Th2,
Th17)
showed either no change in proliferation (Th1) or decrease in proliferation (Th2,
Th17),
compared to the vehicle control. In contrast, Tregs proliferated much more than control
(P < 0.0001). Next, we sought to characterize the changes in cytokine production by
each
effector T cell and Tregs. Compared to the vehicle control, exposure of polarized
effector T
cells to CDC-EVs had little effect on the expression of characteristic cytokine genes,
including Ifnγ and Tnfα (Th1), Il4 and Il13 (Th2), or Il17a and Il17 f (Th17). In
contrast,
exposure of Tregs to CDC-EVs resulted in ~1000-fold increase in expression of Il10,
a key
paracrine agent utilized by Tregs in suppression of inflammation. This response was
specific
to CDC-EVs insofar as it was not recapitulated with dermal fibroblast exosomes.
Concentrations of IL-10 in the culture media of CDC-EV-conditioned Tregs mirrored
the
increases in gene expression.
Summary/Conclusion: CDC-EVs potentiate Tregs by increasing their proliferation
and enhancing production of IL-10. This offers an attractive therapeutic approach
to
inflammatory diseases that relies on harnessing an endogenous mechanism of
immunosuppression.
Funding: NIH T32HL116273
PT15.04 = OP1.04
Prostanoids impair platelet reactivity, thrombus formation and
platelet extracellular vesicle release in patients with pulmonary arterial
hypertension
Aleksandra Gąseckaa
, Marta
Banaszkiewiczb, Rienk nieuwlandc, Edwin van der Pold,
Najat Hajjie, Hubert Mutwilf, Sylwester Rogulaa, Wiktoria
Rutkowskaa, Szymon Darochag, Grzegorz Opolskia, Krzysztof
J. Filipiakf, Adam Torbickig and Marcin Kurzynag
a1st Chair and Department of Cardiology, Medical University
of Warsaw, Warsaw, Poland; bDepartment of Pulmonary Circulation, Thromboembolic
Diseases and Cardiology, Centre of Postgraduate Education Medical, European Health
Centre
Otwock, Poland, Warszawa, Poland; cDepartment of Clinical Chemistry, Amsterdam
UMC, University of Amsterdam, Amsterdam, the Netherlands, Vesicle Observation Center,
Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands;
dDepartment of Clinical Chemistry, Amsterdam UMC, University of Amsterdam,
Amsterdam, the Netherlands; Vesicle Observation Center, Amsterdam UMC, University
of
Amsterdam, Amsterdam, the Netherlands; Department of Biomedical Engineering and Physics,
Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands;
eLaboratory of Experimental Clinical Chemistry, Vesicle Observation Center,
Amsterdam UMC, Locatie AMC, University of Amsterdam, Amsterdam, Netherlands; f1st
Chair and Department of Cardiology, Medical University of Warsaw, Warszawa, Poland;
gDepartment of Pulmonary Circulation, Thromboembolic Diseases and Cardiology,
Centre of Postgraduate Education Medical, European Health Centre Otwock, Poland, Warsaw,
Poland
Introduction: Prostanoids (epoprostenol, treprostinil and iloprost) induce
vasodilation in advanced pulmonary arterial hypertension (PAH) but also inhibit platelet
activation, thereby increasing the risk of bleeding. Therefore, the platelet function
and
extracellular vesicle (EV) concentrations were measured in PAH patients treated with
prostanoids and compared to patients with PAH not receiving prostanoids.
Methods: Venous blood was collected from 42 patients treated with prostanoids
(study group; n = 42, 50 ± 16 years, 70% female) and 38 patients not treated with
prostanoids (control group; n = 38, 55 ± 19 years, 65% female). Platelet reactivity
was
analysed in whole blood by impedance aggregometry using arachidonic acid (AA; 0.5 mM),
adenosine diphosphate (ADP; 6.5 µM) and thrombin receptor-activating peptide (TRAP;
32 µM)
as agonists. In a subset of patients, concentrations of EVs from platelets (CD61+ and
CD62p+; PEVs), leukocytes (CD45+, LEVs) and endothelial cells (CD146+, EEVs) were
measured
in platelet-depleted plasma by flow cytometry (A60-Micro). Platelet-rich thrombus
formation
was measured using a whole blood perfusion system.
Results: Compared to the control group, patients treated with prostanoids had
lower platelet reactivity in response to AA and ADP (p = 0.01) and lower concentrations
of
PEVs and LEVs (p ≤ 0.05). Furthermore, thrombus formation was delayed (p ≤ 0.003)
and
thrombus size was decreased (p = 0.008) on prostanoids. Epoprostenol did not affect
platelet
reactivity in vitro, but decreased the concentrations of CD61+ PEVs (p = 0.04). In
contrast,
treprostinil and iloprost decreased both platelet reactivity in response to AA and
ADP
(p ≤ 0.05) and the concentrations of PEVs (p ≤ 0.08). All prostanoids delayed thrombus
formation and decreased thrombus size (p ≤ 0.04).
Summary/Conclusion: Patients with PAH treated with prostanoids have increased
risk of bleeding both due to impaired platelet aggregation, EV release and thrombus
formation, compared to patients not treated with prostanoids. Antiplatelet effect
of
prostanoids varies: whereas epoprostenol decreases the release of PEVs, treprostinil
and
iloprost impair platelet aggregation.
Funding: AG is supported by the National Science Centre, research project
PRELUDIUM 2018/31/N/NZ7/02260.
EvdP is supported by the Netherlands Organisation for Scientific Research – Domain
Applied
and Engineering Sciences (NWO-TTW), research programmes VENI 15924.
PT15.05 = OP1.05
Nanoflow cytometry identifies an imbalance of epithelium- and
neutrophil-derived extracellular vesicles in the airway environment of paediatric
cystic
fibrosis patients
Brian Dobosh, Vincent Giacalone, Milton Brown,
Lucas Silva, Lokesh Guglani and Rabindra Tirouvanziam
Emory University, Atlanta, USA
Introduction: Progressive lung disease is the leading cause of mortality in
cystic fibrosis (CF), a chronic condition characterized by recruitment of polymorphonuclear
neutrophils (PMNs) into the airways. Newly arrived PMNs are exposed to extracellular
vesicles (EVs) from the airway epithelium and PMNs recruited before them. In controlled
experiments, these EVs were necessary and sufficient to induce pathological changes
including reduced bacterial killing and immunosuppressive activities towards macrophages
and
T-cells. However, children with CF do not always show a high PMN presence in their
airways,
which suggests that the balance between PMN recruitment and the activity of other
cells is
still in flux in early stage disease.
Methods: We utilized spectral nanoflow cytometry to profile the single EV
content of the bronchoalveolar lavage fluid (BALF) from 17 CF children (<6 years of
age).
For nanoflow cytometry, EVs were stained with Di-8-ANEPPs, and with EpCAM, CD66b and
CD115
(to ascertain epithelial, PMN, and macrophage origins, respectively). Violet side
scatter
and/or fluorescence threshold triggering were used for EV detection.
Results: The ratio of neutrophil- to epithelial-derived EVs in CF BALF
correlated positively with the percentage of PMNs that are present in the airways
(p = 0.003, Spearman’s rho = 0.689). This ratio also correlated with the PRAGMA disease
score, which quantifies airway damage by chest computed tomography (p = 0.001,
rho = 0.857).
Summary/Conclusion: Using a method to quantify EVs from specific cell types in
vivo, we demonstrated that the ratio of PMN- and epithelial cell-derived EVs tracks
with
airway damage and neutrophil influx, suggesting a critical interplay between these
cells in
early CF disease. This EV-focused method can be applied to other diseases in which
sampling
cells is difficult. Future experiments will use CF BALF biobanks to strengthen data
presented here.
Funding: CF Foundation (TIROUV15A0), Emory Paediatrics Flow Core.
PT15.06 = OP1.06
The potential of crude extracellular vesicle microRNAs for the
diagnosis of community-acquired pneumonia and for the detection of pneumonia-related
sepsis as a severe secondary complication
Stefanie Hermanna, Florian Brandesb, Benedikt
Kirchnerc, Dominik Buschmanna, Melanie Borrmannb,
Matthias Kleind, Stefan Kotschotee, Michael Bonine, Marlene
Reithmairf, Gustav Schellingb and Michael
Pfafflc
aDivision of Animal Physiology and Immunology, School of Life
Sciences Weihenstephan, Technical University of Munich, Freising, Germany, Freising,
Germany; bDepartment of Anaesthesiology, University Hospital,
Ludwig-Maximilians-University Munich, Munich, Germany, Munich, Germany; cDivision
of Animal Physiology and Immunology, Technical University of Munich, Freising, Germany,
Freising, Germany; dDepartment of Neurology, University Hospital,
Ludwig-Maximilians-University Munich, Munich, Germany, Munich, Germany; eIMGM
Laboratories GmbH, Planegg, Germany, Planegg, Germany; fInstitute of Human
Genetics, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany,
Munich, Germany
Introduction: Circulating cell-free microRNAs (miRNAs), often associated to
extracellular vesicles (EVs), are essential for cell-cell communication in the pathogenesis
of infectious pulmonary disorders. As early pneumonia diagnosis is often clinically
challenging, advances in disease detection could improve outcomes. We characterized
crude EV
miRNAs as potential biomarkers for community-acquired pneumonia and sepsis as a severe
secondary complication.
Methods: 142 individuals were enrolled into our study, subdivided into a
training (volunteer n = 27, pneumonia n = 12, sepsis n = 28) and testing cohort (volunteer
n = 20, pneumonia n = 18, sepsis n = 37). After precipitating crude EVs from sera
(miRCURY
Exosome Isolation Kit-Serum and Plasma) and extracting total RNA, small RNA sequencing
was
performed. miRNAs were selected as biomarker candidates by differential gene expression
analysis (DESeq2) and sparse partial-least-squares discriminant analysis (mixOmics).
Technical and biological validation was performed by reverse transcription quantitative
real-time PCR. Group classification was predicted by partial-least-squares discriminant
analysis. Gene targets and causal networks were identified by ingenuity pathway
analysis.
Results: Differential gene expression analysis revealed 29 significantly
regulated miRNAs in pneumonia compared to volunteers, and 25 miRNAs in pneumonia related
to
sepsis. Based on sparse-partial least discriminant analysis, group separation was
achieved
by 12 miRNAs as discriminators with high sensitivity and specificity (area under the
curve
of the receiver operated curve: volunteer: 0.982, pneumonia: 0.965, sepsis: 0.992).
miR-193a-5p (log2FC = 1.86, padj = 1.49E-6) and miR-542-3p (log2FC = 1.67, padj = 3.29E-5)
differentiated between pneumonia and volunteers and miR-1246 (log2FC = −2.41,
padj = 1.78E-04) between pneumonia and sepsis. Expression levels of miR-193a-5p and
miR-1246
were related to disease severity. miR-542-3p was higher expressed in pneumonia compared
to
volunteers and had equal expression in patient groups. Prediction of group classification
in
the testing cohort was 73.33%. Signalling networks were constructed for “cellular
and
humoral immune response”, “antimicrobial response” and “pathogen influenced signaling”
involving the significantly regulated miRNAs.
Summary/Conclusion: Crude EV miRNAs are potentially novel biomarkers for
community-acquired pneumonia and may help to identify patients at risk for progress
to
sepsis allowing early intervention and treatment.
Funding: This work was supported by a grant from the German Federal Ministry
for Economic Affairs and Energy (protocol number ZF4247001MD6).
PT15.07 = OP1.07
microRNA exosome cargo from induced sputum: new tool for
approaching asthma research
David Sanz Rubioa
, Ana Rosa
Remachab, Laura Pastorb, Elisabeth Verab, Pablo
Cuberob, Jose María Marinc, Victoria Gilb and Marta
Fornerd
aIIS Aragón, HCU Miguel Servet, Zaragoza, Spain;
bIIS Aragón, HCU Miguel Servet, Zaragoza, Spain; cIIS Aragón, HCU
Miguel Servet, University of Zaragoza, Zaragoza, Spain; dCIBERES, HCU Miguel
Servet, Zaragoza, Spain
Introduction: It remains unclear the specific mechanisms that lead to airways
inflammation in asthma and the subsequent remodelling of the airways. Exosomes, small
extracellular vesicles, has become in an important mechanism of cell-to-cell communication
and participate in diverse biological processes including inflammation. In this study,
we
hypothesize that exosomes and their miRNA cargo play an important role in the
proinflammatory status of the upper airway of asthma patients, especially in those
patients
with severe asthma.
Methods: In a pilot study,3healthy subjects had induced sputum using standard
methods. After several centrifugation steps, we were able to isolate exosomes from
sputum
supernatant by both precipitation and Size Exclusion Cromatography (SEC). Exosome
size was
observed with Transmission Electron Microscopy (TEM) and the protein markers CD63
and CD81
were analysed by Western Blot (WB). Then, total RNAs were isolated from sputum exosomes
and
9 miRNAs (miR-103a-p, miR-191-5p, miR-320a, miR-200b-3p, miR-185-5p, miR-223-3p, miR-21-5p,
miR-155-5p, let-7 g-5p), were evaluated by RT-qPCR. After the optimization of the
methodology, 10 healthy adults subjects and 10 patients with persistent moderate-severe
asthma, matched by age and sex were selected and induced sputum was collected.
Results: Exosomes isolated with both methodologies (precipitation and SEC)
were observe under the TEM with a correct range of size. Furthermore, WB assay displayed
a
coherent protein profile for the exosome markers CD63 and CD81. However, SEC displayed
low
signal and the variability of between subjects was to higher. Using the optimized
method of
precipitation, we observed that after normalization, miRNA-320a showed a significant
increased (p = 0.02) in asthma patients compared to control. This miRNA has been linked
with
an active proinflammatory status.
Summary/Conclusion: Our results confirm the presence of exosomes in induced
sputum with promising applications in the field of asthma. The upregulation of exosomal
miR-320a, which is related with inflammation, suggest that exosomes could play a crucial
role in the chronic inflammation of airway described in asthma patients.
PT15.08 = OP1.08
Human nrf2-active multipotent stromal cell exosomes reverse
pathologic delay in the healing of cutaneous diabetic wounds
Joseph Kuhna
, Absara
Hassanb, Sonali Sharmab, Jennifer Kwongb, Montaha
Rahmanb, salma Adamb, Jasmine Leeb, Alvaro Villarreal
Ponceb and Piul Rabbanib
aNYU Langone Health, New York, USA; bNYU Langone
Health, New York, USA
Introduction: Multipotent stromal cells (MSCs) have attracted much attention
for their capacity to accelerate wound healing. Exosomes, nanosized extracellular
vesicles,
may be key to translating MSC therapy. We previously found that nuclear factor erythroid
2-related factor 2 (Nrf2) regulates MSC promotion of diabetic tissue repair. Here,
we
explore a novel role of Nrf2 in exosome biogenesis and investigate whether exosome
treatment
recapitulates the effects MSCs have on healing.
Methods: Exosomes were harvested by differential ultracentrifugation of
conditioned bone marrow derived MSC media. For Nrf2-active exosomes, MSCs were incubated
with potent Nrf2 activator, CDDO-Im. Exosomes and MSCs were vigorously characterized.
Full-thickness humanized-stented wounds were created on adult Leprdb/db diabetic mice
(db/db). Exosomes were injected intradermally and circumferentially to the wound margin.
Results: MSCs adopt an adherent fibroblast morphology, demonstrate robust
osteogenic, chondrogenic, and adipogenic differentiation, express >95% positive MSC
markers (CD44, CD73, CD90, and CD105) and <5% express negative markers (CD45, CD31,
CD14,
CD19, or HLA-DR). Immunoblotting of MSC exosomes shows enrichment for positive exosomal
markers CD81, CD9 and TSG101. Nanoparticle tracking analysis (NTA) shows a nanoparticle
population with mean diameter of 168.0 ± 6.5 nm. Transmission electron microscopy
of
exosomes reveals flattened cup-like spheres. NTA demonstrates that Nrf2-active human
MSCs
increase exosome secretion by 54%, compared to Nrf2-baseline MSCs (p < 0.05). Both
Nrf2-baseline and Nrf2-active exosome treatment significantly reduced closure time
to 15.5
and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p < 0.05).
This reduction eliminated the delay in closure time compared to wounds of C57/B6 mice.
Nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days
compared to untreated C57/B6 wounds. At day 10, exosome-treated db/db wounds have
significant decreases in epithelial gap, expanded granulation tissue, and greater
density of
CD31+ vessels compared to vehicle-treated wounds.
Summary/Conclusion: Enhancing Nrf2 function in MSCs multiplies exosome yield.
Our results demonstrate exosome-based therapies hold tremendous promise and warrant
further
investigation for rapid translation.
Funding: PSF Pilot translational grant, WHS 3 M Fellowship, NYU CTSI
Translational Pilot Project
PT15.09 = OP1.09
Extracellular vesicles from adipose tissue end endothelial cells
of obese humans share miRNA cargos and increase prostate cancer aggressiveness in
conjunction with Twist1
Allison M. Mathiesen, Ryan Huyck, Bronson
Haynes, William McPheat and Anca D. Dobrian
Eastern Virginia Medical School, Norfolk, USA
Introduction: Obesity increases prostate cancer aggressiveness and adipose
tissue (AT) is a rich source of extracellular vesicles (EV) that have been shown to
contribute to pro-oncogenic effects in various malignancies. Twist1 is a key mediator
of
tumour cell metastasis. The goal of this study was to determine molecular and phenotypic
changes of prostate cancer cells in response to EVs from obese human AT and the role
of
different levels of endogenous Twist1.
Methods: EV were harvested from human AT (ATEV) obtained from bariatric
subjects or from AT endothelial cells treated with proinflammatory cytokines (PIC-EV)
to
mimic the obese AT environment. EVs were isolated by ultracentrifugation and characterized
by electron microscopy, NTA and protein markers. We determined the effect of ATEV
and PIC-EV
on PC3-ML prostate cancer cells proliferation and invasion. EV miRNA cargo and transcriptome
of PC3-ML cells treated with ATEV or PIC-EV were assessed using NanoString. To establish
the
contribution of Twist to the EV-related phenotypic and molecular changes in recipient
cells,
we used PC3-ML lines stably overexpressing or deficient in Twist1.
Results: ATEV from obese subjects and EV-PIC from AT endothelial cells both
reduced invasion and increased proliferation in wild-type PC3-ML cells. A molecular
signature showing decreased expression of genes mediating invasion, adhesion and metabolism
supported these functional effects. Also ATEV and EV-PIC shared a subset of miRNA
that
target multiple MMPs, inhibit glycolytic genes and target cell cycle inhibitory genes.
PC3-ML overexpressing Twist1 showed an increase in both proliferation and invasiveness
and
this phenotype was supported by the transcriptomic analysis following EV treatment.
Summary/Conclusion: EV produced by obese AT or by AT endothelial cells share a
subset of miRNA that in conjunction with increased Twist1 expression contribute to
tumorigenesis and metastasis of prostate cancer cells in vitro.
Funding: American Heart Association
PF01: EVS in Metabolism and Metabolic Disease
Chair: Michael Freeman – Cedars-Sinai Medical Centre
PF01.01
Release of larger extracellular vesicles post-acute exercise is
associated with the exercise responder phenotype in youth living with obesity
Taiana Magalhães Pierdoná Martinsa
,
Alexandria Martinb, Patience Obia, Benjamin Bydaka, Ashley
Eadiec, Keith Bruntc, Jonathan McGavockd, Martin
Sénéchale and Ayesha Saleema
aFaculty of Kinesiology and Recreation Management, University
of Manitoba, Winnipeg, Canada; bChildren’s Hospital Research Institute of
Manitoba (CHRIM), Winnipeg, Canada; cFaculty of Medicine, Department of
Pharmacology, Dalhousie University, Saint John, Canada; dDepartment of
Paediatrics and Child Health, Max Rady College of Medicine, University of Manitoba,
Winnipeg, Canada; eFaculty of Kinesiology, University of New Brunswick,
Fredericton, Canada
Introduction: Exercise is associated with various health benefits, including
the prevention and management of obesity and cardiometabolic risk factors. However,
a strong
heterogeneity in the adaptive response to exercise training exists. Differential response
to
exercise training might be mediated by myokines (proteins, nucleic acids, metabolites)
that
can be released directly into the systemic circulation, or packaged within extracellular
vesicles (EVs). The objective of this study was to evaluate if changes in EVs after
acute
aerobic exercise (AE) were associated with the responders phenotype following 6-week
resistance exercise (RE) training.
Methods: This is a secondary analysis of plasma samples from the EXIT trial
(clinical trial #02204670). Eleven sedentary obese youth (15.7 ± 0.5 years, BMI ≥
95th
percentile underwent an acute bout of AE (60% heart rate reserve, 45 min). Blood was
collected before [time (AT) −15, 0 min], during [AT15, 30, 45 min], and after [15 min
(AT60), 75 min (AT120)] exercise. Afterwards, youth participated in 6-week RE programme,
and
were categorized into responders or non-responders (NR) based on changes in insulin
sensitivity (above or below 50 percentile). Primary outcome: EVs were isolated using
size
exclusion chromatography (Izon®) at baseline (AT0), immediately after AE (AT45) and
after
recovery (AT120). EV protein concentration, size, and zeta potential were analysed
in a
single-blind fashion.
Results: Responders had larger EVs (~141.1 nm) as opposed to NRs (~97.3 nm) at
AT0 (p < 0.05) and this pattern was maintained at AT45 and AT120, though not significant
(p = 0.1). NRs displayed differential EV size distribution (peaks at 100 nm or 300 nm),
while EV distribution was highest at 250 nm in responders. No difference in average
zeta
potential or total EV protein yield was observed between groups. An increase in EV
yield
with exercise time and recovery was observed in both groups.
Summary/Conclusion: Our preliminary data suggest that EV size is significantly
increased after an acute bout of AE in obese youth responders. Further research to
delineate
the role of EVs as predictors of exercise adaptation is warranted.
Funding: Funded by DREAM and Research Manitoba.
PF01.02
Using dual-fluorescent reporter mice to track tissue-specific
extracellular vesicles
Andrea Estrada, Gabriella Hehn, Zackary
Valenti, Christopher Allen, Nicole Kruh-Garcia and Dan S. Lark
Colorado State University, Fort Collins, USA
Introduction: Extracellular vesicles (EVs) from tissues like skeletal muscle
(SkM) and adipose tissue (AT) have been implicated in human disease but are understudied.
SkM is likely a major player in EV biology as it accounts for ~35% of total body mass.
Tools
to define cellular EV origin are needed because tissues like SkM are comprised of
a variety
of cell types. Here, we describe our ongoing efforts using the dual fluorescent mG/mT
mouse
as a tool to analyse SkM-myocyte derived EVs.
Methods: Wild-type (WT) and mG/mT mice were used for these studies. mG/mT
mouse cells express membrane-tagged red (mT) or green (mG) fluorescent protein in
the
absence or presence of Cre, respectively. We made SkM myocyte mG expressing mice using
a
mouse expressing Cre on the human skeletal actin promoter. Blood was collected via
cardiac
puncture and platelet-free plasma was obtained via centrifugation. Plasma EVs were
isolated
using Exoquick, Exoquick-TC or size exclusion chromatography. SkM and AT were dissected
into
~5mm chunks, placed in serum-free DMEM and incubated for 24 hours. Tissue-derived
EVs were
isolated using Exoquick-TC. EV abundance was determined with a Horiba ViewSizer. Individual
EVs were analysed with a Cytek Aurora spectral flow cytometer. Settings were optimized
using
polystyrene beads and spectral unmixing was performed to allow detection of mG and
mT.
Results: In WT mice, SkM releases >100 times more EVs than adipose tissue
per unit of mass (p < 0.01 using paired Student’s t-test). Since SkM is also a major
component of total body mass, these data further emphasize the importance of SkM-derived
EVs. SkM-derived EVs from WT mice were not fluorescent (<0.1% of events). EVs from
mG/mT
mouse SkM overwhelmingly expressed mG (>95% of events) with negligible (< 1%)
expression of mT. AT-derived EVs robustly expressed mT but lacked mG.
Summary/Conclusion: These data provide “proof-of-principle” that mG and mT are
readily incorporated into EVs secreted ex vivo. Surprisingly however, plasma EVs from
mG/mT
mice expressed very little mG (~3%) or mT (~ 4%). This observation was confirmed with
three
separate isolation techniques. We are currently exploring possibilities to explain
this
finding, including: 1) modification of EVs post-secretion, 2) clearance of fluorescent
EVs
by the liver or 3) that EVs secreted from tissues remain predominantly in the interstitial
space.
Funding: This work was supported by an Innovative Project Award from the
American Heart Association (18IPA34110052) to DSL.
PF01.03
Endothelial CD36 delivery of FA loaded extracellular vesicles is
critical for thermogenesis.
Terri Pietkaa
, Vivek
Pecheb, Kathryn Pietkab, Curtis Wallsc and Nada
Abumradb
aWashington University in St. Louis, St Louis, USA;
bWashington University in St. Louis, St. Louis, USA; cWashington
University in St. Louis, St. Louis, USA
Introduction: Membrane CD36 facilitates tissue fatty acid (FA) uptake. We
recently found that endothelial cell (EC) CD36 controls muscle and adipose tissue
FA uptake,
and influences the tissue’s metabolic phenotype. The mechanism for CD36-facilitated
FA
uptake is unknown. Here we examined the role of EC CD36 in thermogenesis and in FA
delivery
to brown fat tissue.
Methods: Adult male mice were housed individually, restricted from food during
acute (4 hr) cold exposure (4°C) with core temperature monitored every 30 minutes.
After
4 hours, animals were sacrificed and samples collected for analysis. For cellular
studies,
human microvascular (Lonza) or primary murine microvascular EC were used. For primary
cells,
crude cell pellets from lung homogenates were purified using mouse-CD31 magnetic beads
(Miltenyi). For microscopy studies, alkyne FA (Cayman) was added to cells and to enable
visualization of internalized FAs, click chemistry (Invitrogen) used to label alkyne-FA
with
Alexa 568. For radioactive studies, primary lung EC were serum starved for 5 hrs and
incubated overnight with 3H-oleic acid bound to FA-free BSA (2:1 ratio). Media was
collected, clarified by centrifugation to remove microvesicles and debris. Small
extracellular vesicles (sEVs) were isolated from clarified media using Total Exosome
Isolation reagent (Invitrogen) and counted for radioactivity.
Results: Basal core body temperatures are similar in mice lacking EC CD36
(ecCD36-/-) compared to controls (CD36fl/fl). However, during cold exposure at 4°C,
ecCD36-/- are unable to maintain body temperature (p < 0.001). Plasma free FA are
higher
in cold exposed ecCD36-/- indicating FA clearance by brown fat is impaired. Mitochondrial
function and expression of thermogenic and mitochondrial genes in brown fat from ecCD36-/-
and CD36fl/fl mice were similar. These data suggested that endothelial delivery of
FAs is
necessary for thermogenic maintenance of body temperature.
To examine FA handling by ECs we used alkyne FAs to visualize the process. We found
that
FAs are transferred by microvascular EC through caveolae-mediated transcytosis involving
Src
signalling and Cav-1 phosphorylation. The internalized Cav-1 and CD36 positive vesicles
containing FAs are released as sEVs. To determine the dependence of CD36 on this process,
we
treated primary microvascular EC with radiolabeled FA and found that sEVs secreted
by
CD36-/- cells contain less labelled-FA (p = 0.05).
Summary/Conclusion: Endothelial delivery of FA is critical for thermogenesis.
Our working model for the mechanism of FA uptake by brown adipose tissue is the following:
Endothelial cells transfer the FA through caveolae-mediated transcytosis and secrete
small
extracellular vesicles (sEVs) that help deliver FAs to brown adipocytes.
Funding: This work is supported by NIH grants DK111175 and DK056341.
PF01.04
Nanovesicles from orange juice restore the intestinal functions
altered during diet-induced obesity in mice.
Berger Emmanuellea, Colosetti Pascala, Audrey
Jalaberta, Emmanuelle Meugniera, Oscar P. O. Wiklanderb,
Juliette Jouhetc, Elizabeth Errazurig-Cerdad, Stephanie
Chanona, Armelle Penohata, Dhanu Guptae, Alain
Geloena, Baptiste Panthud, Samir El-Andaloussif, Jennifer
Rieussetg and Sophie Rome
h
aCarMeN Laboratory, Lyon, France; bKarolinska
Institut, Stockholms, Sweden; cCEA-CNRS, Lyon, France; dUniversity of
Lyon, Lyon, France; eDepartment of Laboratory Medicine, Karolinska Institutet,
Sweden, Huddinge, Sweden; fDepartment of Laboratory Medicine, Clinical Research
Centre, Karolinska Institutet, Huddinge, Sweden; gCarMeN laboratory, Lyon,
France; hINRAE, department of Human Nutrition, Lyon, France
Introduction: Diet-induced obesity modifies intestinal permeability leading to
bacteria infiltration and to a decrease in the number of immune cells protecting mucosa.
As
orange consumption is beneficial for human health and used in preventive medicine,
we
determined whether orange juice-derived nanovesicles (ONV) might be recommended as
nutritional strategies for the treatment of intestinal complications associated with
obesity.
Methods: ONV isolated from fresh orange juices were characterized by
lipidomic, metabolomic, microscopy, NTA and for their stability during digestion.
Intestinal
barrier (IB = Caco-2 cells+HT-29 cells differentiated with oleic acid) were treated
with ONV
and co-cultured with adipocytes to monitor IB fat absorption and release. Obesity
was
induced in mice fed for 12 weeks with a high-fat high-sucrose diet (HFHS mice vs standart
chow diet mice). Then half of the HFHS mice were gavaged with 150 micrograms/day for
4 weeks.
Results: ONV did not modify high-fat high-sucrose diet-induced obesity and
insulin resistance but reversed diet-induced gut modifications. Six hours post-gavage,
ONV
accumulated preferentially in jejunum involved in lipid absorption. In jejunum, and
no other
intestinal region, ONV increased villi size, restored immune response and decreased
barrier
permeability in HFHSD mice. In addition, ONV-treated mice had increased expressions
of
ACAT2, ANGPTL4 and DGAT1, but a decreased expression of FABP2, FATP4, MTP vs HFHSD
animals,
which indicated that fat absorption, TG synthesis and chylomicron release were strongly
reduced. Similarly to other plant-derived nanovesicles, these results were likely
associated
with ONV lipid and metabolite compositions (strong enrichment in bioactive phospholipids:
PE, PA, PC, PI and leucine) as ONV did not resist to harsh digestive conditions in
vitro and
were poorly incorporated in enterocytes. As the effects of ONV on the decrease in
TG content
and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate
to these effects.
Summary/Conclusion: ONV are important bioactive compounds of orange juice and
for the first time we demonstrated that they can modulate lipid metabolism in the
intestinal
barrier associated with morphological changes. Interestingly ONV treatment targets
MTP and
ANGPTL4 mRNAs, 2 therapeutic intestinal targets to reduce plasma lipids and for attenuating
inflammation in gastrointestinal diseases. Therefore, ONV might be used to reduce
the
development of dyslipidemia-associated diseases and to restore intestinal functions
in obese
patients.
Funding: Olga Triballat Institut; Benjamin Delessert Institut, INRAE
Institut.
PF01.05
Association, structure, and function of fibronectin in
extracellular vesicles from hepatocytes
Xinlei Li, Ruju Chen, Sherri Kemper and David
Brigstock
Nationwide Children’s Hospital, Columbus, USA
Introduction: We have shown that extracellular vesicles from normal
hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic
stellate cells (HSCs, the principal fibrosis-causing cell in the liver) and hepatocytes.
In
this study, our goal was to determine the molecular nature of the EV components involved
in
cell binding. Fibronectin (FN1) is a key component of extracellular matrix, functioning
in
processes including cell adhesion, differentiation, and wound healing. Two types of
FN1 are
present in vertebrates, of which the soluble plasma FN1 is derived principally from
hepatocytes, while cell-associated FN1 is produced by numerous cell types. Here we
describe
a novel function of plasma FN1 in facilitating binding of hepatocyte EVs to target
cells.
Methods: Differential ultracentrifugation was used to collect EVs released by
parental mouse AML12 hepatocytes, FN1 KO AML12 cells in which FN1 was ablated using
CRISPR-cas9, primary human or mouse hepatocytes, or human HepG2 cells, or from human
or
mouse serum. EVs were characterized by nanosight tracking analysis (NTA), Western
blot,
iodixanol gradient ultracentrifugation, and mass spectrometry. The binding efficiency
of
PKH26-labelled EVs from parental (EV-Hep) or FN1 KO (EV-HepFN1 KO) AML12 cells was
analysed
in hepatocytes or HSCs. Swiss Webster mice were injected with CCl4 for five weeks
to induce
liver fibrosis, with some mice also receiving i.p. administration of EV-Hep or EV-HepFN1
KO
over the last two weeks, followed by determination of hepatic fibrogenic genes by
qRT-PCR.
Results: EV-Hep or EV-HepFN1 KO were 50–200 nm in diameter and positive for
common EV markers (CD63, CD9, flotillin-1). Mass spectrometry showed that FN1 was
the most
abundant protein in EV-Hep and comprised principally the plasma form. The abundant
presence
of EV FN1 was verified by Western blot and co-immunoprecipitation with anti-CD9 or
anti-flotillin-1. Western blot showed that FN1 was also abundant in EVs from primary
human
or mouse hepatocytes, HepG2 cells, and human or mouse serum. FN1 and EV-Hep co-sedimented
at
a density of ~1.15 g/ml. EV-HepFN1 KO yield and size-range were similar to those of
EV-Hep,
suggesting that EV biogenesis is FN1-independent. As compared to EV-Hep, the binding
of
EV-HepFN1 KO to target cells was highly reduced whereas EV binding was independent
of FN1
expression by the target cells themselves. Both EV-HepFN1 KO and EV-Hep were anti-fibrogenic
in vivo but only EV-Hep attenuated collagen 1⍺1 expression in mouse HSCs in vitro.
Summary/Conclusion: FN1 is abundantly associated with hepatocyte EVs and
facilitates EV binding to target hepatocytes or HSCs. Additional studies are needed
to
clarify the functional role of FN1 in mediating EV-Hep anti-fibrogenic actions in
vitro or
in vivo.
PF01.06
Elevated glucose increases soluble and aggregated forms of human
islet amyloid polypeptide in islet-derived extracellular vesicles – Implications in
type 2
diabetes and islet transplantation
Olia Katchanovskia, Georges Graub, Elham
Hosseini-Beheshtic and Lucy Marzban
d
aCollege of Pharmacy, University of Manitoba, Winnipeg,
Canada; bVascular Immunology Unit, Dept. of Pathology, Faculty of Medicine and
Health, University of Sydney, Sydney, Australia; cVascular Immunology Unit,
Faculty of Medicine & Health, University of Sydney, Camperdown, NSW, Australia 2050,
Camperdown, Australia; dUniversity of Manitoba, Canada, Winnipeg, Canada
Introduction: Type 2 diabetes (T2D) is characterized by reduced beta cell mass
and function. Islet amyloid, formed by aggregation of human islet amyloid polypeptide
(hIAPP), contributes to progressive beta cell loss in T2D. Amyloid also forms in human
islets during pre-transplant culture and following transplantation in patients with
type 1
diabetes (T1D) which is associated with graft failure. The cellular mechanisms underlying
islet amyloid formation are still unclear. In this study, we examined the potential
role of
islet-derived extracellular vesicles (EV) in the clearance of soluble and aggregated
(pro)IAPP species from beta cells and amyloid formation.
Methods: Human islets isolated from cadaveric pancreatic donors (n = 4 donors)
and wild-type or hIAPP-expressing (hIAPP+) transgenic mouse islets (n = 4/group) were
cultured in normal (5.5 mM) or elevated (11.1 mM) glucose to form amyloid. EV (exosomes)
were isolated from culture medium using classical centrifugation and ultracentrifugation.
Purified EV were analysed by nanoparticle tracking analysis. Western blot analysis
and
double immunogold transmission electron microscopy were performed to verify the presence
of
EV markers as well as (pro)hIAPP species and oligomers (aggregates).
Results: Human islets formed amyloid during culture with elevated glucose
which was associated with progressive beta cell apoptosis. (Pro)IAPP species were
detectable
in EV released from human islets cultured in normal and elevated glucose. The latter
markedly increased (pro)IAPP content in islet-derived EV. Interestingly, hIAPP aggregates
(oligomers) were present in the majority of EV released from human islets cultured
in
elevated glucose but were not detectable in islets cultured with normal glucose. Similarly,
EV released from hIAPP+ mouse islets which formed amyloid during culture had higher
(pro)IAPP content compared to wild-type islet-derived EV. Moreover, hIAPP oligomers
were
present in EV derived from hIAPP+ islets but not WT islets.
Summary/Conclusion: In summary, our data show that (pro)IAPP species are
present in islet-derived EV and that elevated glucose increases (pro)hIAPP and its
aggregates in EV released from islets. Islet-derived EV may play a key role in the
process
of amyloid formation in T2D and human islet grafts.
Funding: University of Manitoba Research Grants Program (URGP).
on.
PF01.07
Contraction, but not glycolysis, regulates the size of skeletal
muscle EVs secreted ex vivo.
Zackary Valenti, Andrea Estrada, Gabriella
Hehn and Dan S. Lark
Colorado State University, Fort Collins, USA
Introduction: Skeletal muscle (SkM) is a metabolically active tissue and
accounts for ~35% of total human body mass. Acute exercise increases secretion of
extracellular vesicles (EVs), but the mechanisms responsible are unknown. Muscle contraction
increases the demand for ATP which requires intercellular communication in order to
adapt.
We hypothesized that this “metabolic stress” during contraction increases SkM EV
secretion.
Methods: We tested our hypothesis using an ex vivo EV secretion assay. All
studies were approved by the Colorado State University Institutional Animal Care and
Use
Committee. Vastus medialis muscle (SkM) from male C57Bl/6 J mice (n = 6) or female
mT/mG
mice (n = 6) was cut into ~5 mg pieces and added to 12 well plates (~50 mg/well) filled
with
1 ml of serum-free DMEM and placed in a cell culture incubator at 37 C for 24 hours.
SkM
from male mice was treated with 2-deoxyglucose (2-DG) (0.1 nM – 100 mM) to induce
metabolic
stress via inhibition of glycolysis. SkM from female mice was treated with 10uM of
blebbistatin (BLEB), a contraction inhibitor. After incubation, SkM mass was measured
and
conditioned media was centrifuged (3,000 x g for 15 min) to remove cell debris. EVs
were
isolated using ExoQuick-TC. NTA was performed on isolated EVs using a Horiba Viewsizer
3000.
EV secretion was normalized to tissue mass and culture media volume then reported
as
([Particle]/mL/mg tissue). Statistical comparisons for 2-DG experiments were made
using a
repeated measures 1-way ANOVA. BLEB experiments were analysed using a paired Student’s
t-test.
Results: There was a trend towards greater EV abundance (p = 0.07) as a
function of 2-DG treatment, but no effect on EV diameter (p = 0.37). BLEB treatment
did not
alter EV abundance (p = 0.69), but significantly reduced EV mean diameter (p = 0.007;
11%
decrease; DMSO: 114.9 ± 5.1 vs. BLEB: 103.9 ± 4.2).
Summary/Conclusion: Contrary to our hypothesis, inhibition of glycolysis with
2-DG did not stimulate SkM EV secretion. However, BLEB did appear to promote the release
of
small EVs and/or inhibit secretion of larger EVs. Ongoing efforts are focused on testing
other metabolic stressors and defining how blebbistatin promotes small EV secretion.
Funding: American Heart Association Grant to DSL (IPA34110052).
PF02: EVs as delivery vehicles
Chair: Karen Bussard – Thomas Jefferson University
PF02.01
Cell-penetrating sC18 peptide-modified extracellular vesicles
for intracellular delivery
Kosuke Noguchia, Haruka Sumib, Tomoka
Takatani-Nakasec, Ines Neundorfd and Ikuhiko
Nakasea
aDepartment of Biological Science, Graduate School of
Science, Osaka Prefecture University, Sakai-shi, Japan; bCollege of Life,
Environment, and Advanced Sciences, Osaka Prefecture University, Sakai-shi, Japan;
cSchool of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University,
Nishinomiya, Japan; dDepartment of Chemistry, University of Cologne, Köln,
Germany
Introduction: Extracellular vesicles (EVs, exosomes) are nanovesicles
(30–200 nm) secreted from various types of cells. Because of vesicular encapsulation
of
miRNAs and enzymes, the EVs play crucial roles in cell-to-cell communication by delivering
these functional molecules to other cells [1]. On the other hand, the EVs are highly
expected as next generation therapeutic tools due to pharmaceutical advantages such
as
controlled immunogenicity, effective usage of cell-to-cell communication routes, artificial
modification and encapsulation of functional molecules. However, cellular targeting
and
uptake efficacy of the EVs are insufficient to be utilized as therapeutic tools [2,
3]. In
this study, we newly developed EVs decorated with cell-penetrating sC18 or (sC18)2
peptides,
which are derived from the C-terminal domain of the cationic antimicrobial protein,
CAP18,
because the peptides can be efficiently internalized by breast cancer cells. [4, 5].
Methods: All peptides were prepared by Fmoc-solid phase synthesis. Secreted
EVs from CD63-GFP stably expressing HeLa cells were isolated by ultracentrifugation.
Cellular uptake of EVs was analysed using a flow cytometer and a confocal laser microscope.
Encapsulation of saponin in the EV was conducted by electroporation.
Results: sC18 peptide is known as one of cell-penetrating peptides, and
branched structure of sC18 peptides, (sC18)2, further enhances the cellular uptake
[5]. In
this research, we examined the effects of the peptide modification on cellular EV
uptake,
and modification of the sC18 or (sC18)2 peptides on EV membranes was conducted via
stearyl
moiety. As our results, increased macropinocytotic cellular uptake by modification
of the
peptides was successfully attained. Especially, the modification of (sC18)2 peptides
showed
higher cellular uptake and macropinocytosis induction efficacy than that of sC18 peptides.
In addition, anticancer protein, saporin toxin-encapsulated EVs modified with the
(sC18)2
peptides significantly enhanced their biological activity with dependency of
glycosaminoglycan expression on targeted cells.
Summary/Conclusion: The cell-penetrating (sC18)2 peptide-modified EVs shows
high abilities to be effectively internalized by cells and are applicable for intracellular
delivery of therapeutic molecules. This study is expected to contribute to development
of
intracellular delivery techniques based on EVs.
[1] T. Katsuda, et al. Proteomics, 14, 412–425 (2014), [2] I. Nakase, K. Noguchi,
et al.
Sci. Rep. 6, 34937 (2016), [3] I. Nakase, et al. Chem. Commun. 53, 317–320, [4] J.
Hoyer, et
al. Beilstein J. Org. Chem, 8, 1788–1797 (2012), [5] A. Gronewold, et al. ChrmMedChem.
12,
42–49 (2017)
PF02.02
Natural or synthetic: a comparison of the efficiency of
extracellular vesicles vs synthetic carriers for RNA delivery
Daniel E. Murphya
, Maratussholikhah
Nurazizahb, Olivier G. de Jongc, Martijn J. W. Eversb,
Raymond M. Schiffelersc and Pieter Vaderd
aLaboratory of Clinical Chemistry and Haematology, UMC
Utrecht, Utrecht, Netherlands; bUMC Utrecht, Utrecht, Netherlands;
cLaboratory of Clinical Chemistry and Haematology, UMC Utrecht, The Netherlands,
Utrecht, Netherlands; dLaboratory of Clinical Chemistry and Haematology, UMC
Utrecht; Department of Experimental Cardiology, UMC Utrecht, The Netherlands, Utrecht,
Netherlands
Introduction: RNA therapeutics possess high potential which is yet to be
realised, largely due to difficulties involved in delivery to the cytoplasm of target
cells.
Extracellular vesicles (EVs) possess numerous features that may help overcome this
hurdle
and have emerged as a promising RNA delivery vehicle candidate. Despite extensive
research
into the engineering of EVs for RNA delivery, little is known about how their intrinsic
RNA
delivery efficiency compares to current synthetic RNA delivery systems. Using a novel
CRISPR/Cas9 based RNA transfer fluorescent stoplight reporter system, we here compared
the
delivery efficiency of EVs to state-of-the-art DLin‐MC3‐DMA lipid nanoparticles (LNPs).
Methods: EVs were isolated from MDA-MB-231 cells expressing either a targeting
or non-targeting control sgRNA and applied to HEK293 T stoplight+ reporter cells.
LNPs
containing targeting sgRNA were titrated onto HEK293 T stoplight+ reporter cells to
determine the minimum effective dose. LNP and EV particles were characterized using
nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis.
sgRNA
copy number was determined using RT-qPCR.
Results: EVs were 140 ± 7 nm in diameter as measured by DLS and possessed a
negative surface charge of −25.3 ± 3.3 mV. RT-qPCR and NTA analysis indicated that
sgRNA EV
loading was low, with only 1 in 3.8e05 ± 3.5e05 EVs containing a single sgRNA copy.
Nevertheless, EVs containing targeting sgRNA induced significant reporter activation
while
EVs containing non-targeting sgRNA did not. LNPs were 51 ± 0.6 nm in diameter and
possessed
a neutral charge. These particles also induced significant reporter activation when
loaded
with targeting sgRNA. When delivered via EVs, only between 4 to 54 sgRNA copies per
cell
were required to induce statistically significant reporter activation. In contrast,
the
minimal effective sgRNA dose when delivered by LNPs was considerably higher at approximately
9e03 copies per cell.
Summary/Conclusion: MDA-MB-231 EVs deliver RNA in a highly efficient manner
and are functional at sgRNA concentrations several orders of magnitude lower than
those
required for LNP mediated delivery. This underlines the potential of EVs as RNA delivery
vehicles and highlights the need to study the mechanisms by which EVs achieve their
efficiency in order for improved development of RNA therapeutics.
Funding: The work of D.E.M., M.N., M.J.W.E., R.M.S. and P.V. is supported by
the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement
No. 721058. O.G.d.J. is supported by is supported by a VENI Fellowship from the Netherlands
Organisation for Scientific Research (NOW) VI.Veni.192.174.
PF02.03
The role of circulating extracellular vesicles in patients with
chronic chagas disease
Rafael Pedro Madeiraa, Lavínia Maria Dal’Mas
Romerab, Paula de Cassia Buckc, Barbara Maria Iannic,
Fabio Fernandesc, Felix Ramiresc, Charles Madyc and
Ana Claudia Torrecilhasb
aUniversidade Federal de Sao Paulo, Sao Paulo, Brazil;
bUNIFESP, Sao Paulo, Brazil; cUSP, Sao Paulo, Brazil
Introduction: Chagas Disease is a neglected tropical disease (NTD) caused by
the flagellated protozoan Trypanosoma cruzi. It is a major public health problem in
Latin
America, and it is now expanding over the globe through immigration of infected individuals.
Eukaryotic cells release extracellular vesicles (EVs) that circulate in body fluids
and have
an important roles in intercellular communication, both in physiological and pathological
conditions. Objectives. Our study proposes to characterize and to compare the circulating
EVs isolated from plasma of the Chronic Chagas Disease (CCD) patients with healthy
individuals (controls).
Methods: Peripheral blood was collected from patients and controls in the
presence of EDTA and EVs enriched from plasma by differential ultracentrifugation.
The
obtained EVs were characterized and quantified by Nanoparticle Tracking Analysis (NTA)
and
added to human THP-1 cells. After 48 h, the cell supernatants were analysed by ELISA
for the
presence of cytokines.
Results: Lower amounts of EVs were obtained from CCD patients in comparison
with control individuals. However, the same amount of EVs of CCD were more capable
of
inducing cytokines such as IFN-gamma and IL-17 in relation to controls.
Summary/Conclusion: Although less EVs are present in the blood of CCD, these
EVs induce high inflammatory reactions on macrophages suggesting a possible role of
these
EVs in the establishment of chronic disease.
Funding: Supported by FAPESP, CNPq and CAPES.
PF02.04
Extracellular Vesicles – a Trojan horse for therapeutic agent
delivery
Koen Breynea
, Stefano
Ughettob, Lorena Parleac, Bruce Shapiroc and Xandra O.
Breakefieldd
aMGH-Harvard Medical School, Charlestown, USA;
bMGH-Harvard Medical School, charlestown, USA; cNational Cancer
Institute, NIH, Frederick, USA; dMGH-Harvard Medical School, Charlestown, USA
Introduction: Extracellular vesicles (EVs) may prove to be one of the optimal
payload carriers for therapeutic agents. While they travel through the extracellular
space,
the EV’s lipid membrane layer shields their luminal cargo from deleterious external
factors.
When autologous EVs are used to protect this therapeutic cargo, little immunogenic
effects
are expected compared to viral vectors and artificial structures, such as liposomes.
Their
usage is potentially manifold, and they are ubiquitously present in all body tissues
and
fluids. The key is to develop a manageable EV loading agent for adoptive transfer
therapies.
Methods: To exploit the unique properties of EVs, highly positively charged
proteins were used to load them with multiple biomolecules, such as a Cas9 protein
or Dicer
substrate dsRNA as a functional payload and to improve their apparently inadequate
natural
ability to deposit cargo into the cytoplasm of recipient cells.
Results: Highly positively charged proteins can associate with and/or diffuse
through a phospholipid bilayer (Thompson et al. 2012). When these kinds of charged
proteins
are mixed with isolated EVs in vitro, they are loaded into the EVs. The positive charge
of
the protein has the advantage that it can associate with negatively charged agents,
such as
RNA species, and aids the associated molecule to also incorporate into the EV. Moreover,
the
positive charge of the protein helps with cargo delivery, and thus overcoming the
bottleneck
of the EV’s cargo to escape the endosome post-uptake in a recipient cell. Self-quenching
fluorescent lipid dyes demonstrated that discharge of the highly positive EV cargo
into the
cytoplasm is concomitant with lipid mixing between the membrane of EVs and the membrane
of
the recipient cell. When eGFP-expressing microglia were exposed to EVs loaded with
a Dicer
substrate dsRNA able to silence eGFP via the positively charged protein, the uptake
of Dicer
substrate dsRNA was concomitant with a decrease in eGFP expression in the microglia.
A
similar result was achieved when EVs were loaded with Cas9 protein conjugated to the
highly
positively charged protein. Post-uptake of these Cas9-loaded EVs, microglia expressing
anti-eGFP sgRNA (single guide RNA) lead to decreased eGFP expression.
Summary/Conclusion: Our EV delivery technology has the capability of
delivering multiple biomolecules, such as protein and RNA cargo and demonstrates post-uptake
of the EV functionality of the EV delivered cargo in the recipient cell.
PF02.05
Hybrid extracellular vesicles – biomimetic tool for drug
delivery to repair endothelial cell dysfunction
Anna E. Drozdz
a, Magdalena
Surmanb, Małgorzata Przybyłoc and Ewa Stępieńd
aMarian Smoluchowski Institute of Physics, Faculty of
Physics, Astronomy and Applied Computer Science, Jagiellonian University, Kraków,
Poland;
bInstitute of Zoology and Biomedical Research of the Jagiellonian University,
Kraków, Poland; cDepartment of Glycoconjugate Biochemistry, Institute of Zoology
and Biomedical Research, Jagiellonian University, Kraków, Poland; dDepartment of
Medical Physics, M. Smoluchowski Institute of Physics, Faculty of Physics, Astronomy
and
Applied Computer Science, Jagiellonian University, Kraków, Poland, Kraków, Poland
Introduction: Traditional drug delivery systems (DDS) are usually based on
liposomes, micelles or dendrimers. Unfortunately, many DDS cause side effects including
organ toxicity and/or unexpected immune response. In living organisms, extracellular
vesicles (EVs) are responsible for delivering biologically active molecules to distant
cells. In vitro loading of therapeutic compounds into EVs is still not effective and
needs
developing new strategies. For these reasons we aimed to design hybrid extracellular
vesicles (hEVs) with high loading capacity for DDS.
Methods: For hEV synthesis, we used human endothelial derived EVs. Using
freeze/thawing method we fused them with liposomes composed of cholesterol and one
of the
three lipids: DOPC, sphingomyelin or phosphatidylserine. To confirm membrane fusion,
we
applied a spectroscopy ruler – FRET (Förster Resonance Energy Transfer) and CryoTEM
imaging
technique. We characterized hEVs using NTA (for size distribution evaluation), DLS
(zeta
potential) and Western blot (for detection of EVs markers). We evaluated loading efficiency
using calcein as a model drug. Additionally, we performed cytotoxicity tests.
Results: In the CryoTEM imaging, pure and homogenous hEV population with a
diameter of 65 ± 15 nm was detected. Additionally, we observed changes in zeta potential
and
in size distribution after fusion. FRET measurements showed increased fusion efficiency
with
the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein
ratio in
EVs. Additionally, hEV had higher loading efficiency than liposomes and sole EVs and
that
their internalization by endothelial cells did not cause a cytotoxic effect.
Summary/Conclusion: Based on cryo-TEM and FRET, we confirmed that our fusion
method of hybrid EVs is effective and can be applied as a delivery platform for DDS
to
endothelial cells.
Funding: We acknowledge financial support from National Science Centre Poland
(grant no. 2017/25/N/ST5/00831) and the Ministry of Science and Higher Education (grant
no.
2019-N17/MNS/000015).
PF03: EVs of Non-mammalian Organisms
Chair: Yong Song Gho – Department of Life Sciences, Pohang University of Science and
Technology
Chair: Franklin W.N. Chow – Postdoc, Institute of Immunology and Infection Research,
Centre for Immunity, Infection and Evolution, School of Biological Sciences, University
of
Edinburgh
PF03.01
Investigating the effects of oxidative stress on extracellular
vesicle function and cargo loading
Elizabeth Dellara
, Claire M.
Hillb, Jana Terenovab, Monika Gullerovab, Luis Alberto
Baena-Lopezb and David R. F. Carterc
aUniversity of Oxford/Oxford Brookes University, Oxford, UK;
bUniversity of Oxford, Oxford, UK; cOxford Brookes University,
Oxford, UK
Introduction: Extracellular vesicles (EVs) have been demonstrated to be
important mediators of intercellular communication in various contexts, including
in
response to a range of stressors. The functional activity of these EVs in recipient
cells
may, in part, be driven by changes to their biological cargoes. However, the molecular
details of the underlying EV biogenesis and loading processes, and how this may vary
in
different conditions, is poorly understood.
Methods: We first studied the effect of oxidative stress on the functional
activity of EVs in recipient cells using cell viability and mitochondrial membrane
potential
assays in Drosophila S2R+ cells. We then carried out total RNA sequencing of EV and
cellular
RNA under three stress conditions and compared results to existing data in mouse cells.
Further to this we have used a bioinformatic pipeline to identify sequence motifs
enriched
in EVs under stress.
Results: Functional assays indicated changes to cell viability and
mitochondrial membrane potential in recipient cells, which were donor cell-stress
dependent.
Subsequent characterisation of RNA showed an enrichment of ribosomal RNA in EVs relative
to
cells, but no significant changes to other biotypes. Comparative analysis has also
uncovered
a set of genes enriched in EVs under oxidative stress, and a further subset whose
enrichment
may be evolutionarily conserved in mouse. We also identified potential EV-loading
motifs
which may assist in RNA loading specifically under stress.
Summary/Conclusion: We have shown that EVs derived from oxidatively stressed
cells show dose-dependent differences in RNA cargo and identified potential sequence
motifs
that may have a role in its loading. We are now validating the biological significance
of
these findings by combining different in vivo approaches in Drosophila. This will
enable us
to gain insights into the basic mechanisms which govern EV loading in different contexts,
and ultimately the molecular mechanisms underlying EV-mediated intercellular
communication.
Funding: ED and CH are supported by funding from the Biotechnology and
Biological Sciences research Council (BBSRC) [grant number BB/M011224/1] as part of
the
Oxford Interdisciplinary Bioscience Doctoral Training Programme. DC is supported by
the
BBSRC (BB/P006205/1) and LAB-L by Cancer Research UK (C49979/A17516).
PF03.02
Mutant p53 governs tumour microenvironment dynamics via exosomes
and outer membrane vesicles
Ishai Luza, Bibek Bhattaa, Kanaga
Sabapathyb and Tomer Cooksc
aBen-Gurion University of the Negev, Beer-Sheva, Israel;
bNational Cancer Centre, Singapore, Singapore, Singapore;
cBen-Gurion University, Beer Sheva, Israel
Introduction: Mutations in TP53 are considered one of the most frequent
genetic alterations in human cancer. Besides the abrogation of the wild-type (WT)
p53-mediated tumour suppression, a distinct set of missense mutations was reported
to endow
mutant p53 proteins with novel activities termed gain-of-function (GOF). Even though
mutations in TP53 are typically thought to arise in the tumour cells rather than in
the
stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment
are poorly understood.
In the presented studies, focusing on colon cancer as well as on lung cancer microbiome,
we
investigated intercellular interactions mediated by exosomes and outer membrane vesicles
(OMVs) in the context of cancers harbouring mutant p53.
Methods: p
Results: In the colon, tumour cells harbouring mutp53 were found to exert a
non-cell-autonomous effect over macrophages. When exposed to tumour cells harbouring
mutp53,
monocytes became polarized towards a distinguished subset of macrophages characterized
by
TAMs-related markers. The mutant p53 affected TAM were characterized as TNF-αlow/IL-10 high,
over expressing CD-206 and CD163, with decreased phagocytic ability and increased
invasion
and matrix degradation potency. Investigating the exosomal transfer from mutp53 tumour
cells
to macrophages, revealed a mutp53-specific miRs signature led by miR-1246 promoting
the TAM
phenotype and creating an invasive front together with tumour cells. MiR-1246 was
also found
to be the top mutp53-associated miR in a cohort of 57 human colorectal resected tumours.
Separately, in two lung cancer cohorts, we identified a signature of microbiome members
associated with p53 mutations. Acidovorax Temperans, a Gram negative bacterium, was
found to
be abundant in tumours of patients with mutant p53. We found a significant increase
in
tumour volume in animals inoculated with Acidovorax temperans as compared to Sham
treated
animals, and increased lung weight as a percent of total body weight. These preliminary
data
indicate that Acidovorax temperans contributes to lung tumorigenesis in the presence
of
activated K-Ras and mutant p53. OMVs shed by Acidovorax temperans promoted inflammatory
signalling in lung carcinoma cells and elevated CD47 expression on tumour cells and
SIRPα
levels on macrophages.
Summary/Conclusion: Altogether, these findings are consistent with a
microenvironmental role for specific “hot-spot” GOF p53 mutants tightening the interaction
between the tumour cell and the immune compartment in colon cancer. In both colon
and lung
cancer, mutant p53 facilitates cellular interactions within the tumour microenvironmets
mediated by vesicles.
Funding: intramural funding from the National Cancer Institute, National
Institutes of Health.
PF03.03
S. oneidensis vesicle loading is driven by BAR domain protein
and outer membrane cytochrome interplay
Lori Zacharoff and Mohamed El-Naggar
University of Southern California, Los Angeles, USA
Introduction: The metal respiring bacterium S. oneidensis creates outer
membrane extensions and outer membrane vesicles that are sculpted by the novel BAR
domain
protein BdpA. These vesicles and extensions incorporate mutliheme cytochromes involved
in
extracellular electron transfer to metals and electrodes. However, the physiological
relevance of incorporating these cytochromes into the higher order 3D architecture
of a
vesicle or extension is unknown. Given that BAR domains serve as a protein sorting
mechanism
in Eukaryotes, we investigated the pathway crosstalk between BdpA and outer membrane
multiheme cytochromes as means to understand the physiological significance of membrane
architecture.
Methods: o this end, vesicle morphology and content was measured using dry
weights, dynamic light scattering, fluorescence microscopy and comparative proteomics
from
wild type S. oneidensis and deletion strains.
Results: Cells lacking BdpA make large amorphous vesicles that are dense with
protein. In contrast, a strain lacking outer membrane cytochromes recruits less total
protein into smaller vesicles. Proteomics to show that both BdpA and multiheme cytochromes
are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal
relationship.
Summary/Conclusion: in the absence of BdpA, protein crowding has to become the
main driving force of vesiculation and BdpA is essential for efficient incorporation
of
cytochromes. However, multiheme cytochromes are not only vesicular cargo, but are
also
important for shaping and loading vesicles. Both of these situations make it clear
that
vesicles play a role in increasing the respiratory surface area of S. oneidensis cells.
Moving forward, we hope to be able to control Bdpa and cytochrome levels for selective
recruitment of technologically relevant payloads.
PF03.04
Identification and evaluation of potential new biomarkers of
trematode infections in Extracellular Vesicles
Christian M. Sánchez-Lópeza, Mónica Uruburub,
Fernando Cantalapiedrac, María Trelisd, Dolores Bernale,
Ivan Dario Velezb, Carmen Fernández-Becerraf, Hernando A. del
Portillog and Antonio Marcilla
h
aÁrea de Parasitología, Dpt. Farmacia y Tecnología
Farmacéutica y Parasitología, F. Farmàcia, Universitat de València, Spain., Burjassot
(Valencia), Spain; bPrograma de Estudio y Control de Enfermedades Tropicales –
PECET, Universidad de Antioquia, Medellín, Colombia., Medellin, Colombia; cCentre
de Salud Publica de Manises, Manises (Valencia), Spain; dArea de Parasitología,
Dpt. Farmacia y Tecnología Farmacéutica y Parasitología, F. Farmàcia, Universitat
de
València, Spain., Burjassot (Valencia), Spain; eDept. Bioquimica y Biologia
Molecular, F. Ciències Biològiques, Universitat de València, Spain., Burjassot (Valencia),
Spain; fISGlobal, Hospital Clínic – Universitat de Barcelona, Spain., Badalona,
Spain; gISGlobal, Hospital Clínic – Universitat de Barcelona. Institute for
Health Sciences Trias I Pujol (IGTP), Badalona, Spain. Catalan Institution for Research
and
Advanced Studies (ICREA), Barcelona, Spain., Barcelone, Spain; hDepartament de
Farmàcia I Tecnologia Farmacéutica i Parasitologia, Universitat de Valéncia, Spain,
Burjassot (Valencia), Spain
Introduction: Fascioliasis caused by Fasciola hepatica represents a major
economic loss and clinical burden in cattle farming worldwide. Extracellular vesicles
(EVs)
contain pathogen-derived molecules that represent novel biomarkers of disease. In
the
present study, we have identified potential new biomarkers of F. hepatica infection
in EVs
present in sera of infected cattle.
Methods: Parasites and sera were obtained from local abattoirs (Valencia,
Spain, and Medellin, Colombia, respectively). 38 sera from infected and 32 from healthy
animals.
Parasites were cultured, and EVs obtained by size-exclusion chromatography (SEC) and
characterized by NTA, TEM and proteomic profiling.
Recombinant proteins from F. hepatica EVs (enolase and Fh16.5 tegumentary protein)
were
produced, and coupled to magnetic beads. Measurement of bovine IgG antibodies was
performed
using Luminex bead array technology.
Results: A total of 239 proteins were identified associated with EVs as shown
by the presence of typical EV-markers (Tsg101, Alix, CD63). Two parasite proteins,
enolase
and the Fh16.5 tegumentary protein were produced as recombinant proteins and used
for
detection of cattle IgG employing Luminex bead array technology. Interestingly, significant
differences were found in the fluorescence values of both recombinant proteins allowing
discrimination between sera from infected and non-infected cattle. The use of the
Fh16.5
protein generated a highly significant difference between the two groups (p
value = 0.003614); as did enolase (p value was 0.02294).
Summary/Conclusion: This study demonstrates the usefulness of EV proteins as
new biomarkers for early diagnosis of helminth infections using Multiplex assays,
a
technology that may also be applied to other parasite EV molecules.
Funding: Supported by the Conselleria d’Educació, Cultura i Esports,
Generalitat Valenciana, Valencia, Spain (PROMETEO/2016/156 to A.M.) and Spanish Ministry
of
Science and Innovation (RED2018‐102411‐T).
PF03.05
Life stage-specific glycosylation of schistosome-derived
extracellular vesicles
Marije E. Kuipers
a, Esther Nolte-’t
Hoenb, D. Linh Nguyena, Roman I. Koningc, Hermelijn H.
Smitsd and Cornelis H. Hokkea
aDept. of Parasitology, Leiden University Medical Centre,
Leiden, Netherlands; bDept. Biomolecular Health Sciences, Fac. Veterinary
Medicine, Utrecht University, Utrecht, Netherlands; cDept. of Cell & Chemical
Biology, Leiden University Medical Centre, Leiden, Netherlands; dDepartment of
Parasitology, Leiden University Medical Centre, Leiden, Netherlands
Introduction: Glycans play an essential role in pathogen-host interactions.
Larvae and adult worms from Schistosoma mansoni release distinct subsets of glycoconjugates
as excretory/secretory (ES) products. Extracellular vesicles (EVs) are also among
the ES
products. We recently found that schistosomula-derived EVs are glycosylated and bind
human
dendritic cells via C-type lectin receptor (CLR) DC-SIGN, leading to increased IL-10
and
IL-12 release. Here we investigated the glycosylation profile of EVs released by S.
mansoni
adult worms, compared this to schistosomula EVs, and addressed how this may affect
parasite-host interactions via CLRs.
Methods: EVs from cultured S. mansoni parasites were obtained by
ultracentrifugation and purified with iodixanol density gradients. Isolated EVs were
analysed by NTA and cryo EM. N-glycan and lipid glycan content was determined by mass
spectrometry. Density gradient fractions with EVs were loaded onto SDS-page gels followed
by
Western Blot (WB) analysis using anti-glycan monoclonal antibodies (mAbs).
Results: Cryo EM showed that adult worm EVs lacked the long thin filaments
that are characteristic for schistosomula EVs. Additionally, in contrast to schistosomula
EVs, glycolipids could not be detected in the adult worm EVs. Mass spectrometry analysis
showed that the most abundant N-glycans in the adult worm EVs contained GalNAcβ1–4GlcNAc
(LacDiNAc, LDN) motifs, which correspond to previously published overall glycan profiles
of
this specific life stage. Other differences in EV glycosylation between the two life
stages
were observed by WB using anti-glycan mAbs: adult worm EVs showed a paucimannosidic
glycan
motif whereas in the schistosomula EVs Galβ1–4(Fucα1–3)GlcNAc (Lewis X) was detected
in line
with previous MS analysis.
Summary/Conclusion: The adult worm-derived EVs contain life stage-specific
N-glycans and show a distinct glycosylation profile compared to the schistosomula
EVs. The
LDN motifs suggest that adult worm EVs may interact with the macrophage galactose-type
lectin (MGL) on host cells and most likely have other immunological consequences than
the
schistosomula EV-DC-SIGN interaction.
Funding: NWO Graduate School Program[022.006.010] (MEK); the European Research
Council under the European Union’s Seventh 16 Framework Programme [FP/2007–2013]/ERC
Grant
Agreement[No. 337581] (EN-‘tH); ZonMW[Vidi 20972] (HHS); Dutch Lung Foundation
Consortium[5.1.15.015] (HHS)
PF03.06
Isolation and characterization of Extracellular Vesicles from
phloem sap reveals their possible role in defence against phloem-feeding
insects
Christian M. Sánchez-Lópeza, Elisa Garzob,
Alberto Fereresb, Pedro Pérez-Bermúdezc and Antonio
Marcilla
d
aÁrea de Parasitología, Dpt. Farmacia y Tecnología
Farmacéutica y Parasitología, F. Farmàcia, Universitat de València, Spain., Burjassot
(Valencia), Spain; bInstituto de Ciencias Agrarias (ICA), Consejo Superior de
Investigaciones Científicas (CSIC), Madrid, Spain, Madrid, Spain; cDepartamento
de Biología Vegetal, F. Farmàcia, Universitat de València, Spain., Burjassot (Valencia),
Spain; dDepartament de Farmàcia i Tecnologia Farmacéutica i Parasitologia,
Universitat de Valéncia, Spain, Burjassot (Valencia), Spain
Introduction: Phloem plays a central role in plant function, as it is the
responsible for the translocation of photoassimilates from source- to sink-organs,
and a
long-distance route for signals distribution. Due to the sap high nutrient content,
sieve
elements are primary target for plant pathogens and pests.
In this work we aimed to isolate and characterize Extracellular Vesicles (EVs) from
Cucumis
melo phloem sap, derived from plant either exposed or not to the melon aphid, Aphis
gossypii
(Hemiptera: Aphididae).
Methods: Phloem exudates from 5-week-old melon plants, either uninfested or
infested with adults of A. gossypii (n = 15, 4 replicates each), were collected by
cutting
the stem with a sterile razor blade between first and second expanded leaf from the
top. EVs
were isolated by Size Exclusion Chromatography, and analysed by Nanoparticle Tracking
Analysis (NTA) and Transmission Electron Microscopy. EVs proteome was determined by
quantitative mass spectrometry.
Results: EVs from phloem sap were successfully isolated in every condition. No
significant differences were detected among distinct samples, neither in particle
concentration and size by NTA, nor in protein concentration.
Most importantly, a total of 381 different proteins were identified in phloem sap
EVs,
including 152 present in exosome databases (ExoCarta). On top of that, 44 differentially
expressed proteins were identified in EVs derived from aphid infested or uninfested
plants
(p value < 0.05).
Summary/Conclusion: Understanding how plants trigger their defences against
pests and pathogens is important to develop new control measures. The characterization
of
several proteins in EVs from the phloem sap provide valuable information on long distance
signalling in plants.
Moreover, as plants lack an immune system comparable to animals, the different protein
content in phloem sap EVs after exposure to aphids could indicate their important
role in
delivering inducible defences against invading pests and pathogens.
PF03.07
Extracellular vesicles from nematode species Heligmosomoides
bakeri and Trichuris muris contain distinct small RNAs that could enable niche specificity
in the host
Ruby F. Whitea; Sujai Kumarb; Amy
Buckb; María Duque-Correac; Elaine Robertsonb; Kelly
Hayesd; Richard Grencisd; Franklin W.N. Chowb
aUniversity of Edinburgh, EDINBURGH, United Kingdom;
bUniversity of Edinburgh, Edinburgh, United Kingdom; cWellcome
Sanger Institute, Cambridge, United Kingdom; dUniversity of Manchester,
Manchester, United Kingdom
Introduction: Gastrointestinal nematodes are extremely prevalent parasites
that infect most animals and ~24% of human population. Their success as parasites
is
attributed to their ability to secrete diverse molecules that modulate the host immune
system. Extracellular vesicles (EVs) are one of the immune modulatory compounds they
release
that directly modulate host cells. Our goal is to understand how the small RNA (sRNA)
cargo
underpins EV function, using a comparative analysis of EV cargo from diverse nematode
species.
Methods: We first compared how different EV isolation methodologies
(Ultracentrifuge (UC), size fractionation, sucrose gradient floatation) effect the
small
RNAs detected in H. bakeri EVs using different library preparation kits (CleanTag,
TruSeq),
with or without polyphosphatase treatment. We then compared this to small RNA libraries
from
T. muris EVs using comparable methods, UC EV purification, with or without polyphosphatase
treatment and using the CleanTag library preparation kit.
Results: EVs from both species contained miRNAs, however the miRNA gene
familes in H. bakeri and T. muris EVs are distinct. The miRNA content detected in
EV samples
collected by different purification protocols is robust. The largest difference in
detected
miRNAs was found when comparing different library preparation kits. Although both
H. bakeri
EVs and T. muris EVs were dominated by sRNAs derived from intergenic or repetitive
elements
in the parasite genomes, only in H. bakeri EVs were these secondary siRNAs.
Summary/Conclusion: H. bakeri and T. muris EVs contain distinct small RNA
cargos, which may underpin their ability to colonise different host niches, and/or
modulate
the host immune system differently. T. muris EVs do not contain secondary siRNAs,
in
contrast to H. bakeri, however they are dominated by sRNAs derived from intergenic
or
repetitive regions. Comparative analysis of helminth EVs could help pinpoint the sRNAs
involved in cross-species communication.
Funding: Rosetrees Trust, Darwin Trust of Edinburgh
Please provide any keywords if applicable.: Nematode, cross-species
communication, small RNA
PF04: EV Nucleic Acid Biomarkers
Chair: Robert Raffai – University of California & VA Medical Center, San
Francisco
Chair: Phil W. Askenase – Yale University
PF04.01
Circulating non-coding RNA biomarkers of cancers in
extracellular vesicles and serum
Yumiko Koia, Yuta Ibukib, Yasuhiro
Tsutanic, Yukie Nishiyamad, Miyuki Kandae, Daisuke
Uedaa, Ryou-u Takahashif, Junko Tanakag, Morihito
Okadaa and Hidetoshi Tahara
h
aDepartment of Surgical Oncology, Hiroshima University
Hospital, Hiroshima, Japan; bDepartment of Surgical Oncology, Hiroshima
University Hospital, hiroshima, Japan; cDepartment of Surgical Oncology,
Hiroshima University Hospital, Hirohsima, Japan; dDepartment of Cellular and
Molecular Biology, Graduate School of Biomedical & Health Sciences, Hiroshima
University, Hiroshima, Japan, Hiroshima, Japan; eCollaborative laboratory of
Liquid Biopsy, Graduate School of Biomedical & Health Sciences, Hiroshima University,
Hiroshima, Japan, Hiroshima, Japan; fDepartment of Cellular and Molecular
Biology,Graduate School of Biomedical & Health Sciences, Hiroshima University,
Hiroshima, Japan, Hiroshima, Japan; gDepartment of Epidemiology, Infectious
Disease Control and Prevention, Graduate School of Biomedical and Health Sciences,
Hiroshima
University, Hiroshima, Japan; hDepartment of Cellular and Molecular Biology,
Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima,
Japan,
Hiroshima, Japan
Introduction: Extracellular vesicles (EVs) are secreted from various cells
including cancer cells and known to contain protein and small RNAs including miRNA
isoforms
(isomiRs). Therefore we also focused on isomiRs including other small non-coding RNAs
for
biomarker discovery. Although liquid biopsies using small RNAs are promising biomarkers
for
early detection of cancer, current approaches to detecting and analysing miRNAs in
the blood
are still inadequate. Artificial intelligence (AI) data analysis may provide better
algorisms for diagnosing cancer.
Methods: Small RNAs were isolated from serum or purified EVs using a miRNeasy
Mini Kit (Qiagen) and quantified by using the Ion S5™ next-generation sequencing system.
(Thermo Fisher Scientific). EVs were purified using total exosome isolation Reagent
(Invitrogen™). AI data analysis was performed using JMP® Genomics and DataRobot enterprise
AI platform.
Results: Three small RNAs, isomiR of miR-21-5p, miR-23a-3p, and tRF-Lys (TTT)
were significantly upregulated in breast cancer patients compared with the healthy
cancer-free individual. The combination algorithm using these three small RNAs allows
for a
more accurate diagnosis of the area under the curve (AUC) 0.92. To test the possibilities
that these small RNAs are derived from cancer cells, we isolated EVs from the serum
and
performed NGS analysis to profiled serum small RNAs in EVs. Interestingly we found
that two
small RNAs, miR-21-5p and miR-23a-3p, also high in breast cancer EVs, indicating that
these
small RNAs were expected to be derived from cancer cells. In oesophagus cancer, we
also
performed NGS analysis and identified twenty-four miR/isomiRs candidates for diagnostic
biomarkers. A multiple regression model selected miR-30a-5p and two isomiRs (miR-574-3p
and
miR-205-5p). The AUC of the panel index was 0.95. We also performed AI data analysis
and
discovered the novel algorisms that can diagnose breast and oesophagus cancer more
accurately.
Summary/Conclusion: We demonstrated combinations of circulating non-coding
RNAs containing EVs potentially useful for the detection of early-stage breast and
oesophagus cancers. In addition, The DataRobot enterprise AI platform enables us to
the more
accurate diagnosis of cancers at the early stage.
PF04.02
Identification of novel EV-associated miRNAs as toxic biomarkers
in mouse
Ryuichi Onoa
, Yusuke
Yoshiokab, Yuusuke Furukawaa, Noriko Moriyamaa, Erika
Tachiharaa, Miki Uchiyamaa, Hisako Aiharaa, Mie
Narusec, Takahiro Ochiyad, Satoshi Kitajimaa and Yoko
Hirabayashie
aDivision of Cellular and Molecular Toxicology, Center for
Biological Safety and Research, National Institute of Health Sciences (NIHS), Kawasaki,
Japan; bDivision of Molecular and Cellular Medicine, Institute of Medical Science
Tokyo Medical University, chuo-ku, Japan; cCentral Animal Division, National
Cancer Center Research Institute, Tokyo, Japan; dTokyo Medical University,
Shinjuku-ku, Japan; eCenter for Biological Safety and Research, National
Institute of Health Sciences (NIHS), Kawasaki, Japan
Introduction: Recent findings reveal that extracellular vesicles (EVs),
secreted from cells, are circulating in the blood. EVs are classified into exosomes
(40–120 nm), microvesicles (50–1,000 nm) and apoptotic bodies (500–2,000 nm). EVs
contain
mRNAs, microRNAs, and DNAs and have the ability to transfer them from cell to cell.
Recently, especially in humans, the diagnostic accuracy of tumour cell type-specific
EVs as
biomarkers is more than 90%. In addition, microRNAs contained in the EVs are being
identified as specific biomarkers in blood for chemical-induced inflammation and organ
damage.
Therefore, microRNAs contained in the EVs released into the blood from tissues and
organs
in response to adverse events such as chemical substances and medicine are expected
to be
useful as novel biomarkers for toxicity assessment. In this study, we aimed to identify
target organs by comprehensive analysis of EV RNAs in the blood of mice after chemical
exposure to establish a highly sensitive “Next Generation type” toxicity test for
chemical
substances and medicine using EV RNA in blood as a biomarker.
Methods: All animal studies were conducted in accordance with the Helsinki
Declaration and the guidelines approved by the animal care committee of the National
Institute of Health Sciences. C57BL/6 J male mice (12 weeks) were orally dosed with
CCl4
(vehicle, 7, 70 mg/Kg). Serum were separated from blood after 2, 4, 8 and 24 hours
after
CCl4 administration. The serum was centrifuged at 10,000 x g to remove cellular debris
and
subsequently ultracentrifuged 100,000 x g. The pellet is resuspended in PBS and
ultracentrifuged 100,000 x g again. The comprehensive small RNA-seq of collected EVs
were
performed according to the manufacture’s protocols.
Results: We succeeded in isolating more than 10 novel small RNAs, which could
be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride
(CCl4: 7 mg/Kg & 70 mg/Kg). Well known hepatotoxicity biomarkers, miRNA-122 and
miRNA-192 were upregulated more than 1000-fold in the administration of 70 mg/Kg CCl4,
but
not responded in the administration of 7 mg/Kg CCl4.
Summary/Conclusion: These results suggest that mir-122 and mir-192 are mainly
released from liver to blood directly only in the administration of 70 mg/Kg CCl4,
while
novel more sensitive hepatotoxicity biomarkers which responded in the administration
of both
7 mg/Kg and 70 mg/Kg CCl4 should be included in the EV. Our novel biomarkers will
accelerate
a rapid evaluation of chemical substances and medicine in Nonclinical Safety Evaluation.
Funding: Health Sciences Research Grants from the Ministry of Health, Labour,
and Welfare, Japan (H30-KAGAKU-IPPAN-002, H30-KAGAKU-SHITEI-001), the Research on
Regulatory
Science of Pharmaceuticals and Medical Devices (18mk0104073j0103) and JSPS KAKENHI
(26430183
and 18K19315)
PF04.03
Cancer-specific genomic alterations in plasma large EVs from
patients with advanced cancer
Tatyana Vagnera
, Yari
Cianib, Francesco Orlandoc, Mandana Zandiand, Andries
Zijlstrae, Michael Freemand, Ronald Nataled, Edwin
Posadasd, Francesca Demichelisf and Dolores Di Vizioa
aCedars-Sinai Medical Center, West Hollywood, USA;
bDepartment of Cellular, Computational and Integrative Biology (CIBIO),
University of Trento, Trento, Italy, Trento, Italy; cUniversity of Trento,
Trento, Italy; dCedars-Sinai Medical Center, Los Angeles, USA;
eVanderbilt University Medical Center, Nashville, USA; fUniversity of
Trento, Weill Cornell Medical College-New York Presbyterian Hospital, Trento, Italy
Introduction: Advancements in sequencing technologies have allowed analysis of
the genomic landscape of cancer using circulating cell-free(cf) DNA. However, cfDNA
does not
originate only from tumour cells. We recently demonstrated that most of the DNA circulating
in plasma of cancer patients is associated with large EVs (L-EVs), and that L-EV-associated
DNA reflects genomic aberrations of the cells from which L-EVs arise. Since L-EVs
are
specifically released by tumour cells, we explore their potential to report cancer-specific
genomic alterations in patient plasma and compare it to cfDNA.
Methods: Differential ultracentrifugation, tunable resistive pulse sensing,
Qubit dsDNA High Sensitivity assay, capillary electrophoresis, whole exome sequencing
(1500–2000x), targeted sequencing (QIAseqTM), flow cytometry.
Results: We show here that L-EVs in the size range of >1 micrometre are
present exclusively in plasma obtained from cancer patients and absent in plasma from
healthy donors. In agreement with this finding, double-stranded(ds) DNA is detected
only in
L-EV fractions of patient plasma and not in those obtained from healthy donor plasma
using
the same protocol. We also demonstrate that the fragments of dsDNA associated with
circulating L-EV are larger in comparison with cfDNA (>10,000 bp versus ~170 bp).
A
large-scale analysis of L-EV DNA obtained from plasma of patients with metastatic
castration-resistant prostate cancer (mCRPC) as well as with non-small cell lung cancer
(NSCLC) demonstrates that DNA associated with circulating L-EVs reports cancer-specific
genomic alterations in both types of cancer. We further investigate if L-EV-associated
DNA
is intra- or extravesicular and demonstrate that it is present in both forms. We finally
compare the purity of the tumour signal in intravesicular L-EV DNA, total L-EV DNA,
and
cfDNA obtained from patient plasma.
Summary/Conclusion: Our results demonstrate that circulating L-EVs contain
high quality, large molecular weight DNA that contains cancer-specific genomic alterations,
supporting the use of L-EVs as a source of tumour-derived DNA in plasma.
Funding: National Institutes of Health R01CA218526 (DDV); DoD PC150836 (DDV),
Luke Wu-Jei Chang Discovery Fund (DDV), AIRC/CR-UK Accelerator Award A26822 (FD)
PF04.04
Detection of EGFR mutations in exosomal RNA and protein mirrors
disease status in metastatic lung cancer patients
Emma Purcell, Sarah Owen, Emily Prantzalos,
Abigail Radomski, Mina Zeinali, Nayri Carman, Nithya Ramnath and Sunitha Nagrath
University of Michigan, Ann Arbour, USA
Introduction: Epidermal growth factor receptor (EGFR) mutation driven lung
adenocarcinoma (AC) represents a unique subgroup that lends itself to treatment with
oral
EGFR tyrosine kinase inhibitors. Current methods that are used to detect these mutations
(e.g. L858 R or the resistance mutation T790 M) involve invasive tumour biopsies or
blood
circulating tumour DNA (ctDNA) and cell free DNA (cfDNA). The sensitivity of blood
ctDNA and
cfDNA is limited by the frequency of genomic alterations in the EGFR gene; additionally,
ctDNA does not reflect changes in the EGFR protein, against which novel therapies
are in
development. There remains a need to develop blood-based biomarkers that can circumvent
these disadvantages and replace the more standard, invasive tumour biopsies. We propose
the
study of exosomes for treatment monitoring as well as to identify EGFR resistance
related
genomic and proteomic changes.
Methods: We enrolled 10 patients with metastatic lung AC: 8 with EGFR
mutations and 2 without (control). From the patients with EGFR mutant lung AC, we
processed
25 blood samples through the patients’ treatment course, using ultracentrifugation
to
isolate exosomes. We then used both droplet digital PCR (ddPCR) to test exosomal RNA
(exoRNA) for the mutation of interest and western blots to test protein resulting
from exon
19 deletion or L858 R mutations.
Results: From 6 patients with EGFR exon 19 deletion mutations, we detected
identical mutations in exoRNA from 15/19 samples. ExoRNA based mutational load increased
and
mirrored clinical progression in 2 patients. Three patients whose cancer remained
stable
demonstrated a decrease in their exoRNA. One patient had blood drawn only at 2 points
and
was therefore not plotted. ExoRNA from 2 patients with L858 R and T790 M mutations
demonstrated the corresponding mutations; however, exoRNA did not mirror their disease
course. We also demonstrated mutant EGFR protein presence in exosomes from 3 patients.
Finally, we tested cfDNA for EGFR mutations from four matched samples using ddPCR.
We
detected matched mutations in exosomes in all four, while cfDNA mutations were only
detected
in 2/4 patients.
Summary/Conclusion: In summary, we detected EGFR mutations in 19/23 exosome
samples isolated from metastatic lung AC. Our results set the stage for optimization
of
exoRNA methods and inform future experiments relating to exosomal cargo in patients
with
EGFR mutant lung AC.
PF04.05
Identification of plasma-derived, EV-based biomarkers for
glioblastoma
Yevgenia Khodora
, Sudipto
Chakraborttya, Martina Rauscherb, Steven P. Langc, Julia
Smalld, Leonora Balaje, Bob Carterf, Seth Yua
and Johan Skogg
aExosome Diagnostics, Waltham, USA; bExosome
Diagnostics, Munich, Germany; cExosome Diagnostics, Boston, USA; dMGH
and Harvard Medical School, Cambridge, USA; eMassachusetts General Hospital and
Harvard Medical School, Cambridge, USA; fMassachusetts General Hospital,
Cambridge, USA; gExosome Diagnostics, Inc, Waltham, USA
Introduction: Glioblastoma multiforme (GBM) is the most malignant and
aggressive primary brain cancer in adults, with an incidence of 3.2 per 100,000 people.
Currently, diagnosis is only performed via histopathological investigation of a tissue
sample from a GBM lesion, complemented with molecular diagnostics for identification
of
select biomarkers. MRI is the standard of care for follow-up and monitoring of treatment
response. Therefore, development of a “liquid biopsy” to obtain disease-relevant information
from patient’s body fluids is highly desirable.
Methods: We present the results from a clinical study in which extracellular
vesicle (EV)-derived mRNAs and long non-coding RNAs were profiled from the plasma
of GBM
patients and control individuals. We obtained plasma from 8 patients at the time of
initial
diagnosis, and 8 matched controls by sex and age. EV-associated RNA was isolated from
1–2 mL
plasma and RNA-seq was performed using our proprietary pipeline. Sequencing data was
analysed for differential gene expression.
Results: We observed 95 mRNAs as differentially abundant between GBM and
control samples, with 82 mRNAs enriched in GBM samples and 13 mRNAs enriched in control
samples (p < 0.05). Correlation based on differentially abundant mRNAs separated GBM
and
control samples into two unique populations. Eight differentially expressed mRNAs
were
previously identified as part of the mesenchymal GBM subtype. These data, while preliminary,
provide a potential basis for the further development of a non-invasive GBM gene panel
test.
Summary/Conclusion: We have identified a novel RNA signature for GBM from
plasma derived EVs, which differs from previously identified biomarkers isolated from
tissue. Further work will refine this signature to enable detection, characterization,
and
patient monitoring for GBM with minimally invasive techniques.
Funding: Bio-Techne Corporation
PF04.06
Identification of a saliva exosomal RNA signature for Sjogren’s
syndrome
Steven P. Langa, Yevgenia Khodorb, Martina
Rauscherc, Sudipto Chakraborttyb
, Vasisht
Tadigotlab, Robert Kitchend, Mabi Singhe, Andrew
Hoffmanf, Seth Yub, Johan Skogg and Athena
Papase
aExosome Diagnostics, Boston, USA; bExosome
Diagnostics, Waltham, USA; cExosome Diagnostics, Munich, Germany; dMGH
and Harvard Medical School, Boston, USA; eTufts School of Dental Medicine,
Boston, USA; fUniversity of Pennsylvania School of Veterinary Medicine,
Philadelphia, USA; gExosome Diagnostics, Inc, Waltham, USA
Introduction: Sjogren’s syndrome (SS) is a systemic autoimmune disease in
which inflammation progressively damages the moisture producing glands of the afflicted.
4
million Americans are estimated to be suffering from the disease, 90% of which are
women
with an average age of 40. Overlapping symptoms with other health conditions and
co-morbidities make SS particularly difficult to diagnose, with average time to diagnosis
of
3 years. Saliva exosomal RNA profiling has been primarily focused on small RNAs and
has been
limited thus far due to the large contribution of sequencing reads from the oral microbiome.
A non-invasive saliva exosomal RNA (exoRNA) based test capable of diagnosis would
be highly
desirable.
Methods: We began by first developing a novel long RNA-Seq workflow to
selectively enrich and profile human exosomal mRNAs and long non-coding RNAs (lncRNAs)
from
saliva. We then profiled salivary exoRNA obtained from 7 SS patients and 7 healthy
matched
controls. Finally, we performed differential gene expression analysis to obtain an
exoRNA
signature for SS.
Results: RNA-Seq data analysis demonstrated highly efficient enrichment of
human transcriptome, with over 80% of reads mapping to the transcriptome. Further
RNA
biotype analysis showed over 75% of transcriptome reads mapped to protein coding genes
and
lncRNAs. We detected over 10,000 mRNAs and approx.1000 lncRNAs. Differential expression
analysis (DEx) of SS vs. healthy control exoRNA identified 455 upregulated genes,
including
415 mRNAs and 37 lncRNAs (p < 0.05). 120 genes were found to be downregulated in SS,
including 114 mRNAs and 6 lncRNAs. Gene ontology analysis of DEx genes revealed enrichment
of genes involved in various immune system related pathways. Most importantly, principal
component analysis (PCA) resulted in clear separation of SS patients from healthy
controls.
Summary/Conclusion: Our optimized RNA-Seq workflow enables saliva-based liquid
biopsy for biomarker discovery. The gene signature identified in this ongoing study
could
potentially provide a non-invasive molecular means of diagnosing Sjogren’s syndrome.
Funding: Bio-Techne Corporation
PF04.07
Endometrial fluid derived EVs as low invasive diagnostic
biomarkers of implantative endometrium
Jone Ibañez Pereza, Maria Diaz-Nuñezb, Lucia
Lainzb, Maria Iglesiasb, Miren Diezb, Antonia
Expósitob, Juan Jose lozanoc, Monika Gonzalezd, Ana Maria
Aransayd, Esperanza gonzaleze, Felix Royof, Nerea
Subirang, Roberto Matorrash and Juan Manuel
Falcon-Perezi
aHuman Reproduction Unit, Cruces University Hospital,
Biocruces, University of the Basque Country, Bilbao, Spain./Exosomes Laboratory, CIC
bioGUNE, Bizkaia Technology Park, Derio, Spain., Bilbao, Spain; bHuman
Reproduction Unit, Cruces University Hospital, Biocruces, University of the Basque
Country,
Bilbao, Spain., Bilbao, Spain; cBioinformatics Unit, Centre Esther Koplovitz
(CEK), CIBERehd, Barcelona, 08036, Spain., Barcelona, Spain; dGenome Analysis
Platform, CIC bioGUNE, Derio, Bizkaia, 48980, Spain., Bilbao, Spain; eExosomes
Laboratory, CIC bioGUNE, Bizkaia Technology Park, Derio, Spain., Bilbao, Spain;
fExosomes Laboratory, CIC bioGUNE, Bizkaia Technology Park, Derio, Spain./Centro
de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd),
Spain.,
Bilbao, Spain; gDepartment of Physiology, Faculty of Medicine and Dentistry,
University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain./Biocruces Bizkaia
Health
Research Institute, Barakaldo, Bizkaia, Spain, 48903., Bilbao, Spain; hHuman
Reproduction Unit, Cruces University Hospital, Biocruces, University of the Basque
Country,
Bilbao, Spain./Instituto Valenciano de Infertilidad, IVI, Bilbao, Spain., Bilbao,
Spain;
iExosomes laboratory and Metabolomics Platform, CIC bioGUNE, CIBERehd, Bizkaia,
Spain./IKERBASQUE, Basque Foundation for Science, Bizkaia, Spain., Bilbao, Spain
Introduction: Increasing embryo implantation rates has become one of the
greatest challenges in assisted reproduction techniques. Usually an endometrial biopsy
is
done to identify a receptive endometrium, which prevents embryo transfer in the same
cycle,
as it is detrimental for the implantation. The implantation is a complex process,
which
requires a synchrony between the development of the embryo and the endometrium, but
also, an
adequate embryo-endometrial cross talk. The presence of extracellular vesicles (EVs)
as
mediators of this communication has been describe in the endometrial fluid. Therefore,
we
hypothesize that the molecular analysis of the content of the EVs and companion molecules
from endometrial fluid could be a non-invasive method to recognize an implantative
endometrium and consequently improve the implantation rates.
Methods: The objective is to define a simple, sensitive and reproducible
non-invasive EV-based method that allow the quick identification of an implantative
endometrium by means of miRNA analysis. For the establishment of a robust methodology
for
analysing EVs from endometrial fluid in clinical settings, where the sample is limited
and
no sophisticated equipment is available, five different methodologies were compared
in
triplicate. Two of them consisted in the direct extraction of RNA while in the other
three,
before the RNA extraction an enrichment of EVs was done. SmallRNAseq was performed
to
determine the most efficient method. Once the best method was selected, it was applied
in a
set of real samples with different implantation outcome. The content of miRNAs (mainly
associated with EVs) of endometrial fluid samples from women in whom the implantation
was
successful (n = 15) and unsuccessful (n = 15) were analysed.
Results: Our results show that the protocols with a previous enrichment step
of EVs obtained a higher miRNA expression. The results obtained from the differential
analysis of the set of samples with different implantation outcome are being analysed
and it
is expected that the results will be available by the time this communication is
presented.
Summary/Conclusion: This work demonstrates that it is possible to obtain and
analyse EVs and EVs-associated miRNAs from a small volume of endometrial fluid samples,
which allows the use of EV-miRNAs as a low-invasive biomarkers for the detection of
an
implantative endometrium.
Funding: JIP is supported by a predoctoral grant from the Basque
Government.
PF04.08
Small RNA cargo of EVs is affected by hormone treatment in
prostate cancer
Elena S Martens-Uzunovaa, Crystal Cooperb,
James E Richesb, Grant A. Rammc, Guido W. Jenstera and
Carolina Soekmadji
d
aDepartment of Urology, Erasmus Medical Centre, Rotterdam,
Netherlands; bCentral Analytical Research Facility, Institute for Future
Environments, Queensland University of Technology, Brisbane, Australia; cQIMR
Berghofer Medical Research Institute, Brisbane, Australia; dQIMR Berghofer,
Herston, Australia
Introduction: Small RNAs are recently reported as a regulator for prostate
cancer progression to castration-resistant disease. Our previous work has shown that
EVs
protein cargo is affected by male steroid hormone, dihydrotestosterone (DHT). In this
study,
we assess the small RNA cargo of EVs in response to androgen manipulations.
Methods: Androgen receptor-positive LNCaPs are grown in CSS medium to deplete
the androgens. Media were then replaced with vesicle-depleted CSS medium ± 10 nM
dihydrotestosterone (DHT) ± 10 µM Enzalutamide (ENZ) for 48 h. EVs were isolated using
sequential ultracentrifugation (2000 g for 20 min, 10,000 g for 30 min, 100,000 g
for 2 h),
washed once in PBS. Protein and RNA were collected from both parent cells and conditioned
medium to allow direct comparison between S-EVs cargo and cells. Small RNA NGS libraries
were prepared using the Illumina’s TruSeq Small RNA Library Prep kit and single-end
sequenced at a read length of 50 nucleotides (nt). Fastq library files were processed
using
a custom-designed pipeline. Adapters were removed using the Cutadapt tool, trimmed
reads
were mapped with high stringency against ribosomal sequences using Bowtie2. SnoRNA
and tRNA
fragments were identified using the FlaiMapper software. Remaining reads were mapped
against
the human genome hg38 using Bowtie2.
Results: We found that the presence or absence of androgens does not
significantly change the amount of total RNA in small EVs (S-EVs). However, hormone
stimulation altered the small RNA content of S-EVs, in parallel with our previous
published
data on EV protein cargo. DHT increased the abundance of snoRNA in cells, while a
reduction
of snoRNAs was observed in the S-EVs fraction. Interestingly, DHT induced the formation
of
cell filopodia that are not inhibited by androgen inhibitor Enzalutamide. Pathway
analysis
indicates the p53 mediated regulation driven by miRNAs found in S-EVs upon exposure
to DHT.
The expression profile of snoRNA and tRNA fragments in DHT treated cells resembles
results
from clinical prostate cancer specimens.
Summary/Conclusion: Our findings show that androgen manipulation alters both
S-EV derived protein and RNA cargo. Changes in the S-EV RNA profile due to treatment
with
androgens are not identical to small RNA profiles in parental cells, indicating a
specific
sorting mechanism of S-EV small RNA upon androgen manipulation. Further, DHT induces
the
formation of cell filopodia irrespective of Enzalutamide, suggesting cargo selection
of
S-EVs. We conclude that small RNA EV cargo can be utilised to as prostate cancer biomarkers
in androgen targeted treatments.
Funding: US Department of Defense Congressionally Directed Medical Research
Program Prostate Cancer Research Program Idea Development Award [Number: W81XWH-16-1-0736]
for CS.
PF05: EV Protein Biomarkers
Chair: Jennifer Jones – Laboratory of Pathology, National Cancer Institute, National
Institutes of Health
Chair: Patricia Midori Murobushi Ozawa – Department of Cell and Developmental Biology,
Vanderbilt University School of Medicine
PF05.01
Detection of PD-L1 in circulating extracellular
vesicles
Veronica Sancheza
, Daniel
Bachurskib and George Daaboula
aNanoView Biosciences, Boston, USA; bUniversity of
Cologne, Cologne, Germany
Introduction: Cancer immunotherapy, such as PD-L1 blockade, is a method to
eliminate cancer cells. Ectopic expression of PD-L1, on the surface of tumour cells,
has
been associated with tumour persistence and as an important predictor of therapy response.
A
test that, specifically and accurately, detects PD-L1 is critically important in order
to
identify patients that would benefit from these treatments. Emerging evidence has
shown that
extracellular vesicles (EV) can carry immune checkpoint molecules, such as PD-L1,
and whose
expression have been correlated with tumour immunity response. With a multitude of
commercially available antibodies identifying appropriate clones and associated assay
is
important in order to standardize the diagnostic modality used.
Methods: PD-L1 expressing cancer cell lines were used to generate EVs.
PD-L1-MYC vector was transfected to generate an overexpression system. ExoView® sensors
containing different anti-PD-L1 clones were generated. Samples (cell derived and plasma)
were incubated on chips to allow the antibody to bind the antigen on the EV. After
incubation, chips were immunolabeled with fluorescently labelled antibodies against
PDL-1 or
EV associated markers. ExoView R100 reader was used to enumerate the EVs captured
on the
sensor surface and analyse the expression of PDL-1 on single vesicle through fluorescence
imaging. Immunoprecipitation and mass spectrometry (IP/MS) were employed as an orthogonal
method to verify the specificity of the assay.
Results: To study the detection efficiency of the antibodies, engineered
PD-L1-MYC EVs were used. Under these circumstances, all the tested antibodies were
able to
capture EVs. When testing endogenous PD-L1 positive EVs from different cancer cell
lines,
only 28.8 and 73 10 clones consistently bound to EVs. In addition, EVs derived from
plasma
demonstrated to be positive for PD-L1, however, only clone 28.8 was able to immobilize
these
EVs. The results suggested that clone 28.8 could be a potential PD-L1 antibody to
detect
PD-L1 positive EVs originating from various sources. To confirm these results, and
assure
the specificity of the antibody targeted IP/MS was employed.
Summary/Conclusion: In combination with the ExoView platform, anti-PD-L1
antibodies can be screened and potentially used to generate a non-invasive EV-specific
assay
that could detect this protein in patients.
PF05.02
Differences in extracellular vesicle protein cargo is dependent
on head and neck squamous cell carcinoma cell of origin
Heather M. Walline and Chrissy Goudsmit
University of Michigan, Ann Arbour, USA
Introduction: Head and neck squamous cell carcinoma (HNSCC) is the sixth most
common, eighth most fatal cancer worldwide and includes cancers of the oropharynx,
larynx,
hypopharynx, and oral cavity. In 2017, there were over 49,000 new cases and 9,700
deaths
estimated in the USA alone. Despite recent advances in treatment, including radiation,
chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours
develop
resistance and progress. Patients develop metastases or tumours recur locally or regionally;
the 5-year overall survival rate for HNSCC is only 40–50%. Factors that contribute
to poor
survival for patients with HNSCC include late stage diagnosis, lack of reliable markers
for
early stage detection, high level of biologic heterogeneity, and local recurrence
and
distant metastases after treatment.
Methods: This study used 8 representative HPV-positive and HPV-negative HNSCC
cell lines, one HPV-transformed cell line. and two non-cancer oral keratinocyte cell
lines.
EVs were isolated using differential ultracentrifugation and PEG
precipitation/ultracentrifugation. EVs were characterized by TEM, NTA, and Wes protein
analysis for reported EV markers. EV and whole cell lysates were assessed by LC-MS/MS
analysis using the Tandem Mass Tag-10plex kit. Cluster analysis was performed on the
fold-change peptide spectrum matches (PSM) for the EVs from the HNSCC lines compared
to the
EVs from the normal keratinocyte line (NOKsi). Protein was measured using a capillary-based
electrophoresis instrument.
Results: CD9 and AnnexinV were detected in all of the EV lysates tested, while
Calnexin was detected in all of the whole cell lysates and none of the EV samples
tested.
Selected proteins STAT3, HLA-A, Tenascin, E-Cadherin, β Catenin, Cytokeratin 19, EPHA2,
and
CD59, and HPV-related markers p16, p53, RB, Cyclin D1, and EGFR were tested using
the WES
platform. EVs from HPV-positive cell lines showed higher protein levels compared to
EVs from
HPV-negative cell lines in STAT3, HLA-A, and Tenascin. Only KERT19 demonstrated lower
protein levels in EVs from HPV-negative cell lines. Of the common HPV-associated HNSCC
markers: EGFR, p53, RB, Cyclin D1 and p16, only EGFR was positive in any the EVs tested.
The
remaining proteins queried, E-Cadherin, β Catenin, EPHA2 and CD59 showed varying protein
levels in EVs from both HPV positive and HPV-negative cell lines.
Summary/Conclusion: Our findings suggest that these proteins may be potential
HNSCC EV markers that may be 1) selectively included in EV cargo for export from the
cell as
a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment,
or 2) representative of originating cell composition, which may be developed for diagnostic
or prognostic use in clinical liquid biopsy applications.
PF05.03
Validation of antibodies on western blot for extracellular
vesicles from biological human samples and cancer cell conditioned media
Kengo Horie, Richard Zieren, Liang Dong, Sarah
Amend and Kenneth Pienta
The Brady Urological Institute, Johns Hopkins University School of
Medicine, Baltimore, USA
Introduction: One of the major challenges in Extracellular vesicles (EVs)
research is to prove the particles that are isolated are true EVs, rather than other
co-isolated contaminants, like lipoproteins. ISEV recommends using multiple assays
to
characterize EVs. This study aims to validate the positive and negative protein markers
for
extracellular vesicles from plasma, urine and prostate cancer cell conditioned media
(CCM).
Methods: Membrane and cytosolic fractions of MCF7 cells served as positive and
negative controls for all antibodies validated. EVs were isolated from plasma of healthy
volunteers, urine of healthy volunteers and CCM of PC-3 cells using differential
ultracentrifugation. Eight protein markers were assessed: positive markers CD63, CD81,
CD9,
Flotillin1 (Flot1), Alix and Tumour susceptibility gene 101 (TSG101), negative marker
calnexin (CANX), and contaminant markers Apo-A1 for plasma and THP for urine. Tetraspanins
are small transmembrane proteins expressed in EVs. Flot1 is membrane protein that
forms
microdomains in the plasma. Alix and TSG101, an accessory protein of the endosomal
sorting
complex required for transport, are involved in the biogenesis of EVs. They are positive
markers for EVs. CANX is in the membrane of the endoplasmic reticulum. Apolipoprotein-A1
(Apo-A1) is the protein components of lipoproteins, therefore it is marker of contamination
for Plasma EV. Tamm-Horsfall protein (THP) is contamination marker for Urine EV, because
it
is most abundant protein in human urine.
Results: All antibodies were validated in the correct positive and negative
control, thus confirmed as usable and reliable antibodies for Western Blot. In Plasma
EV,
CD63, CD9, CD81 and Flot1 were positive and CANX and Apo-A1 were negative. In Urine
EVs,
CD9, CD63, Flot-1, Alix and TSG101 were positive and CANX and THP were negative. In
CCM EVs,
CD9, CD63, Flot1, Alix and TSG101 were positive and CANX was negative.
Summary/Conclusion: We confirmed a high degree of EV purity from 3 sample
types: urine, plasma, and CCM. Of particular importance, we confirmed that EVs isolated
from
biologic patient samples, plasma and urine, had low contamination. Future work will
use
these methods to confirm purity of EV samples prior to addition analysis, such as
examining
EV cargo and biologic significance.
PF05.04
Proteomic study of mesenchymal stem cells derived exosomes
modified using miR.
Miriam Morente-Lópeza, Juan
Fafián-Laborab
, Jesús Mateosc, Mónica
Carrerac and María Arufed
aPredoctoral student, A Coruña, Spain;
bEpigenetics & Cellular Senescence Group, Blizard Institute, Barts and The
London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark
Street,
London E1 2AT, UK, London, UK; cIIM, Vigo, Spain; dUniversity of La
Coruña, A Coruña, Spain
Introduction: The project we are working on is to modify the immunogenic
profile of human CMMs from the umbilical cord stroma through its stable transfection
with
anti-miR-21-5p, and therefore of the exosomes that these cells generate, for use in
free-cell therapy to treat inflammatory process.
Methods: EVs released from a primary culture of human umbilical cord
mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal
stem
cells miR21-/- modified through stable lentiviral transfection were isolated by
ultracentrifugation processes, characterized by transmission electron microscopy (TEM)
and
measured by nanoparticles tracking analysis (NTA). Protein extraction from EVs was
made
using RIPA buffer and after checking protein integrity the total EV proteins. We performed
a
shotgun proteomic study using a TMT (10-plex) label of the total miR21-/- exosomes
protein
comparing it with normal exosomes. After labelling the LTQ-Orbitrap platform of ProteoRed
was needed for fraction injections and data acquisition. Proteome Discoverer 2.2 (Thermo)
was used for protein processing and quantification.
Results: A total of 1.861 proteins were identified at least with a unique
peptide and we have able to establish the proteomic profile of miR21-/- exosomes against
normal exosomes. We found out several protein modulated by miR21 and related to
inflammation.
Summary/Conclusion: We have able to establish the proteomic profile of
miR21-/- exosomes against normal exosomes focusing on proteins involving inflammation
process. All those results seem indicate that exosomes could be modified, which could
be
used as an anti-inflammatory free-cell therapy.
Funding: ProteoRed Concept Test Project Grant.
PF05.05
A novel extracellular vesicle isolation method used to discover
urine liver disease biomarkers
Shannon Pendergrasta
, Anna
Markowskab, Ashok Reddyc, Phillip Wilmarthc, Scott
Nauglerd, Yiyi Chene, Lina Gaoe, Thuy Ngof,
Charles Thomasg and Christie Binderg
aYmir Genomics, Cambridge, USA; bYmir Genomics,
Cambridge, USA; cProteomics Shared Resources, Ohsu, Portland, USA;
dDivision of Gastroenterology and Hepatology, Ohsu, Portland, USA;
eDepartment of Biostatistics, Ohsu, Portland, USA; fKnight CEDAR
Technology, Ohsu, Portland, USA; gRadiation Medicine, Knight Cancer Institute,
Ohsu, Portland, USA
Introduction: Hepatocellular carcinoma (HCC) is the 6th most common cancer
worldwide and the 3rd most common cause of cancer death; additionally, its incidence
is
increasing. While outcomes for early HCC are superior to those for late stage disease,
early
detection of HCC remains a challenge. Current guidelines have suboptimal sensitivity
and
specificity. In this pilot study, we hypothesize that urine extracellular vesicles
(EVs) may
identify candidate biomarkers towards the development of an inexpensive, widely accessible
screening assay for the early detection of HCC.
Methods: Urine samples from 20 healthy subjects, 19 subjects with cirrhosis,
and 19 subjects with cirrhosis plus HCC were collected and processed using YMATRIX
columns
to isolate EV-associated protein and miRNA. Protein was analysed using a tandem mass
tag
method on a Thermo Scientific Orbitrap Fusion mass spectrometer with Comet/PAWS and
EdgeR
processing. miRNA was analysed using a targeted Firefly microarray from Abcam. Differential
expression and predictive modelling for the presence of HCC and cirrhosis was performed
to
identify candidate miRNA and protein biomarkers.
Results: For miRNA, 39 samples were eligible for analysis after low expression
filtering. We used pair-wise ratios of 34 cancer-associated miRNAs by gradient boosting
of
decision trees to develop a predictive model for HCC. Our best model had a sensitivity
and
specificity of 0.93 and 0.92 respectively using 6 miRNAs to distinguish HCC from cirrhosis.
All samples were eligible for protein analysis. Based on differential expression and
biologic relevance, we identified 10 protein candidate biomarkers. Interestingly,
we found
liver-selective proteins and known HCC/cirrhosis plasma/tissue markers, demonstrating
proof-of-concept for the method.
Summary/Conclusion: Urine extracellular vesicles contain liver-selective
proteins and known liver disease serum biomarkers as well as novel miRNA and protein
biomarkers that are significantly up-regulated in disease samples. The described candidate
biomolecules may be easily accessible biomarkers with which to develop a sensitive
and
specific universal screening diagnostic for the early detection of cirrhosis and HCC.
Funding: Funded by: Ymir Genomics LLC
Christie Binder Medical Research Foundation Early Career Investigator Grant
CEDAR Seed Pilot Award
PF05.06
Evaluation of plasma extracellular vesicles in Rheumatoid
Arthritis and Systemic Lupus Erythematosus
Samantha McElyea, Ethan Pickerill, Andrea
Martin and Robert Benschop
Eli Lilly and Company, Indianapolis, USA
Introduction: Circulating extracellular vesicles (EVs) are known to carry
biologically active molecules which contribute to homoeostatic and pathologic functions.
EVs
and their cargo have been associated with the development and perpetuation of autoimmune
(AI) diseases such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE).
Understanding the content of EVs and how they correlate with disease state, activity,
and
response to therapy could provide a valuable tool for patient tailoring in AI diseases.
Methods: Samples were collected after independent review board approval and
patient informed consent was obtained. EVs were isolated from EDTA plasma from patients
with
RA (n = 20) or SLE (n = 20) and from age- and sex-matched healthy controls (n = 20).
EV size
and concentration were assessed by Nanoparticle Tracking Analysis (NTA), miRNA content
by
NanoString, and protein content by Luminex 41-plex Immunoassay.
Results: NTA revealed a higher concentration of EVs in RA (p = 0.042), but not
SLE (p = 0.278) plasma compared to healthy controls. Additionally, the size of EVs
from RA
and SLE samples compared to those of healthy controls were significantly larger (p = 0.0074
and p = 0.0004, respectively). Analysis of miRNA identified differential expression
of 8 and
11 miRNA in RA and SLE EVs, respectively, compared to healthy controls. All 8 species
that
changed in RA EVs were also differentially expressed in SLE EVs. Furthermore, when
we
analysed the protein content, we found that 2 proteins in RA EVs and 4 proteins in
SLE EVs
were differentially expressed compared to healthy control EVs. Of these analytes,
MDC was
downregulated in EVs from both RA and SLE samples compared to those of healthy controls
(p = 0.04 and p = 0.0004, respectively).
Summary/Conclusion: Plasma EVs from patients with RA and SLE displayed altered
size, miRNA and protein content compared to EVs from healthy controls. Additional
analyses,
correlating findings with disease metrics, will be performed and may reveal biomarkers
with
relevance to clinical outcomes. Identification and analysis of EVs circulating in
patients
with AI diseases may present a new paradigm for future biomarker strategies.
PF05.07
Using extracellular vesicles as a template for autoimmune
diagnostic screening
Anne Sophie Schoua
, Rikke
Bækb, Kim Varmingb, Rikke Katrine Jentoft Olsenc and
Malene M. Møllerd
aDepartment of Clinical Immunology, Aalborg University
Hospital, Vodskov, Denmark; bDepartment of Clinical Immunology, Aalborg
University Hospital, Aalborg, Denmark, Aalborg, Denmark; cResearch Unit for
Molecular Medicine, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark;
dDepartment of Clinical Immunology, Aalborg University Hospital, Aalborg,
Denmark
Introduction: The peptidergic G-protein coupled receptors (GPCRs) are
cell-signalling transmembrane proteins, which in their native form comprise of seven
segments embedded in the cell membrane. This structural advancement is believed to
be
maintained in extracellular vesicles (EVs). In autoimmune diseases, the presence of
autoantibodies towards GPCRs is not uncommon, and to detect plasma autoantibodies,
EVs
carrying GPCR will be used as template in a novel microarray screening tool.
Methods: Purified EVs from HEK293 cells were printed on different types of
surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic 96 well
plates. Five different print buffers were tested in a multiplex assays. Spots containing
EVs
were stained with biotinylated antibodies (CD9, CD63, CD81, ADRβ2, Hsp70, EPCAM and
Flotilin-1) followed by binding of Cy5-labelled streptavidin and visualized microarray
scanner.
Results: The outcome of these experiments was promising, as some of the chosen
printing buffers showed increased tendencies to bind EVs. The EV presence was verified
with
a panel of markers known to be present on small EVs. In addition, the EV content of
the
adrenergic beta-2 receptor (ADRβ2-receptor), which is a GPCR of interest in autoimmune
diseases, was verified in some of the experimental setups.
Summary/Conclusion: The approach of using EVs as template in a screening tool
possesses the potential to easily screen for autoimmune illness markers in diagnostic
purposes. Using the microarray technology allows the screening to be multivariate,
specific
and highly sensitive.
PF05.08
Circadian variation of extracellular vesicles secreted in urine:
Analysis of time point collection and normalization strategy.
Luca Mussante
a, Sabrina La
Salviaa and Uta Erdbrüggerb
aUniversity of Virginia School of Medicine, Charlottesville,
USA; bUniversity of Virginia, Charlottesville, USA
Introduction: Urinary extracellular vesicles (uEVs) are an ideal source of
biomarkers for kidney and urogenital diseases. Despite the great deal of interest
generated
by uEVs, little is known about its collection time and normalization approach. The
majority
of the studies on uEVs focus on spot urine collection based on the assumption that
it
accurately reflects the renal function, although time point of collection is not
standardized. Therefore the practice to collect spot urine does not allow for calculating
and standardizing accurately the uEV excretion rate which may vary during the day.
In
addition, no research has been carried out yet to show the quantitative and qualitative
difference of uEVs between spot urine and 24 h collections.The aim of this study is
to
compare uEVs excreted in all single voids during a 24 hour collection period and compare
it
with 24 hour collection performed.
Methods: uEVs were enriched by differential centrifugation and electron
microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse
sensing
and imaging flow cytometry were used to quantify uEVs and associated markers variation
during the 24 hour. Creatinine, urine osmolality and particle concentration were used
to
normalize the assessed analytes.
Results: Electron microscopy showed a heterogeneous population of EVs and
western blot confirmed the presence of EV markers (TSG101, ALIX and CD9). RNA was
extracted
by a column-based method (miRNA extraction kit Qiagen) and cel-39 miRNA was spiked
in each
sample. A multiparametric detection of nephron markers podocalyxin, aquaporin-2 and
uEVs pan
tetraspanins (CD9 + DC63 + CD81) was performed utilizing imaging flow cytometry. Whereas
the
uEV composition did not change across the 24 hours analysis, the quantity of uEVs
and
related markers fluctuated during the day depending on the hydration and excretion
rate.The
results of a 24 hour urine collection reflected the average results of all single
voids over
a 24 hr period. Creatinine and particle count normalization failed to normalize
“outliers”.
Summary/Conclusion: This study represents the very first report which compares
single void urine versus 24 hour uEV analysis. We concluded that the 24 hour collection
is
the preferred choice for a robust and rigorous assessment of uEVs and its associated
markers.
PF05.09
Porcine body fluids differ in small extracellular vesicle
counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle
sources for proteomic analyses
Helena Kupcova Skalnikova
a, Jakub
Cervenkab, Jaromir Novaka, Karolina Turnovcovac, Bozena
Levinskaa, Jana Juhasovaa, Stefan Juhasa and Petr
Vodickaa
aInstitute of Animal Physiology and Genetics, Czech Academy
of Sciences, Libechov, Czech Republic; bInstitute of Animal Physiology and
Genetics CAS, v. v. i. Libechov, Libechov, Czech Republic; cInstitute of
Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic
Introduction: Extracellular vesicles (EVs) released from cells to body fluids
are extensively studied as potential carriers of nucleic acid and protein biomarkers,
particularly in cancer research. Our aim was to compare small EV content in body fluids
of
the biomedical porcine model to assess their suitability for subsequent proteomic
analyses.
Methods: Small extracellular vesicles were enriched from freshly collected
porcine blood (plasma), cerebrospinal fluid (CSF) and semen by ultracentrifugation.
Size,
quantity and quality of isolated particles were characterized by transmission electron
microscopy, flow cytometry and western blots. Protein composition of seminal plasma-derived
vesicles was analysed using LC-MS/MS (tripleTOF with SWATH quantification).
Results: Seminal plasma yielded twice the number of small EVs than the blood
plasma. Approximately 13times less EVs were obtained from CSF compared to seminal
plasma.
Proteomic analysis of seminal plasma EVs resulted in approx. 1500 identified proteins
including proteins involved in exosome biogenesis and transport (74 of the 100 most
frequently identified proteins in EVs according to Exocarta database).
Summary/Conclusion: Porcine seminal and blood plasma (single ml volumes) are
reasonable sources of EVs for proteomic analyses. In contrast, EV counts in CSF are
very
low. Techniques for the EV enrichment and proteomic analysis implemented in this study
may
be applied to biomarker discovery in porcine model of diseases as well as adopted
to other
species, including human.
Funding: Operational Program Research, Development and Education
(CZ.02.1.01/0.0/0.0/16_019/0000785), Czech Science Foundation (19–01747 S), National
Sustainability Programme I. of the Czech Ministry of Education, Youth and Sports
(LO1609).
PF05.10
Identification of potential pancreatic cancer therapeutic
targets in the cargo of extracellular vesicles released by human macrophages
Cristina Xavier
a, Inês
Castrob, Hugo R. Cairesb, Dylan Ferreirac, Bruno
Cavadasb, Luisa Pereirab, Lúcio L. Santosc, Maria josé
Oliveirad and Maria Helena Vasconcelose
ai3 S – Instituto de Investigação e Inovação em Saúde,
Universidade do Porto, Portugal, Porto, Portugal; bi3 S (Instituto de
Investigação e Inovação em Saúde, Universidade do Porto), Porto, Portugal; cIPO –
Instituto Português de Oncologia, Porto, Portugal, Porto, Portugal; di3 S –
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; Tumour
&
Microenvironment Interactions Group, INEB – Instituto Nacional de Engenharia Biomédica,
Universidade do Porto;, Porto, Portugal; ei3 S – Instituto de Investigação e
Inovação em Saúde, Universidade do Porto; Cancer Drug Resistance Group – IPATIMUP
–
Instituto de Patologia e Imunologia Molecular da Universidade do Porto; FFUP – Faculdade
de
Farmácia da Universidade do Porto, Porto, Portugal
Introduction: Macrophages contribute to therapy inefficiency in pancreatic
ductal adenocarcinoma (PDAC), possible through secretion of extracellular vesicles
(EVs). We
aimed to understand the impact of EVs released by macrophages on PDAC cellular response
to
gemcitabine (GEM) and to identify novel targets for therapeutic intervention.
Methods: EVs released by polarized pro-inflammatory or anti-inflammatory
macrophages, derived from human healthy blood donors, were isolated by differential
centrifugation. EVs were characterized by Nanoparticle Tracking Analysis, Transmission
Electron Microscopy and Western Blot. Response to GEM was analysed in PDAC cells with
or
without prior co-culture with EVs, using SRB assay. Protein content of EV’s cargo
was
assessed by proteomic analysis. Potential candidates interfering with GEM response
were
identified and confirmed using human recombinant proteins (rh) or specific pharmacological
inhibitors. In addition, tissue samples from 3 metastatic PDAC and from healthy
non-pathological tissues were immunohistochemically stained for the identified proteins,
as
well as for CD68, a macrophage lineage marker. Using The Cancer Genome Atlas (TCGA)
database, we assessed the association of the identified candidates with the overall
survival
of 176 PDAC patients.
Results: Our results showed that large EVs (10 k-centrifugation pellet) shed
by the distinctly polarized macrophages decreased BxPC3 PDAC cellular sensitivity
to GEM, in
an EVs-concentration dependent manner. Proteomic analysis of those EVs identified
Chitinase
3-like 1 (CHI3L1) and Fibronectin (FN) as one of the most abundant proteins. Both
EVs and
rhCHI3L1 or rhFN induced GEM resistance, involving the ERK signalling pathway. Moreover,
Pentoxyfilline and Pirferidone, two FDA-approved drugs inhibitors of CHI3L1 and FN,
respectively, increased PDAC cellular sensitivity to GEM. Immunohistochemistry data
confirmed that CHI3L1 and FN are expressed in the stroma of human PDAC tumour samples,
associated to the presence of macrophages, and have no relevant expression in healthy
pancreas. Using TCGA, we also found an association between CHI3L1 and FN gene expression
with PDAC patients’ overall survival, response to GEM and macrophage infiltration.
Summary/Conclusion: This work highlights the relevance of EVs shed by human
macrophages to GEM response and identified CHI3L1 and FN as potential therapeutic
targets in
PDAC.
Funding: CPR Xavier is supported by FCT, Portugal (SFRH/BPD/122871/2016). The
work was financed by FEDER through the COMPETE 2020 – POCI, Portugal 2020, and by
FCT in the
framework of the project “Institute for Research and Innovation in Health Sciences”
(POCI-01-0145-FEDER-007274).
PF06: EVs in Reproduction and Pregnancy
Chair: Carlos Salomon, MSc, DMedSc, PhD – Exosome Biology Laboratory, Centre for
Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal
Brisbane and Women’s Hospital, The University of Queensland
PF06.01
In vivo derived bovine blastocyst secrete different population
of extracellular vesicles compared to vitro produced counterparts
Miguel Gutierrez, Joel Cabezas, Daniel Veraguas, Fidel Ovidio Castro
and Lleretny Rodriguez-Alvarez
Universidad de Concepcion, Chillan, Chile
Introduction: In vitro produced (IVP) bovine embryos secrete extracellular
vesicles (EVs) that participate in cell communication. Cargo and characteristics of
EVs
(size mean: SM, size mode; SMo and concentration: C) vary during preimplantation development
according to the origin and competence of embryos. In vivo produced (IVV) embryos
have
better developmental potential than IVP. Here we aim to evaluate if IVV embryos secrete
different population of EVs compared to IVP counterparts.
Methods: For IVV embryos, heifers were superovulated and inseminated with
commercial semen. Embryos were recovered at morula stage and cultured individually
in EVs
depleted media (DOFd) until day7. IVP embryos were produced by in vitro fertilization
and
cultured in groups (25 zygote per well) until morula stage, at this point were cultured
further individually in SOFd. At day 7, all embryos were classified according to
developmental stage and culture media (CM) were collected from embryos that reached
the
blastocyst stage. Blastocysts were transferred to fresh media until day 11 to assess
their
post-hatching competence. CM from competent embryos, (diameter > 270 μm) were used
for
EVs analysis (IVV: n = 26; IVP: n = 30). EVs were analysed using NTA. Data were analysed
by
Wilcoxon test or Pearson correlation. Statistic signification for p < 0.05.
Results: At day 7 IVV embryos had a greater diameter than IVP (IVV: 190.7 μm;
IVP: 165.7 μm; p < 0.05). However, IVP embryos reached a bigger diameter at day 11
(IVV:
289.9 μm; IVP: 381.9 μm; p < 0.05). Isolated particles from CM of both groups were
classified as EVs by their morphology using TEM and by the expression of surface markers
CD9, CD63 and CD81. EVs parameters for IVV were: SM = 149.6 nm; SMo = 116.4, C = 1.2
x108xml
while for IVV: SM = 124.7 nm; SMo = 93.5 and C = 1.5x108xml. Both SM and SMo were
statistically different between groups. Also SM and SMo positively correlate with
embryo
diameter at day 7.
Summary/Conclusion: IVV embryos secrete bigger EVs than IVP, what may point to
a higher competence of in vivo derived embryos. This coincides with our previous finding
that competent IVP embryos secret bigger EVs compare to non-competent IVP embryos
(Mellisho
et al., 2019).
Funding: Supported by Fondecyt 1170310.
PF06.02
Extracellular vesicles as messengers in early embryo-maternal
communication
Joanna Najmula and Monika M. Kaczmarek
Institute of Animal Reproduction and Food Research of Polish Academy of
Sciences, Olsztyn, Poland
Introduction: Behind the beauty of each pregnancy, stands an incredible
complexity. The most critical stage of the pregnancy, unfortunately marked by a high
rate of
pregnancy loss, is the embryo implantation. Its success depends on a proper embryo
development synchronised with timely acquired uterine receptivity. Extracellular vesicles
(EVs) are considered now as important players in embryo-maternal communication. Despite
the
ongoing effort, still little is known about precise role of EVs in modulation of
embryo-maternal milieu. Thus, the overarching aim of this study was to determine the
effect
of the delivery of uterine EVs to primary porcine trophoblast (pTr) cells in vitro.
Methods: Several methods (e.g., Western blot, NTA) were used to characterize
EVs isolated from porcine uterine lumen during early pregnancy (day [D] 12 and D16).
TaqMan
Array Cards were applied to profile miRNAs carried by uterine EVs. Ingenuity Pathway
Analysis tools were used to identify in silico biological pathways and functions governed
by
detected miRNAs. Expression of putative targets of selected miRNAs was tested using
qPCR
after in vitro delivery of uterine EVs to pTr cells.
Results: Careful characterisation confirmed that uterine lumen is enriched
with a diverse population of EVs caring miRNAs. Interestingly, 36 out of 79 detected
miRNAs
showed difference in abundance between tested days of pregnancy and half of them was
exclusively detected on D16. Identified miRNAs were characterized as potent regulators
of
cellular development, growth, proliferation, and movement, in addition to their involvement
in organismal and embryonic development. The expression of 20 genes identified as
a possible
miRNA targets was tested after EVs delivery to pTr cells in vitro. Both down- (e.g.,
PTGER4)
and up-regulated (e.g., LIFR) genes were found (p < 0.05); involved in the same molecular
and cellular functions enriched by detected miRNAs.
Summary/Conclusion: Uterine lumen is enriched with EVs transporting miRNAs,
which may have an impact on proper embryo development and implantation. In addition,
delivery of uterine EVs to the trophoblast cells affect the expression of miRNA targets
involved in embryo-maternal communication. Altogether, these results show an important
role
of EVs and their cargo during early pregnancy in pigs.
Funding: Grants: 0041/DIA/2014/43, 2014/15/B/NZ9/04932,
2016/21/N/NZ9/03443
PF06.03
Arrdc4-dependent extracellular vesicle biogenesis is required
for sperm maturation
Natalie J. Foot
a, Macarena Bermudez
Gonzalezb, Kelly Gembusc, Jarrod Sandowd, Diana
Tranb, Andrew Webbd, Rebecca Robkerb and Sharad
Kumarc
aUniSA, Adelaide, Australia; bUniversity of
Adelaide, Adelaide, Australia; cUniversity of South Australia, Adelaide,
Australia; dWalter and Eliza Hall Institute, Melbourne, Australia
Introduction: Extracellular vesicles (EVs) are emerging as important players
in cell to cell communication in reproductive systems. Notably, EVs have been found
and
characterized in the male reproductive tract, however direct functional evidence for
their
importance in mediating sperm function is lacking. We have previously demonstrated
that
Arrdc4, a member of the alpha-arrestin protein family, is involved in EV biogenesis
and
release. Here we show that Arrdc4-mediated EV biogenesis is required for proper sperm
function.
Methods: EVs were harvested from wild type and Arrdc4-/- epididymal cells
using differential ultracentrifugation, then characterised using Nanoparticle Tracking
Analysis and transmission electron microscopy. Sperm motility was measured using Computer
assisted sperm analysis and ImageJ. Fertilisation capacity was measured using the
following
assays: Capacitation-associated tyrosine phosphorylation, calcium ionophore induced
acrosome
reaction, Zona pellucida binding assay and In vitro fertilization with time-lapse
imaging of
embryo development. Immunohistochemistry was also used to visualise two pronuclei
formation
and blastocyst morphology. Arrdc4-/- sperm was supplemented with wild type EVs in
the above
assays to assess whether they could restore function.
Results: Sperm from Arrdc4-/- mice develop normally through the testis but
fail to acquire adequate motility and fertilization capabilities through the epididymis,
as
evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida
binding and production of two-cell embryos. We observed a significant reduction in
EV
production by Arrdc4-/- epididymal epithelial cells, and addition of wild type EVs
to
Arrdc4-/- sperm dampens the acrosome reaction and restores zona pellucida binding.
Summary/Conclusion: These results indicate that Arrdc4 is important for proper
sperm maturation through the control of extracellular vesicle biogenesis.
Funding: NHMRC
PF06.04
Maternal EV-derived myomiRs are associated with the development
of large-for-gestational age babies in pregnancies complicated by gestational
diabetes
Margeurite Kennedy
a, Sarah
Cartlandb, Nigel Simpsonb, Eleanor Scottb and Karen
Forbesb
aUniversity of Leeds, Leeds, UK; bUniveristy of
Leeds, Leeds, UK
Introduction: Gestational diabetes (GDM) is among the most common pregnancy
complications. Despite treatment, up to 25% of pregnancies complicated by GDM result
in
infants being born large-for-gestational-age (LGA). This not only causes problems
at birth
but predisposes offspring to developing cardio-metabolic disease in adulthood. There
are no
treatments for LGA as the cause is unclear, although it is associated with altered
placental
vascular development. microRNAs (miRNAs) regulate placental development; they are
produced
within cells but can be released into the circulation inside EVs, which in turn can
be
transported into target cells and tissues to influence cellular processes.
We aimed to characterise circulating EVs in pregnancies complicated by GDM-LGA and
determine if EV-derived miRNAs have the potential to influence placental development.
Methods: Maternal serum and plasma samples were collected from women with
pregnancies complicated by GDM at 24–32 weeks gestation; placental tissue was collected
at
delivery and birth outcomes recorded. Serum and plasma EVs were isolated and characterised
by electron microscopy (shape), nanoparticle tracking analysis (NTA; size/concentration),
and Western blotting (EV-enriched proteins). miRNA QPCR arrays were performed on EVs.
miRNAs
were quantified in placental tissue via QPCR.
Results: EM and Western blotting confirmed isolation of EVs and NTA revealed
no significant difference in size/concentration in GDM-LGA pregnancies (n = 7) compared
to
GDM-AGA (n = 13; p > 0.05). Several EV miRNAs were altered in maternal circulation
in
GDM-LGA compared to GDM-AGA (n = 7/group; >twofold-change; p < 0.05), including four
skeletal muscle-specific “myomiRs”: miR-1-3p, miR-133a-3p, miR-133b, and miR-499a-3p
(all
increased). All four myomiRs were present in placenta but only miR-1-3p was significantly
altered in GDM-LGA compared to GDM-AGA (n = 12–14/group; p < 0.05).
Summary/Conclusion: EV-bound myomiRs could have predictive value for aberrant
foetal growth in cases of GDM. miR-1-3p regulates vascular development in other systems,
so
we propose that miR-1-3p contributes to LGA by influencing placental vascular development,
however further work is required to establish this.
Funding: Medical Research Council, University of Leeds Doctoral
Scholarship
PF06.05
A new type of nanovesicles in seminal plasma: the
myelinosomes
Celia Ravel
a, Anne -Sophie
Neyroudb, Nicolas Bourmeysterc and Marina Yefimovad
aCHU de Rennes, Rennes, France; bCHU de Rennes,
Rennes, France; cUniversité de Poitiers, Poitiers, France; dRussian
Academy of Sciences/Sechenov Institute, Saint petersbourg, Russia
Introduction: Seminal plasma is particularly rich in extra cellular vesicles.
Myelinosomes are membranous organelles described throughout the seminiferous epithelium
of
the testis but never reported in semen. The aim of this study was to look for the
presence
of myelinosome vesicles in human seminal plasma.
Methods: Because of the viscosity of seminal gel and its water-holding
capacity, classical transmission electron microscopy does not seem to be an optimal
technique to reveal the presence of myelinosomes in this fluid. Cryo-electron microscopy
is
a technique that allows visualization of nanosized structures without prior fixation
or
addition of heavy metals for contrast. The sample is therefore visualized as close
to its
native state as possible. Using standard myelinosome preparation from TM4 Sertoli
cells, we
first analysed the appearance of “standard” native myelinosomes by cryo EM and then
compared
it with the vesicles from human seminal plasma samples.
Results: We have specified by cry-EM the morphological aspect of “standard”
myelinosomes isolated from the culture media of TM4 Sertoli cells. The vesicles with
the
same morphological appearance were revealed in human seminal plasma specimens.
Summary/Conclusion: Myelinosomes are membranous organelles found in the
seminiferous epithelium of the testis and secreted by the somatic Sertoli cells in
the lumen
of the seminiferous tubules.The preparations from human seminal plasma contains a
population
of large EV (average diameter 200 nm) whose morphological appearance resemble those
of
myelinosomes. Defining the specific biomarkers and functionalities of myelinosomes
in human
seminal plasma are the concerns to be addressed in our further research.
Funding: CHU de Rennes
Univ Rennes Inserm, Irset (Institut de recherche en santé, environnement et travail)
–
UMR_S 1085,
PF07: EVs in Kidney, Urinary Tract, and Related Diseases
Chair: Luca Musante – University of Virginia School of Medicine
Chair: Dylan Burger, PhD – Chronic Disease Program, Ottawa Hospital Research
Institute
PF07.01
Tuberous sclerosis complex axis controls extracellular vesicles
production and protein content
Prashant Kumar
a, Fahad
Zadjalia, Ying Yaoa and John J. Bisslerb
aUTHSC, Memphis, USA; bUTHSC, Eads, USA
Introduction: More than one million patients world-wide suffer from tuberous
sclerosis complex (TSC) and have mutations in either TSC1 or TSC2 genes. Together,
the TSC
proteins regulate mTORC1 activity. All TSC patient post-mortem samples exhibit renal
disease
and 40% of patients with TSC experience a premature loss of renal function. Mouse
and human
studies are incongruity with the second somatic hit mechanism of disease, because
of the low
percentage of cystic cells exhibiting loss of TSC expression. We posited that the
loss of a
TSC protein expression may alter extracellular vesicle (EV) biology and contribute
to
disease.
Methods: We used CRISPR/CAS9 to disrupt the Tsc2 gene in mouse inner medullary
collecting duct (mIMCD) cells, and isolated EVs using gel filtration from the isogenic
cell
lines. We characterized the EVs using tunable resistive pulse sensing (TRPS), dynamic
light
scattering (DLS), transition electron microscopy (TEM), and wester blot analysis.
We further
performed mass spectroscopy on the EV proteins.
Results: Loss of the Tsc2 gene in mIMCD cells induced a greater than
three-fold increase in EV production compared to the same cells having an intact Tsc
axis.
Electron microscopy confirmed the purity and spherical shape of EVs. Both TRPS and
DLS
demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately
90% of the EVs were in the 100–250 nm size range. Western blot analysis using proteins
isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane
proteins CD63, CD81 and CD9, and the primary cilia-related Hedgehog signalling-related
proteins Arl13b. Proteomic analysis of EVs identified a significant difference between
the
Tsc2-intact and Tsc2-deleted cells that correlated well with the increased production.
Summary/Conclusion: EVs may be involved in tissue homoeostasis and cause
disease by overproduction and altered protein content. The EVs released by renal cyst
epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis
and in developing potential therapeutic strategies.
Funding: DoD grant W81XWH-14-1-0343
PF07.02
Extracellular vesicles derived from human amniotic fluid stem
cells: characterization and therapeutic effect in a model of chronic kidney
disease
Charmi Dedhiaa, Paola Aguiaria, Astgik
Petrosyana, Jo-Anna Reemsb, Hasmik Solayana, Johnny
Akersc, Mya Thud, Sargis Sedrakyand and Laura
Perin
d
aChildren’s Hospital Los Angeles, Los Angeles, USA;
bUniverisity of Utah, Sallt Lake sity, USA; cVisicell Medical, San
Diego, USA; dChildren’s Hospital Los Angeles-University of Southern California,
Los Angeles, USA
Introduction: We have shown that EVs derived from amniotic fluid stem cells
(AFSC) of mouse origin present therapeutic effect in an animal model of chronic kidney
disease, Alport Syndrome (AS). In light of clinical translation, we isolated AFSC-EVs
of
human origin, characterized their cargo and evaluated thier therapeutic effect in
vivo.
Methods: Human clonal AFSC were derived from amniotic fluid collected after
volunteer donors provided consent. EVs were obtained from AFSC and identity and purity
were
assessed by RNA-seq and proteomics. Potency of hAFSC-EVs was evaluated by performing
in vivo
studies. EV biodistribution was evaluated by MRI and therapeutic effect by measuring
renal
function and mice life-span. Bulk RNA-seq was performed on glomeruli obtained from
injected
and non-injected mice to identify potential EV regulating targets.
Results: Proteomic profiling identified 675 intact proteins and RNA-seq data
identified 2,535 miRs in hAFSC-EVs. hAFSC-EV “fingerprint” was assessed by performing
GO
analysis on the 100 most highly expressed proteins and miRs. The results identified
pathways
involved in tissue homoeostasis such as mTOR pathway, TGFβ and VEGF pathways. When
injected
in vivo into AS mice, biodistribution studies showed that hAFSC-EVs localized in the
kidney,
corrected proteinuria. No side effects (including teratoma) were noted in the treated
mice.
RNA-seq of glomeruli obtained from treated AS mice showed similar gene expression
patterns
to wilt type mice, by cluster analysis. Our data indicated that hEVs highly modulated
pathways involved in collagen and matrix deposition remodelling, in addition to downstream
targets of VEGF, FGF, TNF, angiotensin and preserved glomerular cells structure and
function.
Summary/Conclusion: Our protocol for hEVs derivation is reproducible and
allows derivation of EV lots with the same identity (specific cargo of proteins and
miRs)
and potency (present therapeutic effect in AS). hAFSC-EVs modulated signalling pathways
that
are central to maintaining glomerular homoeostasis and preserved glomeruli structure
with
improved kidney function. This suggests the possibility of using hAFSC-EVs as a new
therapeutic option for treating renal failure in humans.
Funding: -Intramural CHLA funding
-Pilot Imaging CHLA Core Funding
PF07.03
A millifluidic in vitro model of glomerular filtration to test
the regenerative effect of extracellular vesicles under dynamic conditions
Linda Bellucci and Benedetta Bussolati
University of Torino, Torino, Italy
Introduction: Recent studies have shown that stem cell-derived extracellular
vesicles (MSC-EV) therapy improves renal outcomes in models of acute ad chronic renal
disease. However, to better investigate the molecular mechanisms of EV-induced regeneration,
and to define new EV sources, devices that mimic 3D organ architecture and flow conditions
are needed. The aim of our work is to evaluate the regenerative potential of naïve
and
engineered EV in a millifluidic in vitro 3D model of glomerular damage in continuous
perfusion.
Methods: Methods: we set a millifluidic in vitro 3D model of glomerular
filtration, a three-layers structure composed by human podocytes and glomerular endothelial
cells, and, in between, of a basement membrane of Collagen type IV. The barrier thus
formed
is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows
continuously at a certain flow rate. We reproduced different pathological conditions
and
tested the localization and effect of EVs in a dynamic system.
Results: Results: we obtained a standardized protocol and an adequate
configuration of the milli-fluidic circuit subject to continuous reperfusion. Renal
damage
was induced by doxorubicin or by hypoxia-reperfusion injury. We evaluated uptake,
cargo
transfer and effect of naïve and miRNA engineered MSC-EVs or of Klotho engineered
ineffective EVs administered into the dynamic co-culture system. EVs were able to
pass
through the system and to deliver to podocytes pro-regenerative factors, promoting
survival
and limiting permeability.
Summary/Conclusion: In conclusion, we highlighted the effects of MSC naive EVs
as well as of engineered EVs in a model of glomerular damage under EV continuous
perfusion.
Funding: This study was supported by Regione Piemonte POR FESR 2014/2020 –
Bando Piattaforma Tecnologica Salute e Benessere – Project “Terapie Avanzate per Processi
Fibrotici Cronici (EVER).
PF07.04
Extracellular vesicles from kidney cancer tissue and comparison
of two quantitative methods
Richard Zieren
a, Liang
Donga, Kengo Horiea, Sarah Amenda, Theo de
Reijkeb, Phillip Pierorazioa and Kenneth Pientaa
aThe Brady Urological Institute, Johns Hopkins University
School of Medicine, Baltimore, USA; bAmsterdamUMC, University of Amsterdam,
Amsterdam, Netherlands
Introduction: Worldwide, renal cell carcinoma (RCC) is 8th most common cancer
in men and 10th most common in women. New biomarkers are needed to aid RCC-diagnosis,
provide prognostic information, and to predict response to modern targeted therapies.
Extracellular vesicles (EVs) are an emerging source of cancer biomarkers because all
cells,
including cancer cells, secrete EVs into biofluids as blood and urine. However, benign
cells
contribute to EV populations isolated from blood and urine reducing the disease-specificity.
We have developed a protocol for EV isolation directly from human RCC tissue that
can
increase tumour-specificity of biomarkers.
Methods: We obtained technical and biological replicates from normal kidney
tissue and clear cell RCC tissue. Serum-free media was incubated with the specimens.
A
combination of differential centrifugation, filtration, and ultracentrifugation was
used for
EV isolation. EVs were quantitated using two methods, allowing for comparison between
NanoSight NS300 and NanoFCM. TEM was used to determine presence of intact vesicles
in the EV
samples. Presence of EV protein markers (CD81, CD63, flotillin-1), and absence of
cellular
debris (calnexin), were assessed using Western Blot.
Results: Particle concentrations for the technical replicates of normal kidney
EVs were 1.44 × 1010 ± 2.51 x 109 p/mL (mean ± SD) measured by NanoSight, and
1.67 × 1010 ± 6.89 x 109 p/mL (mean ± SD) measured by NanoFCM. Vesicle concentrations
for
replicates RCC EVs were 1.80 × 1010 ± 3.59 x 109 p/mL (mean ± SD) measured by NanoSight
and
1.68 × 1010 ± 4.18 x 109 p/mL (mean ± SD) measured by NanoFCM. Among different patients,
we
observed an acceptable biological variance in EV counts. A head-to-head comparison
of
NanoSight and NanoFCM demonstrated differences in total particle counts of less than
fivefold. NanoFCM performed better in measuring particle size distribution. Small
EVs were
visible on TEM images in all technical and biological replicates. EV markers were
positive
in all samples and calnexin was negative.
Summary/Conclusion: We optimized EV isolation from RCC tissue and normal
kidney. The product of our protocol contains small EVs in high abundance. The protocol
can
contribute to study tumour microenvironment and biomarker discovery in kidney cancer.
Funding: This work was supported by Cure for Cancer foundation (Amsterdam, The
Netherlands), the Prostate Cancer Foundation, the Patrick C. Walsh Prostate Cancer
Research
Fund, the William and Carolyn Stutt Research Fund, and the National Cancer Institute.
PF07.05
Tetraspanins on serum derived-EVs enable detection of renal cell
carcinoma
Md Khirul Islam
a, Parvez
Syeda, Janne Leivoa, Bert Dhondtb, Kim
Petterssona and Urpo Lamminmäkia
aUniversity of Turku, Turku, Finland; bLaboratory
of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University,
Belgium; Cancer Research Institute Ghent, Belgium, Ghent, Belgium
Introduction: Renal cell carcinoma (RCC) is a lethal urological cancer with an
incidence rate which accounts for about 3% of all human cancers. Nearly 40% of RCC
patients
are diagnosed with either locally invasive or metastatic disease. Added to this, there
is a
dearth of reliable biomarkers for early diagnosis. These facts highlight the urgent
need for
novel non-invasive diagnostic tools. Extracellular vesicles (EVs) are considered a
promising
biomarker target for diagnosis of various malignancies. However, investigation of
EVs
typically demands isolation of them from body fluids, which is difficult and time-consuming
process. The aim of our study was to develop EV-based assay for the early and non-invasive
detection of RCC using a highly sensitive nanoparticle-aided time-resolved fluorescence
immunoassay (TRFIA).
Methods: EVs from the serum of renal cell carcinoma (RCC), bladder cancer
(BlCa), benign and healthy samples were captured with biotinylated
anti-tetraspanin-antibodies (CD63 or CD81) immobilized on streptavidin coated microtitration
wells. The captured EVs were detected using 95 nm europium dyed nanoparticles (polysterene
beads packed with ~30,000 Eu3+ chelates) conjugated with anti-tetraspanin-antibodies
(CD63
or CD81). Isolated EVs-derived from four-cancer cell lines was taken to validate this
TRFIA.
Serum samples from RCC (n = 14), BlCa (n = 14), benign prostate (n = 14), and healthy
(n = 10) controls were analysed. This study was conducted following the guidelines
of
Helsinki Declaration. Participants had given written informed consent.
Results: This TRFIA can measure purified-EVs with high sensitivity. The
CD63-CD63 assay enabled significant discrimination of RCC patients from BlCa (4.3-fold,
p = 0.001), benign (fivefold, p = 0.0007) and healthy (2.8-fold, p = 0.010) controls.
Similarly, the CD81-CD81 assay also showed a discrimination of RCC patients from BlCa
(6.3-fold, p = 0.001), benign (3.7-fold, p = 0.020) and healthy (1.8-fold, p = 0.003)
samples.
Summary/Conclusion: The results obtained from this study suggest that the EVs
derived from the serum of patients with pathological conditions display varying amounts
of
tetraspanins. Detection of such varied expression of tetraspanins using non-invasive
techniques may play a major role in the early detection of RCC. Further validation
with
large cohort of samples is required.
Funding: DPMLS-Graduate school, University of Turku, Finland.
PF07.06
Extracellular vesicles as a source of cancer biomarkers in the
urine of urothelial carcinoma patients: characterization of a two-step protocol for
the
isolation of urine-derived EVs
Ana Margarida Barreiraa, Manuel Castanheira
Oliveirab, Avelino Fragab, Maria josé Oliveirac, Maria
Helena Vasconcelosd, Ricardo Ribeiroe and Hugo R.
Caires
f
ai3 S – Instituto de Investigação e Inovação em Saúde,
Universidade do Porto, Portugal; Cancer Drug Resistance Group – IPATIMUP – Instituto
de
Patologia e Imunologia Molecular da Universidade do Porto;, Porto, Portugal;
bi3 S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto,
Portugal; Tumour & Microenvironment Interactions Group, INEB – Instituto Nacional
de
Engenharia Biomédica, Universidade do Porto; Department of Urology, Centro Hospitalar
do
Porto;, Porto, Portugal; ci3 S – Instituto de Investigação e Inovação em Saúde,
Universidade do Porto, Portugal; Tumour & Microenvironment Interactions Group, INEB
–
Instituto Nacional de Engenharia Biomédica, Universidade do Porto;, Porto, Portugal;
di3 S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto;
Cancer Drug Resistance Group – IPATIMUP – Instituto de Patologia e Imunologia Molecular
da
Universidade do Porto; FFUP – Faculdade de Farmácia da Universidade do Porto, Porto,
Portugal; ei3 S – Instituto de Investigação e Inovação em Saúde, Universidade do
Porto, Portugal; Tumour & Microenvironment Interactions Group, INEB – Instituto Nacional
de Engenharia Biomédica, Universidade do Porto; Laboratory of Genetics and Instituto
de
Saúde Ambiental, Faculdade de Medicina da Universidade de Lisboa, Porto, Portugal;
fi3 S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto;
Cancer Drug Resistance Group – IPATIMUP – Instituto de Patologia e Imunologia Molecular
da
Universidade do Porto, Porto, Portugal
Introduction: Urothelial carcinoma (UC) is a malignant cancer that affects the
urothelial cells, representing 90% of all bladder tumours. At diagnosis 75% of bladder
cancers are non-muscle invasive tumours. Importantly, upon transurethral resection
of the
bladder tumour, nearly 35–80% of these patients will experience disease relapse and
10–20%
will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive
follow-up. Currently, this is performed through the frequent use of highly invasive
cystoscopy and the low sensitivity urine cytology. Thus, innovative liquid biopsy-based
biomarkers that circumvent these drawbacks are highly desirable for improved UC clinical
management. Here, we AIM to implement a protocol for the isolation and characterization
of
extracellular vesicles (EVs) from UC patients’ urine samples.
Methods: A two-step protocol involving ultracentrifugation (UCt) and by
size-exclusion chromatography (SEC) was optimized for urine samples. The isolated
urine-derived EVs from 9 UC patients were then characterized according to their size,
concentration (NTA), morphology (TEM), protein amount (Lowry method), presence of
EV-associated and disease-associated protein markers (Western blot).
Results: Isolated urinary EVs from UC patients had a size ranging from 50nm to
400 nm with characteristic EV morphology, express EV-associated markers as CD63 and
HSP70
and were negative for cell debris markers. The recovery yield and purity of isolated
EVs
following each isolation technique was characterized. Upon UCt, SEC was required to
deplete
most of the EV-associated THP and albumin protein contaminants. Some disease-associated
protein markers were highly enriched in isolated urinary EVs compared to crude urine.
Summary/Conclusion: Taken together, these results indicate that a two-step EV
isolation protocol was properly implemented and validated in UC patients’ urine samples.
Notably, several EV-associated disease biomarkers were detected in the urine of UC
patients.
This EV-based liquid biopsy might provide the means for real-time monitoring of residual
disease and relapse in UC patients.
PF08: EVs in Cancer Pathogenesis
Chair: Jeffrey Franklin – Vanderbilt University Medical Center
Chair: Aurelio Lorico – Roseman University of Health Sciences
PF08.01
Extracellular vesicles are involved in the expansion of the
mesenchymal signature in glioblastoma tumours
Thomas Simon
a, Rosemary
Lanea, Christos Toliasa, Giles Critchleyb, Nicolas A.
Stewartc and Georgios Giamasa
aUniversity of Sussex, Brighton, UK; bBrighton and
Sussex University Hospitals, Brighton, UK; cUniversity of Brighton, Brighton,
UK
Introduction: Glioblastoma multiforme (GBM) is a very aggressive type of brain
tumour. Different GBM molecular subtypes (proneural, mesenchymal and classical) often
co-coexist within the same tumour, with the mesenchymal subtype driving the tumour
progression. Recently, our lab demonstrated that the cargo of extracellular vesicles
(EVs)
could mirror the molecular background of the GBM cells from which they were derived.
Altogether, we believe that GBM cell-derived EVs can be directly involved in the expansion
of the mesenchymal signature in tumours, thus supporting GBM aggressiveness.
Methods: Non-mesenchymal (T98 & U138) GBM cells were “primed” using EVs
derived from mesenchymal-like (U87 & LN18) GBM cells. EV-primed GBM cells were then
co-cultured with their non-primed counterparts to determine whether the mesenchymal
signature can “spread” from cell to cell via EVs. Effect on cell proliferation, migration
and invasion (in hyaluronic acid hydrogels) was assessed following EV treatment and
co-culture. The expression of mesenchymal GBM markers was measured by western blotting.
Further mass spectrometry analysis of cell and EV content was undertaken to describe
potential underlying mechanisms.
Results: Co-culture with EV-primed GBM cells significantly increased
proliferation and hydrogel invasiveness of non-mesenchymal cells. Interestingly, the
stimulating effect of co-culture was even stronger on the proliferation of EV-primed
GBM
cells. Moreover, further proteomic analysis revealed that expression of mesenchymal
GBM
markers such as CD44 was increased in non-mesenchymal cells following co-culture.
Summary/Conclusion: Our data suggest that EVs from mesenchymal GBM cells can
be uptaken by GBM cells from different subtypes, thus stimulating tumour progression.
Overall, we think the present study provides with new insights for the understanding
of GBM
recurrence and the development of potential therapeutic strategies.
Funding: This work is supported by Action Against Cancer, The Colin McDavid
Family Trust, The Rothschild Foundation, The Bernard Sunley Foundation, The Searle
Memorial
Charitable Trust, Mr Alessandro Dusi and Mr Milan Markovic.
PF08.02
Inhibition of extracellular vesicle release in triple negative
breast cancer
Niamh McNamee
a, Mariadelva
Catalanob, Anindya Mukhopadhyab and Lorraine O’
Driscollb
aTrinity College Dublin, Dundalk, Ireland;
bTrinity College Dublin, Dublin, Ireland
Introduction: Triple-negative breast cancer (TNBC) is the most aggressive form
of breast cancer. Previously we reported that the heterogenous population of EVs released
from TNBC cells promotes the growth and aggression of recipient cells. Here we investigated
if, by using compounds proposed to inhibit EV release i.e. calpeptin and Y27632 (to
block
those budding at cell membrane) and GW4869 and manumycin A (to block EVs from MVBs),
we
could reduce the associated transmission of aggressive phenotype.
Methods: EVs were separated from medium conditioned by TNBC cell line
Hs578Ts(i)8, using a discontinuous optiprep density gradient, after the cells were
treatment
for 48 hrs with the compounds listed above. EVs (pooled fractions 3–9 with a density
range
of 1.03–1.16 g/mL) were characterised by NTA, BCA, lipid assay, immunoblot, TEM and
flow
cytometry. To investigate the functional effects of the EVs released, proliferation
and
migration assays were performed on Hs578T and MDA-MB-468 cells using the EV to cell
ratios
of 1 × 105 EVs/3x103 cells, 1 × 106 EVs/3x103 cells, 1 × 107 EVs/3x103 cells to evaluate
dose-response. EV-TRACK ID EV190109 (Score of 88%).
Results: GW4869 significantly (p = 0.035) decreased EV release from
Hs578 Ts(i)8 cells. Manumycin A and a combination of calpeptin and Y27632 (Combo)
decreased
EV release, but significance was not reached. Conversely, calpeptin and Y27632 actually
increased EV release; but not significantly. Of the reduced numbers of EVs released
following GW4869 treatment, HLA-DR+ EVs were significantly (p = 0.032) enriched. None
of the
EVs analysed significantly changed Hs578 T or MDA-MB-468 growth rates. However, EVs
from
cells treated with calpeptin (p = 0.007), GW4869 (p = 0.025), manumycin A (p = 0.01)
and
Combo (p = 0.001) caused significant reduction in MDA-MB-468 migration compared to
the
effects of EVs from untreated cells. Similarly, EV from cells treated with GW4869
(p = 0.011), and Combo (p = 0.018) caused significant reduction in Hs578 T migration.
Summary/Conclusion: While GW4869 was the only compound that caused a
significant decrease in quantities of EV released, the EVs that continued to be released
following treatment with GW4869 or calpeptin and Y27632 significantly reduced migration
of
both recipient cell lines.
Funding: PhD funding: 1252 TCD Scholarship and Carrick Therapeutics Ltd
PF08.03
Extracellular vesicles from highly metastatic lung cancer cells
induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity
in a
16-day mature bronchial epithelium
Ikjot Singh Sohal, Zulaida Soto-Vargas and
Andrea Kasinski
Purdue University, West Lafayette, USA
Introduction: Epithelial-to-mesenchymal (EMT) transition plays an integral
role in cancer metastasis, which is responsible for as much as 90% of cancer mortality.
Cancer exosomes induce EMT in bronchial epithelial cells, however, the epithelial
cells
inhibit EMT when allowed to form a mature epithelial barrier with apical-basal polarity.
It
is not known if cancer-derived extracellular vesicles (EVs) can induce EMT and more
importantly, barrier disruption in a mature epithelium. Here, we show that EVs from
a highly
metastatic lung cancer cell line (Calu6) are) are not only sufficient to induce EMT
in
non-tumorigenic bronchail epithelial cells (BEAS-2B), but are also capable of disrupting
a
16-day mature bronchial epithelial barrier by significantly reducing TEER, inducing
sixfold
increase in permeability and complete loss of E-cadherin at cell-cell tight junctions.
Methods: BEAS-2B and Calu6 EVs were characterized using electron microscopy,
NanoSight and western blotting for exosome-specific features. For permeability studies,
BEAS-2B cells were cultured in transwell for 16 days to establish an intact epithelium
–
confirmed by measuring TEER (trans-epithelial electrical resistance). Intact BEAS-2B
monolayers were treated with Calu6 EVs at 1, 10 and 20 μg/ml for 24 hrs, and barrier
intactness and permeability were evaluated by measuring TEER, apical-basolateral
translocation of dextran beads and confocal imaging of tight junctions (E-cadherin).
For EMT
experiments, BEAS-2B cells treated with Calu6 EVs at 1 and 10 μg/ml were evaluated
for
E-cadherin and Vimentin levels by qRT-PCR and western blot after 48 hrs.
Results: BEAS-2B and Calu6 EVs were enriched in 50–200 nm size range, and CD9
and CD81 were enriched in the EV fraction in contrast to the cell lysate and vice
versa for
GP96. Calu6 EVs significantly impaired 16-day mature BEAS-2B monolayer’s barrier properties,
which at the highest dose caused 33% reduction in TEER from 27.0 ± 2.5 to 18.2 ± 3.2 Ω.cm2
(n = 4). This was further confirmed by ~sixfold increase in dextran beads’
apical-basolateral translocation in 30 min (14.8 ± 7 ng/ml in control vs 87.3 ± 51 ng/ml
in
treated) (n = 3) and complete loss of E-cadherin expression at cell-cell tight junctions
(n = 3). At the transcript level, Calu6 EVs induced significant downregulation of
E-cadherin
by 36% and upregulation of Vimentin (mesenchymal marker) twofold (n = 3) in BEAS-2B
cells,
indicating transition into mesenchymal phenotype.
Summary/Conclusion: We demonstrated the involvement of EVs derived from highly
metastatic lung cancer cells in inducing EMT in bronchial epithelial cells and epithelial
barrier disruption – the initial stage of the intravasation process.
PF08.04
Grp78 plays a crucial role in the extracellular vesicle-promoted
radioresistance of irradiated head and neck cancer cells
Michael J. Schneider
a, Klaudia
Winklera, Rosemarie Kella, Simone Moertlb and Michael
Atkinsona
aHelmholtz Centre Munich, Munich, Germany;
bFederal Office for Radiation Protection, Munich, Germany
Introduction: Small EVs released from irradiated head and neck squamous cell
carcinoma (HNSCC) cells increase resistance of recipient HNSCC cells to radiation
in vitro.
We have identified the Glucose-regulated protein 78 (Grp78), a chaperone protein of
the
HSP70 family which is involved in cellular stress responses and associated with worse
survival in head and neck cancer patients, as an essential component of the EV-mediated
radioresistance.
Methods: Small EVs were isolated from conditioned medium from irradiated and
non-irradiated BHY HNSCC cells by combined microfiltration (0.22 µm) and differential
ultracentrifugation. Grp78 surface expression was measured by proteomic analysis,
immunoblotting and bead-FACS. Radiation resistance of BHY cells was determined by
a
clonogenic survival assay.
Results: Increased Grp78 was identified on the surface of EVs from irradiated
cells. The increase in EV Grp78 correlated with increased Grp78 expression at the
donor cell
surface. The Grp78 content of recipient cells also increased upon transfer of EVs
from
irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival.
To
check a potential role of elevated Grp78 in radiation resistance we overexpressed
Grp78.
Here the modest (3x) overexpression of Grp78 was sufficient to confer an enhanced
radioresistant phenotype to the BHY cells. A correlation between Grp78-dependent increase
of
radioresistance and activation of the Akt pathway is yet to be determined.
Summary/Conclusion: Our results suggest a pivotal role for EV-transferred
Grp78 in modulating the radiation response of recipient HNSCC cells. Radiation directly
increases the cellular and vesicular Grp78 levels, and subsequent EV-mediated transfer
leads
to enhanced Grp78 levels and radioresistance in recipient cells. This study provides
new
mechanistic insights into the effects of EVs in radiation response and elucidates
an
interesting target protein and novel strategies for the improvement of radiotherapy.
PF08.05
3D modelling of EV release in progressing prostate
cancer
Liliia Paniushkina
a, Martin
Wolfb, Krisztina V. Vukmanc, Laura Bianciardid, Natasa
Zarovnid, Edit Buzáse, Strunk Dirkb and Irina
Nazarenkof
aInstitute for Infection Prevention and Hospital
Epidemiology; Medical Centre – University of Freiburg, Faculty of Medicine, University
of
Freiburg, 79106 Freiburg, Germany., Freiburg, Germany; bCell Therapy Institute,
Spinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI-TReCS), Paracelsus
Medical
University (PMU), Salzburg, Austria, Salzburg, Austria; cDept. of Genetics, Cell-
and Immunobiology, Semmelweis University, Budapest, Hungary, Budapest, Hungary;
dExosomics S.p.A., Siena, Italy, Siena, Italy; eSemmelweis University,
Department of Genetics, Cell and Immunobiology, MTA-SE Immune-Proteogenomics Extracellular
Vesicle Research Group, Budapest, Hungary and HCEMM_SE Extracellular Vesicle Research
Group,
Budapest, Hungary; fInstitute for Infection Prevention and Hospital Epidemiology;
Medical Centre – University of Freiburg, Faculty of Medicine, University of Freiburg,
79106
Freiburg, Germany, Freiburg, Germany
Introduction: The modelling of cancer progression should be capable to
translate acquired knowledge of cell behaviour to the real human body conditions.
However,
the extracellular vesicles (EVs) isolated from 2D cell models are commonly exploited
in
research. Taking into account the specificity of the prostate cancer (PC) environment,
and a
strong need of early diagnosis of castrate-resistance by prostate cancer (CRPC) patients,
we
suggest in-depth profiling of different EV subtypes isolated from 3D culture as a
new tool
to model the progressing PC.
Methods: Cells from hormone-resistant prostate carcinoma 22-RV1 line were
cultured in 2D and 3D conditions, using 3D CoSeedisTM. ACD plasma controlled for haemolysis
and remaining platelets was taken from patients with PC and CRPC. The 40 fractions
of 4 EV
subtypes from cell culture and plasma were obtained by differential centrifugation
(DC)
followed by iodixanol density gradient purification. Each of the fractions was measured
by
Nanoparticle Tracking Analysis (NTA), Tunable Resistive Pulse Sensing (TRPS) followed
by
ELISA. For that, CD63 and CD9 were used as EV markers, ApoB and ApoA1 for lipoprotein
contaminants control, and CD3, CD41 and PSMA as tissue-specific biomarkers for determination
of fractions containing EVs of different origin. EV-contained fractions were subjected
to
Next Generation Sequencing (NGS).
Results: In 3D conditions, the 22-RV1 cells produce up to 1000-times higher EV
number than in 2D. Size and density distribution of EVs derived from 3D cultures but
not of
2D resembled plasma EVs. Size distribution and biomarker expression among different
EV
subtypes allowed distinguishing between PC and CPRC-derived samples, indicating a
potential
to translate these results into clinics for early CPRC detection.
Summary/Conclusion: This work demonstrates a new approach to study the
secretome of a progressing PC under 3D conditions. The profiles of EV subtypes produced
by
cancer cells growing in a 3D spatial architecture resemble the profiles of plasma
EVs and
can serve a useful tool for the establishment of new biomarkers.
Funding: European Union’s Horizon 2020 research and innovation programme under
the Marie Sklodowska-Curie grant agreement No 722148.
PF08.06
Multiplex analysis of renal cell carcinoma cell extracellular
vesicles to identify potential clinically relevant markers
Timothy Traynor
a, Bryce
Killingsworthb, Joshua A. Welshc, Aleksandra Dakicb,
Jason Savageb, Kevin Camphausend, Maria Merinoe, Marston
Linehanf and Jennifer Jonesb
aLaboratory of Pathology, National Cancer Institute, National
Institutes of Health, Gaithersburg, USA; bLaboratory of Pathology, National
Cancer Institute, National Institutes of Health, Bethesda, USA; cLaboratory of
Pathology, National Cancer Institute, National Institute of Health, Bethesda, USA;
dRadiation Oncology Branch, National Cancer Institute, National Institutes of
Health, Bethesda, USA; eCenter for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, USA; fUrologic Oncology Branch, Center
for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,
USA
Introduction: Renal cell carcinoma (RCC) is the most common primary renal
neoplasm, with over 80,000 cases in the US alone each year. Early detection of RCC
leads to
consistently better patient outcomes, and extracellular vesicles (EVs) isolated from
patient
samples may prove to be a valuable clinical tool in the future. EVs are abundant in
blood
and urine and show a large amount of heterogeneity but are difficult to analyse due
to their
small size and difficulty in isolation. Here, we employ a multiparametric analysis
of EV
surface markers to identify a set of markers that may prove clinically relevant in
future
studies.
Methods: RCC cell lines VOK111, VOK130, and VOK151 were cultured in flasks
containing 500 mL of EV-depleted media (10% FBS, centrifuged 18 hr x 100,000 g). When
cells
reached ~75% confluency, the conditioned media was collected and spun at 2,500 g for
10 mins
two times to deplete any remaining debris, leaving ~ 450 mL of media. This media was
concentrated to a final volume of ~ 7 mL using a PALL jumbosep 100 kDa MWCO filter.
This
concentrate was purified from protein by using an Izon qEV-10 column, collecting 5 mL
fractions. Protein content of each fraction was analysed using A280 absorbance while
concentration and diameter distribution were determined through nanoparticle tracking
analysis (NTA). Pooled samples made of the three most concentrated fractions were
concentrated to a final volume of ~190 µL using the PALL Microsep 100 kDa filter and
then
used for analysis in the Miltenyi MACSPlex exosome kit. Flow cytometric data were
generated
by the CytoFLEX S and analysed using FlowJo and MPAPASS software. These positive signals
were verified through bead-only controls and titrations.
Results: The MPAPASS software allowed for heatmap generation, data reduction,
clustering and visualization of expression patterns. Of the 11 detection antibodies
used
across 39 capture beads, CD276, CD26, CD82, Beta-2 Microglobulin, and CD151 were found
to be
prevalent in these RCC EVs. These markers were found to be co-expressed particularly
with
CD63, CD81, and CD29.
Summary/Conclusion: The use of multiplex analysis allowed for detection of
five distinctive surface markers found to be prevalent in EVs collected from RCC cell
lines.
These results demonstrate the utility of multiplex analysis and MPAPASS software for
identifying potential markers of interest and provide proteins that are worth exploring
further. The next steps to this work will be developing custom multiplex arrays that
tailor
capture and detection of EVs specifically for RCC pathology.
Funding: This work was supported by the NCI CCR Intramural Research Programs
and by the 2019 Ruth Anne Cafrtiz Award via FNIH.
PF08.07
Low molecular weight protein tyrosine phosphatase (LMWPTP)
carried by colorectal cancer cells-derived extracellular vesicles as a player in
tumour-educated human fibroblast
Stefano P. Clerici, Sílvio Consonni and Carmen
V. Ferreira-Halder
University of Campinas – UNICAMP, Campinas, Brazil
Introduction: Extracellular vesicles (EVs) are double-membrane-bound
nanovesicles released by cells playing a key role as mediators of intercellular
communication. Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) is upregulated
in
several cancers type, including colorectal cancer (CRC), and it has been correlated
with
aggressiveness, chemoresistance and poor prognostic.
Methods: The aim of this study was to determine whether CRC cells release
LMWPTP-enriched-EVs and influence tumour microenvironment-associated cells as a
representative tumour education. CRC cells, HCT116 and HT29, were cultured in serum-free
medium for 24 hours. Conditioned medium was concentrated by ultrafiltration (MWCO
10 kDa)
and EVs were isolated by Total Exosome Isolation Reagent (Invitrogen). EVs were
characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy
(TEM) and western blotting (WB). LMWPTP levels were analysed by WB and sandwich-ELISA.
To
evaluate tumour education, HFF-1 fibroblasts were used as recipient cells. The uptake
of EVs
(PKH26 fluorescently labelled EVs), proliferation (viability) and migration (wound
healing
assay) were analysed in a co-culture model of CRC-derived EVs and HFF-1.
Results: NTA showed a higher concentration of EVs released by HT29. HCT116 and
HT29 EVs displayed a mean diameter around 140 nm and a cup-shaped morphology. Isolated
EVs
were positive for EVs-markers CD81 and TSG101 and negative for GM130 a non-EVs marker.
HT29
lineage as well as derived-EVs are LMWPTP-enriched in comparison to HCT116 cells and
EVs.
Upon incubation, fluorescently HCT116 and HT29 derived EVs were internalized into
HFF-1
cells in a perinuclear region. EVs derived from both cells increased the viability
and
proliferation of HFF-1 cells. Intriguingly, EVs derived from HT29 promoted cell
migration.
Summary/Conclusion: In conclusion, for the first time, we showed that LMWPTP
can be carried by EVs derived from CRC cells and LMWPTP-enriched-EVs can modulate
biological
aspects of HFF-1 fibroblast. Overall, our findings point LMWPTP out as important player
in
tumour-educated fibroblast.
Funding: This study was supported by São Paulo Research Foundation (FAPESP) –
Grants: 2018/03593-6 and 2015/20412-7.
PF08.08
Exosomal miR-181a Inhibition by Vincristine and Prednisone in
Paediatric Acute Lymphoblastic Leukaemia.
Sarah R. Vaiselbuha and Shabirul
Haque
b
aStaten Island University Hospital at Northwell Health,
Manhasset, USA; bThe Feinstein Institutes for Medical Research, Manhasset,
USA
Introduction: Vincristine and prednisone are standard agents in treatment of
paediatric acute lymphocytic leukaemia (P-ALL). Mechanistically, vincristine induces
apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic
receptors and inhibits DNA synthesis, both of which lead to apoptosis. The effect
of these
agents on exosomal micro-RNA expression and its functional regulation is not yet
investigated. Elevated levels of miR-181a in circulating exosomes (nanoparticles)
has been
shown to lead to progression in several cancers, including ALL. We have previously
shown
that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating
of
miR-181a expression and silencing of exosomal miR-181a reverses this exosome-induced
cell
proliferating effect. The objective is to investigate the effect of vincristine and
prednisone on exosomal mi-R181a expression in ALL.
Methods: JM1, SUP-B15, and NALM-6 leukaemic cell lines were treated in vitro
with vincristine (0.1 to 4.0 µM) and prednisone (0.1 to 12.0 µM) in exo-free medium
and
apoptosis was measured by MTS assay. Total RNA of exposed cell lines was isolated
and cDNA
was prepared for miR-181a analysis. Expression of miR-181a was analysed by q-PCR.
Exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation
method. Purity and particle size of exosomes were confirmed by western blot and nanoparticle
tracking analysis (NTA) assay respectively. Total exosomal RNA was isolated from exosomes
(Exo-RNA) by Trizol method. Synthesis of cDNA was carried out with the miScript II
RT kit
(Qiagen).
Results: Vincristine and prednisone promote apoptosis in leukaemia cell lines
(JM1 and SUP-B15) in a dose-dependent manner. Both cellular and exosomal miR-181a
expression
was down-regulated by vincristine and prednisone exposure in all three leukaemia cell
lines
(JM1, SUP-B15, and NALM-6). These observations demonstrate that cellular miR-181a
down
regulation in the parental cells is stable and can be transferred to exosomes, confirming
the concept that exosomes are the fingerprint of parent cells.
Summary/Conclusion: Our data suggest that the vincristine and prednisone
anti-proliferative effect in P-ALL maybe induced by another yet unexplored pathway,
that
suppresses miR-181a at a cellular and exosomal level in P-ALL, resulting in apoptosis.
Funding: This project is supported by the DiMartino Family Foundation.
PF08.09
Secreted extracellular vesicles from renal cell carcinoma
cells
Anatoliy Samoylenko, Artem Zhyvolozhnyi, Eslam
Abdelrady, Naveed Ahmad, Genevieve Bart and Seppo Vainio
Oulu University, Oulu, Finland
Introduction: Clear cell Renal Cell Carcinoma (ccRCC) represents the most
common form of kidney cancer and is among the most lethal of all genitourinary cancers.
Despite surgery and medication therapy, most patients with metastatic ccRCC have a
poor
prognosis. Intratumoural hypoxia is a key factor involved in renal cancer progression
and it
is known to promote secretion of EVs by many types of tumour cells.
Methods: RCC-derived Renca cells, embryonic kidney derived UB cells, and
primary mouse hepatocytes were used in the study. EVs were purified from cell culture
media
by gradient ultracentrifugation, sequential ultracentrifugation and Exo-spin™ columns.
Before EV isolation cells were kept for 24 h either under normoxia or hypoxia (1%
oxygen).
EVs were analysed by transmission electron microscopy with negative staining and
immunolabeling, by nanoparticle tracking analysis (NTA) and Western blotting. Cells
proliferation and viability were assayed by live cell imaging using IncuCyte ZOOM
(Essen
BioScience), cell metabolic activity by Seahorse XF Analyser (Agilent), RNA expression
by
qPCR and ddPCR. Proteins were identified by ultra-performance liquid chromatography-mass
spectrometry (UPLC-MS). RNA libraries were made using NEBNext small RNA library prep
kit,
and sequenced on NextSeq550 (Illumina).
Results: We showed that hypoxia induced production of EVs by RCC cells, and
characterized differences in protein and RNA content of EVs generated by Renca cells
cultured under normoxic and hypoxic conditions. We also showed that RCC-produced vesicles
modify key features of tumorigenesis (gene expression, metabolic activity, motility,
and
growth) of target cells. These data were obtained by using two target cell types:
model
mouse kidney cells and primary mouse hepatocytes, which represent typical site of
RCC
metastasis with an exceptionally poor prognosis. We proposed that a possible mechanism
of EV
action in RCC is related to changes in caveolin-1 function. We also tracked Renca-derived
EVs in a chick embryo model and in a novel kidney organoid co-culture assay developed
by our
group (Xu et al., 2017).
Summary/Conclusion: Hypoxia may influence tumorigenic properties of RCC by
changing rates of production and composition of EVs.
Funding: The study was supported by Finnish Cancer Foundation grants.
PF08.10
Exosomes synthesizing HER2 miRNA and engineered to adhere to
HER2 on tumour cells surface exhibit enhanced anti-tumour activity
Lei Wang
a, Xusha Zhoua,
Weixuan Zoua, Yinglin Wub, Jing Zhaoc, Xiaoqing
Chend and Grace Guoying Zhoud
aGuangzhou Medical University, Guangzhou, China (People’s
Republic); bSchool of Basic Medical Sciences, Guangzhou Medical University,
Guangzhou, China (People’s Republic); cShenzhen International Institute for
Biomedical Research, Shenzhen, China (People’s Republic); dShenzhen International
Institute for Biomedical Research, Shenzhen, China (People’s Republic)
Introduction: Exosomes are small extracellular vesicles averaging 100–150 nm
in diameter. They serve as a means of intercellular communication. Typically they
consist of
structural proteins as well as selected proteins, miRNAs, mRNAs, and long noncoding
RNAs.
Thus in an earlier report this laboratory designed a miRNA targeting a major herpes
simplex
virus regulatory protein. As predicted by the nucleotide packaging signal the miRNAs
were
packed in exosomes and on exposure to infected cells significantly reduced virus yields.
HER2 (human epidermal growth factor receptor 2) plays an important role in the neoplasia
of
some breast cancers. The protein is exhibited on the cell surface and is the target
of
therapeutic antibodies.
Methods: Firstly, we report on the construction of a miRNA targeting the
synthesis of HER2 both in cells constitutively expressing HER2 and in cells transfected
with
a plasmid encoding HER2. Secondly, we report that the miRNA targeting the synthesis
of HER2
reduced the viability of HER2 positive cancer cells both in cell culture and in implanted
tumours. Lastly, we enhanced the anti-tumour activity of the exosomes by binding to
the
exosome surface a ligand with affinity for the HER2 on the surface of tumour cells.
Results: The 293-miR-HER2 exosomes package with miRNA designed to block HER2
synthesis and deliver to cells. These exosomes kill cancer cells dependent on HER2
for
survival but have no effect on cells lacking HER2 or which were engineered to have
HER2 but
do not depend on it for survival. The 293-miR-XS-HER2 exosomes carry in addition a
peptide
which enables the exosome to adhere HER2 on the surface of the cancer cells. In consequence,
these exosomes preferentially enter and kill cells exhibiting HER2 on their surface.
The
exosomes with 293-miR-XS-HER2 are significantly more effective in shrinking the size
of
HER2-positive tumours implanted in mice than the 293-miR-HER2 exosomes.
Summary/Conclusion: Our studies indicate that exosomes carrying miRNA against
HER2 have no effect on HER2 negative cells it was nevertheless desirable to increase
the
uptake of exosomes carrying the HER2 miRNAs by HER2-positive tumour cells. To this
end we
modified the exosomes to exhibit on their surface a peptide that bound the exosomes
to the
HER2 on the surface of cancer cells. In consequence, we significantly enhanced the
uptake of
exosomes carrying the miRNAs directed against HER2 by HER2 positive cells.
Funding: These studies were supported by grants from Shenzhen Overseas
High-Calibre Peacock Foundation KQTD2015071414385495, Shenzhen Science and Innovation
Commission Project Grants JCYJ20170411094933148, JCYJ20180306173333907 to Shenzhen
International Institute for Biomedical Research.
PF08.11
Systematic characterization of ovarian cancer-derived exosomes
unveil miRNAs interfering with CD8 + T cell activation
Xiang Nan
a, Sammy
Ferri-Borgognob, Jianting Shenga, Xin Wangc, Samuel
Mokb and Stephen Wongc
aHouston Methodist Hospital Research Institute, Weill Cornell
Medical College, Houston, USA; bMD Anderson Cancer Center, Houston, USA;
cHouston Methodist Research Institution, Weill Cornell Medical College,
Houston, USA
Introduction: CD8+ tumour-infiltrating lymphocytes (TIL) have been widely
reported to correlate with cancer patient survival, including ovarian cancer. Even
with the
presence of TILs, immunotherapy has limited success in ovarian cancer. Understanding
the
interaction between CD8+ TIL and tumour cells is thus important. Our hypothesis is
that
tumour-derived exosomes are released and taken up by CD8+ TIL such that specific miRNAs
contained within modulate physiological processes that inhibit CD8 + T cell activation.
We
aim to identify miRNAs carried in tumour-derived exosomes that inhibit CD8 + T cell
activation in ovarian cancer.
Methods: We purified exosomes from nine ovarian cancer cell lines and stocked
in high concentration. Interferon-gamma (IFN-gamma expression screening was performed
after
3 days of co-incubation of tumour derived exosomes, CD8 + T cells, and activators
in
conditioned medium. Cell counts and viability were tested by trypan blue staining
at day 0
and day 3. RNA-seq for exosomes were generated to identify miRNAs critical in
differentiation effects on CD8 + T cell activations. MicroRNA target matching uncovered
target mRNAs while enriched pathway analysis predicted potential signalling pathways
involved.
Results: Our IFN-gamma screening results indicated the exosomes exhibit
different behaviours in interfering CD8 + T cell activation owing to different donors.
Exosomes derived from PEO.1 and OVCA432 cells have consistent polarized results in
IFN-gamma
expression. Exosomes derived from PEO.1 remained a low IFN-gamma expression and from
OVCA432
stayed at relatively high level. Small RNAs profiling analysis between the two cell
lines
identified 56 miRNAs (p < 0.05), and 13 miRNAs have been reported with validated
targeting information, and 10 out of 13 have targets involved in immune signalling.
210 mRNA
targets were uncovered by target matching. CMap search identified complex connections
among
mRNAs with the top 20 enriched pathways actively involved in cell cycle and immune
related
behaviours.
Summary/Conclusion: Our IFN-gamma screening identified crucial miRNAs in
ovarian cancer exosomes interfering CD8 + T cell activation. Computational modelling
on both
experimental and public multi-omics datasets predicted promising signalling pathways
of
tumour-immune crosstalk for functional validation.
Funding: T.T. & W.F. Chao Foundation, John S Dunn Research Foundation
PF08.12
Irradiation of breast cancer cells alters the quality of DNA
cargo in the exosomes that they produce
Sheila Spada, Paul Zumbo, Doron Betel, Tuo
Zhang, Nils-Petter Rudqvist and Sandra Demaria
WEILL CORNELL MEDICINE, New York, USA
Introduction: Irradiation of breast cancer cells with an immunogenic dose
(8GyX3) leads to accumulation of cytosolic DNA that is sensed by cGAS leading to interferon
type I (IFN-I) signalling via cGAS/STING pathway [1–3]. We previously showed that
tumour-derived exosomes (TEX) secreted by irradiated (8GyX3) (RT-TEX) but not untreated
(UT-TEX) TSA carcinoma cells carry DNA that stimulates the production of IFN-I in
recipient
dendritic cells (DC) via the cGAS/STING pathway [4]. Moreover, mice vaccination using
RT-TEX, but not UT-TEX, elicited anti-tumour immune response inhibiting tumour growth
[4].
Here, we hypothesized that the differential ability of RT-TEX and UT-TEX to activate
IFN-I
in recipient DCs is due to qualitative differences in DNA cargo of RT-TEX compare
to
UT-TEX.
Methods: The length of DNA purified from TEX and from the cytosolic fraction
of TSA cells was measured by Agilent Bioanalyzer. The DNA cargo of TEX was analysed
by
whole-genome sequencing (WGS) and whole-genome bisulphite sequencing. The percentage
of
methylation of total DNA in TSA cells was quantified by 5-methyl cytosine DNA Elisa
kit.
Results: DNA fragments with size between 60 and 250 bp were enriched in RT-TEX
compared to UT-TEX, as well as in the cytosolic fraction of irradiated compared to
mock-treated TSA cells. WGS revealed that the entire genome was represented in TEX
DNA
cargo, regardless of RT. More than 99% of TEX DNA was of nuclear origin, but mitochondrial
DNA was increased in RT-TEX. Interestingly, we found that RT decreases the level of
methylation in both exosomal and total DNA in TSA cells compared to the controls.
Summary/Conclusion: These data support the hypothesis that immunogenic RT
alters some characteristics of the exosomal DNA cargo, mirroring molecular changes
occurring
in parent irradiated breast cancer cells. The enrichment in DNA fragments of 60–250
bp in
RT-TEX is intriguing considering that cGAS is optimally activated by DNA in this length
range [5]. We are currently investigating which features of the cargo DNA that differ
between UT-TEX and RT-TEX may explain the differential ability to induce IFN-I pathway
activation in recipient DCs. The identification of a DNA signature associated with
the
ability of TEX to activate the cGAS/STING pathway could provide a circulating biomarker
of
the RT-driven immunogenic tumour response.
References
1. Vanpouille-Box et al., Nature Communication, 2017
2. Mackenzie et al., Nature, 2017
3. Harding et al., Nature, 2017
4. Diamond et al., Cancer Immunology Research, 2018
5. Du et al. Science, 2018
PF08.13
IRE1 inhibition modulates immune phenotype of triple negative
breast cancer cells and cancer-derived extracellular vesicles
Dagmar Quandt and Matthew Griffin
Regenerative Medicine Institute (REMEDI) at CÚRAM Centre for Research in
Medical Devices, School of Medicine, National University of Ireland Galway, Galway,
Ireland
Introduction: Triple negative breast cancer (TNBC) is among the most difficult
cancer subtypes to treat and continues to cause a high number of cancer-related deaths
annually. Extracellular vesicles (EVs) transfer cell type-specific cargo and have
important
implications in disease initiation, therapy and outcome. Upon treatment of cancer
cells with
low-dose chemotherapy, released EVs are able to transfer phenotypic traits to other
cancer
cells. New treatment strategies for TNBC, like inhibitors of the ER stress pathway
(IRE1)
might impact on EV biogenesis, cargo delivery and response of cells in the cancer
microenvironment. Our aim is to identify immune modulatory alterations in breast cancer
cells and cancer derived EVs upon treatment with inhibitors of the ER stress pathway.
Methods: Human TNBC cell lines were treated with IRE1 inhibitor MKC8866 and
cells were analysed for immune modulatory surface markers, like HLA-I, B7-H molecules
and
different integrins. Mitochondrial and lysosomal activities were investigated by the
use of
a Mito- and Lysotracker and analysed by ImageStream (ISX) technology. Extracellular
vesicles
were isolated from cell culture supernatants by sequential centrifugation, quantified
by
Nanoparticle tracking (NTA) and characterized by Exosome bead array. Single EV analysis
of
total cell free supernatants and of isolated EVs was performed by ISX and marker positive
EVs were quantified for absolute fluorescence signals and total amount by objectives/ml.
EV
uptake into T cells was investigated by the use of different EV labelling strategies.
Results: Several immune relevant surface markers (HLA-I and CD54) are
downmodulated by IRE1 inhibition across different cell lines. Cell surface expressed
CD63
and B7-H3 show cell line specific downmodulation profiles upon IRE1 inhibitor treatment.
Other immunomodulatory marker such as B7-H1 and B7-H4, integrin CD29, cell
adhesion-promoting CD146 and stemness/metastasis marker (CD44 and SSEA) are unaltered
on
IRE1 treated breast cancer cells. Cancer cell derived EVs were tetraspanin positive
(CD9,
CD63, CD81), similar in number and showed differential expression of immune markers
upon
IRE1 treatment. Mitochondrial and lysosomal activities were unaltered under IRE1 inhibition,
whereas cell proliferation was diminished. No breast cancer-derived EV uptake of externally
labelled EVs into healthy T cells could be detected.
Summary/Conclusion: Ongoing analyses focus on the multicolour analysis of
multiple markers on single EVs by imaging flow cytometry and on the functional impact
of
cancer derived EVs on T cells delivered by EV receptor binding.
Funding: Dagmar Quandt is supported by the SFI (CÚRAM Research Centre,
13/RC/2073), the European Regional Development Fund and the Dr. Werner
Jackstädt-Stiftung.
PF09: EVs in Blood Disorders
Chair: Uta Erdbrügger – University of Virginia
Chair: Larry Harshyne – Thomas Jefferson University
PF09.01
Comparison of three isolation protocols to search extracellular
vesicles signature in sickle cell disease patients
Cristiane Maria de Souza
a, Irene
Pereira dos Santosb, Carolina Lanarob, Sara Teresinha Olalla
Saadb and Fernando Ferreira Costab
aUniversity of Campinas – UNICAMP, Indaiatuba, Brazil;
bUniversity of Campinas – UNICAMP, Campinas, Brazil
Introduction: Sickle cell disease (SCD) is an inherited disorder characterized
by chronic haemolysis and continuous activation of different cell types. Extracellular
Vesicles (EVs) were described to be at increased levels in SCD patient’s plasma compared
to
healthy subjects and were associated with several clinical manifestations such as
leg ulcers
and stroke. SCD patient’s plasma has increased concentrations of haem, free-Hb and
other
proteins and lipoproteins as chronic haemolysis consequence. Here, we report the comparison
of three mostly used isolation protocols to search EV signature in SCD patient’s plasma
by
flow cytometry.
Methods: Blood samples were obtained from SCD patients (n = 3) following
Wisgrill et al., (2016) protocol. Three different EV isolation protocols were used:
differential centrifugation (DC), ultracentrifugation (UC) and size-exclusion chromatography
(SEC). Lactadherin and calcein-AM were used to detect phosphatidylserine (PS)+ vesicles
and
membrane integrity, respectively. Platelet-derived EVs (PEVs), endothelial-derived
EVs
(EEVs), leucocyte-derived EVs (LEVs) and monocyte-derived EVs (MEVs) were quantified.
Silica
beads were used to define EVs gate and samples were acquired in the CytoFLEX cytometer
platform.
Results: The quantification of PEVs in UC, DC and SEC samples was,
respectively, 31x106, 8,5x106 and 9,7x106 events/mL mean, EEVs was 6,4x106, 1 × 106
and
4,3x106 events/mL mean, LEVs was 2x106, 6 × 105 and 1,3x106events/mL mean and MEVs
5,7x105,
6,5x105 and 3,7x105 events/mL mean. UC samples demonstrated a higher concentration
of EVs,
which could be more useful to functional studies than DC and SEC, however, it took
more time
to separate than DC. DC was the fastest method to separate EVs from plasma, being
useful to
study large patients cohorts, but showed the smallest overall number of EVs. SEC also
demonstrated high capability to detect EVs in plasma and the possibility of obtaining
a
purer sample, although it is the most expensive and time-consuming method among all
tested.
All EVs populations were detected in the three protocols tested.
Summary/Conclusion: In summary, all protocols tested were efficiently to
detect EVs in SCD patient’s plasma and the definition of the best protocol may vary
based on
the research aim and time and budget available.
Funding: FAPESP 2014/00984-3.
PF09.02
Extracellular vesicles from patients with sickle cell disease
disrupt gap junctions
Gabrielle Lapping-Carr, Joanna Gemel, Yifan
Mao and Eric Beyer
University of Chicago, Chicago, USA
Introduction: Aberrant cell-cell interactions involving the endothelium are
central to the pathophysiology of sickle cell disease (SCD), including acute chest
syndrome
(ACS), a deadly and unpredictable complication. We previously demonstrated that the
plasma
of SCD patients contains increased circulating small extracellular vesicles (EVs)
compared
to controls and that those vesicles can disrupt endothelial integrity in vitro by
affecting
adherens junctions and VE-cadherin. The current study was designed to examine the
effects of
those EVs on other cellular junctions including tight (zonula occludens 1, ZO-1) and
gap
junctions (connexin43, Cx43) and to test the hypothesis that the junctions would be
more
severely affected by EVs isolated from patients during an episode of ACS than by ones
isolated from the same patient at baseline.
Methods: We identified subjects with SCD in our biobank who had plasma
isolated at baseline and at the beginning of an admission for ACS. EVs were isolated
from
platelet free plasma using established methodologies. To determine the effects on
endothelium, cultures of human microvascular endothelial cells were treated with EVs
for 48
h and studied by immunofluorescence, immunoblotting and RT-qPCR. Gap junction-mediated
intercellular communication was assessed following microinjection of Lucifer yellow
and
neurobiotin.
Results: The distribution and abundance of ZO-1 at the plasma membrane were
minimally affected by SCD EVs. While baseline EVs did not affect the distribution
of Cx43,
EVs isolated during an episode of ACS caused loss of Cx43 from the plasma membrane.
The
integrated intensity of Cx43 membrane staining was decreased by ~20% following treatment
with ACS EVs. Cx43 protein decreased on average by 32%, Cx43 mRNA levels by 21% and
neurobiotin transfer by 67–94% in cells treated with ACS EVs, compared to baseline
EVs.
Summary/Conclusion: Circulating EVs in SCD affect multiple components of
endothelial junctions. Gap junctions composed of Cx43 are the most sensitive of the
cell-cell junctions, since their abundance and function are reduced by ACS EVs even
when the
endothelial monolayer appears intact. Cx43-mediated intercellular communication may
be an
early and sensitive event in the endothelial disturbance caused by EVs in SCD patients.
Funding: NIH UL1 TR000430, Comer Hospital RBC Race Funds, Ted Mullin
Fund.
PF09.03
The effects of platelet concentrate storage time on
extracellular vesicle interactions associated with fibrin clot formation
in-vitro
Jamie Nash
a, Christine
Saundersb, Amanda Daviesa and Philip Jamesa
aCardiff Metropolitan University, Cardiff, UK;
bWelsh Blood Service, Velindre University NHS Trust, Cardiff, UK
Introduction: Platelet concentrates (PCs) have been utilised for decades to
prevent bleeding in thrombocytopenic patients and to stop active bleeding. The storage
of
PCs however is a logistical challenge due to the limited 7 day shelf life under standard
conditions. During storage, platelets undergo a number of mechanical and biochemical
changes
contributing to the short shelf life of a PC. These changes are collectively known
as the
platelet storage lesion. Platelet extracellular Vesicles (PEVs) are known to increase
throughout PC storage, due to an increase in platelet activation. As PEVs have previously
been shown to be pro-coagulant and increase in Annexin V binding over PC storage.
The aim
was to investigate the effect of PC storage time on extracellular vesicle interactions
on
fibrin clot formation.
Methods: PCs were sampled on alternate days up to 10 days of storage and
centrifuged to achieve acellular plasma. The plasma was subjected to ultracentrifugation
(100,000xg) to pellet EVs. The size and concentration of EVs was assessed using Nanoparticle
tracking analysis software, followed by a western blot to confirm EVs were of platelet
origin. The PEVs were added at a fixed number to a control pooled plasma sample with
added
thrombin and tissue plasminogen activator. The time to clot and 50% lysis time were
recorded
by using the turbidometry of the plasma over time.
Results: EVs isolated from the PC were confirmed to be of platelet origin by
western blot using CD41 as a marker of platelet origin and CD9 as an EV marker. PEVs
caused
a significant increase effect on the fibrin clot formation (P < 0.001) when compared
to
the control plasma. PEVs also had a significant effect (P < 0.01) on the fibrinolysis
time, extending the time taken to lyse the clot. The time point during storage of
PEV
isolation had no significance (P > 0.05) on the fibrin clot.
Summary/Conclusion: PEVs from PCs significantly enhance fibrin clot formation
and extend clot lysis time, irrespective of PC storage time. Trauma patients could
benefit
from a PC unit with high PEV numbers; however, more research into the potential negative
effects of high PEV numbers, including surplus platelet activation in oncology patient
groups, is required.
Funding: Knowledge Economy Skills Scholarships (KESS 2) is a pan-Wales higher
level skills initiative led by Bangor University on behalf of the HE sector in Wales.
It is
part funded by the Welsh Government’s European Social Fund (ESF) convergence programme
for
West Wales and the Valleys.
PF09.04
Characterization of miRNA from serum derived exosomes in a mouse
tibia fracture model of Complex Regional Pain Syndrome
Jason Wickman
a, Seena
Ajitb, Ahmet Sacanc, Peyman Sahbaied, David
Clarke, Tian‐Zhi Guoe and Renee Jean-Toussaintb
aDrexel University College of Medicine, Roxborough, USA;
bDrexel University College of Medicine, Philadelphia, USA; cDrexel
University, Philadelphia, USA; dVeterans Affairs Palo Alto Health Care System
Anesthesiology Service, Palo Alto, USA; eStanford University School of Medicine
Department of Anesthesiology, Perioperative & Pain Medicine, Stanford, USA
Introduction: Complex regional pain syndrome (CRPS) is a debilitating chronic
disease that occurs after trauma to the periphery and is intimately associated with
nerve
injury. Its presentation is often described as an injury that is disproportional to
the
inciting event and manifests neuropathic pain, systemic inflammation, and immune
dysregulation. Owing in part to our poor understanding of disease aetiology, current
treatments for CRPS are insufficient and as a disease of exclusion there is a lack
of
quantitative diagnostic markers. Exosomes are small extracellular vesicles (sEVs)
30–100 nM
in size which provide a means of cellular communication through their cargo molecules
(protein, miRNA, mRNA, lipids), and have demonstrated promise in uncovering mechanisms
of
disease manifestation and identifying potential diagnostic markers. We have shown
previously
that CRPS patients have differential expression of several miRNAs in serum derived
sEVs as
compared to healthy controls, but little is known on how this compares to the established
mouse tibia fracture model of CRPS.
Methods: Mice undergoing fracture were anesthetized and subjected to a
unilateral tibia fracture followed by casting of the injured limb. After confirming
the
establishment of pain hypersensitivity, serum samples were collected from fracture
model and
control mice three weeks post-injury. sEVs were isolated by differential centrifugation
and
characterized using nanoparticle tracking analysis, transmission electron microscopy
and
western blotting. RNA-seq analysis is being performed to identify differentially expressed
miRNAs.
Results: Nanoparticle tracking analysis showed no significant difference in
the number or size of sEVs present in the serum from the fracture model and control
mice.
RNA-seq is ongoing and differential miRNA expression in sEVs from fracture model will
be
compared to control samples. Comparative studies identifying miRNAs that are common
between
CRPS patients and the rodent model will facilitate the development of correlational
outcomes
between preclinical and human studies.
Summary/Conclusion: Identification of similarities and differences between
CRPS patients and animal models will aid in directing future studies at clinically
relevant
aspects of CRPS aetiology and identifying potential diagnostic markers for CRPS
patients.
PF09.05
Extracellular Vesicle-based liquid biopsy in Acute Myeloid
Leukaemia: a reliable source of residual disease biomarkers?
Hugo R. Caires
a, Pedro
Nunesa, Alexandra Teixeirab, Manuel Sobrinho-Simõesc,
José Eduardo Guimarãesc and Maria Helena Vasconcelosd
ai3 S – Instituto de Investigação e Inovação em Saúde,
Universidade do Porto; Cancer Drug Resistance Group – IPATIMUP – Instituto de Patologia
e
Imunologia Molecular da Universidade do Porto;, Porto, Portugal; bi3 S –
Instituto de Investigação e Inovação em Saúde, Universidade do Porto; Fish Immunology
and
Vaccinology Group, IBMC – Instituto de Biologia Molecular e Celular, Universidade
do Porto,
Porto, Portugal, Porto, Portugal; ci3 S – Instituto de Investigação e Inovação em
Saúde, Universidade do Porto; Cancer Drug Resistance Group – IPATIMUP – Instituto
de
Patologia e Imunologia Molecular da Universidade do Porto; Serviço de Hematologia
Clínica do
Centro Hospitalar de São João, Porto; FMUP – Faculdade de Medicina da Universidade
do
Porto;, Porto, Portugal; di3 S – Instituto de Investigação e Inovação em Saúde,
Universidade do Porto; Cancer Drug Resistance Group – IPATIMUP – Instituto de Patologia
e
Imunologia Molecular da Universidade do Porto; FFUP – Faculdade de Farmácia da Universidade
do Porto, Porto, Portugal
Introduction: Acute myeloid leukaemia (AML) is an haematopoietic stem cell
disorder with a poor 5-year survival rate. Monitoring of Measurable Residual Disease
(MRD)
in AML patients receiving chemotherapeutic treatment is useful to assess therapy response
and predict relapse. Indeed, many different leukaemia associated immunophenotypic
protein
markers (LAIPs) are presently useful to detect MRD. Nevertheless, their analysis currently
requires invasive bone marrow aspirates, thus severely hindering real-time monitoring
of the
disease. Therefore, alternative peripheral blood-based methods are highly desirable
for an
easy, real-time and cost-effective monitoring of AML progression.
This work AIMs was to assess the feasibility of a peripheral blood EV-based liquid
biopsy
method for AML disease monitoring, based on the detection of LAIPs with a known negative
impact on the prognosis of AML.
Methods: The profile of EVs isolated from 12 paired samples from AML patients’
blood plasma collected at diagnosis, complete remission (and some at relapse) was
compared
and correlated with clinical data. For that, a size-exclusion chromatography (SEC)
method
was optimized to isolate the circulating EVs from the blood plasma. The EVs of the
12 paired
AML patients’ blood samples were then characterized according to their size (DLS/NTA),
morphology (TEM), protein-to-lipid ratio (Lowry/Sulpho phosphovanillin assay), surface
charge (Zeta-Sizer) and protein cargo (Western blot).
Results: SEC allowed the isolation of size-resolved plasma-derived EVs from
the peripheral blood of AML patients. Isolated EVs had a size ranging from 30 nm to
300 nm
with an intact morphology, expressing EV-associated markers such as HSP70, CD63, CD81
and
CD9. Size-resolved EVs also had a differential expression of Mitofilin, Actinin-4,
Syntenin-1 and Annexin-XI proteins. Several LAIPs were detected in the isolated EVs
and
their relative abundance changed throughout the stage of the disease.
Summary/Conclusion: Our preliminary data shows that AML patients’ circulating
EVs carry relevant immunophenotypic protein markers, which might predict AML clinical
outcome.
Funding: FCT – Foundation for Science and Technology (Portugal), project
POCI-01-0145-FEDER-030457.
PF10: Cell-EV Interaction, Uptake, and Fusion
Chair: Terri F. Bruce, MD, PhD – Clemson University
PF10.01
Profiling mRNAs of parental prostate cancer cells with different
phenotypes and their daughter extracellular vesicles using the NanoString low RNA
input
nCounter assay
Liang Dong, Richard Zieren, Kengo Horie, Sarah
Amend and Kenneth Pienta
The Brady Urological Institute, Johns Hopkins University School of
Medicine, Baltimore, USA
Introduction: Cell plasticity regulated by the balance between the
epithelial-to-mesenchymal transition (EMT) and MET is critical in the metastatic cascade.
Extracellular vesicles (EVs) may play an important role in this balance by shuttling
molecular cargos into recipient cells. This study aims to evaluate the feasibility
of
profiling mRNAs of parental prostate cancer (PCa) cells with different phenotypes
and their
daughter EVs using the NanoString low RNA input nCounter assay.
Methods: PC3-Epi and PC3-EMT cell lines representing epithelial and
mesenchymal phenotype, respectively, were generated from original PC3 cell line. The
cell
culture supernatant was first pre-cleared for any dead cells and debris by centrifugation
at
1000 × g for 20 min. Without disturbing the pellet, the supernatant was then transferred
to
a fresh ultracentrifuge tube and centrifuged at 10,000 × g for 20 min at 4°C. The
remaining
supernatant was then centrifuged to isolate the EVs at 100,000 × g for 120 min at
4°C. The
EVs pellet was further washed in 1× PBS followed by a second centrifugation at 100,000 × g
for 120 min at 4°C. The final EVs pellet was resuspended in 1× PBS for subsequent
characterization (transmission electron microscopy, nanoparticle tracking analysis
and
Western blot) and nCounter assays. The total RNA of cells and their daughter EVs were
assayed by the nCounter PanCancer Progression Panel to determine expression of 770
selected
mRNAs. The NanoString nCounter Low RNA Input Kit with the multiplex 770-gene primer
pool was
used for the pre-amplification of mRNA and overnight hybridization with the PanCancer
Progression panel. Each sample type was submitted to the assay in biological triplicate.
Results: When comparing all 12 samples, Eisen Cluster analysis separated all
the cells and all EVs into two groups, regardless of their phenotypes. In subgroup
analysis,
the expression patterns between PC3-Epi and PC3-EMT cells were significantly different.
CLEC2B, KDR, CRIP2, IL13RA2, CC2D1B were significantly upregulated in PC3-EMT cells,
while
CXCL8, EPCAM, ESRP1, TGFB2, CDH1, S100A14, OVOL2 were significantly downregulated
in PC3-EMT
cells. The expression patterns between PC3-Epi and PC3-EMT EVs were also significantly
different. TBX1, CAV1, COL4A1, SLC35A3, MYC, ITGB2, TIMP4, CAMK2B, PTGDS, P3H2, ITGB6,
VIM,
STAT3 were all significantly downregulated in PC3-EMT cell derived EVs.
Summary/Conclusion: The NanoString low RNA input nCounter assay can provide
reliable mRNA expression profiling of EVs. The mRNA expression patterns are very different
between cells and their daughter EVs. Both cells and EVs with different phenotypes
have
different gene expressions.
PF10.02
Cancer cell-derived EVs containing alphaV beta6 Integrin
regulate CD163, IL-6 and IL-10 levels in peripheral blood mononuclear cells
Nicole M. Naranjo, Israa Salem, Shiv Ram
Krishn, Larry Harshyne, Douglas Hooper and Lucia Languino
Thomas Jefferson University, Philadelphia PA, USA
Introduction: Extracellular vesicles (EVs) mediate communication in the tumour
microenvironment and play an important role in cancer progression. Previously, we
have shown
the enrichment of alphaV beta6 integrin in small extracellular vesicles (sEVs) isolated
by
differential ultracentrifugation and iodixanol density gradient from PC3 prostate
cancer
cells. We have also shown in the past that alphaV beta6-positive sEVs induce peripheral
blood mononuclear cell (PBMC) polarization by increasing the expression of pro-tumorigenic
M2 markers, such as CD163 and CD204. Finally, we have demonstrated that down-regulation
of
alphaV beta6 integrin up-regulates the STAT1-Interferon Stimulated Genes (ISGs) pathway
in
cancer cells and in sEVs released by them.
Methods: In order to investigate whether prostate cancer cell-derived
vesicular STAT1 has a causal effect in PBMC polarization, we down-regulated alphaV
beta6 and
STAT1 in prostate cancer cells derived sEVs using siRNA as well as CRISPR-Cas9 strategies.
The sEVs isolated from these cells were used to analyse M2 polarization by measuring
the
levels of CD163 in PBMC.
Results: The results show that sEVs lacking alphaV beta6 inhibit CD163 levels
in PBMC in a STAT1-independent manner. Analysis of cytokines released by PBMC upon
incubation with sEVs lacking alphaV beta6, show that PBMC selectively up-regulate
the levels
of IL-10 and IL-6, which are predominantly anti-tumorigenic cytokines. In contrast,
sEVs
lacking alphaV beta6 do not upregulate pro-angiogenic cytokines, such as VEGF.
Summary/Conclusion: These findings suggest that cancer cell-derived sEVs
containing alphaV beta6 integrin promote a pro-tumorigenic PBMC phenotype in the tumour
microenvironment by regulating CD163, IL-6 and IL-10 levels.
Funding: NIH-R01 CA-224769, P01 CA-140043 (LRL). This project is also funded,
in part, under a Commonwealth University Research Enhancement Program grant with the
Pennsylvania Department of Health (H.R.) (LRL). Israa Salem, PhD Student, is supported
by a
fellowship from the Saudi Arabian Ministry of Education
PF10.03
T cell activation by allogeneic EVs and allogeneic MHC
cross-dressed cells in vitro and in vivo
Aurore Prunevieille
a, Bruno Adonai
Gonzalez Nolascob and Gilles Benichoub
aCenter for Transplantation Sciences, Department of Surgery,
Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA, boston,
USA;
bCenter for Transplantation Sciences, Department of Surgery, Massachusetts
General Hospital and Harvard Medical School, Boston, MA, USA, Boston, USA
Introduction: The recognition of donor-MHC molecules by recipient T cells
triggers the immune response leading to rejection of allografts. Our recent studies
have
documented the presence of high numbers of recipient APCs displaying donor-MHC molecules
(cross-dressed) on their surface in the lymphoid organs of mice after skin, heart
or
pancreatic islet transplantation. In addition, we have reported that acquisition of
allogeneic MHC molecules by host APCs (MHC cross-dressing) is mediated by donor-derived
extracellular vesicles (EVs) trafficking through blood and lymphatic vessels (Marino
et al.
Science Immunology, 2016). In the present study, we investigated the ability of allogeneic
EVs and allo-MHC-cross-dressed cells to initiate a T cell alloresponse in vitro and
in
vivo.
Methods: EVs were isolated (using differential centrifugation) from BALB/c
Bone Marrow Derived Dendritic Cells (BMDCs). These EVs were used to cross-dress B6
splenocytes in vitro. The transfer of donor MHC class I and II on B6 cells was analysed
by
imaging flow cytometry. Next, T cells from B6 mice were cultured in vitro with either
allogeneic BMDC-derived BALB/c EVs or B6 spleen cells cross-dressed with allogeneic
BALB/c
MHC. Alternatively, 2 × 109 BALB/c or B6 BM derived EVs or 20 × 106 BALB/c BM cells
were
injected IV to B6 mice. In both cases, the T cell response was assessed by activation
markers detection, INFg production and cell proliferation.
Results: APCs cross-dressed with allogeneic MHC molecules can trigger a
pro-inflammatory direct alloresponse by T cells in vitro and in vivo. On the other
hand,
allogeneic EVs alone were only able to induce early T cell activation but not proliferation
in vitro. Furthermore, injection of mice with allogeneic EVs alone could induce some
but
suboptimal alloresponse in vivo and only when administered with complete Freund’s
adjuvant.
Summary/Conclusion: Blocking donor EVs release and subsequent recipient APC
cross-dressing may represent a promising target to selectively inhibit anti-donor
T cell
inflammatory responses thus achieving long-term allograft survival.
Funding: R01DK115618.
PF10.04
Antifungal antibiotic activity of outer membrane vesicles from
adherent Lysobacter enzymogenes C3 against therapeutic and biocontrol targets.
Paul Meers and Kunal Shah
Rutgers University, New Brunswick, USA
Introduction: Lysobacter enzymogenes is a predatory gram negative bacterial
species being studied for biocontrol activity against fungi. Planktonic L. enzymogenes
C3
produces outer membrane vesicles (OMV) harbouring small molecule antifungal antibiotics
(Meers et al. 2018). We show here that the more biologically relevant surface-associated
C3
exerts remote antifungal activity via OMV as well. The results have important consequences
regarding the natural mechanism of biocontrol of fungal pathogens by C3 as well as
isolation
and delivery of therapeutically relevant antifungal compounds.
Methods: OMV were isolated from scraped adherent C3 culture on agar by similar
methods to Meers et al 2018. OMV were stained in some cases with fluorogenic SYTO
9 DNA
stain for microscopic observation. Fungal growth was monitored via turbidity readings
in
liquid culture or photomicrographs on agar. C3 was also grown on polycarbonate filter
membranes with defined pore sizes to monitor growth of fungal cells on the opposite
side.
Vesicles were also labelled with an amine-reactive probe Alexa-555 and washed 4x by
sedimentation. Binding of labelled OMV to fungal cells was observed by epifluorescence
microscopy.
Results: SYTO 9-stained vesicles from surface-adherent C3 were similar to
previously observed ~130 nm vesicles (Meers et al., 2018). The isolated vesicles inhibited
growth of Saccharomyces cerevisiae or Candida albicans in liquid cultures at similar
potency
and were active against the filamentous species Fusarium subglutinans grown on agar
or maize
leaves. C3 cultures grown on filters with 400 nm pore size but not 30 nm were able
to
inhibit the hyphal growth of F. subglutinans on the opposite side. Similarly C3 on
filters
with a 200 nm pore size were able to inhibit growth of C. albicans. Observation of
fluorescently-labelled C3 OMV after interaction with C. albicans showed binding specifically
to hyphae or pseudohyphae and for F. subglutinans to the growing hyphal tips.
Summary/Conclusion: The OMV of C3 specifically bind and inhibit the growth of
fungal hyphae of various species without direct C3 cell contact. These data elucidate
mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal
antibiotics. Meers et al. (2018) Appl. Env. Microbiol. 84(20), e01353-18.
Funding: Funded by Rutgers University Byrne Seminar and Master of Business and
Science programs.
PF10.05
Elucidating the cellular uptake and tissue distribution
mechanism of cell derived vesicles, a novel therapeutic carrier
Hui-Chong Lau
a, Jae Young
Kima, Jin-Hee Parka, Jun-Sik Yoona, Min Jung
Kanga and Seung Wook Ohb
aMDimune Inc, Seoul, Republic of Korea; bMDimune
Inc, Seattle, USA
Introduction: Cell derived vesicles (CDVs) are emerging as a novel therapeutic
carrier. One of the crucial factors in the development and therapeutic applications
of CDVs
is to understand the precise mechanism by which vesicles find and enter the target
cells. In
this study, we aim to investigate the uptake mechanism of CDVs produced from natural
killer
(NK) cells using a manufacturing process established at MDimune Inc. Both in vitro
uptake
assay and in vivo distribution analysis were performed to provide precise insights
into how
CDV exert its effect at the cellular level.
Methods: NK cells were mainly used to produce CDVs. Breast cancer cells,
BT549, and human and rodent endothelial cells, with a varying degree of ICAM-1 expression,
were used to determine the effect of LFA-1 expressed on the surface of NK-CDVs in
cellular
uptake using FACS and confocal imaging analysis. Next, various inhibitors for uptake
pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated
endocytosis, were used to understand the underlying mechanism of cellular uptake of
CDVs.
Biodistribution profile of CDVs were characterized using both normal and tumour xenograft
models by IVIS imaging.
Results: Using a recently established manufacturing process, we demonstrate
that NK-CDVs can efficiently enter the target cells. This study also shows that the
cellular
uptake depends on the molecular interaction between ICAM-1 and LFA-1. In vivo distribution
profile of NK-CDVs are also assessed using various tumour models. Furthermore, we
present a
cellular uptake mechanism involved in the entrance of CDVs into the target cells.
Summary/Conclusion: This study demonstrates that the CDVs produced at the
manufacturing scale can be easily taken up by cells via specific cellular pathways.
This
finding will facilitate the development of more efficient therapeutics for cancer
and other
debilitating diseases.
PF10.06
Myofibroblasts-derived microvesicles increase dermal fibroblasts
collagen production through PLGF-1
Syrine Arif, Sebastien Larochelle and
Véronique J. Moulin
CHU de Québec – Université Laval, LOEX, Québec, Canada
Introduction: A proper wound healing of the skin involves angiogenesis,
extracellular matrix (ECM) remodelling and re-epithelialization. These three mechanisms
require well-organized interactions between different cell populations. A key role
in this
context is played by myofibroblasts (Wmyo), a cell population mainly differentiated
from
dermal fibroblasts. These cells contract wound edges and synthesize new ECM. We previously
showed that myofibroblasts predominantly produces microvesicles (MVs) and can favour
angiogenesis. However, proteomic analysis of MVs from our previous studies indicated
some
molecules that can potentially be implicated in ECM remodelling. In this study, we
evaluated
whether myofibroblasts-derived MVs could affect dermal fibroblasts who are highly
responsible for ECM regulation.
Methods: MVs were isolated by differential centrifugation of medium collected
from Wmyo cells. Number and size of MVs were characterized by transmission electron
microscopy and NanoSizer. Multiplex assays of 45 cytokines were evaluated in MVs samples,
Wmyo and MVs-depleted medium. To examine the interaction of MVs with fibroblasts,
we
evaluated the uptake of MVs isolated from Wmyo transduced with a fluorescent protein.
We
then treated fibroblasts cultures with MVs or a selected cytokine for 5 days and evaluated
collagen production. Lastly, we neutralized the selected cytokine in MVs samples before
evaluating collagen production.
Results: PLGF-1 was the cytokine detected in MVs samples in large amount
(0.88 ± 0.63 pg/µg proteins in MVs). Fibroblasts treated with MVs or PLGF-1 significantly
stimulated pro-collagen I level production with a fold change of 1.80 ± 0.18 and
2.07 ± 0.18. Moreover, the neutralization of PLGF-1 present in MVs significantly inhibited
the production of pro-collagen I by dermal fibroblasts.
Summary/Conclusion: Our results indicated that MVs influence fibroblasts
pro-collagen production through PLGF-1 signalling.
Funding: This work was supported by Natural Sciences and Engineering Research
Council of Canada (NSERC) (RGPIN2014-04404); les Fonds de recherche du Québec-Santé
(FRQS)
via the research centre funding grant; the Quebec Cell and Tissue and Gene Therapy
Network–ThéCell (a thematic network supported by FRQS).
PF10.07
Structural insights on fusion mechanisms of extracellular
vesicles with model plasma membranes
Fabio Perissinottoa, Valeria Rondellib,
Beatrice Senigagliesic, Paola Broccab, Laszlo almasyd,
László Bottyánd, Daniel Merkeld, Barbara Sartorie, Heinz
Amenitsche, Mario Gimonaf, Eva Rohdeg, Loredana
Casalish and Pietro Parisse
i
aInstitut de Biologie de Lille INSERM, Lille, France;
bUniversità di Milano, Milano, Italy; cUniversità di Trieste,
Trieste, Italy; dCentre for Energy Research, Budapest, Hungary;
eTechnical University of Graz, Graz, Austria; fGMP Unit at Paracelsus
Medical University, Salzburg, AUSTRIA, Salzburg, Austria; gGMP Unit, Spinal Cord
Injury and Tissue Regeneration Centre Salzburg (SCI-TReCS) and University Institute
for
Transfusion Medicine, Paracelsus Medical University Salzburg, Salzburg, Austria;
hElettra-Sincrotrone Trieste S.C.p.A., Trieste, Italy, Trieste, Italy;
iElettra Sincrotrone Trieste S.C.p.A., Trieste, Italy
Introduction: Extracellular vesicles (EVs) represent a potent intercellular
communication system. While their functional biological properties are more and more
investigated, the biophysical aspects of their interaction with recipient cells are
often
overlooked. Small size (30 to a few hundred nanometres in diameter) of EVs and their
heterogeneous origin still pose a great challenge for their isolation, quantification
and
biophysical/biochemical characterization. In particular the complex network of interactions
between differently classified EVs and recipient cells remains to be further revealed.
Here
we deeply investigate the fusion mechanism between EVs and a model plasma membrane
system by
an interplay of different structural/morphological techniques to get a molecular description
of the interaction helping to clarify the role of different membrane compartments
on the EVs
uptake mechanism.
Methods: Standardized protocols and Good Manufacturing Practice conditions
were employed to derive highly stable vesicles of defined size and reproducible molecular
profiles from Umbilical Cord multipotent Mesenchymal Stem (Stromal) Cells. After a
thorough
biophysical and biochemical characterization of EVs non-contact liquid imaging Atomic
Force
Microscopy (AFM) and, in parallel, Neutron Reflectometry (NR), as well as Small Angle
Neutron Scattering (SANS) experiments were performed on EVs to determine their interaction
with model plasma membranes in the form both of supported lipid bilayers and suspended
unilamellar vesicles of variably complex composition.
Results: We observed that EVs tend to fuse with the model membranes with a
preferential interaction with the external layer of the fluid membrane. Moreover we
revealed
a stronger interaction with the liquid ordered domains, strengthening the hypothesis
of a
critical role of lipid rafts in fusion mechanisms.
Summary/Conclusion: Our results on the analysis of the interaction of EVs with
artificial lipid membranes could provide insights on the internalization mechanisms
of EVs.
The approach shown here can be further extended to convey incremental complexity,
adding
glycolipid and membrane proteins to the model lipid bilayers. This approach combined
with
data on the specific biological function of each EV subpopulation as retrieved by
standard
functional assays, will turn useful to select the crucial molecular aspects of EVs
internalization by cells.
Funding: The authors acknowledge funding from the European Regional
Development Fund Interreg V-A Italia–Austria 2014–2020 (EXOTHERA ITAT1036).
PF11: EVs as Intrinsic Medicines
Chair: James G. Patton, PhD – Vanderbilt University
Chair: Chantal Boulanger – Université de Paris, Paris Cardiovascular Research
Centre
PF11.01
Platelet enhanced plasma: a novel extracellular vesicle enriched
resuscitation fluid?
Joshua MJ Price
a and Paul
Harrisonb
aUniversity of Birmingham, Salisbury, UK;
bUniversity of Birmingham, Birmingham, UK
Introduction: Platelet-derived extracellular vesicles (PEV) are the most
abundant circulating extracellular vesicle (EV) and exhibit platelet-like properties,
hence
the original term “platelet dust”. Direct phenotyping of EV surface markers within
biofluids
is challenging often requiring time-intensive purification steps that can significantly
alter resultant EV population characteristics. The ExoView™ (NanoView Biosciences)
specifically captures EV sub-populations and was used to characterise the EV content
of
platelet free plasma (PFP) and a potential novel haemostatic agent designed for the
treatment of severe trauma and haemorrhage, Platelet Enhanced Plasma (PEP).
Methods: Freeze-thaw cycling of platelet rich plasma/expired platelet
concentrates was followed by centrifugation to remove platelet remnants and yeilded
PEP. PFP
controls were prepared by double centrifugation (2000 g for 20 minuntes followed by
13,000 g
for 2 minutes). Rotational Thromboelastometry (ROTEM) and Calibrated Automated Thrombography
(CAT) were used to assess EV driven haemostasis and thrombin generation. A dilutional
and
hypothermic model of coagulopathy was designed to assess PEP. EV capture arrays comprised
of
anti-CD41, anti-CD63, anti-CD81 and anti-CD9 were used (ExoView™, NanoView Biosciences).
Captured vesicles underewent interferometric imaging and were quantified, sized and
further
probed with fluorescent tetraspanin markers, Annexin-V and intravesicular markers.
Results: PEP is highly procoagulant, exhibits enhanced thrombin generation and
can restore haemostasis in a dilutional model of coagulopatic whole blood. PEP can
be
generated from expired platelet concentrates, potentially allowing for upscalable
production. The predominant vesicle population were PEV with a large CD41/CD9 population
that contained a smaller sub-population of phosphatidyserine positive procoagulant
vesicles.
PFP as expected has a much lower number of PEV and a CD81 positive EV population.
Summary/Conclusion: PEP is a unique resuscitation fluid containing high PEV
levels for the potential treatment of severe trauma and haemorrhage. Exoview measurements
can be performed in unpurified plasma and may be useful for measuring circulating
EV in
health and disease.
Funding: Defence and Security Accelerator, DSTL
PF11.02
Therapeutic effect of exosomes in mice model of
Autism
Daniel Offen
a, Reut
Horeva, Nisim Peretsa, Ehud Maromb, Uri Danonb
and Yona Gefenb
aTel Aviv University, Tel Aviv, Israel; bStem Cell
Medicine LTD., Jerusalem, Israel
Introduction: During the recent decade, exosomes that derived from mesenchymal
stem cells (MSC-exo) have been spotlighted as a promising therapeutic target for various
clinical indications, including neurological disorders. We have previously shown that
intranasal administration of MSC-exo, cross the BBB and significantly ameliorate
autistic-like behavioural phenotype in BTBR and SHANK3 animal models of autism, representing
a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (ASD).
Our
objective is to study the mechanism of action and the cellular pathways in which the
MSC-exo
activate their target, we performed RNA sequencing analysis of primary neurons isolated
from
SHANK3 mice treated with MSC-exo.
Methods: Primary neuronal cell cultures were prepared from newborn SHANK3
homozygotes mice model of autism. Cultures were treated with MSC-exo (10^7 particles/ul),
isolated from human adipocytes, followed by RNA sequencing. The alterations in gene
expression between the treated and intact neurons were analysed for gene ontology
and
pathways and were also compared to proteomics analysis of the MSC-exo in order to
find
regulatory proteins that may lead to these differences.
Results: Bioinformatic analysis revealed several up-regulators proteins that
might be responsible for the increase in anti-inflammatory and protective factors
seen in
the mice neurons treated with MSC-exo. One of them is BDNF which is known as an essential
growth factor responsible for neuroprotection and neurogenesis. Importantly, no difference
in the genetic expression of cancer-related genes was identified following MSC-exo
treatment
indicating for their safety.
Summary/Conclusion: Our data suggest that adipocyte-derived MSC-exo carry
therapeutic potential in ASD via alternation in gene-expression related mainly to
immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis.
The beneficial effects of the exosomes treatment in mice models is being translated
into a
novel, easy to administer, a therapeutic strategy to reduce the symptoms of ASD.
Funding: Stem Cell Medicine, LTD. Israel
PF11.03
miRNAs enriched in extracellular vesicles isolated from plasma-
and serum-based autologous blood-derived products for osteoarthritis therapy
Alexander Otahal
a, Olga
Kuten-Pellab, Karina Kramera, Christoph Stotterb, Zsombor
Laczac, Stefan Nehrera and Andrea De Lunaa
aCenter for Regenerative Medicine; Danube University Krems,
Krems, Austria; bCenter for Regenerative Medicine, Krems, Austria;
cOrthosera GmbH, Krems, Austria
Introduction: Autologous blood-derived products gain increasing focus in
regenerative medicine, especially in orthopaedics and osteoarthritis therapy. This
disease
is characterised by cartilage degradation and inflammation among other symptoms, which
are
targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved.
Citrate-anticoagulated platelet rich plasma (CPRP) is often clinically applied to
stimulate
soft and hard tissue healing. Recently, cell-free alternatives to CPRP including hyperacute
serum (hypACT™ serum) have been developed. CPRP and hypACT™ serum contain specific
profiles
of growth factors, however, they also contain extracellular vesicles (EVs) that harbour
signal molecules including miRNA.
Methods: EVs were enriched by ultracentrifugation (UC) followed by size
exclusion chromatography (SEC) to obtain purified EVs. Particle size and concentration
of
each fraction was measured by nanoparticle tracking analysis (NTA). Fractions with
the
highest amount of particles were pooled and concentrated via UC, before miRNA expression
was
assessed via screening with a panel of 372 miRNA-specific primer pairs by RT-qPCR.
Presence
of EVs was confirmed by cryo-electron microscopy.
Results: The EV concentration tended to be lower in hypACT™ serum than in CPRP
as determined via NTA. Similarly, lower diversity of miRNA species was found in hypACT™
serum than CPRP EVs. Around 90% of detected miRNAs were found in both blood products,
whereas only 30% of miRNAs were shared between EVs from CPRP and hypACT™ serum. While
miRNAs
such as miR-101 were consistently depleted in EVs compared to the corresponding blood
product, others like miR-27a were in enriched in hypACT™ EVs, but not CPRP EVs, indicating
release of specific miRNAs via EVs in response to clotting.
Summary/Conclusion: Although the purification resulted in high loss of EVs, we
identified specific miRNAs enriched in EVs from CPRP and hypACT™ serum. Their functional
spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation,
inhibition of tissue remodelling via matrix degrading enzymes as well as preventing
senescence. This renders blood product derived EVs as interesting candidates for in
vitro
and in vivo testing with respect to cartilage regeneration.
Funding: The work was jointly supported by the European Fund for Regional
Development (EFRE) and the Fund for Economy and Tourism of Lower Austria, grant number
WST3-F-5030664/003-2017.
PF11.04
Protective role of shiitake mushroom-derived exosome-like
nanoparticles in D-galactosamine and lipopolysaccharide-induced acute liver injury
in
mice
Baolong Liu, Xingyi Chen and Jiujiu Yu
University of Nebraska Lincoln, Lincoln, USA
Introduction: Fulminant hepatic failure (FHF) is a rare, life-threatening
liver disease with poor prognosis. New therapeutic interventions are urgently needed
to
treat this disease. Administration of D-galactosamine (GalN) and a low dose of
lipopolysaccharide (LPS) triggers acute liver damage in mice, which simulates many
clinical
features of FHF in humans and therefore is widely used to investigate the molecular
mechanisms and potential therapeutic interventions of FHF. Recently, suppression of
the
nucleotide binding domain and leucine rich repeat related (NLR) family, pyrin domain
containing 3 (NLRP3) inflammasome was shown to alleviate the severity of LPS/GalN-induced
liver injury in animal models. Therefore, the goal of this study was to identify
food-derived exosome-like nanoparticles (ELNs) with anti-NLRP3 inflammasome function
to
potentially control FHF.
Methods: Seven commonly consumed mushrooms were used to extract ELNs, which
were examined for anti-NLRP3 inflammasome activities in primary macrophages.
Results: It was found that these mushrooms contained ELNs composed of
biomolecules including RNAs, proteins, and lipids. Among these mushroom-derived ELNs,
only
shiitake mushroom-derived ELNs (S-ELNs) strongly inhibited NLRP3 inflammasome activation
by
blocking the inflammasome assembly. This inhibitory effect was specific for the NLRP3
inflammasome because S-ELNs had no impact on activation of the Absent in Melanoma
2 (AIM2)
inflammasome. S-ELNs also inhibited the secretion of interleukin (IL)-6 and both protein
and
mRNA levels of the Il1b gene in macrophages. Remarkably, pre-treatment of S-ELNs protected
mice from LPS/GalN-induced acute liver injury.
Summary/Conclusion: Therefore, S-ELNs, identified as potent inhibitors of the
NLRP3 inflammasome, represent a new class of agents with the potential to combat FHF.
PF11.05
Approaches to assess clinically available exosomes’ quality and
safety
Kevin C. Hicok
a, Kenneth
Pettineb and Timothy Moseleyc
aDirect Biologics LLC, La Jolla, USA; bDirect
Biologics, Medical Director, Fort Collins, USA; cCSO, Direct Biologics LLC, La
Jolla, USA
Introduction: Recent adverse events resultant from an exosome product use in a
Nebraska clinic, highlight the importance of assuring product quality and safety standards.
An often-overlooked safety risk is ancillary reagents remaining within a finished
product.
When processes to obtain exosomes utilize cow proteins such as FBS or bovine sera
albumin,
failure to adequately remove these can result in significant adverse allergic reactions.
We
evaluated 3 different exosome products to test the hypothesis that purity of some
products
may not be consistent with actual product quality and safety profiles claimed.
Methods: Three different exosome products (manufacturer A, B, and C) were
prepared per their instructions for use. Sample source identity was blinded from assaying
scientists. An independent CRO service was used to conduct the experiments to ensure
unbiased assay execution and data collection. Exosome suspensions were sampled undiluted
for
bovine protein content using commercially available bovine secretome protein arrays
from Ray
Biotech. A total of 30 different proteins found in bovine serum were quantified.
Results: Six of 30 proteins were not detected in any sample. 8 of 24 array
antibodies were found to cross react with human antigens. Of the 16 bovine proteins
that
were acceptable for analysis, manufacturers A, B, and C exosomes contained 15 of 16
proteins,13 of 16 proteins, and 0 of 16 proteins, respectively. Concentrations of
individual
bovine proteins ranged from 0.2 to 1,220.1 ng/mL.
Summary/Conclusion: These results indicate manufactures A and B are selling
potentially dangerous products. The successful implementation of exosome products
into the
clinic requires equivalent demonstrations of safety and quality. This requires adopting
strict quality standards and safety testing during their production. Physicians must
require
safety data prior to clinical use.
PF11.06
Engineering pro-healing EV cargo using a closed-system
bioreactor.
Michael J. Van Kanegan
a, Benjamin
Buehrera, Jeffrey Gimbleb, Trivia Frazierb and John
Ludlowa
aZenBio, Inc, Research Triangle Park, USA;
bObatala Sciences, Inc, New Orleans, USA
Introduction: Chronic wounds, including diabetic ulcers and pressure ulcers,
are difficult and expensive to treat. While tissue engineering approaches have largely
failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived
extracellular vesicles (EVs) may provide several unique advantages. ZenBio, Inc has
developed a methodology to generate commercial-scale stem cell-derived exosomes using
a
closed-system hollow fibre bioreactor capable of continuous EV production. Additionally,
we
have shown that by manipulating the cellular environment, we can improve the pro-healing
capacity of the EVs.This technology leverages the complex healing capabilities of
stem cells
without the obstacles of replicating cells.
Methods: We have demonstrated that a mild heat shock resulted in EVs enriched
for stress-response proteins and increased pro-healing activities in vitro. We extended
this
innovative approach to include stimulating adipose stem cells with combinations of
heat
shock and growth factors to generate differential extracellular vesicle packaging
that
enhances pro-healing activity. To monitor reproducibility across lots and batches,
we
rigorously characterized tuned EVs for particle size and number as well as surface
marker
and cargo composition.
Results: Our results using tuned EVs showed efficacy using cellular models of
inflammation, motility, vascularization, collagen production and metalloprotease activity.
We utilized an established murine model of pressure ulcers to assess the in vivo efficacy
of
the tuned EVs. These studies showed a single injection into the wound site activated
a more
rapid wound closure, increased collagen deposition and reduced dermal thickness compared
to
saline control.
Summary/Conclusion: These data strongly support our hypothesis that EVs may be
selectively modified to improve their wound healing activity by modulating the culture
or
tissue microenvironment. Future studies will use chronic wound models to determine
optimal
dosing and routes of administration.
Funding: NIH-NIA SBIR (2R44AG058351)
PF11.07
Comprehensive analysis of methods and outcome reporting in
preclinical animal studies of mesenchymal stem cell derived extracellular vesicles:
A
systematic review
Alvin Tieu
a, David
Allanb, Duncan Stewartc, Mitchell Slobodianc, Dean
Fergussonb, Joshua Montroyb, Dylan Burgerd and Manoj
Lalub
aClinical Epidemiology Program and Regenerative Medicine
Program, Ottawa Hospital Research Institute, Ottawa, Canada; bClinical
Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, Canada; cOttawa
Hospital Research Institute, Ottawa, Canada; dChronic Disease Program, Ottawa
Hospital Research Institute, Ottawa, Canada
Introduction: Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs)
can reduce inflammation, promote healing and improve organ function thereby providing
a
potential “cell-free” therapy. Prior to clinical translation, there is a critical
need to
synthesize existing preclinical evidence supporting their efficacy. This systematic
review
provides the most comprehensive evidence map of methods, safety and efficacy for MSC-EV
research to date.
Methods: MEDLINE and Embase were systematically searched for in vivo
interventional studies using MSC-EVs. Two reviewers extracted data for: 1) methodology,
2)
study design, 3) intervention details and 4) efficacy/adverse events.
Results: After screening 754 articles, 206 studies met our eligibility
criteria. MSC-EVs were used to treat a variety of diseases including renal (16%),
neurological (12%) and cardiac (10%) conditions. Benefits were described in 95% of
studies
across all organ systems and adverse effects were seen in only three studies; two
showing
tumour growth. However, several key methodological concerns were evident. Based on
size
criteria for EV subtypes (exosomes/small EVs ~30-150 nm, microvesicles ~150-1000 nm)
only
60% of studies used appropriate nomenclature. Ultracentrifugation (70%) and isolation
kits
(23%) were the most common isolation methods despite marked differences in yield and
purity.
EVs were inconsistently dosed by protein (68%), particle number (16%) or cell count
(4%),
hindering inter-study comparisons. Two-thirds of studies used xenogeneic EVs suggesting
immunocompatibility. Techniques to determine size, protein markers and morphology
was highly
heterogeneous, and only 12 and 4 studies met the characterization standards recommended
in
the MISEV 2014 and 2018 guidelines, respectively. Finally, 50% of studies did not
incorporate randomization which represents a high risk for bias and only a quarter
performed
biodistribution studies.
Summary/Conclusion: This systematic review reveals extensive heterogeneity in
methods and intervention details for animal studies of MSC-EVs. Nonetheless, nearly
all
studies showed significant benefits in a wide range of distinct conditions. The knowledge
gaps we identified highlight important opportunities for improving preclinical design
and
the need for more standardized approaches in this growing field of EV therapeutics.
PF11.08
MSC-exosomes as next generation therapeutics for atopic
dermatitis
Yong Weon Yi
ExoCoBio Inc, Seoul, Republic of Korea
Introduction: Atopic dermatitis (AD) is a systemic inflammatory disease with
unknown cause. Recent approval of a targeted therapy, dupilumab, opens new era of
AD
management. However, current therapeutic options for AD are only targeting inflammation,
a
component of AD vicious cycle including itching and barrier disruption. Human mesenchymal
stem cells (MSCs) have been highlighted as a novel therapy for suppressing allergic
progress
of AD in clinical studies. Unfortunately, phase III clinical study of human umbilical
cord
blood MSCs for AD was failed with unknown reason. Previously, our group reported that
exosomes derived from human adipose tissue-derived MSCs (ASC-exosomes) alleviated
the
pathological symptoms in a murine AD model with concomitant reduction of inflammation.
Methods: Our group has further investigated the therapeutic effects of human
ASC-exosomes in an alternative murine AD model with skin barrier defects. Large scale
isolation of ASC-exosomes was performed by tangential flow filtration and isolated
ASC-exosomes were characterized according to the recommendation by the ISEV. The protein
and
lipid cargo were also analysed.
Results: We found that ASC-exosomes induced restoration of skin barrier by
inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines.
In addition, ASC-exosomes suppressed the expression of itching-causing cytokines.
Transcriptomic analysis of AD skin lesions revealed that ASC-exosomes reversed the
abnormal
expression of genes functioning in skin barrier function, lipid metabolism, and cell
cycle.
Summary/Conclusion: Taken together, ASC-exosomes could be a promising
cell-free therapeutic option for the treatment of AD, which affecting inflammation,
skin
barrier function, and itching.
PF11.09
Cell derived vesicles: unravelling the science of novel vesicles
with therapeutic promises
Hui-Chong Laua, Ji Hye Leea, Dongwoo
Hana, Jin-Hee Parka, So-Yeon Kimb, Jae Young
Kima and Seung Wook Oh
c
aMDimune Inc, Seoul, Republic of Korea; bMDimune
Inc, Seoul, Republic of Korea; cMDimune Inc, Seattle, USA
Introduction: Cell derived vesicles (CDVs) are nanosized vesicles produced by
serially extruding cells through small pores. A growing number of studies have implicated
their therapeutic potentials, with superior yield compared to other extracellular
vesicles
(EVs). However, two key objectives remain to be accomplished to demonstrate the utility
of
CDVs in clinical applications. First, a manufacturing process has to be developed
to allow a
large-scale production of CDVs. Next, these novel vesicles need to be thoroughly
characterized at multiple levels.
Methods: Manufacturing-scale extruders were developed to allow extrusion of
large volume of cell suspension in a single process. CDVs with approximately 150–200 nm
in
diameter were obtained by a serial extrusion. Crude samples were then purified using
the
tangential flow filtration method to further remove cellular impurities. Finally,
physical
and biochemical characteristics of purified CDVs were analysed using DLS, NTA, Cryo-EM,
and
FACS analysis. Additionally, CDVs were subject to multi-omics profiling to comprehend
our
understanding in molecular contents of CDVs. Both mesenchymal stem cells (MSCs) and
natural
killer (NK) cells were used for this study.
Results: In this study, we first demonstrate that the large-scale extruder
efficiently produce CDVs with consistent quality at the scale that are compatible
for
clinical applications. Surface marker and membrane composition analyses show that
the CDVs
are primarily formed using plasma membrane of source cells, with characteristic cellular
markers enriched on the surface. Comprehensive profiling of molecular components reveals
the
unique properties of CDVs as well as the underlying mechanism of formation of CDVs.
Summary/Conclusion: Recently, we have established a manufacturing process to
enable clinical applications of CDVs. This study also highlights key molecular features
of
CDVs that can be harnessed to offer a powerful tool for regenerative and anticancer
medicine.
PF11.10
Antifibrotic properties of extracellular vesicles derived from
human induced pluripotent stem cells
Milena Pawa, Kinga Polanskaa, Marcin
Piejkoa, Sylwia Kedracka-Krokb, Ruba Hammadc, Dawid
Wnuka, Toni Cathomenc, Zbigniew Madejad, Ewa K.
Zuba-Surmad and Sylwia Bobis-Wozowicz
e
aDepartment of Cell Biology; Faculty of Biochemistry,
Biophysics and Biotechnology; Jagiellonian University, Krakow, Poland;
bDepartment of Physical Biochemistry; Faculty of Biochemistry, Biophysics and
Biotechnology; Jagiellonian University, Krakow, Poland; cInstitute for
Transfusion Medicine and Gene Therapy, and Centre for Chronic Immunodeficiency, Medical
Centre – University of Freiburg, Freiburg, Germany; dDepartment of Cell Biology,
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow,
Poland, Kraków, Poland; eDepartment of Cell Biology, Faculty of Biochemistry,
Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland, Krakow, Poland
Introduction: Fibrosis is a pathological condition resulting from abnormal
healing of various tissues. It is triggered by activation of fibroblasts and their
subsequent transition to myofibroblast. In consequence, excessive deposition of
extracellular matrix proteins leads to impaired organ function. To revert this process,
we
employed extracellular vesicles (EVs) derived from human induced pluripotent stem
cells
(hiPSCs). As a model system, we used human cardiac fibroblasts (hCFs), since heart
fibrosis
constitutes a serious socio-economic problem worldwide.
Methods: We isolated EVs from conditioned media from three hiPSC lines using
ultrafiltration combined with size exclusion chromatography methods. Next, we analysed
the
EVs by NanoSight, transmission electron microscopy, mass spectrometry and Western
blot
methods. Finally, we treated TGF-b-stimulated hCFs with hiPSC-EVs and evaluated expression
of fibrosis-related genes using real-time qPCR, Western blot and fluorescence
microscopy.
Results: We detected anti-fibrotic properties of hiPSC-EVs exerted on hCFs
pre-stimulated with TGF-b. The EVs significantly decreased the expression levels of
ACTA2,
FN, TNC, SNAI2, COL1A1 and reduced the number of myofibroblasts. The canonical profibrotic
TGF-b-dependent SMAD2/3 pathway was significantly attenuated in response to
EV-treatment.
Summary/Conclusion: In this study we demonstrated strong anti-fibrotic
function of hiPSC-EVs. Our findings can further be exploited for future medical applications
to treat fibrotic diseases, such as heart fibrosis.
Funding: This work was supported by the project SONATA12:
UMO-2016/23/D/NZ3/01310 from the National Science Centre of Poland to SBW.
PF11.11
Induced pluripotent stem cells- derived extracellular vesicles
ameliorates D-galactosamine and lipopolysaccharide induced acute liver failure
Ying luo, Peng Wang and Yingtang Gao
Tianjin Third Central Hospital affiliated to Nankai University, Tianjin,
China (People’s Republic)
Introduction: Liver failure is among the most causes of death in patients with
liver disease. Promoting liver regeneration will help patients with liver failure
recover on
their own. Extracellular vesicles (EVs) can released by induced pluripotent stem cells
(iPSCs) through paracrine effects and play a pivotal role in inter-cellular communication
in
the treatment of disease. In this study, we investigated whether the iPSCs-EVs have
therapeutic effects on acute liver failure.
Methods: The iPSCs-EVs were isolated by ultracentrifugation and identified
using nanoparticle tracking analysis, transmission electron microscopy and Western
blotting.
The isolated iPSCs-EVs were administrated D-galactosamine-injured HepRG cells in vitro
and
tail intravenously injected into D-galactosamine and lipopolysaccharide induced acute
liver
failure model mice in vivo, respectively. The anti-apoptosis role and potential mechanism
were evaluated using flow cytometry and immunofluorescence staining. And Alanine
transaminase (ALT) and aspartate transaminase (AST) in serum, H&E staining and TUNEL
staining were explored the effect of iPSCs-EVs on liver injured and liver function.
Finally,
high throughput sequencing of small RNAs was performed to investigate miRNA expression
profiles in iPSCs-EVs and iPSCs.
Results: The iPSCs-EVs that were all 50–200 nm, double-layered and oval or
round cellular vesicles and expressed the marker proteins CD63, TSG101 and HSP70.
In vitro,
the iPSCs-EVs treatment inhibited HepRG apoptosis induced with D-galactosamine in
a time-
and dose-dependent manner and promote the proliferation of hepatic stem cells. In
vivo
results showed that iPSCs-EVs significantly alleviated liver failure, improved liver
function and prolonged the survival period. TUNEL assay showed that iPSCs-EVs suppress
apoptosis of hepatocytes. Moreover, miRNA expression profiles analysis found that
miR17-92a
cluster and miR302-367 cluster were enriched in iPSCs-EVs and iPSCs.
Summary/Conclusion: These findings indicated that iPSCs-EVs could ameliorate
D-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte
apoptosis, which will be benefit for therapy of liver disease in the future.
Funding: Natural Science Foundation of Tianjin Municipal Science and
Technology Commission under grant [No.17JCYBJC26100], Key Research Project of Tianjin
Health
Commission under grant [No.16KG150].
.
PF11.12
MSC-derived extracellular vesicles promote human cartilage
regeneration by control of autophagy
Suzy Varderidou-Minasian
a, Lucienne
Vonkb, Sanne van Dooremalenc, Nalan Livd, Judith
Klumpermane, Daniël Sarisf and Magdalena Lorenowiczc
aCenter for Molecular Medicine & Regenerative Medicine
Centre University Medical Centre Utrecht I Uppsalalaan 8 I 3584CT Utrecht I The Netherlands,
UTrecht, Netherlands; bDepartment of Orthopaedics, University Medical Centre
Utrecht, PO BOX 85500, 3508 GA, Utrecht, the Netherlands, Utrecht, Netherlands;
cCenter for Molecular Medicine& Regenerative Medicine Centre University
Medical Centre Utrecht, Utrecht University, Utrecht, Netherlands; dCenter for
Molecular Medicine, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG,
Utrecht, the Netherlands, Utrecht, Netherlands; eCenter for Molecular Medicine,
University Medical Centre Utrecht, Utrecht University, Utrecht, Netherlands;
fDepartment of Orthopaedics, University Medical Centre Utrecht, Utrecht
University, Utrecht, Netherlands
Introduction: Osteoarthritis (OA) is a rheumatic disease leading to chronic
pain and disability with no effective treatment available. Recently, allogeneic human
mesenchymal stromal/stem cells (MSC) entered clinical trials as a novel therapy for
OA.
Increasing evidence suggests that therapeutic efficacy of MSC depends on paracrine
signalling. Here we investigated the role of bone marrow MSC-derived extracellular
vesicles
(BMMSC-EVs), an important component of MSC secretome, in cartilage repair.
Methods: To test the effect of BMMSC-EVs on OA cartilage inflammation the
TNF-alpha-stimulated human OA chondrocytes were treated with BMMSC-EVs and inflammatory
gene
expression was measured by qRT-PCR after 48 h. To access the impact of BMMSC-EVs on
cartilage regeneration the BMMSC-EVs were added to the regeneration cultures of OA
chondrocytes, which were analysed after 4 weeks for glycosaminoglycan content by DMMB
and
qRT-PCR. Paraffin sections of the regenerated tissue were stained for proteoglycans
(safranin-O) and type II collagen (immunostaining).
Results: We show that BMMSC-EVs promote cartilage regeneration in vitro.
Treatment of OA chondrocytes with BMMSC-EVs induces production of proteoglycans and
type II
collagen and promotes proliferation of these cells. MSC-EVs also inhibit the adverse
effects
of inflammatory mediators on cartilage homoeostasis. Our data show that BMMSC-EVs
downregulate TNF-alpha induced expression of pro-inflammatory COX-2, pro-inflammatory
interleukins and collagenase activity in OA chondrocytes. The anti-inflammatory effect
of
BMMSC-EVs involves the inhibition of NFκB signalling, activation of which is an important
component of OA pathology.
Autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular
organelles and macromolecules, is essential to maintaining chondrocytes survival and
differentiation. The expression of autophagy regulators is reduced in osteoarthritic
joints,
which is also accompanied by increased chondrocyte apoptosis. Our preliminary data
indicate
that BMMSC-EVs carry mRNA of natural autophagy inducers and promote autophagy in OA
chondrocytes. Therefore, we hypothesize that MSC-EVs exert their beneficial effects
on
cartilage regeneration by restoring the expression of autophagy regulators.
Summary/Conclusion: In summary, our findings indicate that BMMSC-EVs have
ability to promote OA cartilage repair by reducing the inflammatory response and stimulation
of OA chondrocytes to produce extracellular matrix, the essential processes for restoring
and maintaining cartilage homoeostasis. Thus, MSC-EVs hold great promise as a novel
therapeutic for cartilage regeneration and osteoarthritis.
Funding: S. V-M. is supported by the grant (2018-1-261469) from Dutch
Arthritis Foundation and M.J. L. by the grant (11600.420) from ZonMw.
PF11.13
Large-scale preparations of small extracellular vesicles from
conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly
established Graft-versus-Host-Disease model in batch dependent manners
Rabea Madela, Verena Börgerb, Robin
Dittrichb, Michel Bremerc, Lambros Kordelasd, Sven
Brandaua, Carsten J. Kirschninga and Bernd
Giebel
e
aUniversity Hospital Essen, Essen, Germany;
bInstitute for Transfusion Medicine, University Hospital Essen, 45147 Essen,
Germany, Essen, Germany; cUniversity of Heidelberg, Heidelberg, Germany;
dDepartment of Bone Marrow Transplantation, University Hospital Essen, 45147
Essen, Germany, Essen, Germany; eHeidelberg, Heidelberg, Germany
Introduction: Extracellular vesicles (EVs) harvested from supernatants of
humane adult bone marrow-derived mesenchymal stem/stromal cells (MSCs) can suppress
acute
inflammatory cues in a variety of different diseases, including Graft-versus-Host
Disease
(GvHD) and ischaemic stroke. Furthermore, they can promote regeneration of affected
tissues.
Following a successful clinical treatment attempt of a steroid refractory GvHD patient,
we
intend to optimize MSC-EV production strategies for further clinical applications.
As we
observed functional differences of independent MSC-EV preparations in vitro, we aimed
to
adopt an in vivo GvHD model for the more advanced functional testing of different
MSC-EV
preparations.
Methods: To this end we set up a bone marrow transplantation mouse model in
which endogenous bone marrow was myeloablated by ionizing irradiation (IIR). GvHD
was
induced by the transplantation of major histocompatibility mismatched allogeneic
spleen-derived murine T cells. If not treated otherwise, myeloablated mice developed
severe
GvHD symptoms.
Results: The GvHD symptoms were effectively suppressed, when MSC-EV
preparations were applied at 3 consecutive days, which exerted immune modulatory effects
in
a mixed-lymphocyte reaction assay. MSC-EV preparations lacking in vitro immune modulating
activities, however, hardly improved the symptoms of the GvHD mice. Thus, our results
demonstrate that not all MSC-EV preparations harvested from adult bone marrow-derived
MSCs
contain the same therapeutic potential.
Summary/Conclusion: Thus, successful transplantation of MSC-EVs into the
clinics requires a platform allowing identification of MSC-EV preparations with sufficient
therapeutic, most probably immune modulating activities.
Funding: This research was funded by SEVRIT Leitmarkt LifeScience.NRW
LS-1-1-051 g.
PF11.14
Milk extracellular vesicles can repair malnutrition-induced gut
barrier dysfunction
Mohamed Karim H. Maghraby
a, Bo
Lib, Lijun Chic, Catriona Lingd, Abderrahim
Benmoussae, Patrick Provostf, Andrea Postmusg, Abdirahman
Abdih, Agostino Pierroc, Celine Bourdonc and Robert
Bandsmac
aDepartment of Nutritional Sciences, School of Graduate
Studies, University of Toronto, mississauga, Canada; bThe Hospital for Sick
Children, Toronto, Canada; cTranslational Medicine Program, Hospital for Sick
Children, Toronto, Canada; dDepartment of Nutritional Sciences, School of
Graduate Studies, University of Toronto, Toronto, Canada; eCentre de recherche du
CHU Sainte-Justine, Montreal, Canada; fDepartment of Microbiology-Infectious
Disease and Immunity and Faculty of Medicine, Université Laval, Québec, Canada;
gFaculty of Medical Sciences, University of Groningen, Groningen, Netherlands;
hKEMRI-Wellcome Trust Research Programme, Kilifi, Kenya
Introduction: Malnutrition impacts approximately 50 million children worldwide
and is linked to 45% of global mortality in children below the age of five. Severe
acute
malnutrition (SAM) is associated with intestinal barrier breakdown and epithelial
atrophy.
Extracellular vesicles including exosomes (EVs; 30–150 nm) can travel to distant target
cells through biofluids including milk. Since milk-derived EVs are known to induce
intestinal stem cell proliferation, this study aimed to examine their potential efficacy
in
improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction.
Methods: Mice were fed either a control (18%) or a low protein (1%) diet for
14 days to induce malnutrition. From day 10 to 14, they received either bovine milk
EVs
enriched using differential ultracentrifugation and sucrose gradient purification
or control
gavage and were sacrificed on day 15, 4 hours after a Fluorescein Isothiocyanate (FITC)
dose. Tissue and blood were collected for histological and epithelial barrier function
analyses.
Results: Mice fed low protein diet developed intestinal villus atrophy and
barrier dysfunction. Despite continued low protein diet feeding, milk EV administration
improved intestinal permeability, intestinal architecture and cellular proliferation.
Summary/Conclusion: Our results suggest that EVs enriched from milk should be
further explored as a valuable adjuvant therapy to standard clinical management of
malnourished children with high risk of morbidity and mortality.
Funding: CB was generously awarded a catalyst grant from The Centre for Global
Child Health at the Hospital for Sick Children to support this work.
PF11.15
The impact of spheroids culture on mesenchymal stem cells and EV
production
Gina D. Kusuma
a, Anqi
Lib, Dandan Zhua, Hannah McDonaldb, Daniel
Chambersc, Jessica Frithd and Rebecca Limb
aHudson Institute of Medical Research, Clayton, Australia;
bThe Ritchie Centre, Hudson Institute of Medical Research, Clayton, Australia;
cQueensland Lung Transplant Service, The Prince Charles Hospital, Brisbane,
Queensland, Australia, Brisbane, Australia; dDepartment of Materials Science and
Engineering, Monash University, Clayton, Australia
Introduction: Mesenchymal stem/stromal cells (MSCs) are now widely believed as
bio-factories releasing bioactive products responsible for their therapeutic effect,
i.e.
cytokines, chemokines, and extracellular vesicles (EVs). MSCs are highly sensitive
to
physical stimuli from their surrounding microenvironment and can change their
characteristics in response to their environment. The application of 3D spheroids
cell
culture allows MSCs to adapt to their cellular niche environment which, in turn, influences
their paracrine signalling activity. We aim to determine how 2D and 3D culture
microenvironments can modulate the EV production and investigate their anti-fibrotic
activity.
Methods: For 2D culture, bone marrow-derived MSCs were cultured on standard
tissue culture plastic. For 3D culture, MSCs were aggregated into spheroids using
non-adherent 96-well plates and cultured with addition of 0.25% methylcellulose. To
collect
conditioned media, both 2D and 3D MSCs were cultured using serum free medium for 4 days.
EVs
were isolated by serial ultracentrifugation and were characterised on ExoView platform
which
allows simultaneous detection of particle size and expression of CD81/CD63/CD9. Cell
lysates
were collected for miRNA isolation and qRT-PCR was performed to analyse expression
of
candidate miRNAs. To model the progress of lung fibrosis, human lung fibroblasts (HLFs)
were
cultured with TGF-β1 to induce fibroblast activation, subsequently exposed to 2D and
3D EVs,
and collagen production was measured. Further, 2D and 3D MSC-EVs were added into human
lung
MSCs isolated from healthy and IPF patients and cell proliferation was assessed using
MTS
assay.
Results: 2D and 3D MSC-EVs have similar EV characteristics in terms of
particle size and EV tetraspanin markers expression. ExoView analysis showed expressions
of
CD81/CD63/CD9 and average particle diameters of <100 nm. On a cellular level, we
identified a panel of anti/pro-fibrotic miRNAs which are differentially expressed
in 2D and
3D MSCs. 2D and 3D MSC-EVs have similar anti-fibrotic activity shown by their ability
to
reduce collagen deposition in HLF cultures. Both 2D and 3D MSC-EVs could promote cell
proliferation on IPF lung MSCs but no overall effect on healthy lung MSCs.
Summary/Conclusion: This concept of engineering the cellular microenvironment
to promote EV production is as yet untouched and we foresee that in 3D cultures, we
can
culture MSCs for longer timeframe and therefore maximising the overall EV production
process. The outcome presents future potential for 3D culture of MSC to increase the
efficiency and feasibility of scalable EV production.
PF11.16
Outer Membrane Vesicles from Photobacterium damselae subsp.
piscicida: characterization and antigenic potential
Alexandra Teixeira, Inês Loureiro, Nuno M. S.
Santos and Ana Do Vale
i3 S – Instituto de Investigação e Inovação em Saúde, Universidade do
Porto; Fish Immunology and Vaccinology Group, IBMC – Instituto de Biologia Molecular
e
Celular, Universidade do Porto, Porto, Portugal, Porto, Portugal
Introduction: Photobacterium damselae piscicida (Phdp) is a Gram-negative
bacterium that causes a septicaemia in > 20 fish species worldwide. It represents
a major
drawback for aquaculture, whose importance has been sharply growing as a food supplier.
Given the Phdp massive mortality and widespread antibiotic resistance, an effective
vaccine
is highly needed. Extracellular products (ECPs) have an essential role in Phdp virulence,
containing important antigens. However, the ECPs’ identity remain undisclosed. In
our
efforts to dissect their composition, we found that they contain high amounts of outer
membrane vesicles (OMVs). These particles are potent weapons for bacteria and are
being
explored in the field of vaccinology, since OMVs present antigens in native conformations
and are strongly immunogenic, without requiring adjuvants. This potential associated
to the
urgent need for an anti-Phdp vaccine prompted us to isolate and characterize the OMVs
shed
by Phdp.
Methods: In order to harvest high amounts of pure Phdp OMVs, a reproducible
optimized protocol was developed: the bacteria-free supernatant from a Phdp overnight
culture is concentrated, dialysed and ultracentrifuged to collect the OMVs.
Results: Analysis of the obtained OMVs preparations by Transmission Electronic
Microscopy and Dynamic Light Scattering indicate that the main population of vesicles
has
sizes around 30–50 nm. Proteomic analysis of the vesicles revealed the presence of
the
apoptogenic AB toxin AIP56 that is known to play a major role in Phdp virulence, a
putative
pore-forming toxin, a putative adhesin/invasin and several Outer Membrane Proteins
(OMPs),
including a 64kDa OMP, predicted to be involved in iron acquisition, and 5 other OMPs
(18–34kDa), with an OmpA-like structure that may act as adhesins. Moreover, preliminary
in
vivo studies suggest that some of those proteins may have important roles for virulence,
since injection of knock-out strains in sea bass induced a decreased mortality comparing
to
the wt strains.
Summary/Conclusion: Our findings suggest that OMVs are a promising vaccine
candidate and we are currently studying their biological activities and determining
the
antigenic potential of the identified proteins.
Funding: This work was financed by FEDER – Fundo Europeu de Desenvolvimento
Regional funds through the COMPETE 2020 – Operacional Program for Competitiveness
and
Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT –
Fundação
para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior
in the
framework of the project POCI-01-0145-FEDER-030018 (PTDC/CVT-CVT/30018/2017).
PF12: Advances in Characterization of EV-associated Molecules:
Omics
Chair: Marta Adamiak – Cardiovascular Research Centre, Icahn School of Medicine, Mount
Sinai
Chair: Juan M Falcón-Pérez – CIC bioGUNE
PF12.01
Comparison of extracellular vesicle isolation methods from small
volumes of urine for radiation metabolomics analysis.
Meth Jayatilake
a, Charles P.
Hinzmana, Brian Fishb, Marjan Boermac, Meetha
Medhorab and Amrita Cheemaa
aGeorgetown University Medical Center, Washington, USA;
bMedical College of Wisconsin, Milwaukee, USA; cUniversity of
Arkansas for Medical Sciences, Little Rock, USA
Introduction: Whole body exposure to high doses of ionizing radiation (IR) can
potentially be lethal if radiation injury is not diagnosed and treated expeditiously.
When
considering a non-invasive approach for the identification of biomarkers of IR exposure,
we
and others have studied molecules in plasma, serum, saliva, and urine. However, these
matrices can potentially have significant background noise, obscuring potential biomarkers
of biological importance. Extracellular vesicles (EVs) are fast becoming a platform
for
biomarker discovery in radiation research as well as in other pathologies. However,
no
groups have investigated the use of metabolomics to analyse EVs derived from urine
in the
context of IR exposure. Furthermore, the dominant protocols for EV isolation from
urine
require a large (up to 30 mL) amount of starting volume, which may not be available
for many
studies. The aim of this study was to optimize EV isolation from rat urine and assess
radiation-induced alterations in urine EV number and metabolic content.
Methods: As a proof of concept, we compared and optimized several EV isolation
methods on small volumes of urine from male WAG/RijCmcr rats exposed to 0 Gy or 13 Gy
X-rays
to the whole body except the hind leg. Starting with either 500 µL or 1000 µL of urine,
we
isolated EVs using ultracentrifugation (UC) with filtration, size exclusion chromatography
(SEC), and a proprietary bead-based isolation method developed by a 3rd party provider.
EV
samples were characterized using nanoparticle tracking analysis. Metabolomics profiles
were
measured using LC-QToF-MS.
Results: We found that SEC resulted in the highest yield of EVs from as little
as 500 µL of urine, while UC was the poorest performing. LC-QToF-MS analysis revealed
that
SEC and UC had the most consistent identification of features, whereas the bead-based
method
contained artefacts likely as a result of the extraction method. We next used SEC
to isolate
EVs from a larger cohort of rats exposed to IR and analysed with MS. EV metabolic
content
will eb related to differences in survival and organ function between sham and irradiated
groups.
Summary/Conclusion: We conclude that SEC is the preferred method for isolating
EVs from small volumes of urine for broad-based mass spectrometric analysis, and that
the EV
metabolome may be a sensitive and specific early indicator of radiation injury.
Funding: The authors would like to acknowledge the Metabolomics Shared
Resource at Georgetown University which is supported by the Cancer Centre Support
Grant
Developmental Funds Award as well as by NIAID grant U01 AI133561.
PF12.02
MALDI-TOF MS fingerprinting of potential oesophageal cancer
biomarkers in exosomes isolated by LEAPS
Xiaodan Dai
a, Qingfu
Zhub, Yuchao Chenc and Fei Liua
aWenzhou Medical University, Wenzhou, China (People’s
Republic); bSchool of Ophthalmology & Optometry, School of Biomedical
Engineering, the First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang
325035, China, Wenzhou, China (People’s Republic); cSchool of Ophthalmology &
Optometry, School of Biomedical Engineering, Wenzhou Medical University, Wenzhou,
Zhejiang
325035, China; The First Affiliated Hospital, Wenzhou Medical University, Wenzhou,
Zhejiang
325035, China; Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou,
Zhejiang 325001, China, Wenzhou, China (People’s Republic)
Introduction: There is growing evidence that contents (including RNA and
proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction
of cancers. Here we aim to identify potential protein markers for oesophageal cancer.
Methods: Using our newly developed label-free exosome automated preparation
system (LEAPS), exosomes were isolated from 20 ml culture medium of various oesophageal
cancer cells with different differentiation profiles and different sources of metastasis.
Exosomes from 20 µl plasma of cancer patients at different clinical stages or with/without
relapse and healthy controls were also prepared by LEAPS. Matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to directly
analyse
exosomes. Protein identities of exosomal fingerprint peaks were tentatively assigned
by
correlation with top-down and bottom-up proteomics.
Results: Start from 20 ml culture medium or 20 µl plasma, high-quality
exosomes rapidly isolated by LEAPS are sufficient for MALDI-TOF mass spectrometry.
It seemed
that poorly differentiated cells showed more exosome release. MALDI-TOF MS fingerprints
of
exosomes in cells is cell line specific. MS profiles from poorly differentiated cells
showed
more peaks than that from highly differentiated cells. Fingerprints also allowed
classification of cancer cell lines through software mathematical analysis. We identified
different numbers of significantly differentially expressed peaks in exosomes of various
cancer cells. Fingerprints of exosomes derived from the poorly differentiated cells
showed
more elevated peaks. Top four peaks (2,427 m/z, 3,596 m/z, 4,458 m/z, 4,537 m/z) were
commonly down-regulated in exosomes of most cancer cells. Top four protein peaks (2,268 m/z,
5,713 m/z, 6,255 m/z, 6,337 m/z) that might be correlated to the differentiation profile
of
cancer cells were also identified. MALDI-TOF MS detection of exosomes in the plasma
and
clarifying identities of potential biomarker peaks will be done in the future.
Summary/Conclusion: The combination of LEAPS and MALDI-TOF mass spectrometry
provides a fast and high-throughput tool for exosomal marker discovery. Potential
biomarker
identified in exosomes derived from oesophageal cancer cells or from plasma of cancer
patients by this tool might be useful in cancer diagnosis and prognosis.
Funding: This research was supported by research funding provided by the
National Natural Science Foundation of China (31741036, 31700702, 21904098), the Zhejiang
Provincial and Ministry of Health Research Fund for Medical Sciences (WKJ-ZJ-1910),
the
Wenzhou Medical University (89218012, 89219012) and the Zhejiang Provincial Natural
Science
Foundation (LQ18H120006).
PF12.03
Fraction-based Proteomic profiling of serum extracellular
vesicles derived from cervical cancer patients
Raphatphorn Navakanitworakul
a,
Piyatida Molikaa, Sittiruk Roytrakulb, Thanawat
Pitakpornpreechac and jitti Harnprasertpongd
aDepartment of Biomedical Sciences, Faculty of Medicine,
Prince of Songkla University, Hat Yai, Thailand; bProteomic Research Laboratory,
National Center for Genetic Engineering and Biotechnology, Thailand Science Park,
Khlong
Luang, Thailand; cDepartment of Biochemistry, Faculty of Science, Prince of
Songkla University, Hat Yai, Thailand; dDepartment of Obstetrics and Gynecology,
Faculty of Medicine, Prince of Songkla University, Hat yai, Thailand
Introduction: Current evidence indicates that extracellular vesicles (EVs) can
release from most of cell types and affect adjacent or distant cells by circulating
in all
bodily fluids. Proteomic analysis of EVs from clinical samples is complicated by the
low
abundance of EV proteins relative to highly abundant circulating proteins. Size exclusion
chromatography (SEC) has been overcome as a method to deplete protein contaminants
and
enrich EVs.
Methods: we collected serum of healthy women and cervical cancer patients with
stage I–III and then counted concentration and size distribution of the EVs using
nanoparticle tracking analysis (NTA). Differential ultracentrifugation combined with
SEC was
used to isolate and purify EVs from contaminant proteins. Isolated EVs were investigated
their characteristic based on morphology using transmission electron microscope (TEM)
and on
expression of CD63, CD81, CD9 protein markers using western blot analysis. Fraction
no.8–10
of isolated EVs in among sample groups were profiled by nano-liquid chromatography
tandem
mass spectrometry (nanoLC-MS/MS) analysis.
Results: NTA shows that the concentration of EVs is increased in patients
compared with healthy women. Proteome profiles of EVs isolated by SEC were compared
in each
fraction. Moreover, we detected molecular evidence for fraction-specific molecular
pathways
in connection with cancer progression and complied a set of protein signatures that
closely
reflect the associated clinical pathophysiology.
Summary/Conclusion: These unique features in each fraction among sample groups
would be the informative considering in order to select for further analysis as in
vitro.
Funding: Faculty of Medicine, Prince of Songkla University, Thailand
PF12.04
Isolation methods of exosomes optimized for biomarker screening
based on proteomic analysis using 2-D gel electrophoresis
Eunjoo Kima, Eun-Sook
Choi
a, Hasan Md Al Faruquea, Jung-Hee Kima
and Mi Young Leeb
aDaegu Gyeongbuk Institute of Science and Technology (DGIST),
Daegu, Republic of Korea; bSoonchunhyang University, Asan City, Republic of
Korea
Introduction: Recently, diagnostic biomarkers from exosomes by proteomic
analysis have been reported, but it is required to optimize the isolation protocol
to screen
out more effective biomarkers. For serum-originated exosomes, it has been also reported
to
isolate them selectively, however, it is observed that a different method resulted
in
different protein profiles in 2-D gel electrophoresis.
Methods: We isolated exosomes by two discrete methods, using
ultracentrifugation and magnetic separation. Before ultracentrifugation and magnetic
separation, precipitation using polymer materials was perforemd. The isolation of
exosomes
by these two methods followed by comparison of their size, total vesicle number, morphology,
and protein markers. To identify protein biomarkers, proteomic analyis using 2-D gel
electrophoresis was performed.
Results: Both methods induced enrichment of exosome-specific proteins, but
protein profiles in each exosome fraction was totally different. The protein profiles
showd
that the magnetic seperation following a polymer-based precipitation step was more
efficient
to screen out candidate biomarkers, which showed nearly 200 protein profiles originated
from
exosomes.
Summary/Conclusion: In our study, magnetic separation of exosomes from serum
fraction was optimized for 2-D gel electrophoresis to observe identifiable biomarkers.
PF12.05
An extracellular small RNA-seq data processing pipeline
optimized for high-performance computing
Chenghao Zhu and Angela Zivkovic
Department of Nutrition, UC Davis, Davis, USA
Introduction: A variety of RNA species is found in extracellular biofluids
such as blood, bile, and urine, carried by extracellular particles including extracellular
vesicles (EVs) and lipoproteins (e.g high density lipoproteins (HDLs)). The extracellular
RNA (exRNA) carried by EVs and HDLs is of great interest for two reasons: 1) the exRNA
within different carriers could be diagnostic of the state of the tissues from which
the
particles originate, and 2) exRNA has been shown to affect gene expression in target
cells.
Although the origin and functions of exRNAs remain largely unknown, there is growing
interest in exRNA research for the development of diagnostics and new therapeutic
targets.
Small RNA sequencing is widely used to estimate the abundance of exRNAs in biofluid
samples.
Methods: Here we present a data processing pipeline for extracellular small
RNA sequencing. Sequencing data are pre-processed through quality control, and then
aligned
to the endogenous genome to obtain the gene counts for various RNA biotypes, including
microRNA, tRNA, rRNA, PIWI-interacting RNA, long non-coding RNA (lncRNA) and protein
coding
RNA. It also aligns sequencing reads to exogenous databases, including the ribosomal
RNA
sequence database SILVA, and all sequenced bacteria genomes available on Ensembl,
to
estimate the abundance of exogenous genes.
Results: We analysed a publicly available small RNA-seq dataset of HDL from
three Systemic lupus erythematosus (SLE) patients and three healthy controls using
this
pipeline. The miRNA hsd-mir-93, lncRNA AL137186.2 and AC108050.1 were elevated in
SLE
patients compared to controls. Exogenous RNA reads mapped to Bacteroidetes were also
elevated in SLE patients.
Summary/Conclusion: Our pipeline is able to process exRNA sequencing data and
estimate the abundance of major exRNA species, as well as exogenous RNA taxonomy.
The
pipeline is optimized for the job scheduler SLURM, and can therefore utilize the full
computational power of high-performance computers. The pipeline is publicly available
on
github (www.github.com/zhuchcn/exceRNApipeline).
Funding: NIH/NIA R01AG062240, NIH UG3 CA241694-01
PF12.06
Characterization of extracellular vesicles (EVs) derived from
Peripheral Blood Mononuclear Cells (PBMCs) of Inflammatory Bowel Disease (IBD)
patients
Maria Emilia Scharrig Fernandez, Waldo
Spessott and Claudio Giraudo
Thomas Jefferson University, Philadelphia, USA
Introduction: IBD is a chronic hyperinflammatory disorder that severely
compromises the intestines. The aetiology of IBD is poorly understood. However, it
has been
associated with a dysregulation of the immune system and gut microbiota and with genetic
and
environmental factors. Cumulative evidence indicates that EVs play an essential role
in
modulating immune responses. Recent research suggests that EVs derived from dendritic
cells,
saliva and intestinal epithelial cells may be involved in the progression of IBD
inflammation. However, little is known about the contribution of immune cells-derived
EVs
with this pathology. The goal of this study is to shed light on the contribution of
PBMC-derived EVs on IBD pathogenesis.
Methods: Here we characterized and compared the composition of EVs derived
from PBMCs of 3 IBD patients and 1 Healthy control. EVs were isolated by differential
centrifugations from the supernatant of PBMC activated with CD3-CD28 beads for 6 days
in
serum-free media. Size and concentration were analysed using a Nano Sight 300 instrument,
while the presence of known EVs markers (CD63, CD81, Hsp70) was analysed by immunoblotting.
Whole EVs proteome was performed by MS/MS and functional-enrichment analysis was done
using
FunRich with Uniprot database.
Results: Proteomics analyses identified a total of 1299 proteins in the four
groups. Of those, 673 (51.8%) were present in both the IBD patients and control. This
group
of protein was composed of several Ras-related proteins, eukaryotic initiation factors,
granzyme, CD9, tubulin, and serpins among others. Patients’ EVs shared 46 proteins
in common
such as proteasome subunit beta type-10, T cell receptor beta, and the amine oxidase
containing copper 3. Interestingly, each patient sample had a unique group of proteins.
Among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type-3,
and
signalling lymphocytic activation molecule (SLAMF1).
Summary/Conclusion: These preliminary studies show that the EV composition
from PBMCs of IBD patients is specific and differs from a healthy control. This exclusive
composition has the potential to be used as a biomarker for diagnostics and progression
of
the disease, and it could also provide new insights into our understanding of the
cellular
pathways involved in the pathogenesis of IBD. The studies were performed with corresponding
IRB approvals.
PF12.07
Proteomic analysis of exosomes isolated using precipitation and
column-based approaches
Tânia Soares Martins
a, Rui
Marçalob, Cristóvão B. da Cruz e Silvac, Tânia Melod,
Dário Trindadeb, Ilka Martins Rosaa, Franscisco Amadod,
Odete A. B. da Cruz e Silvab and Ana Gabriela Henriquesb
aiBiMED – Institute of Biomedicine, Aveiro, Portugal;
bNeuroscience and Signalling Group, Department of Medical Sciences, Institute
of Biomedicine (iBiMED), University of Aveiro, 3810–193 Aveiro, Portugal, Aveiro,
Portugal;
cLaboratório de Instrumentação e Física Experimental de Partículas – LIP, Av.
Elias Garcia 14–1°, 1000–149 Lisboa, Portugal, Lisboa, Portugal; dDepartment of
Chemistry, QOPNA (Organic Chemistry Natural and Agrofood Products and LAVQ-REQUIMTE),
University of Aveiro, Aveiro, Portugal, Aveiro, Portugal
Introduction: Exosomes are a subtype of small extracellular vesicles (EVs)
involved in various physiological and pathological processes with huge potential as
biomarker resources or as therapeutic tools. Although several exosome isolation approaches
are available, complementary studies focusing on optimizing the methods for human
blood-derived exosomes isolation and method-specific comparative exosomal proteomic
profiles
will be of clinical value.
Methods: Blood-derived EVs were isolated through precipitation- and
column-based methods and characterized by transmission electron microscopy, nanoparticle
tracking analysis and western blot analysis. Serum-derived exosomal proteomes were
analysed
by mass spectrometry (MS). The resulting proteomes were then overlapped with the proteomes
obtained from exosome-related databases, to determine the % of similar content. In
addition,
bioinformatic analysis, including Gene Ontology (GO) was carried out.
Results: Both methodologies tested isolated particles with the expected
morphology and size range, although the column-based method isolated a higher number
of
particles. About 75% of the exosomal proteins identified through MS overlapped with
the
proteomes extracted from the databases. GO terms were similar for the proteomes isolated
from the column- and precipitation-based methodologies. The top 3 GO terms identified
for
Molecular Function were ion binding, peptidase activity and enzyme regulator activity
and
for Biological Process were immune system process, transport and response to stress.
Further, partial least square analysis revealed a clear segregation of proteomes obtained
by
the distint methodologies and complementary statistical analysis revealed the proteins
differently expressed.
Summary/Conclusion: No major differences were found in the top 3 biological
processes and molecular function based on GO analysis. Nonetheless, the two approaches
result in different EVs yields and significant proteome differences were identified.
Characterization of distinct methods for blood-derived exosomes isolation can be useful
in
the context of EVs potential in disease diagnostics/therapeutics.
Funding: The work was supported by an Alzheimer’s Association grant
(2019-AARG-644347), Instituto de Biomedicina (iBiMED)- UIDB/04501/2020, FCT, COMPETE
program, QREN, European Union; “pAGE”(CENTRO2020-CENTRO-01-0145-FEDER-000003), Centro
2020
program, Portugal 2020, European Union. AGH is supported by the FCT stimulus of scientific
employment (CEECIND/01803/2017). TSM is supported by a FCT PhD grant
(SFRH/BD/145979/2019).
PF13: Separation and Concentration
Chair: Metka Lenassi – University of Ljubljana, Faculty of Medicine, Institute of
Biochemistry
Chair: Fuquan Yang – Institute of Biophysics, Chinese Academy of Sciences
PF13.01
From total plasma EVs to immuno-captured neuronal EVs – yield,
purity and enrichment
Maja Mustapic
a, Joseph M.
Blommerb, David A. Routenbergc and Dimitrios
Kapogiannisd
aNIH/National Institute on Aging, Baltimore, USA;
bNational Institute on Aging, Intramural research program, Laboratory of
Clinical Investigations, Baltimore, USA; cMeso Scale Diagnostics, LLC.,
Rockville, USA; dLaboratory of Clinical Investigation, National Institutes of
Aging, Baltimore, USA
Introduction: We and others are developing biomarkers for neurodegenerative
diseases using neuronal-enriched EVs immunocaptured from a suspension of total plasma
EVs.
Here we assess how the isolation method for total EVs affects the yield, purity and
enrichment of neuronal EVs.
Methods: For N = 5 subjects, total EVs were isolated by EV precipitation
solution (ExQ), EV precipitation solution plus bipartite resin columns (ExU) and Size
Exclusion Chromatography (qEV) from 0.5, 0.5 and 2.7 ml plasma, respectively. Then,
neuronal-enriched EVs were immunoprecipitated using anti-L1CAM antibody. In total
and L1CAM
EVs, we measured particle concentration by Nanoparticle Tracking Analysis, protein
concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins
CD81, CD63 and CD9 on intact EVs.
Results: For total EVs, yield followed the order of ExQ > qEV > ExU,
assessed by particle (p < 0.0001) and protein concentrations (p < 0.001). L1CAM EVs
immunocaptured after ExQ showed 16-fold higher particle (p < 0.001) and fivefold higher
protein (p < 0.01) concentrations compared to L1CAM EVs after ExU, and 41-fold higher
particle (p < 0.001) and 11-fold higher protein (p < 0.001) concentration compared
to
L1CAM EVs after qEV. L1CAM EVs after EV precipitation (ExQ) showed 48, 35 and 30-fold
higher
CD81, CD63, and CD9 concentrations (p < 0.001) compared to L1CAM EVs after ExU, and
59,
43 and 36-fold higher CD81, CD63, and CD9 concentrations (p < 0.001) compared to L1CAM
EVs after qEV. L1CAM EVs following 3 different methods had equal purity assessed by
ratios
of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations
(p = ns).
Summary/Conclusion: L1CAM EV immunocapture preceded by ExQ exceeded the yield
of immunocapture preceded by ExU or qEV. Recovered L1CAM EVs showed equal purity by
particle/protein and tetraspanin/particle metrics. Neuronal enrichment results will
be
available by the time of ISEV. Immunoprecipitation following ExQ, often considered
impure,
purifies final isolates as effectively as more onerous methods typically considered
purer.
Balancing sensitivity, purity and scalability is essential for implementation of blood
biomarkers in the clinical setting and may be achieved by combining techniques.
Funding: This research was supported in part by the Intramural research
Program of the NIH, National Institute on Aging.
PF13.02
Characterisation of breath exosomes: towards non-invasive
diagnosis
Deanna Ayupova
a, Renee
Gorehamb and Paul Teesdale-Spittlec
aSchool of Chemical and Physcial Sciences, Victoria
Univeristy of Wellington, Wellington, New Zealand; bUniversity of Newcastle,
Newcastle, Australia; cSchool of Biological Sciences, Victoria Univeristy of
Wellington, Wellington, New Zealand
Introduction: Breath-derived exosomes present new potential for non-invasive
diagnosis of lung cancer. However, breath-derived exosomes have not been well characterized
and methodology for their purification has not been optimised. In order to exploit
their
potential for diagnosis, it is first necessary to develop methods that reproducibly
provide
high quality pure exosomes from breath. In this study, we optimise methods for their
isolation and characterise them in comparison to exosomes derived from cell culture
models.
Methods: In order to characterize exosomes from exhaled breath condensate
(EBC) it was first necessary to optimize methods for isolation of pure, intact, and
high
quality exosomes. To this end, isolation methods were optimised on cell-derived exosomes
and
then applied to EBC, yielding high quality exosomes from size exclusion chromatography
(SEC). EBC exosomes were compared with those from A549 and WI-cells using DLS, TEM,
and
Cryo-SEM. An immunoblotting-grid technique was used to validate the presence of
exosome-specific markers CD63 and CD81. Protein content of exosomes were quantified
and
compared.
Results: SEC-based isolation was more effective at isolation of pure and
intact exosomes than ultracentrifugation, with the highest purity exosomes obtained
in the
middle fractions of the exosome-containing eluate. Exosomes from EBC had a size range
(45–100 nm), protein content (20–40 ug/mL) and molecular markers typical of cell-derived
exosomes.
Summary/Conclusion: Breath-derived exosomes isolated through size exclusion
chromatography are sufficiently pure for diagnostic purposes and are phenotypically
similar
to exosomes derived from other sources. We foresee their use in non-invasive diagnostics
for
lung cancer as an important future application.
PF13.03
Ligand-based Exosome Affinity Purification (LEAP) is a rapid and
reproducible method for the enrichment of functional EVs
Jancy Johnson
a, Chantelle
Blythb, Sam Lawc, Angus Testerb and Gregor
Lichtfussb
aExopharm Ltd/University of Melbourne, Parkville, Australia;
bExopharm, Melbourne, Australia; cExopharm Ltd, Melbourne,
Australia
Introduction: Platelet-derived extracellular vesicles (pEVs) represent the
next generation of therapeutic biologics as they enable a more refined and targeted
approach
when compared to crude blood derivatives currently used for treating diseases such
as
cancer, thrombocytopenia and chronic wounds. However, development of an EV-based therapeutic
is hindered by the lack of a scalable, validated and reproducible purification process.
In this study, pEVs were isolated from activated platelet concentrates and purified
using
Exopharm’s Ligand-based Exosome Affinity Purification (LEAP) technology to produce
a
functionally active EV therapeutic.
Methods: Platelet concentrates (n = 20) were obtained from the Australian Red
Cross Blood Service and were activated by Exopharm’s proprietary process. Activation
was
verified by measuring CD62p using flow cytometry. The resulting platelet releasate
(500 ml)
was subjected to LEAP purification to isolate pEVs. For characterization, protein
concentration was determined by a bicinchoninic acid assay, microfluidic resistive
pulse
sensing (MRPS) was used to perform a particle count and transmission electron microscopy
(TEM) enabled visualization of EV morphology. Key EV markers were detected using Mass
Spectrometry (MS) and Western Blots.
To confirm biological activity, human dermal fibroblasts were subjected to serum starvation
for 16 hours before treatment with pEVs (15 µg/ml). Cell growth was recorded by the
real-time xCelligence system and differences in proliferation were statistically analysed
using a one-way ANOVA.
Results: MRPS and TEM both revealed isolated pEVs to be 100–300 nm in size.
The final product was positive for platelet markers (CD41, CD62p) and key EV markers
(Tsg101, Alix, CD63). Treatment with purified pEVs significantly increased proliferation
in
serum-starved fibroblasts over 48 hours.
Summary/Conclusion: Exopharm’s LEAP technology is a rapid and reproducible
purification process which produces pEVs that adhere to MISEV 2018 guidelines and
are
functionally active.
Funding: All funding was through Exopharm Ltd (ASX:EX1)
PF13.04
A Novel but simple method to obtain purified exosomes by
one-step ultracentrifugation
Fengmei Pi
a, Yangwu
Fangb, Yi Wangb and Peixuan Guoa
aExonanoRNA, Columbus, USA; bExonanoRNA, Foshan,
China (People’s Republic)
Introduction: Exosomes are Extracellular vesicles (EVs) that are derived from
endosome membrane. They are usually 50–150 nm in diameter, actively secreted in most
living
cells. Originally, exosomes were thought to act as cellular garbage disposals. Recent
studies showed that exosomes not only can serve as biomarkers for diagnosis, but also
can be
used as an ideal delivery vehicle for drugs in therapeutics. Exosomes are natural
carrier
for mRNA, miRNA, siRNA, protein, DNA and peptide for long distance intercellular
communication. Isolation of exosomes is challenging due to their small size and
heterogeneity. Traditional differential ultracentrifugation method is still the gold
standard for exosome purification. To further explore the potentials of exosomes being
as
the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to
purify
them in high quality at high yield.
Methods: Here, we report a novel method to obtain intact shape, high- quality
and high purity exosomes with one-step ultracentrifugation by using “Exojuice”.
Results: Data of nanoparticle tracking analysis (NTA) and western blotting
showed “Exojuice” can yield exosomes with a simpler method to obtain higher purity
exosomes
in comparison to previous method of cushion ultracentrifugation using Optiprep.
Summary/Conclusion: Our method can be used to purify exosomes from cell
culture medium, serum, urine, saliva, and other biofluids.
PF13.05
A straightforward device to extract apoplastic fluid from
succulent fruits for higher purity of extracellular vesicles
Tuong Nguyen
a, Wei Duana
and Phuong Tranb
aSchool of Medicine and Centre for Molecular and Medical
Research, Deakin University, Waurn Ponds, Victoria, Australia, Waurn Ponds, Australia;
bSchool of Medicine and Centre for Molecular and Medical Research, Deakin
University, Waurn Ponds, Victoria, Australia, Warun Ponds, Australia
Introduction: Edible plants are emerging as a sustainable source for
extracellular vesicle (EV)-based drug delivery vehicles. However, current isolation
methods
(e.g. grinding or squeezing) may cause destruction of plants’ biostructures, and in
turn
leads to unwanted effects in downstream applications and complicates the study of
nanovesicles – cell. Therefore, we designed a simple device that allows the extraction
of
apoplastic fluid (AF) from succulent fruits, facilitating EV isolation as well as
effective
downstream applications.
Methods: An inner filter tube was designed to extract AF with a determined
membrane pore size. AF was collected by low-speed centrifugation method and then filtered
to
eliminate the impurities from the cytoplasm and damaged cells. Minced juice (MJ) was
homogenized by a blender and then centrifuged to remove large fragments. Subsequently,
the
differential centrifugation method was employed to extract EVs from AF and MJ.
Fourier-transform infrared spectroscopy (FTIR), nanoparticle tracking analysis (NTA),
and
transmission electron microscopy (TEM) were performed to discriminate AF, MJ and their
EVs.
Results: The “spectroscopic” protein-to-lipid (P/L) ratio of AF (1.23 ± 0.03)
is significantly lower than that in MJ (1.27 ± 0.01), showing the higher lipid contents
in
AF, which may result from the loss of lipids in MJ obtained from grinding or juicing
methods. Similarly, FTIR showed the difference in P/L ratio between AF and its EVs
(1.23 ± 0.03 and 1.29 ± 0.02, respectively). NTA showed the sharper peak and smaller
vesicle
size in the following order: MJ (173.0 ± 21.3 nm), AF (154 ± 11.0 nm), AF-derived
EVs
collected at 40,000 × g and 10,000 × g (108.5 ± 2.4 nm and 95.7 ± 3.1 nm, respectively).
Furthermore, TEM study indicated that the collected EVs exhibited a typical lipid
bilayer of
extracellular nanovesicles.
Summary/Conclusion: By using a reusable filter device, we successfully
isolated AF from succulent fruits, paving the way to collect plant EVs without an
interference of significant biodestruction or damaged cells, hence improving the purity
of
EVs and facilitating downstream applications. Moreover, this method is straightforward,
reproductive, and can be potentially used in a large-scale production.
Funding: Tuong N.G. Nguyen was supported by Deakin University Postgraduate
Research Scholarship (DUPR). Dr. Phuong H-L Tran is the recipient of the Australian
Research
Council’s Discovery Early Career Researcher Award (project number DE160100900).
PF13.06
Method to simultaneously capture multiple classes of intact
extracellular RNA carriers including extracellular vesicles and lipoprotein
particles
Hannah E. Houtsa, Emily R. Mallickb, Joanne K.
Agusc, Chenghao Zhud, Bonita H. Powelle, Yiyao
Huangf, Dominik Buschmanng, Tanina Arabb, Juan Pablo
Tosarh, Micheal Paulaitisi, Carlito Lebrillaj, Wyatt
Vreelandk, kenneth witwerl, Angela Zivkovicd and
Brian V. Hong
aUniversity of California, Davis, Davis, USA;
bDepartment of Molecular and Comparative Pathobiology, Johns Hopkins University
School of Medicine, Baltimore, USA; cDepartment of Nutrition, University of
California Davis, Davis, USA; dDepartment of Nutrition, UC Davis, Davis, USA;
eJohns Hopkins University School of Medicine, Baltimore, USA;
fDepartment of Molecular and Comparative Pathobiology Neurology, The John Hopkins
University School of Medicine, Baltimore, USA; gDivision of Animal Physiology and
Immunology, School of Life Sciences Weihenstephan, Technical University of Munich,
Freising,
Germany, Freising, Germany; hUniversidad de la República, Institut Pasteur de
Montevideo, Montevideo, Uruguay; iDepartment of Ophthalmology, The John Hopkins
University School of Medicine, Baltimore, USA; jDepartment of Chemistry,
University of California Davis, Davis, USA; kNational Institute of Standards and
Technology, gaithersburg, USA; lDepartment of Molecular and Comparative
Pathobiology and Department of Neurology, The Johns Hopkins University School of Medicine,
baltimore, USA
Introduction: Extracellular particles including extracellular vesicles (EVs),
lipoproteins, and free proteins are carriers of extracellular RNA (exRNA), which has
been
shown to regulate cellular function. Because these particles have different physiological
origins, they have different RNA signatures, so the first step to understanding the
biology
of exRNA is to isolate individual particle fractions with high purity and efficiency.
Current methods for isolating EVs are optimized for increased yields and purity of
EV
fractions but typically require multiple millilitres of starting plasma and do not
capture
the other exRNA carrier particle types. Methods that can capture EVs from low starting
plasma volumes and can also capture other exRNA carriers simultaneously are needed
for
analysing samples from previously conducted large cohort studies, biorepositories,
and in
populations where sample volume is limiting.
Methods: We have developed a method adapted from lipoprotein isolation that
requires only 500 µl of starting plasma, and uses brief ultracentrifugation (UC) followed
by
fast protein liquid chromatography (FPLC) to capture 6 classes of purified exRNA carriers
including EVs, LDL, HDL, lipidated albumin, proteins, and VLDL/chylomicrons. We have
validated successful capture of EVs by Microfluidic Resistive Pulse Sensing (MRPS,
Spectradyne), transmission electron microscopy (TEM), and Single Particle Interferometric
Reflectance Imaging System (SP-IRIS; ExoView) with optional fluorescence.
Results: We have observed 1.02 × 1010 particles per ml from a 1 ml FPLC
fraction of EVs measured from 25,000 events by MRPS, confirming that EVs are being
captured
by this method. There were also 1.26 × 1010 particles/ml and 2.70 × 1010 particles/ml
in the
two subsequent 1 ml fractions that are known to contain lipoprotein particles, though
these
were measured from 1,500 events each. By TEM we confirmed these observations that
EVs are
eluting before lipoprotein particles with some EVs eluting later in fractions containing
lipoproteins.
Summary/Conclusion: These results confirm the efficacy of the method in
isolating multiple exRNA carrier fractions simultaneously from a single 500uL plasma
sample,
making it amenable for the analysis of exRNA in samples from large cohort studies,
biorepositories, and vulnerable populations such as the elderly and young children.
Funding: NIH/NIA R01AG062240; NIH UG3 CA241694-01
PF13.07
Optimizing the isolation of placental mesenchymal stromal
cell-derived extracellular vesicles in a 3D bioreactor system
Leora Goldbloom-Helzner
a and Aijun
Wangbaaaaa
aUC Davis, Davis, USA; bUC Davis Medical Center,
Sacramento, USA
Introduction: Extracellular vesicles (EVs) derived from placental mesenchymal
stromal cells (PMSCs) have the potential to provide neuroprotection at sites of injury.
However, a rate limiting step in EV research is the low yield, high technical time,
and high
cost of current isolation procedures. To address this inefficiency, we cultured PMSCs
in a
unique bioreactor system to increase the absolute yield of EVs per mL of media and
per cell.
Future studies will determine if this system can improve PMSC EV yield without altering
the
demonstrated neuroprotective properties of PMSC-EVs.
Methods: PMSCs were cultured in the bioreactor for ten weeks. EV-conditioned
media was collected weekly and EVs were isolated through differential centrifugation.
Nanoparticle tracking analysis (NTA) measured EV size and concentration. Western blots
tested for normal EV markers (CD9, CD63, and CD81, Calnexin(-)) and enzyme-linked
immunosorbent assays (ELISA) measured levels of characteristic growth factors in conditioned
media including vascular endothelial growth factor (VEGF), brain-derived neurotrophic
factor
(BDNF), and hepatocyte growth factor (HGF).
Results: EVs remained consistent until week eight, after which a decrease in
both EV size and concentration was seen. Western blots revealed normal positive expressions
of CD9, CD63, and CD81 and negative expressions of calnexin. Levels of VEGF, BNDF,
and HGF
were comparable after 10 weeks. Cost analysis revealed an overall increase in EV yield
for
shorter labour time and lower material cost.
Summary/Conclusion: This initial study uses a bioreactor system for a unique
source of cells and has brought us closer to optimizing PMSC EV isolation protocols
for
increased yield, lower cost and time commitment, and maintained sample purity. Preliminary
data suggests the EV phenotype and cell secretome are consistent with those present
in
current culture settings. Future experiments will assess the preserved neuroprotective
properties of the PMSC EVs.
PF13.08
A novel method for isolating extracellular vesicles from cell
culture media and plasma using polyethylenimine
Catherine R. Taylor, Alexander Macpherson,
Sheena Fry, Sebastien R. Fournier, Jeremy Roy, Andrew Joy, Gabriel Wajnberg, Ngoc-Nu
Mai,
David Barnett, Anirban Ghosh, Stephen M. Lewis and Rodney J. Ouellette
Atlantic Cancer Research Institute, Moncton, Canada
Introduction: Due to their ability to transport DNA, RNA, and protein cargoes
between cells, extracellular vesicles (EVs) are becoming popular for biomarker discovery
as
well as for therapeutic delivery. Here we describe the development of a novel precipitation
method for the isolation of EVs from cell culture media and plasma that is based on
polyethylenimine (PEI), an inexpensive, water-soluble, and biocompatible cationic
polymer.
PEI is a group of hydrophilic cationic polymers that are synthesized as either linear
or
branched forms of varying molecular masses (1,000 to 750,000 Da) and are widely used
in the
biomedical field as a coating and transfection agent.
Methods: Linear and branched PEI of varying molecular weights (MW) were tested
for their ability to precipitate EVs from either conditioned culture media (CCM) or
human
plasma. Isolated EVs were characterized by Western blotting and nanoparticle tracking
analysis (NTA). The small RNA profile of EVs isolated using PEI from human plasma
was
analysed by NGS and EV-specific miRNAs were confirmed by digital droplet PCR (ddPCR).
Mass
spectrometry (MS) was used to analyse the proteome of PEI-captured EVs from plasma.
HEK293
cells producing GFP+ EVs were used to optimize conditions for release of EVs from
both
linear and branched PEI by fluorescent spectrophotometry and flow cytometry
measurements.
Results: Linear and branched PEI were both able to precipitate EVs as
determined by Western blotting for EV protein markers; however, branched PEI with
MW >
10,000 Da and linear PEI with MW > 25,000 Da were more efficient for EV precipitation
than lower MW forms. Despite its known ability to bind nucleic acids PEI was unable
to
capture cell-free DNA from plasma, although RNA and in particular EV-associated miRNAs
such
as mir-142-3p were recovered. MS revealed that PEI enriches extracellular exosome
proteins
from plasma. EVs captured from CCM by PEI could be released from the complex using
heparin
or high salt conditions.
Summary/Conclusion: PEI has an unexpected preference for associating with EVs
compared to nucleic acids in complex biological samples and has a hitherto unrecognized
application for EV precipitation.
Funding: Atlantic Canada Opportunities Agency
PF13.09
Assessment of various extracellular vesicle isolation methods
through chemical analysis with surface-enhanced Raman spectroscopy
Alyssa Powell
a, Hanna J.
Kostera, Dina Phama, Andrew Birkelandb and Randy
Carneya
aDepartment of Biomedical Engineering, University of
California, Davis, Davis, USA; bUC Davis Medical Center, Sacramento, USA
Introduction: There is ongoing debate about which is the most appropriate
method for isolation of EVs, with most labs using some combination of differential
ultracentrifugation (UC), size-exclusion chromatography (SEC), and/or density gradient
ultracentrifugation (DG). Here we applied a surface-enhanced Raman spectroscopy (SERS)
analysis platform to compare chemical composition of the isolate from each method
against
lipoprotein standards to assess the relative purity of the EV preps.
Methods: 2–3 mL of plasma was separated from whole blood collected from head
and neck cancer patients. Each sample was split into 3 batches and EVs were isolated
by
either UC, SEC, or DG. Following isolation, samples were incubated on commercial SERS
substrates and Raman spectra were collected. Lipoprotein standards were purchased
and also
measured for comparison. Using principle component analysis (PCA), spectra were analysed
for
chemical variability.
Results: SERS analysis of SEC, UC, and DG isolated EVs were chemically
distinguishable using simple PCA. The chemical changes could in large part be attributed
to
fitting the differences in spectra to lipoprotein standards. We found that UC isolated
populations clustered with the high-density lipoproteins (HDL), SEC populations with
the
low-and very low-density lipoproteins (LDL, VLDL), and DG populations were more variable,
but mainly clustered together with the high-density-lipoproteins (HDL).
Summary/Conclusion: This set of experiments matches our expectation that
various lipoprotein would contaminate EV preps according to their relative size and
density
distributions. No single isolation method could separate pure EV samples. This study
also
illustrates the utility of label-free SERS analysis for rapid chemical characterization
of
EVs.
PF13.10
Bioreactors: lessons to develop an extracellular vesicle
factory
Vanessa Chang, Priscila Dauros-Singorenko,
Lawrence W. Chamley, Colin L. Hisey and Cherie Blenkiron
The University of Auckland, Auckland, New Zealand
Introduction: High density mammalian cell culture systems (bioreactors)
provide valuable advantage for large scale production of secreted products such as
Extracellular Vesicles (EV). However, optimisation of design selection, handling and
operational costs can be quite challenging. Here we provide our experience with a
CELLine
Bioreactor system.
Methods: Cultures of 6 adherent cell lines were established in CELLine AD 1000
bioreactors and propagated for up to 19 weeks. Media was changed twice weekly and
cells shed
into serum-free conditioned medium were counted and assessed for viability. NanoEVs
were
isolated by sequential centrifugation (2000 g – 10,000 g – 100,000 g) and Size Exclusion
Chromatography (SEC). NanoEVs were characterised in their protein (BCA) and particle
(Nanoparticle Tracking Analysis) amount, EV markers (Western blotting) and morphology
(Transmission electron microscopy, TEM).
Results: The viability of shed cells varied between cell lines and through
time, suggesting a changing dynamic during reactor establishment and continuous growth
phases, that was specific to each cell line. HDFa, BT20 and BT474 consistently shed
mainly
dead cells (60–100%), as opposed to MCF7 and MDA-MB-231 which predominantly shed live
cells.
SEC fractionation of nanoEVs identified a dominate EV-rich peak and significant quantities
of smaller proteins, highlighting the need for further purification. NanoEV yields
from each
3–4 day culture averaged 2–7 × 1010 particles, representative of yields obtained from
cells
grown in 8 to 28 conventional T175 tissue culture flasks. EV markers and TEM confirmed
the
protein profiles and morphology of EVs obtained from bioreactors.
Summary/Conclusion: High density bioreactor cultures offer a physiologically
relevant, cost and space efficient approach to produce significant amounts of EVs,
providing
sufficient material for numerous experimental uses. In our hands, with careful twice
weekly
management, they can be propagated for up to 19 weeks without significant changes
to the
EVs.
Funding: Breast Cancer Foundation NZ
PF13.11
Rapid and efficient isolation of extracellular vesicles by
Phosphate−DNA−Cholesterol functionalized defected Metal Organic Framework
Bo Li
a, Weilun Pana and
Lei Zhengb
aDepartment of Laboratory Medicine, Nanfang Hospital,
Southern Medical University, Guangzhou, China (People’s Republic); bDepartment of
Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515,
China, Guangzhou, China (People’s Republic)
Introduction: Extracellular vesicles (EVs) have potential applications for
clinical theranostics. Ultracentrifugation is most commonly adopted to the EVs isolation,
which is recommended as a gold standard method. However, ultracentrifugation is
time-consuming and expensive equipment requirement, resulting in the co-isolation
of
contaminants such as protein aggregates. Therefore, our aim is to develop a rapid
and
efficient platform to isolate heterogonous EVs based on the insertion of lipid molecules
into the EVs membrane to avoid co-isolation of non-membranous protein particles.
Methods: Herein, a defected nanoscale functional Metal Organic Framework (MOF)
was constructed as an efficient platform for EVs isolation. Typically, one single-stranded
DNA was designed and modified with a phosphate group at the 5ʹ-end and cholesterol
at the
3ʹ-end to form a capture DNA named Phosphate−DNA−Cholesterol (PDC). The phosphate
group
forms a strong covalent bond with the designed defeated site of Zr (IV) in MOF UiO-66-NH2
and the cholesterol inserts into the phospholipid bilayer to capture EVs without
non-membranous particles contamination. The formed MOF−Phosphate−DNA−Cholesterol−EVs
(MOF@PDC@EVs) system was further treated with DNase I for DNA hydrolysis to give high
pure
EVs.
Results: A rapid and efficient isolation platform of EVs based on a defected
MOF functionalized with Phosphate-DNA-Cholesterol (MOF@PDC) has been constructed
successfully. Compared with Ultracentrifugation, MOF@PDC platform promises to isolate
size
heterogeneous EVs i) without non-membranous particles contamination, maintaining EVs
intact
membrane structure, protein components, and biological functions; ii) with the ability
to
capture EVs with 78% isolation efficiency; iii) makes EVs isolation process simple
and fast,
which could be finished in 40 minutes without requirement of the expensive equipment.
Summary/Conclusion: In conclusion, this rapid and efficient platform is
suitable for isolation EVs from biological fluid for downstream protein analysis.
This work
opens a new perspective in MOF-based separation researches and may shed light on further
studies towards EVs isolation.
Funding: This study is supported by the National Natural Science Foundation of
China (81702100), the Major Program of Health Care and Innovation of Guangzhou Project
(201704020213, 201604020015).
PF14: EVs as Delivery Vehicles
Chair: Pieter Vader – Laboratory of Clinical Chemistry and Haematology, UMC Utrecht;
Department of Experimental Cardiology, UMC Utrecht, The Netherlands
Chair: Riccardo Alessandro – University of Palermo School of Medicine
PF14.01
Site-specific integration enables rapid cell engineering for the
development of precision extracellular vesicle therapeutics
Shelly Martin, Aaron Sulentic, Leonid
Gaidukov, Ke Xu, Gauri Mahimkar, Chang Ling Sia, Nuruddeen Lewis, Scott Estes, Kevin
Dooley
and Jonathan Finn
Codiak BioSciences, Cambridge, USA
Introduction: Incorporation of pharmacologically active molecules on the
surface or the lumen of extracellular vesicles (EVs) is an important strategy for
maximizing
the therapeutic potential of EVs. Genetic engineering of producer cells by introducing
DNA
through random or site-specific integration are promising strategies for creating
engineered
EVs. Long-term stability with consistent transgene expression in the EV producer cells
and
therapeutic potency of resulting engineered EVs are crucial for biomanufacturing.
We present
a comprehensive study to investigate stability of transgene expression and potency
of two
potential therapeutic engineered EVs derived from stably selected pools transfected
by
either random integration (RI) or site-specific integration (SSI).
Methods: Producer cells were engineered to make EVs displaying interleukin 12
(IL12) or interferon gamma (IFNg) by RI or nuclease-mediated SSI into AAVS1 locus.
Following
puromycin (puro) selection, long-term cellular stability and transgene expression
without
selective pressure was investigated. EVs were generated from stable cell pools at
0, 1, and
2 months post-thaw and purified by density gradient ultracentrifugation. Purified
EVs were
biochemically characterized by NTA, BCA, Western blot, and cholesterol quantitation.
Transgene expression and biological activity of EVs displaying IL12 and IFNg were
assessed
by AlphaLISA and in vitro reporter assays.
Results: Transfection by SSI resulted in faster recovery in puro selection
compared to RI. All stable cell pools, regardless of integration method, resulted
in
comparable cell culture performance, EV yield, and lipid and protein content at all
time
points tested. The engineered EVs also demonstrated long-term stability of IL12 and
IFNg
transgene expression and in vitro activity from both integration strategies.
Summary/Conclusion: Both methods for generating stable cell lines were
comparable in terms of cell stability, transgene expression, EV titre and potency,
with SSI
having the advantage of speed, allowing for more rapid iteration cycle times. Thus,
both
methods are suitable for the precision engineering of therapeutic EVs. This work
demonstrates feasibility to manufacture therapeutic engineered EVs from stable cells
from
either integration strategy for clinical development.
PF14.02
Transport of outer membrane vesicles as a model therapeutic
delivery system in pathogenic and commensal bacteria
Shannon M. Collins
a, Spencer
Morosb, Leah Paschb, Samuel Agroc and Angela
Brownc
aDepartment of Chemical and Biomolecular Engineering, Lehigh
University, Warrington, USA; bDepartment of Bioengineering, Lehigh University,
Bethlehem, USA; cDepartment of Chemical and Biomolecular Engineering, Lehigh
University, Bethlehem, USA
Introduction: Outer membrane vesicles (OMVs) in Gram-negative bacteria have
been shown to be important carriers of biomolecules, including toxins and other virulence
factors, peptidoglycan, and nucleic acids. It has been shown that OMVs play an important
role in the delivery of these biomolecules to host cells and bacterial cells. While
many
thorough studies have explored OMV delivery to host cells, few studies have explored
the
mechanisms of delivery of OMVs to bacterial cells. Our goal was to study the delivery
of
OMVs to other bacterial cells. Specifically, we were studying the oral pathogen
Aggregatibacter actinomycetemcomitans (A.a.), a Gram-negative organism associated
with
localized aggressive periodontitis, to study the process by which vesicles from this
organism communicate with other bacterial cells. Overall, we want to understand the
roles
specific surface components of OMVs play in the transport of these OMVs to other bacterial
cells.
Methods: We studied OMVs from two strains of A.a.: JP2, a highly pathogenic
strain, and 33384, a natural commensal strain. AF488-labelled OMVs were incubated
with fresh
bacterial cultures. Association of the OMVs with the bacterial cells was quantified
using
flow cytometry. To examine the role of surface-associated DNA in this process, DNA
was
digested with DNase, and the amount of surface-bound DNA was quantified with the membrane
impermeable DNA stain, TOTO-1.
Results: Using flow cytometry, we observed JP2 OMVs were delivered to 33,384
cells, and at a lesser amount to JP2 cells. Alternatively, 33,384 OMVs associated
readily
with JP2 cells, more than to 33,384 cells. This suggests that the delivery of OMVs
to
bacterial cells may be a targeted delivery mechanism. Furthermore, we hypothesized
surface-associated DNA may play a role in this interaction. We next digested the
surface-associated DNA on the OMVs with DNase, and observed a decrease in association
between the OMVs and bacterial cells. This supports our hypothesis that DNA on the
surface
of the OMVs plays a role in association. Current experiments are investigating this
interaction in more detail.
Summary/Conclusion: We have demonstrated that OMVs are selectively delivered
to bacterial cells, and surface-associated DNA plays a role in this process. We propose
to
investigate this process to further understand OMVs delivery to bacterial cells.
Funding: R03DE025275 & R21DE027769.
PF14.03
Utilizing a Gaucher’s disease cell line for the evaluation of a
novel exosome-based replacement therapy
Annie K. Brown
a, Jiayi
Zhangb, Brendan Lawlerb and Biao Lub
aSanta Clara University, San Jose, USA; bSanta
Clara University, Santa Clara, USA
Introduction: Engineered nano-scale exosomes have great potential as new and
targeted delivery vehicles for the treatment of Gaucher’s disease, the most common
lysosomal
storage disease. Recently, we have reported the design, production, and isolation
of
exosomes loaded with lysosomal β-glucocerebrosidase (GBA). People suffering from Gaucher’s
disease do not have functional GBA, which results in toxic build-up of undegraded
substrates
within the cell.
Methods: To evaluate the efficacy of this exosome-based therapy, a human
Gaucher’s disease model is required. Here, we have utilized near-haploid human cells
(Hap1)
modified via CRISPR-Cas9 to model Gaucher’s disease in vitro. These cells contain
a 479bp
insertion in the 6th exon of the GBA gene, resulting in non-functional GBA. PCR, enzyme
activity assays, and flow cytometry have been employed to confirm the diseased genotype
and
phenotype.
Results: Characterization of GBA-knock out cells shows a total loss of GBA
enzyme activity. Further characterization demonstrates a normal growth rate but an
increased
number of lysosomes, indicating a diseased phenotype.
Summary/Conclusion: The utilization of a human GBA-knock out cell line will
enable the evaluation of the efficacy of our engineered exosomes. Disease models will
be an
important resource for the evaluation of new biologic therapeutics, including exosomes.
Funding: We would like to acknowledge the Santa Clara University School of
Engineering for their support.
PF14.04
ThRxosomes: a novel exosomes based theranostic for lung
cancer
Akhil Srivastava
a, Narsireddy
Amreddya, Natascha Riedingerb, Rheal Townerc, Anupama
Munshia and Rajagopal Ramesha
aThe University of Oklahoma Health Science Center, Oklahoma
City, USA; bOklahoma State University, Stillwater, USA; cOklahoma
Medical Research Foundation, Oklahoma City, USA
Introduction: Chemotherapy is the first-line of treatment for lung cancer.
However, inefficient bio-distribution and reduced accumulation of drugs in the tumour
results in treatment failure. Therefore, improved drug delivery and diagnostic systems
are
warranted. Herein, we propose a novel theranostic system “ThRxosomes” where exosomes
are
loaded with Super Paramagnetic Iron Nanoparticles (SPIONs) conjugated to an anticancer
drug
via a pH-responsive linker for controlled release. We hypothesize that ThRxosomes
will exert
profound anticancer tumour activity that can be concurrently be monitored by Magnetic
Resonance Imaging (MRI).
Methods: ThRxosomes were produced by combining normal human lung fibroblast
(MRC9) cell-derived exosomes with SPIONS conjugated to and anti-cancer drug (Chemodrug
or
miRNA) via a pH cleavable linker. The physical and biological properties of ThRxosomes
were
determined using Transmission Electronic Microscopy (TEM), Nanotracker-Analysis (NTA),
Inductively Coupled Plasma Mass Spectrometry (ICPMS), Western blotting, cell viability,
and
MRI.
Results: Exosomes used in preparing ThRxosomes were 130 nm in size with a
typical lipid bilayer structure, and were positive for CD63, CD81, Flotillin and negative
for Annexin A1 confirming presence and purity of exosomes. Charge analysis, TEM, and
ICMPS
data showed successful loading of SPION-drug conjugate. Biological studies showed
selective
and enhanced drug release under acidic condition (pH 5.5) compared to drug release
at pH
7.2. Cell uptake and viability studies demonstrated increased uptake and killing of
ThRxosome-treated human A549 lung cancer cells compared to MRC-9 cells. In vivo studies
demonstrated accumulation and detection of SPIONs by MRI in in-situ tumours of A549
tumour-bearing mice.
Summary/Conclusion: Our study demonstrates ThRxosomes will produce profound
anticancer activity in lung cancer that is measurable by MRI.
Funding: The study was supported by grants from the Department of Defence
(W81XWH-18-1-0637 & W81XWH-19-1-0647), Department of Veterans Affairs Merit Grant
(101BX003420A1), by the National Cancer Institute Cancer Center Support Grant (P30CA225520),
Oklahoma Center for Advanced Science and Technology (OCAST; HR18-088) and Presbyterian
Health Foundation Bridge Grant.
PF14.05
Exosome-modified nanoparticles as an alternative delivery system
for small RNAs in cancer therapy
Petro Zhupanyn
a, Alexander
Eweb, Thomas Büchc and Achim Aignera
aIndependent Division for Clinical Pharmacology at
Rudolf-Boehm-Institute for Pharmacology and Toxicology, Faculty of Medicine, University
of
Leipzig, Germany, Leipzig, Germany; bDr., Leipzig, Germany;
cRudolf-Boehm-Institute for Pharmacology and Toxicology, Faculty of Medicine,
University of Leipzig, Germany, Leipzig, Germany
Introduction: Gene knockdown by RNA interference (RNAi) is an alternative,
non-invasive method for inhibiting proliferation or promoting apoptosis in tumour
cells.
This technique allows the specific targeting of key signalling proteins or mutated
genes.
Most of the available transfection compounds suffer from rather profound cytotoxicity
in
vitro. The aim of our study was to establish a novel targeted small nucleic acid delivery
system to the cells, with good cellular biocompatibility and applicability for in
vivo
studies. For this aim, we used native, cell own vesicles-exosomes. Since exosomes
are known
to transport peptides and different RNAs between cells and tissues, these unique,
small
extracellular vesicles (EV) may also be useful as transport vehicles for therapeutic
siRNA.
Methods: As detected by multiple cell surface protein expression analysis,
exosomes carry specific surface expression markers, allowing the cellular uptake by
the most
of tissues. We established an EV purification protocol from tumour cell culture supernatants
and a strategy for the efficient EV loading with our test siRNAs or antimiRs. Here
we used
the combination of polyethylenimine (PEI)-complexation of the RNAs with ultrasound
treatment
for their loading into the EVs. Our EV-modified, ultrasound-treated nanoparticles
were
tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines
or by
RT-qPCR gene expression analysis.
Results: More efficient cellular siRNA uptake was observed upon
EV-modification of our PEI/RNA nanoparticles, accompanied by efficient inhibition
of gene
expression. Biological efficacies were retained also after storage for several days
at room
temperature. The monitoring of the EV-based particles by FACS revealed a different
time
resolution of cellular uptake and nucleic acid release compared to the classically
formulated PEI-nanoparticles. In an in vivo therapy study in tumour xenograft-bearing
mice,
high biocompatibility, significant biological knock-down and tumour inhibition were
observed
after injection of anti-Survivin siRNAs formulated in our ECV-modified PEI
nanoparticles.
Summary/Conclusion: Our data demonstrate the usability of ECV-modified
nanoparticles as efficient delivery system for small RNAs in cancer therapy.
PF14.06
Microglial extracellular vesicles as therapeutic vector for
neuroinflammation
Giulia Marostica
a, Annamaria
Finardib and Roberto Furlana
aSan Raffaele Scientific Institute, Milan, Italy;
bSan Raffaele Scientific Institute, milan, Italy
Introduction: Microglia is considered an eligible target against the
progressive multiple sclerosis (MS), but currently available therapies do not allow
its
efficient targeting. As many cell types, microglia communicate with the neighbouring
cells
through a complex system of extracellular vesicles (EVs) exchange. Recently my group
described that microglia derived-EVs, engineered to encapsulate IL4, are taken up
by
microglia itself, mediating a phenotype switch to a protective phenotype. In vivo
studies
suggest that these EVs can ameliorate established neuroinflammation, thus making them
a
promising drug-delivery tool to target CNS in MS. My project focuses on understanding
the
mechanism of action and the signalling pathway of EVs delivery and to exploit this
knowledge
to specifically deliver different potential therapeutic molecules. For this purpose,
we
decided to characterize the EVs through TRPS technology.
Methods: A murine microglia cell line (BV2) was engineered to stably
overproduce endogenous IL4. This cell line was cultured in exosome-depleted RPMI and
stimulated with PMA(20 mg/mL) for 30 min. EVs isolation was carried out by collecting
supernatant and subjecting it to consequential centrifugation of 300 g,10 min, RT
and
2000 g,20 min, 4°C. The resulting supernatant was filtered (5 µm) and ultracentrifuged
at
100,000 g for 2h at 4°C. The EVs pellet was re-suspended in ice-cold PBS.
Results: The EVs analysis with TRPS shows two populations of EVs, one with a
mean diameter of 60–80 nm and a broad zeta potential ranging from −10 mV to −60 mV,
while
the second population has a mean diameter of 120–140 nm and a zeta potential of −10/-20 mV.
This difference can be consistent with the different pathway formation of exosomes
and
microvesicles. We demonstrated in vivo the strong phenotypic change induced by our
EVs to
resting microglia in a dose- and time-dependent effect. Then, impairing the physiological
procedure of the endosome acidification, the effect of our EVs on recipient cells
is higher.
Thus, suggesting an endocytic pathway for the internalization of the vesicles. We
further
demonstrate with gradient ultracentrifugation the capability of our formulation to
vehicle
endogenous IL4 inside the vesicles. Even if some protein is co-purified in the procedure,
we
know that the half-life of this cytokine is too short to elicit a strong in vivo response.
Consequently, we assume that the anti-inflammatory effect of our EVs in vivo is a
result of
the IL4 internalized in our formulation.
Summary/Conclusion: These data help us understand more in detail the process
of internalization and phenotype change mediated by these EVs. Our next goals are
to
discriminate between different internalization pathways and further validate the efficacy
of
our therapy on the EAE mouse model.
PF14.07
Targeting IL-3Rα on tumour-derived endothelial cells blunts
metastatic spread of triple negative breast cancer via extracellular vesicle
reprogramming
Cristina Grange
a, Tatiana
Lopatinab, Claudia Cavallaric, Victor Navarrod, Isabella.
Castellano Castellanoe, Giovanni Camussie and Maria Felice MF
Brizzie
aDeparment of Medical Sciences,University of Torino, Turin,
Italy; bDepartment of Medical Science, University of Turin, Turin, Italy;
cDepartment of Medical Sciences University of Turin, Turin, Italy;
d2i3T Scarl University of Turin, Turin, Italy; eDepartment of Medical
Sciences, University of Torino, Turin, Italy
Introduction: The lack of an approved targeted therapy and the early onset of
metastasis highlight the need for new treatments for triple-negative breast cancer
(TNBC)
patients. Interleukin-3 acts as an autocrine factor for tumour-endothelial-cells (TEC),
and
exerts pro-angiogenic paracrine action via extracellular vesicles (nEVs). IL-3Rα blockade
on
TEC changes TEC-EV (anti-IL-3R-EVs) microRNA cargo and promotes the regression of
established tumour vessels. As TEC are the doorway for “drug” entry into tumours,
we have
aimed to assess whether IL-3R blockade on TEC impacts tumour progression via their
unique EV
cargo.
Methods: 27 human TNBC samples, MDA-MB-231, MDA-MB-453 and MCF10 cell lines
were evaluated for the expression of IL-3Rα. nEVs and anti-IL-3R-EVs were characterized
by
electron-microscopy, MACSPlex-Exosome-Kit and western blot. Proliferation, migration,
apoptosis and sphere formation were evaluated. SCID mice were used for in vivo
experiments.
Results: We noticed that, besides TEC and inflammatory cells, tumour cells
from 55.5% of the human TNBC samples expressed IL-3Rα. MDA-MB-231 and MDA-MB-453,
but not
MDA10 cells, expressed IL-3Rα. In vitro, nEVs provide survival and migratory signals,
while
anti-IL-3 R-EVs promoted apoptosis as well as reduced cell viability and migration
of human
TNBC cell lines. In vivo anti-IL-3 R-EV treatment induced vessel regression in established
tumours formed of MDA-MB-231 cells and almost abolished the spread of liver and lung
metastasis. Moreover, decreased β-catenin and TWIST1 were found in tumours from animals
treated with anti-IL-3 R-EVs. In addition, anti-IL-3 R-EVs reduced lung metastasis
that was
generated via the intravenous injection of MDA-MB-231 cells. nEVs that were depleted
of
miR-24-3p (antago-miR-24-3p-EVs) were effective as anti-IL-3 R-EVs in down-regulating
TWIST1
and reducing lung-vessel density and metastatic lesions in vivo.
Summary/Conclusion: Overall, these data provide the first evidence that
IL-3 Rα is highly expressed in TNBC cells, TEC and inflammatory cells, and that IL-3 Rα
blockade on TEC impacts tumour progression.
Funding: AIRC, associazione Italiana per la Ricerca sul Cancro.
PF15: Evs in Cancer Metasis and Tumour Angiogenesis
Chair: Elena S Martens-Uzunova – Department of Urology, Erasmus Medical Centre
Chair: Richard Zieren – The Brady Urological Institute, Johns Hopkins University
School of Medicine
PF15.01
Statins decrease the implantation and invasion of high-grade
serous ovarian cancer cells induced by exosomes by altering its composition and its
cell
uptake and the endocytic traffic at the metastatic niche.
Mauricio A. Cuello
a, Sumie
Katob, Francisca Liberonab and Paula Mancillab
aPontificia Universidad Católica de Chile, Santiago, Chile;
bDivision Gynaecology and Obstetrics, School of Medicine, Pontificia
Universidad Católica de Chile, Santiago, Chile
Introduction: High-grade serous ovarian cancer (HGSOC) is the deadliest
gynaecologic cancer. Its lethality is explained for late diagnosis at advanced stages
and
frequent recurrences despite achieving complete response with standard therapy. Most
of
recurrences occurs at abdominal cavity with multiple metastasis. Therefore, identifying
key
determinants of metastatic process remains as priority to find better therapies. Current
evidence assigns a central role of the exosomes in conditioning the metastatic niche
in
epithelial cancers. Recently, we demonstrated that statins reduce metastasis in HGSOC
in
preclinical models. Here, we decided to study the effects of statins on HGSOC-derived
exosomes and its capability to condition the metastatic niche.
Methods: Exosomes were isolated from HeyA8 ovarian cancer cell line and
primary tissue cultures established from advanced-stage HGSOCs (with signed informed
consent
and IRB approval) by differential ultracentrifugation and quantified by nanoparticle
tracking analysis (NTA). Enriched-cancer initiating cells (CIC) spheroids were established
from HeyA8 cells by using stem-selecting conditions. The paracrine effect of exosomes
on CIC
migration/invasion was studied using either 3D migration or Boyden chamber invasion
assays.
Previous to exosome isolation, HeyA8 cells were treated with simvastatin (5uM, 24 h)
or
solvent and proteins involved in exosome biogenesis/uptake (Alix, Tsg101), its trafficking
(Rab7a, Rab27a) and in conditioning the metastatic niche (EMMPRIN) were measured by
immunoblotting.
Results: Exosomes isolated from HeyA8 cells or HGSOCs enhance the metastatic
potential of HeyA8-established spheroids in 3D migration or Boyden chamber invasion
assays.
Upon simvastatin treatment, we observed a significant reduction in migration/invasion
induced by equivalent number of exosomes in HeyA8-derived CICs. Under same treatment,
we
observed a significant decrease in protein levels of Alix and Tsg101 and an increase
in the
inactive forms of Rab7a and Rab27a in HeyA8 cells. We also observed a decrease in
EMMPRIN
levels in HeyA8-derived exosomes.
Summary/Conclusion: Here, we demonstrated a paracrine effect of HGSOC-derived
exosomes that favour the metastasis process. In addition, we demonstrated that simvastatin
reduces metastasis induced by cancer-derived exosomes. Such an effect is partially
explained
by changes in the expression of proteins involved in exosome biogenesis/uptake, its
endocytic trafficking and in the content of proteins conditioning the metastatic niche.
Thus, simvastatin arises as potential therapeutic target to improve outcomes in this
disease.
Funding: This research was supported by Fondecyt granted to Mauricio A.
Cuello
PF15.02
Label-free optical imaging and characterization of
cancer-associated extracellular vesicles in tissues
Marina Marjanovic
a, Sixian
Youb, Janet Sorrellsb, Jaena Parkb, Edita
Aksamitienec, Yi Sund, Haohua Tuc and Stephen
Bopparte
aUniversity of Illinois at Urbana-Champaign, Urbana, USA;
bDepartment of Bioengineering, University of Illinois at Urbana-Champaign,
Urbana, USA; cBeckman Institute for Advanced Science and Technology, University
of Illinois at Urbana-Champaign, Urbana, USA; dElectrical and Computer
Engineering, University of Illinois at Urbana-Champaign, Urbana, USA;
eDepartments of Electrical and Computer Engineering, and Bioengineering,
University of Illinois at Urbana-Champaign, Urbana, USA
Introduction: Cancer-associated extracellular vesicles (EVs) visualized in the
tumour microenvironment have been identified as a potential biomarker for cancer-related
tissue changes. Analyses of EVs have traditionally been performed in cells or isolated
EVs,
with no temporal or spatial information that could be critically important for elucidating
their roles in carcinogenesis. Since the unperturbed distribution and organization
of EVs in
the tumour microenvironment is associated with their cellular function and can potentially
serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing
EVs
in freshly isolated tissue specimens. Currently, only fluorescent labelling methods
enable
visualization and tracking of EVs. We used a custom label-free multimodal multiphoton
optical imaging system to detect and characterize EVs and classify them using their
optical
signatures both in isolated tissues and in situ tumours.
Methods: Label-free multimodal multiphoton imaging was used to provide
simultaneous, co-registered structural and functional images of EVs in untreated samples.
Heterogeneous populations of EVs could be identified from their unique optical
signatures.
Results: The intrinsic metabolic and structural properties of EVs enabled
reliable visualization and optical characterization of EVs from cell cultures and
in situ
imaging of tumour-bearing rats. Unique optical signatures were then used for identification
of cancer-related EVs in tissues from human breast cancer patients, and their density
was
found to be highly correlated with clinical diagnosis. In the current study, EVs were
isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer
patients.
Analysis of EV content showed higher concentration of NAD(P)H in EVs isolated from
cancer
subjects, than from healthy subjects, which reflects the reprogramming of cellular
metabolism in carcinogenesis.
Summary/Conclusion: These results suggest a potential label-free optical
methodology to detect and characterize EVs by their optical signatures, which can
be
utilized as possible diagnostic and prognostic biomarkers for cancer.
Funding: This research was conducted under protocols approved by the IACUC and
IRB at the University of Illinois and Carle Foundation Hospital, and supported by
funding
from NIH.
PF15.03
Novel potential anticancer therapies based on interference with
nuclear entry of cancer cell-derived extracellular vesicle components in recipient
cells
Mark Santos
a, Aurelio
Loricob, Simona Fontanac, Jana Karbanovád, Gyunghwi
Woob, Tony Huynhb, Kevin Huangb, Riccardo
Alessandroe, Germana Rappab and Denis Corbeilf
aTouro University College of Medicine, Hendersom, USA;
bTouro University Nevada College of Medicine, Henderson, USA;
cPalermo University School of Medicine, Palermo, Italy; dTechnische
Universität Dresden, Dresden, Germany; eUniversity of Palermo School of Medicine,
Palermo, Italy; fTissue Engineering Laboratories, Technische Universität Dresden,
Dresden, Germany
Introduction: The intercellular communication mediated by extracellular
vesicles (EVs) in the tumour microenvironment plays an important role in tumour progression.
Experimental evidence indicates that EVs derived from highly metastatic cells influence
the
behaviour of less aggressive cancer cells. We have previously described a novel
intracellular pathway where a fraction of endocytosed EV-associated proteins and nucleic
acids is transported into the nucleoplasm of the host cell via a subpopulation of
late
endosomes penetrating into nucleoplasmic reticulum (NR). Here, we better characterize
this
pathway and report that it is required for the induction of an aggressive behaviour
induced
by EVs released from highly metastatic SW620 colon cancer cells in isogenic primary
cancer
cells.
Methods: Super resolution-structured illumination microscopy and
magnetic-based co-immunoisolation studies were employed to identify the protein components
of the nuclear pathway and to monitor the entry of EV-containing late endosomes into
the
nucleoplasmic reticulum. Human SW480 carcinoma cells expressing ER-GFP and Rab7-RFP
were
exposed to EVs from SW620 cells and then live imaged.
Results: We have previously reported that the tripartite protein complex,
containing VAP-A, ORP3 and Rab7 orchestrates the localization of EV-carrying late
endosomes
into NR. We now report that silencing of ORP3 or VAP-A, but not its homologue VAP-B,
reverses the pro-metastatic changes induced by EVs isolated from metastatic cells
on their
non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding
and
blebbing. Moreover, we found that certain nuclear pore complex proteins and importin-beta1
are co-immunoisolated with ORP3, VAP-A and Rab7 suggesting the formation of a large
protein
complex at the entry of nuclear pores.
Summary/Conclusion: Interfering with the mechanisms regulating this novel
intracellular pathway may find therapeutic applications particularly in EV field and
oncology.
PF15.04
Educated osteoblasts regulate breast cancer proliferation via
small extracellular vesicles
Karen Bussard and Alison Shupp
Thomas Jefferson University, Philadelphia, USA
Introduction: Breast cancer commonly traffics to bone, where breast cancer
cells (BCCs) can survive undetected for years before metastatic outgrowth. In bone,
BCCs
interact with surrounding stromal cells, including osteoblasts (OBs), to shape the
metastatic niche. Our lab discovered there are at least two subpopulations of OBs
in the
bone-tumour niche, based on protein marker expression. One group, “educated osteoblasts”
(EOs) have engaged in crosstalk with BCCs whereas another group, naïve OBs, have not.
We
have novel evidence that EOs regulate BCC proliferation. The purpose of this study
was to
determine if extracellular vesicles (EVs) produced by EOs play a role in regulating
BCC
proliferation. We hypothesized EVs produced by EOs would decrease BCC proliferation.
Methods: EO-derived small EVs from culture media were isolated via
ultracentrifugation and characterized EVs for size, protein marker expression, and
density
floatation to validate the purity of EV samples. The functionality of EO-derived EVs
on BCC
proliferation was examined using EdU and checkpoint proteins p21 and p27. BCC protection
from chemotherapy induced cell death was also examined.
Results: We found that EVs produced by EOs, but not naïve OBs, decreased both
triple negative and ER-positive BCC proliferation in a concentration dependent manner.
Furthermore, using an EdU assay, we found that exposure to EO-derived EVs induces
BCCs to
undergo cell cycle arrest. Interestingly, the cell cycle arrest was reversible and
BCC
proliferation was restored upon removal of EO-derived EVs. In addition, exposure to
EO-derived EVs leads to increases in BCC expression of the G1 checkpoint proteins,
p21 and
p27.
We next wanted to investigate proliferative signalling pathways that may be deregulated
in
BCCs following exposure to EO-derived EVs. We found that EO-derived EVs reduce BCC
levels of
ERK1/2.
Because our data indicate EO-derived EVs induce sustained cell cycle arrest in BCCs,
we
desired to know if EO-derived EVs protected BCCs from chemotherapy-induced cell death.
We
found that BCCs exposed to EO-derived EVs and the chemotherapy drug, doxorubicin,
have
decreased cell death compared to BCCs exposed only to doxorubicin.
Summary/Conclusion: Altogether, our data suggest EOs play a crucial role in
bone-tumour microenvironment by regulating BCC proliferation.
Funding: Supported by NIH R00 CA178177 and Commonwealth of Pennsylvania –
Department of Health SAP 4100072566 for KMB.
PF15.05
Phosphorylation of Tyrosine 23 in annexin A2 is essential for
its association with exosomes and for imparting invasive and proliferative capacity
to
other cells
Priyanka Prakash Desai
a, Pankaj
Chaudharyb, Xiangle Sunb and Jamboor Vishwanathaa
aUNT Health Science Center at Fort Worth, Fort Worth, USA;
bUNT Health Science Center, Fort Worth, USA
Introduction: Triple negative breast cancer (TNBC) accounts for 15%-20% of all
breast cancer cases. The lack of targeted-based therapies highlights the importance
of
studying TNBC. Elevated levels of Annexin A2 (AnxA2), a Ca+2-dependent phospholipid
binding
protein, has been correlated with worse overall survival in TNBC patients. Our previous
data
implicate that exosomal AnxA2 is involved in creating a pre-metastatic niche and
facilitating metastasis in TNBC. Moreover, N-terminal phosphorylation of Tyrosine
(Tyr) 23
in AnxA2 has been implicated in regulating several AnxA2 activities in cancer progression.
Here, we demonstrated that N-terminal phosphorylation of AnxA2 at Tyr23 is important
for its
association with exosomes which imparts invasive and proliferative phenotype to other
cells.
Hence, dissecting the regulatory pathway will be critical for verifying the value
of AnxA2
as a therapeutic, diagnostic or prognostic marker in TNBC.
Methods: pN1-EGFP plasmids expressing the constitutive phosphomimetic
(AnxA2-Y23E) and non-phosphomimetic mutant (AnxA2-Y23 F) gene expressing mutation
at Tyr23
site were overexpressed in MDA-MB-231 TNBC cells. Mutant cells were experimentally
validated
for AnxA2 specific functions like migration, invasion and proliferation. Exosomes
were
isolated from the mutant phosphomimetic (exo-AnxA2-Y23E-GFP) and non-phosphomimetic
(exo-AnxA2-Y23 F-GFP) cells and were analysed for exosomal surface expression of AnxA2
by
immunoprecipitation and flowcytometry. CAL-148 breast adenocarcinoma epithelial cells
were
treated with exo-AnxA2-Y23E-GFP and exo-AnxA2-Y23 F-GFP to analyse the rate of invasion
and
proliferation by transwell invasion and proliferation assay, respectively. Transfer
of
exosomal AnxA2 in CAL-148 was studied using immunofluorescence and its implications
on
signalling pathways were studied by Western blot.
Results: MDA-MB-231 phosphomimetic TNBC mutant cells showed increased
migratory, invasive and proliferative capacity compared to non-phosphomimetic TNBC
mutant
cells. Exo-AnxA2-Y23E-GFP had elevated surface AnxA2 expression compared to
exo-AnxA2-Y23 F-GFP. CAL-148 cells treated with Exo-AnxA2-Y23E-GFP showed high migratory,
invasive and proliferative characteristics, with a higher expression of p-AnxA2(Tyr23),
p-Src(Tyr416) and p-Paxillin(Tyr31) compared to Exo-AnxA2-Y23 F-GFP treated cells.
Summary/Conclusion: N-terminal phosphorylation of Tyr23 in AnxA2 in MDA-MB-231
TNBC cells (Phosphomimetic mutant cells) is essential for its association with exosomes
and
for conferring increased invasive and proliferative capacity to other breast cancer
cells.
Funding: The above study is funded by National Institute of Health RO1CA220273
and NIMHD’s U54MD006882 to Dr.J.K.Vishwanatha.
PF15.06
A novel method for epithelial-derived extracellular vesicle
isolation and enrichment in patients with advanced prostate cancer
Arpit Rao, Helene Barcelo and Bharat
Thyagarajan
University of Minnesota, Minneapolis, USA
Introduction: Evaluation of changes in prostate cancer biology is difficult
due to presence of lymph nodal or bony metastatic disease in a majority of patients.
A
number of liquid biopsy assays have shown clinical utility in prostate cancer, but
are
limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability
to
perform transcriptome sequencing (cell-free DNA-based assays). Epithelial-derived
extracellular vesicles (epi-EV)-based assays are uniquely positioned overcome both
these
limitations as EVs are abundantly secreted into the blood and have RNA-cargo that
mirrors
the cell of origin. However, a reliable method to enrich for epi-EVs is currently
lacking.
Methods: Plasma was isolated from the peripheral blood collected from 15
patients with metastatic prostate cancer enrolled in an institutional biobanking study
before initiation of systemic antineoplastic therapy. EVs were isolated from 500 µl
of
plasma using a commercially available qEV 35 size exclusion column (Izon Inc.). Without
subjecting the EVs to any physical stressors such as centrifugation, CD61 magnetic
beads
were used to fractionate the EVs into CD61+ (platelet derived) and CD61- (non-platelet
derived) fractions. Multiparameter flow cytometry was used to evaluate EVs that expressed
CD9 and EpCAM and were negative for Calnexin. Nanotracking Analysis (NTA) was used
to
quantify both total EV and CD61+ and CD61- fractions in all patient samples.
Results: The average ± standard deviation of total EVs obtained from the 15
patients was 6.39x10^11 ± 4.60x10^11 EVs/ml of plasma (coefficient of variation [CV]:
72%)
while the average and standard deviation of CD61- EVs was 1.24x10^10 ± 3.37x10^10
(CV:
271%). The CD61- EV fraction represented a variable amount of the total EVs in prostate
cancer patients ranging from 0.0001% to 32.21%. Multiparameter flow cytometry showed
that
over 80% of total EVs were CD9+ and calnexin-, suggesting an endosomal origin for
a vast
majority of the EVs in these plasma samples. However, the proportion of EVs expressing
EpCAM
(marker of epi-EVs) was higher among the CD61- fraction (5% – 30%) as compared to
the
CD61+ fraction (0.04% – 1%).
Summary/Conclusion: Our novel method was able to isolate and enrich the epi-EV
from the plasma of advanced prostate cancer patients. Correlation between clinical
characteristics and EV quantity is being evaluated to identify the reason(s) for large
variations in CD61- EV fraction. Future studies are planned to use our method in improving
the sensitivity of EV-based assays and increase the RNA yield to facilitate transcriptome
sequencing.
Funding: This work was funded by grants from Randy Shaver Community and
Research Fund, Minnetonka, MN.
PF15.07
Exosomes drive medulloblastoma metastasis in a MMP2 and EMMPRIN
dependent manner
Hannah K. Jackson
a, Franziska
Linkea, Ian Kerrb and Beth Coylec
aChildren’s Brain Tumour Research Centre, School of Medicine,
University of Nottingham Biodiscovery Institute, Nottingham, UK; bSchool of Life
Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK;
cChildren’s Brain Tumour Research Centre, School of Medicine, University of
Nottingham Biodiscovery Institute, Nottingham, UK
Introduction: Recurrent/metastatic medulloblastoma (MB) is a devastating
disease with an abysmal prognosis of less than 10% 5-year survival. The secretion
of
extracellular vesicles (EVs) has emerged as a pivotal mediator for communication in
the
tumour microenvironment during metastasis. The most investigated EV’s are exosomes,
nanovesicles secreted by all cell types and able to cross the blood-brain-barrier.
Matrix
metalloproteinases (MMPs) are enzymes secreted by tumour cells that can potentiate
their
dissemination by modification of the extracellular matrix. We hypothesise that exosomal
MMP2
and its inducer EMMPRIN could enhance metastasis of MB.
Methods: Proliferation, invasion and migration assays were used to evaluate
the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour
cell-derived exosomes. Gelatin zymography and western blotting were performed to confirm
MMP2 functional activity in cell lines and exosomes. Nanoscale flow cytometry was
used to
measure surface exosomal EMMPRIN levels. Exosomal MMP2 and EMMPRIN were modulated
at the RNA
level.
Results: Number of exosomes is directly related to the migratory behaviour of
parental MB cell lines (p < 0.01). Notably, functional exosomal MMP2 and EMMPRIN levels
also correlate with this. Furthermore, exosomes from metastatic cell lines conferred
enhanced migration and invasion on their matched isogenic primary (non-metastatic)
cell line
pair by ~3.8-fold (p < 0.01). Exosomes from metastatic cell lines also conferred
increased migration on poorly migratory foetal neuronal stem cells.
Summary/Conclusion: Together this data suggests that exosomal MMP2 and EMMPRIN
may promote medulloblastoma metastasis and supports analysis of exosomal MMP2 and
EMMPRIN
levels in patient cerebral spinal fluid samples.
Funding: The James Tudor Foundation, The Children’s Brain Tumour Research
Centre, The University of Nottingham Life Sciences
PF15.08
Anti-migratory effect of Nm23-H1 via exosomes
Vasu Saini, Yung Hou Wong and Siu Nam Sun
Hong Kong University of Science and Technology, Hong Kong, Hong Kong
Introduction: Exosomes secreted from cancer cells harbour the potential to
regulate intracellular signalling and promote metastasis. Wherein, metastasis suppressor
genes (MSGs) play a pivotal role in regulating such signalling cascades. However,
the
regulation gets hampered due to low expression of MSGs under metastatic conditions.
Nm23-H1,
product of first identified metastasis suppressor gene NME1, is significantly downregulated
under metastatic conditions. Nm23-H1 serves as a regulator of small GTPases. Several
evidences have highlighted an involvement of small GTPases (such as Rab5, Rab7 and
Rab27) in
the biogenesis of exosomes. In addition, bacterial homolog of Nm23 has been shown
to
interact with Rab5 and Rab7. However, experimental evidence supporting a relationship
between exosomes and Nm23-H1 is lacking. Our current focus is to deduce the relationship
between exosomes and MSGs.
Methods: Breast cancer cell lines were used to assess the effect of exosomes
isolated from highly metastatic cells (MDA-MB-231 cells) on lower/non metastatic cells
(MCF-7 cells). NME1 was overexpressed in MDA-MB-231 cells and subsequently used to
isolate
exosome fractions. Equivalent amount of isolated exosome fractions from MDA-MB-231
cells and
MDA-MB-231/NME1 cells were utilized to access their effect on migration and difference
in
exosome markers.
Results: We observed an enrichment of Nm23-H1 in the exosomes isolated from
MDA-MB-231 cells upon overexpression of NME1. Proteinase K protection assay confirmed
the
packaging of Nm23-H1 inside the exosomes isolated from MDA-MB-231/NME1 cells and excluded
the possibility of membrane association of Nm23-H1. Additionally, overexpression of
Nm23-H1
led to a significant reduction in the ability of MDA-MB-231 exosomes to stimulate
movement
of MCF-7 cells as confirmed by wound healing assays. Our data also highlights a clear
reduction in the protein levels of exosome markers such as CD63, CD9 and Alix in the
exosome
fraction isolated from MDA-MB-231/NME1 cells as compared to MDA-MB-231 cells. Interestingly,
Rab7A, a protein involved in the endosome-lysosome fusion was also present in lower
amount
in the exosomes isolated from Nm23-H1 overexpressing cells.
Summary/Conclusion: Our data highlights an anti-migratory effect of Nm23-H1
via exosomes. These findings support a regulatory role of Nm23-H1 in the packaging
or
release of exosomes in highly metastatic breast cancer cells, and further suggest
that
metastasis suppressor proteins may be involved in the regulation or packaging of exosomes.
Additional studies will be required to decipher the downstream signalling of Nm23-H1
which
affects the biogenesis of exosomes as well as to assess the effect of Nm23-H1 overexpression
on the content of exosomes. These insights could help us delineate the complex exosome
biogenesis pathway and provide new potential drug targets for exosome regulation.
Funding: Supported by SBI17SC04 and ITCPD/17-9
PF16: EVs in Tissue Injury and Repair
Chair: Cristina Grange – Deparment of Medical Sciences,University of Torino
Chair: Felix Kim – Thomas Jefferson University
PF16.01
Moderate exercise has beneficial effects on mouse ischaemic
stroke by enhancing the functions of circulating endothelial progenitor cell-derived
exosomes via activation of the miR-126/BDNF/PI3 k pathway
Jinju Wang, Shuzhen Chen and Ji Bihl
Wright State University, Dayton, USA
Introduction: Exosomes (EXs) are emerging as novel players in the beneficial
effects induced by exercise on vascular diseases. Our recent study has revealed that
moderate exercise enhances the function of circulating endothelial progenitor cell-derived
exosomes (cEPC-EXs) on protecting endothelial cells against hypoxia injury. In this
study,
we aimed to investigate whether exercise-regulated cEPC-EXs contribute to the beneficial
effects of exercise on ischaemic stroke (IS).
Methods: C57BL/6 mice performed moderate treadmill exercise (10 m/min, 4-wks)
before IS induced by middle cerebral artery occlusion surgery. Acute injury was evaluated
at
day 2 by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra
and
neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor
functions at day 28. The correlations of cEPC-EXs and their carried miR-126 with
neurological parameters were analysed. The underlying mechanism of the effects of
cEPC-EXs
isolated from exercised mice was explored in a hypoxia neuron model. Cellular miR-126
level,
apoptosis, axon growth ability and gene expressions (cas-3, BDNF and Akt) were measured.
Results: 1) Exercised mice had a smaller infarct volume on day 2, which was
associated with decreased cell apoptosis and cleaved cas-3 level, and a higher microvessel
density than those in control; 2) The elevated cEPC-EX level positively correlated
with
tEPC-EXs in ischaemic brain of exercised mice on day 2. The upregulated miR-126 level
positively correlated with the numbers of tEPC-EXs in ischaemic brain; 3) The numbers
of
cEPC-EXs and their carried miR-126 level negatively correlated with the infarct volume,
cell
apoptosis and positively correlated with the microvessel density in the peri-infarct
area on
day 2; 4) Exercised mice had decreased infarct volume, increased microvessel density,
promoted angiogenesis/neurogenesis and improved sensorimotor functions on day 28,
accompanying with upregulated levels of BDNF, p-TrkB/TrkB and p-Akt/Akt; 5) cEPC-EXs
of
exercised mice protected neurons against hypoxia-induced apoptosis and compromised
axon
growth ability which were blocked by miR-126 and PI3 k inhibitors.
Summary/Conclusion: Our data suggest that the protective effects of moderate
exercise intervention on the brain against MCAO-induced ischaemic injury are ascribed
to
cEPC-EXs and their carried miR-126.
Funding: This work was supported by American Heart Association
(18POST33990433) and NIH (1R01NS102720).
PF16.02
Syndecan-1 regulates alveolar type 2 epithelial cell senescence
mediating through extracellular vesicles during lung fibroproliferation
Tanyalak Parimon
a, Changfu
Yaoa, Adam Aziza, Stephanie Boraa, Marilia Zuttion1
Zuttiona, Dianhu Jianga, Melanie Koenigshoffb, Cory
Hogaboama, Paul Nobela, Barry Strippa and Peter
Chena
aCedars-Sinai Medical Center, Los Angeles, USA;
bUniversity of Colorado, Denver, USA
Introduction: Alveolar type 2 epithelial cell (AT2) senescence is implicated
in the pathogenesis of lung fibrosis, a progressive fatal condition. Syndecan-1, a
heparan
sulphate proteoglycan, is overexpressed by AT2 cells of human idiopathic pulmonary
fibrosis
(IPF) and bleomycin-injured WT mice and the overexpression of syndecan-1 is profibrotic.
Moreover, syndecan-1 deficient (Sdc1-/-) mice had less lung fibrosis after bleomycin
injury.
We reported that extracellular vesicles (EVs) in bronchoalveolar lavage (BAL) of
bleomycin-injured WT mice augmented lung fibrosis whereas the Sdc1-/- -BAL-EVs attenuated
the process. Moreover, WT-BAL-EVs expressed lower level of anti-fibrotic miRNAs (miR-34b-5p,
−142-3p, −144-3p, and −503-5p) compared to the Sdc1-/–BAL-EVs. These miRNAs targeted
genes
in the cellular senescence pathway indicating that syndecan-1 altered microRNA profiles
in
the BAL-EVs to promote cellular senescence during lung fibrogenesis. We investigate
how
syndecan-1 regulates AT2 senescence through EVs.
Methods: Bleomycin was intratracheally given into WT and Sdc1-/- mice. At day
21, lungs were processed for single-cell RNA sequencing (scRNAseq) and western blot
(WB).
EVs were isolated using ultrafiltration centrifugation method. Human (A549) and mouse
(MLE-15) lung epithelial cell lines were used for in vitro experiments.
Results: scRNAseq analysis indicated while bleomycin stimulated an
overexpression of cellular senescence-specific genes on AT2 cells of WT mice, these
genes
were significantly downregulated on Sdc1-/- AT2 cells. Senescence proteins, p16 and
p21,
were also less expressed in the lungs of Sdc1-/- than of the WT mice by WB. To determine
the
functional effects of EVs in BAL, A549 cells were treated with human IPF or control
lung
wash-EVs and evaluated for beta-galactosidase activity. We found that IPF-EVs markedly
increased beta-galactosidase enzymatic activity. Corroborating with these data,
bleomycin-injured BAL-WT-EVs also significantly upregulated senescence marker, p21,
by WB on
MLE15 cells whereas Sdc1-/- -BAL-EVs inhibited p21 expression.
Summary/Conclusion: Our data indicate that syndecan-1 regulates lung fibrosis
through the senescence signalling pathway on AT2 cells. Furthermore, syndecan-1 controls
AT2
senescence mediating through extracellular vesicles in the BAL. Lastly, the most likely
cargo molecules mediating this process are microRNAs.
Funding: This research was funded by the National Institute of Health
(NIH)/National Centre for Advancing Translational Sciences UCLA-CTSI-KL2-UL1TR001881
(TP),
R01 HL13707 (PC), and the Parker B Francis Foundation Fellowship (CY).
PF16.03
Immortalized cardiosphere-derived cell EV-associated piRNA,
imEV-Pi, protects against ischaemic injury in the heart
Alessandra Ciullo, Ahmed Ibrahim, Liang Li,
Chang Li, Weixin Liu and Eduardo Marbán
Smidt Heart Institute, Cedars Sinai Medical Center, Los Angeles, USA
Introduction: Cardiosphere-derived cells (CDCs) are a population of
heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and
clinical
settings. CDCs function by secreting extracellular vesicles (EVs), lipid-bilayer
nanoparticles laden with bioactive molecules. Recently our group developed a strategy
for
immortalizing CDCs (imCDC) that retains their therapeutic potential and enhances CDC
function indirectly through their secreted EVs. imCDC show a different RNA content(miRNA,
mRNA, rRNA, tRNA and piRNA) compared to primary CDC. In particular, we focus on Piwi
RNAs
(PiRNAs), small RNAs bound by Piwi proteins, important regulators of both the epigenome
and
transcriptome. We seek to explore the role of a PiRNA highly enriched in imCDC-EVs
(imEV-Pi).
Methods: EVs are prepared by conditioning cells for 24 hrs in serum-free basal
media, in hypoxic culture. After 24 hrs conditioned medium is cleared of cellular
debris and
EVs isolated using ultrafiltration by centrifugation (ufc). Fractions were analysed
in terms
of particle size, number, and concentration and piRNA content. In vitro, bone marrow
derived-macrophages (BMDM) were exposed to imCDC-EV, imEV-Pi and control and transcriptomic
profile and potentially activated pathways were assessed. In vivo, 8–10 week-old
Wistar-Kyoto female rats received 10^10 imCDC-EV, imEV-Pi, scramble or vehicle intracoronary
20 minutes after ischaemia-reperfusion(I/R). Cardiac Troponin I levels, scar size
and
monocytes were assessed at 24 and 48 hrs.
Results: By small-RNA sequencing analysis we found that piRNAs are enriched in
both CDC-EV and imCDC-EV. imCDC show a different PiRNA composition compared to primary
CDC.
imEV-Pi was identified as one of the most highly-expressed non-coding RNAs (the number
of
reads were 35X higher in imCDC-EV compared to CDC-EV). In vitro, imExo-Pi-conditioned
BMDM
exhibit a different transcriptomic profile compared with control, with upregulation
of
pathways involved in the inflammatory response, cell death, and cell-to cell signalling.
In
vivo, imEV-Pi is cardioprotective, as shown by reduced scar size and lower cardiac
troponin
levels compared to vehicle- and scramble-injected animals at 48 hrs post I/R. imEV-Pi
only
minimally alters neutrophil counts profile in blood but it alters monocytes profile
with a
decreased number at 24 hrs and an increase at 48 hrs.
Summary/Conclusion: We posit that imEV-Pi is a key determinant of imCDC-EV
therapeutic efficacy. Our results indicate that target cells may be macrophages/monocytes,
given that imEV-Pi exposure modifies their composition and mRNA profile both in vitro
and in
vivo.
Funding: This work was supported by funding from the National Institutes of
Health (NIH R01 HL124074 (to EM)
PF16.04
Enhancement of extracellular vesicles from umbilical stem cell
in hair follicle regeneration
Yi-You Huang
National Taiwan University, Taipei, Taiwan (Republic of China)
Introduction: Extracellular vesicles (exosomes, EVs) are cell membrane
particles (30–200 nm) secreted by virtually cells. During intercellular communication
in the
body, secreted EVs play crucial roles by carrying functional biomolecules (e.g., microRNAs
and enzymes) into other cells to affect cellular function, including disease progression
and
tissue regenerations. Literature previously reported that the macropinocytosis pathway
contributes greatly to the efficient cellular uptake of EVs. The activation of growth
factor
receptors, such as epidermal growth factor receptor (EGFR), induces macropinocytosis.
In
this study, we demonstrated the effects of EVs on demal papilla and hair follicle
regeneration.
Methods: Identification of distinct nanoparticles and subsets of extracellular
vesicles from umbilical cord blood stem cell by asymmetric flow field-flow
fractionation.
Results: The effects of EVs from umbilical cord blood stem cell on the
propagation of demal papilla and hair follicle regeneration were observed.
Summary/Conclusion: The enhancement of extracellular vesicles from
umbilicalcord blood stem cell the propagation of demal papilla and hair follicle
regeneration were observed and confirmed.
PF16.05
Mechanisms of host resistance to plasma membrane damage induced
by pneumolysin attack
Joana M. Pereira
a, Sara
Alvesa, Didier Cabanesb and Sandra Sousaa
ai3 S – Instituto de Investigação e Inovação em Saúde,
Universidade do Porto, Portugal; Cell Biology of Bacterial Infections Group, IBMC
–
Instituto de Biologia Molecular e Celular, Portugal, Porto, Portugal; bi3 S –
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; Molecular
Microbiology Group, IBMC – Instituto de Biologia Molecular e Celular, Portugal, Porto,
Portugal
Introduction: Bacterial pore-forming toxins (PFTs) are major virulence factors
produced by pathogens. PFTs target host plasma membrane (PM) and create transmembrane
pores,
which allow uncontrolled flux of ions and small molecules across the PM disrupting
cellular
homoeostasis. To survive, cells display poorly understood repair mechanisms to recover
the
cell homoeostasis. Several mechanisms were proposed to participate in cell recovery:
exocytosis of cortical lysosomes; endocytosis of PFTs pores; PM blebbing and shedding.
Methods: We used increasing concentrations of purified PLY to intoxicate
cells. PM permeability was assessed by flow cytometry using propidium iodide dye.
Cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy.
Extracellular vesicles released during PM repair were isolated by high-speed centrifugation
and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy
(TEM) and mass spectrometry/liquid chromatography analysis.
Results: PLY triggers a complete reorganization of the actomyosin cytoskeleton
inducing the formation of cortical actomyosin bundles at sites of PM remodelling.
These
structures assemble upon loss of PM integrity and disassemble as PM recovers. We detected
the release of microvesicles during the recovery of PM integrity. Vesicle population
is
heterogeneous with sizes ranging from 100 to 500 nm, with the majority of them measuring
100–200 nm. Vesicle proteomic analysis revealed that they contain PLY, suggesting
they
participate in pore removal, proteins involved in vesicle trafficking, PM repair and
exosome
biogenesis.
Summary/Conclusion: Our data demonstrate that cells are able to recover from
the damage induced by sub-lytic concentrations of PLY. Actomyosin cytoskeleton undergo
massive changes with the assembly of cortical bundles possibly at sites of PM damage.
We
showed that cells produced extracellular vesicles during the process of repair. We
are now
focusing on understanding the biogenesis of those vesicles and its importance during
the
process of repair.
Funding: This work received funds from FEDER through the COMPETE 2020 –
Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal
2020,
and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/Ministério
da
Ciência, Tecnologia e Ensino Superior under the project PTDC/BIA-CEL/30863/2017. J.P.
acknowledge FCT I.P., Portugal for fellowship SFRH/BD/143940/2019.
PF17 = OP2 Oral with Poster Session 2: Cancer and
Technology
Chair: Lizandra Jimenez – Postdoctoral Research Fellow, Vanderbilt
University
PF17.01 = OP2.01
Development of scalable processes to produce therapeutic
mesenchymal stromal cell-derived extracellular vesicles and their
characterization
Raquel M. S. Cunha
a, Elga
Vargasb, Filipa Piresc, Cecília Caladod, Joaquim
Cabrale, Cláudia Silvae and Ana
Fernandes-Platzgummere
aInstituto Superior Técnico, University of Lisbon, Lisbon,
Portugal; bFaculty of Medicine, National University of Colombia, Bogotá, Bogotá,
Colombia; cInstituto Superior de Engenharia de Lisboa, Lisboa, Lisboa, Portugal;
dInstituto Superior de Engenharia de Lisboa, Lisboa, Portugal;
eDepartment of Bioengineering and iBB – Institute for Bioengineering and
Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal,
Lisboa,
Portugal
Introduction: Despite of high expectations, mesenchymal stromal cell
(MSC)-based therapies still lack efficacy, partially due to loss of cell viability
and
function upon administration. MSC-derived extracellular vesicles (MSC-EV) emulate
the
regenerative potential of MSC, shifting the field towards cell-free therapies. Clinical
applications require the establishment of a scalable and GMP-compliant processes for
the
production and isolation of MSC-EV, combined with robust characterization platforms.
Methods: To develop a well-established process for the production of
therapeutic MSC-EV, we compared different MSC sources (bone marrow, adipose tissue,
umbilical cord matrix), culture media compositions (DMEM supplemented with foetal
bovine
serum (Thermo Fisher Scientific), DMEM supplemented with human platelet lysate (AventaCell
Biomedical) and StemPro MSC SFM Xeno Free medium (Thermo Fisher Scientific)) and culture
parameters (oxygen tension and shear stress) in two different culture platforms (2D
static
tissue culture flask vs 3D dynamic spinner vessels). Subsequently, MSC-EV were isolated
by
ultracentrifugation or a commercially available isolation kit and characterized according
to
ISEV guidelines.
Results: MSC derived from different sources/donors were able to grow under
normoxia and hypoxia in 2D T-flasks and 3D spinner vessel culture systems, while maintaining
their immunophenotype and differentiation potential, according to the minimal criteria
defined by the ISCT. The time point for pre-conditioning and collection of conditioned
medium for MSC-EV isolation was also optimized for both 2D and 3D culture systems.
MSC-EV
were characterized according to MISEV 2018 guidelines, using techniques as NTA, protein
and
lipid quantification, western blot, imaging and Fourier-Transform Infrared Spectroscopy
(FTIR). The results indicate that MSC-EV derived from different sources/donors have
similar
size distribution, however, EV yields tend to be higher for the 3D culture system.
Of
notice, several spectral regions were identified by FTIR, enabling the detection of
differences in the biomolecules present in MSC-EV, MSC-conditioned media and cells
produced
under different conditions.
Summary/Conclusion: In summary, this study contributes to the establishment of
a scalable process for MSC-EV production.
PF17.02 = OP2.02
Evaluation of three different isolation methods for small
extracellular vesicles from human plasma in prostate cancer diagnosis
Bairen Pang
a, Ying Zhua,
jie nia, Xupeng Baia, Julia Beretova, Valerie
Wasingera, David Maloufb, Joseph Buccib, James
Thompsonb, Peter Grahamb and Yong Lia
aUNSW Sydney, Sydney, Australia; bSt George
Hospital, Sydney, Australia
Introduction: Extracellular vesicles (EVs) have great potential in prostate
cancer (PCa) diagnosis and progression monitoring to complement the inaccurate prostate
specific antigen (PSA) screening and invasiveness of tissue biopsy. However, current
methods
cannot isolate pure EVs and therefor EVs characteristics remain largely unknown. In
order to
develop an accurate approach for EV isolation, we aimed to compare three emerging
methods
with different characteristics of small EVs (sEVs) from human PCa plasma samples and
to
choose the best one for diagnostic and functional studies.
Methods: PCa patients and age-matched healthy controls (HC) plasma (n = 6 in
each group) were used to isolate sEVs with 3 different isolation methods including
commercial ExoQuick Ultra Kit, qEV 35 and qEV 70 size exclusion chromatography (SEC).
Isolated sEV were characterized by nanoparticle tracking analysis, immunoblotting,
cyrogenic
electron microscopy, flow cytometry (FC) and proteomics analysis. For FC characterizing
surface marker expression, the sEVs were further purified by CD9 and CD81 commercial
immunoaffinity magnetic beads . Lipoprotein was captured by streptavidin biotinylated
ApoB
magnetic beads to measuring the lipoprotein contamination.
Results: The sEV size, morphology, surface protein and protein cargo with
proteomics were analysed between the three isolation methods. sEVs isolated from SEC
methods
had a lower particle size, protein amount, protein/sEV marker ratio and ApoB+/sEV
marker
ratio than those from ExoQuick Ultra method. In addition, sEVs isolated from qEV35
demonstrated a significantly higher sEV content, more up-regulated and down-regulated
PCa
proteins from proteomics but lower sEV marker/protein ratio and a higher protein
contamination than those from qEV70. Furthermore, sEV marker signal also showed a
good
correlation with particle numbers instead of protein content in all the methods.
Summary/Conclusion: qEV 70 method demonstrated better performance in isolating
relatively pure sEVs from human plasma; qEV35 has the better performance in isolating
samples with higher sEV content; ExoQuick Ultra isolated samples with closely sEV
content to
the qEV35 but with the highest non-sEV protein contaminations. People can choose higher
sEV
content or higher sEV purity according to the downstream analysis.
Funding: St. George Hospital Cancer Research Trust Fund, UNSW
Sydney-University International Postgraduate Award, Cancer Institute NSW Early Career
Fellowship.
PF17.03 = OP2.03
Multiplexed surface protein profiling of tumour-derived
extracellular vesicles by an electrokinetic sensor
Sara Cavallaro
a, Vasiliki
Arapib, Lorenca Berishac, Petra Håågb, Christiane
Stillera, Kristina Viktorssonb, Rolf Lewensohnb, Amelie
E. Karlströma, Jan Linnrosa and Apurba Devd
aKTH Royal Institute of Technology, Stockholm, Sweden;
bKarolinska Institute, Stockholm, Sweden; cPolitecnico di Torino,
Turin, Italy; dUppsala University, Uppsala, Sweden
Introduction: Small extracellular vesicles (sEVs) (30–200 nm in diameter) are
secreted by most cells, including tumour cells. They have attracted interest as biomarker
for cancer diagnostics based on liquid biopsies, because they are abundant in body
fluids
and their content (proteins, RNAs and other cargos) reflects their parent cells. Moreover,
sEVs may also be used for treatment monitoring, as recent studies suggested that the
expression levels of certain markers may change during therapy, reflecting tumour
response.
For cancer diagnostics and therapeutic purposes in clinical settings, it is important
to
have a device which allows multiplexed measurements, in order to scan a large number
of
markers simultaneously and compare the expression levels of different patients, or
same
patients at different treatment stages, in a time efficient manner.
Methods: Herein, we propose a multiplexed platform for label-free detection
and surface protein profiling of sEVs. The technique is based on the electrokinetic
phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta*
change
upon sEV binding on functionalized microcapillary surfaces. For the purpose, we used
sEVs
derived from lung cancer cells. In its current form, the platform can measure up to
5
channels simultaneously, however, it can be further expanded.
Results: Having demonstrated that our electrokinetic sensor successfully
detects sEVs in a specific way, we tested its ability to measure the expression level
of
membrane proteins. The analysis showed that it could detect differences in the expressions
of EGFR on sEVs, with a sensitivity of 10%. We then extended the platform for multiplexed
analysis, by connecting and measuring four capillaries, functionalized with different
capture probes, simultaneously. For the purpose, we targeted specific tumour markers,
i.e.
EGFR, and exosomal tetraspanin family proteins, such as CD9 and CD63. The results
showed
successful multiplexed EV detection.
Summary/Conclusion: Being the sensor suitable for multiplexed sEV detection,
we shall present our investigation on a set of pleural effusion samples collected
from a
cohort of lung-cancer patients with different genetic makeup.
Funding: Erling Persson Foundation.
PF17.04 = OP2.04
Optimized immunocapture methods for the direct detection of EV
tumour associated proteins in biological fluids: playing around with biophysics
Carmen Campos Silva
a, Yaiza Cáceres
Martellb, María del Puerto Moralesc, Estela Sánchez
herrerod, Atocha Romero Alfonsod, Ricardo Jara Acevedoe,
María Yáñez Móf and Mar Valés Gómezb
aSpanish National Centre for Biotechnology, Madrid, Spain;
bSpanish National Centre for Biotechnology (CNB-CSIC), Madrid, Spain;
cInstituto de Ciencia de Materiales de Madrid (ICMM-CSIC), Madrid, Spain;
dInstituto de Investigación Sanitaria Hospital Universitario Puerta del Hierro,
Madrid, Spain; eImmunostep S.L, Salamanca, Spain; fCentro de Biología
Molecular Severo Ochoa (CBM-SO), Madrid, Spain
Introduction: Extracellular vesicles (EVs) are released to biological fluids
from different tissues and organs and they contain molecules proposed as biomarkers
for
multiple pathological conditions. However, most EV biomarkers have not been validated
due to
the lack of sensitive techniques compatible with high-throughput analysis required
for
routine screenings. Using immunocapture techniques, combining antibodies against
tetraspanins and candidate tumour-specific markers we have recently optimized several
assays
that greatly facilitate EV characterization.
Methods: We have improved flow cytometry and ELISA assays, increasing
substantially the sensitivity for EV detection. Using DLS, EM and analytical
ultracentrifugation, we have characterised the biophysical basis of this enhancement.
The
final methodology can be performed in any laboratory with access to conventional flow
cytometry or ELISA reader.
Results: Using combinations of antibodies specific for the tetraspanins CD9,
CD63 and CD81, it is possible to detect EVs in minimal volumes of urine and plasma
samples
without previous enrichment. Additionally antibodies against other less abundant markers,
like the epithelial marker EpCAM, have been used to capture and identify EVs directly
in
minimal volumes of urine or plasma with sensitivity higher than Western Blot analysis
of
isolated EVs. Furthermore, we demonstrate that additives altering the biophysical
properties
of an EV suspension, increased detection of tumour antigens in these immune-assays.
Summary/Conclusion: The development of sensitive, high-throughput methods,
easily translatable to clinical settings, as ELISA and flow cytometry described here,
opens
a new avenue for the systematic identification of any surface marker on EVs, even
scarce
proteins, using very small volumes of minimally processed biological samples. These
methods
will allow the validation of EV biomarkers in routine liquid biopsy tests.
Funding: MINECO, IMMUNOTHERCAN, TENTACLES, Immunostep
PF17.05 = OP2.05
Normalized extravesicular protein expression profiles on
antibody microarrays reveal protein associations in EVs of organotropic and metastatic
breast cancer cell lines
Molly L. Shen, Rosalie Martel, Lucile
Alexandre, Philippe Decorwin-Martin, Grant Ongo, Andy Ng, Lorenna Oliveira and David
Juncker
McGill University, Montreal, Canada
Introduction: When EV subpopulations are enriched on antibody microarrays and
probed for their surface proteins, the detection signal is biased towards abundant
subpopulations as it is dependent on both the protein expression level and the number
of EVs
captured. To address this challenge, we developed a novel normalization approach allowing:
1) the estimation of a target signal independent of EV subpopulation size through
dye-based
EV quantification, and 2) the assessment of subpopulation target enrichment relative
to the
population average by leveraging TIM4 as an unbiased, lipid-based EV capture. Here,
we
investigated the expression of cancer-associated proteins, particularly
metastasis-associated integrins (ITGs), in breast cancer EVs with varying metastatic
potential and organotropism.
Methods: The relative protein enrichment profiles for various EV
subpopulations were established from EVs of SkBr3 (HER2+), T47D and MCF-7 (ER+PR+),
BT549
and MDA-MB-231 (triple negative) breast cancer cell lines, as well as five
MDA-MB-231-derived cell lines of four different organotropisms (brain, bone, lung,
liver)
using our custom antibody microarrays with our normalization approach.
Results: As expected, HER2 was broadly detected in HER2+ SkBr3 EVs.
Interestingly, HER2- T47D and MCF-7 EVs also expressed HER2 where it was highly enriched
in
its EpCAM+ subpopulations. ITG α6, β3 and β4 were only found in triple negative and
organotropic EVs with ITG β3 and β4 differentially enriched based on the organotropism.
The
population average of MDA-MB-231 and lung-tropic EVs had high expression of ITG β4,
where
subpopulations of CD44+ EVs showed positive enrichment while CD9+ and CD63+ EVs showed
negative enrichment. ITG α5, β3 and β4 were absent in the bone-tropic CD81+ EV
subpopulation, a profile atypical in other organotropisms. Lastly, EGFR was negatively
enriched in Tetraspanin+ subpopulations in MDA-MB-231 EVs, but positively enriched
in these
subpopulations in organotropic EVs, especially for brain-tropism.
Summary/Conclusion: Following normalization, we were able to quantify specific
protein associations, uncovering a multitude of co-enrichment profiles that characterize
specific metastatic and organotropic cell lines. Notably, we found enrichment signatures
that distinguish between different organotropisms derived from the same parental cancer
line.
Funding: This research was supported by the Natural Sciences and Engineering
Research Council of Canada (NSERC) and the Genome Canada Distruptive Innovation program.
PF17.06 = OP2.06
Heparan sulphate proteoglycans are required for EV-mediated
delivery of multiple growth factors
Sara Veiga, Alex Shephard, Alex Cocks, Aled
Clayton and Jason Webber
Cardiff University, Cardiff, UK
Introduction: The tissue microenvironment surrounding tumours is complex and
the cross-talk between cancer and non-cancer cells is essential for tumour growth
and
progression. We have previously shown that heparan sulphate proteoglycans (HSPGs),
on the
surface of prostate cancer EVs, are required for delivery of TGFβ and initiation of
a
disease-supporting fibroblast phenotype. However, HSPGs are known to bind numerous
growth
factors, so here we have explored the repertoire of such proteins tethered to EVs
by
HSPGs.
Methods: EVs were isolated from DU145 prostate cancer cell conditioned media
by ultra-centrifugation onto a sucrose cushion. Vesicular HSPGs were modified either
by
removal of heparan sulphate (HS) glycosaminoglycan (GAG) chains using the enzyme Heparinase
III (HEPIII), or attenuation of HSPG core protein expression using shRNAs to knockdown
specific HSPGs within the parent cell. Differences in proteins present in control
vs
modified EVs were identified by a sensitive protein array, based on proximity-ligation
technology, and selected targets validated by ELISA. Functional delivery of growth
factors
by EV-associated HSPGs to recipient fibroblasts is being explored using a variety
of in
vitro techniques.
Results: Proteome analysis identified 49 targets that bind to HS-GAG chains,
and also 108 different proteins that showed altered expression following the loss
of one or
more HSPGs from EVs. Using ELISA, we have been able to quantify selected candidates
on wild
type vesicles, some of these are lost following HS-digestion. We were also able to
validate
proteins on HSPG-deficient vesicles. Gene ontology analysis suggests that EV HSPG-mediated
delivery of growth factors is important for control of processes such as angiogenesis,
tumour invasion and immune regulation. Functional validation of proteins identified
is
ongoing.
Summary/Conclusion: Here we demonstrate that HSPGs play a key role in loading
of EVs with a complex assortment of growth factors, and therefore subsequent EV-mediated
growth factor delivery. We anticipate that loss or damage of EV-associated HSPGs will
result
in attenuation of EV induction of a tumour-supporting fibroblast phenotype.
Funding: Cancer Research Wales
PF17.07 = OP2.07
Robust exosomal biomarker panel discovery in ovarian cancer
using machine learning approaches and studying miRNA & miRNA-target
interactions
Paritra Mandal
a, Tyler
Sloneckib, Brian C. Deanb, R. Kenneth Marcusb, William
Bridgesb and Terri F. Bruceb
aClemson University, Pendleton, USA; bClemson
University, Clemson, USA
Introduction: Ovarian cancer (OC) is the fifth leading cause of cancer-related
death in women, partly due to difficulty in early diagnosis. Extracellular vesicles
(EVs)
show promise for use in early diagnostics of OC. Here, EVs from cervical mucus (CM)
of
ovarian cancer patients were used for discovery of OC biomarkers for diagnostics.
Machine
learning was used to mine EV miRNA data to develop an OC biomarker panel (validation
via The
Cancer Genome Atlas). Examination of the miRNA targets reveal that the panel is a
sufficiently accurate predictor of OC.
Methods: EVs from the CM of 48 patients (15 high-grade serous, 24 low-grade, 7
benign) were isolated for small RNA-sequencing. The top differentially expressed miRNAs
were
used in a random forest and “voom” (variance modelling at the observational level)
model.
Unsupervised approaches were used and then vetted against patient symptomology data.
A TCGA
ovarian cancer dataset (n = 100) was used for validation.
Results: An OC biomarker panel of 10 microRNAs (voom: 96.55% accuracy; random
forest: 88% accuracy) was generated. The panel consists of members from the mir-200
family
and the mir-16 family, among others. The miRNA targets are associated with molecular
functions and pathways specific in OC progression.
Summary/Conclusion: Our method has identified EV miRNA biomarkers that may be
crucial for early, non-invasive detection of OC. Data science has been used to develop
a
feedback system integrating biochemical experiments, smaller datasets, and previously
available data to identify and verify a biomarker panel for OC diagnostics.
Funding: Support from the National Science Foundation, Eppley Foundation for
Scientific Research, Gibson Foundation, Prisma Health System and ITOR Biorepository
are
gratefully acknowledged.
PS01: EVs in Infectious Diseases and Vaccines
Chair: Susmita Sil – University of Nebraska Medical Centre
PS01.01
Ethanol and HIV-induced exosome from hepatocytes activate
hepatic stellate cells
Raghubendra S. Dagur, Murali Ganesan, Moses
New-Aaron, Larisa Poluektova and Natalia Osna
University of Nebraska Medical Center, Omaha, USA
Introduction: Liver disease has become a significant cause of morbidity and
mortality among HIV patients. Alcohol exposure can further exacerbate liver damage
by
activating hepatic stellate cells (HSCs), leading to hepatic fibrosis or cirrhosis,
often
seen at all levels of alcohol exposure among people with HIV. Due to the potentiating
effects of alcohol on HIV-induced hepatocytes (Hep) damage, as well as the effect
of ethanol
in HSC-mediated extracellular remodelling, it is imperative to understand the interplay
of
Hep and HSCs. Here, we focus on the exosomes released by HIV-and ethanol exposed Hep
and how
these exosomes modulate the functional behaviour of HSCs.
Methods: Human hepatocyte Huh7.5CYP2E1 [hepatoma cells stably transfected with
CYP2E1 designated as RLW cells] were infected with HIV in the presence or absence
of alcohol
metabolite, acetaldehyde using the acetaldehyde-generating system (AGS). The conditioned
medium was collected from 4 groups of cells: untreated, HIV-, AGS- and HIV+AGS.
Quantification of exosomes number and size were evaluated with ZetaView or Nanosight
and
further characterized for exosome markers following the guideline from Minimal information
for studies of EVs 2018 (MISEV2018). The human hepatic stellate LX-2 cell line was
exposed
to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory
markers IL-1β, IL-6, TNFα, and fibrotic markers ACTA2, and TIMP1 using quantitative
PCR. We
also analysed exosome miRNA content in primary human hepatocytes (PHH), which potentially
regulates the function of recipient cells by “programming” their inflammation/fibrosis
status. The network analysis for mRNA and miRNA were carried out using Gene ontology
consortium, and mirror 2.0 and DAVID bioinformatics resources 6.8.
Results: AGS treatment further enhanced the release of HIV-induced exosome
from hepatocytes. Size distribution assessed by zeta view or Nanosight revealed that
approximately 85–90% of particles distributed in the range of 50 to 200 nm, with a
peak at
~90 nm. Enriched expression of HIV protein P24 was observed in fractions F9-F12. Western
blotting of hepatocyte-derived exosome demonstrated positivity for exosome-enriched
proteins
Alix, TSG 101 and CD9 specifically in F2-F8 fractions and negative for endoplasmic
reticulum
protein calnexin. The uptake of Hepatocyte-derived exosomes by HSCs was apparent as
demonstrated by immunofluorescence. The internalization of hepatocyte-exosome induced
activation of HSCs as evidenced by increased expression of pro-inflammatory IL-1β,
IL-6,
TNFα markers in the latter cells.
Summary/Conclusion: We conclude that AGS treatment in HIV-infected hepatocytes
potentiates the release of exosomes, which, following uptake by the HSCs, leads to
their
activation.
Funding: This work is supported by NIH‐1 R01 AA027189‐01A1.
PS01.02
Antimicrobial peptide LL-37 induces neutrophil-derived
extracellular vesicles with antibacterial potential and protects murine sepsis
Yumi Kumagai, Taisuke Murakami, Kyoko Kuwahara
and Isao Nagaoka
Juntendo University, Bunkyo-ku, Japan
Introduction: Extracellular vesicles (EVs) released from immune cells or other
host cells upon microbial infection modulate the immune response and thereby regulate
the
infection. Sepsis is a life-threatening multiple organ dysfunction caused by systemic
dysregulated inflammatory response to infection. Nevertheless, numerous therapeutic
trails
concerning immune dysfunction have still been disappointing outcomes. We have previously
shown that LL-37, a human cathelicidin antimicrobial peptide, improves the survival
of
caecal ligation and puncture (CLP) septic mice. Here, we investigated the induction
of EV
release by LL-37 and functions of LL-37-induced EVs in murine sepsis.
Methods: EVs were isolated from peritoneal exudates of CLP mice and the
supernatant of LL-37-stimulated mouse bone marrow neutrophils by differential centrifugation
or size exclusion chromatography. Isolated EVs were analysed by flow cytometry, western
blotting, and nano particle analysis. Neutrophil-derived EVs were injected into CLP
mice to
assess the protective function of EVs against septic mice. The antibacterial activity
of EVs
was evaluated by incubating with Escherichia coli.
Results: In CLP mice, LL-37 augmented the level of EVs. EVs from
LL-37-injected CLP mice contained higher amounts of neutrophil-derived antibacterial
proteins (lactoferrin and CRAMP, cathelicidin-related antimicrobial peptide) and exhibited
higher antibacterial activity compared to EVs from PBS-injected CLP mice. Furthermore,
LL-37
stimulated mouse bone marrow neutrophils to release EVs with antibacterial potential,
and
administration of the LL-37-induced EVs reduced the bacterial load and improved the
survival
of CLP mice.
Summary/Conclusion: LL-37 induces the release of antimicrobial EVs from
neutrophils in CLP mice, thereby reducing the bacterial load and protecting mice from
lethal
septic condition.
PS01.03
Identification of miRNA profiles of serum exosomes in active
tuberculosis
Qian Qiua
, Wei Xiongb,
Song Yangc and Xiaofeng Yanc
aResearch Institute of Tuberculosis, Chongqing Public Health
Medical Center, chongqing, China (People’s Republic); bDepartment of Geriatrics,
Southwest Hospital, Army Medical University, Chongqing, China (People’s Republic);
cChongqing Public Health Medical Center, Chongqing, China (People’s
Republic)
Introduction: Tuberculosis (TB) has exceeded HIV as the most lethal infectious
disease globally for two consecutive years, mainly due to difficulties in achieving
early
and definitive diagnosis, and timely treatment. Exosomes carrying RNA, particularly
miRNA,
have demonstrated their functional and diagnostic potential in diseases including
TB.
However, few published studies have explored whether exosomal miRNAs could be used
for
diagnosis of TB. Thus, more systematic and comprehensive study of exosomal miRNAs
with
regard to their potential as non-invasive TB biomarkers is still urgently needed.
Methods: We searched the Gene Expression Omnibus database for datasets
published before December 2019, and performed meta-analysis on available exosomal
miRNA
profile data for healthy control (HC) and active TB clinical specimens . Reprocessing
next
generation sequencing data under uniform parameters and utilizing state-of-the-art
bioinformatics analysis.
Results: We identified many distinct up-regulated and down-regulated
differentially expressed exosomal miRNA across multiple studies, and further screened
the
top 10, which might provide a potential panel for differentiation of HC and TB. We
classified all differentially expressed miRNAs into six expression patterns and identified
two persistently up-regulated miRNA (hsa-miR-140-3p, and hsa-miR-423-3p) as potential
markers during TB progression. Moreover, the differential expressed exosomal genes
that we
screened from the datasets were consistent with the genes overlapped with predicted
mRNA
targets of differentially expressed miRNA. Pathway and function analysis further
demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism
and apoptosis/necrosis.
Summary/Conclusion: Our findings demonstrated the selective packaging of RNA
cargoes into exosomes under different stages of Mycobacterium tuberculosis (Mtb) infection,
as well as facilitating development of potential targets for the diagnosis, prevention
and
treatment of TB.
Funding: 1. Natural Science Foundation of Chongqing of China under Grant No.
cstc2019jcyj-msxmX0028.
2. Scientific Research Foundation of Science and Health Joint Medicine of Chongqing
of
China under grant No. 2019QNXM038.
3. Youth Innovation Fund of Chongqing Public Health Medical Center under grant No.
2019QNKYXM01.
PS01.04
The new therapy to treatment experimental Acute Chagas
Disease
Lavínia Maria Dal’Mas Romera, Rafael Pedro Madeira, Patricia Xander
and Ana Claudia Torrecilhasa
Universidade Federal de Sao Paulo, Sao Paulo, Brazil
Introduction: Trypanosoma cruzi is a protozoan parasite that causes Chagas
disease, a relevant source of morbidity in Latin America, which has spread to many
countries
as result of immigration of the people from endemic areas. Many studies have been
showed
that trypomastigote forms of T. cruzi release extracellular vesicles (EV) that increase
parasite infection. Objectives. Here, we aim to test if previous immunization with
EVs in
adjuvant can generate a protective immune response by decreasing the effects of EVs
in
experimental Chagas disease.
Methods: Female BALB/c mice were immunized by intra peritoneal (ip)
administration with 5 × 105 or 107 EVs isolated from trypomastigotes forms, with aluminium
hydroxide adjuvant (AlOH). Injections were administered intravenous in 3 doses during
45 days (15 days interval). After immunization, mice were infected intra-peritoneally
with
500 trypomastigotes forms. Parasitaemia was quantified by counting motile parasites
in fresh
blood sample drawn from lateral tail veins. Mortality and weight were analysed during
the
infection. In control group, the mice were immunized with AIOH.
Results: The immunization with EVs with AlOH decreased the blood parasitaemia
and the animals survived, while all animals died in the group AlOH alone. The animals
immunized with EVs had an increase of F4/80+ CD11b+ and CD80/CD86 expression in cells
isolated from the peritoneum.
Summary/Conclusion: These results indicate that T. cruzi EV antigens can
induce an immune response that controls the development and establishment of the
experimental Chagas disease.
Funding: FAPESP, CNPq and CAPES.
PS01.05
Characterization of outer membrane vesicle-carried proteins as
pathogenicity factors from Acinetobacter baumannii
Lucía Giaconea, Guillermo Repizoa, Tania
Cebrero-Cangueirob, Jerónimo Pachónb, María Eugenia
Pachón-Ibáñezb and Jorgelina Moran
Barrioa
aInstitute of Molecular and Cellular Biology of Rosario
(IBR). Faculty of Biochemistry and Pharmacy, CONICET, National University of Rosario
(UNR),
Argentina., Rosario, Argentina; bInstitute of Biomedicine of Seville (IBiS),
University of Seville/CSIC/University Hospital Virgen del Rocío, Seville, Spain.Department
of Medicine, University of Seville, Spain., Sevilla, Spain
Introduction: Acinetobacter baumannii (Ab) is a nosocomial pathogen, of major
concern due to its multi-drug resistance (MDR) and the recent appearance of hyper-virulent
strains in the clinical setting. The World Health Organization included Ab as a critical
priority pathogen for the development of novel antibiotics. Ab pathogenesis is associated
with a multitude of potential virulence factors (VF) that remain poorly characterized.
There
is growing evidence that outer membrane vesicles (OMV) are used as vehicles to transport
bacterial proteins that contribute to set up the conditions for the infections. In
the
present work we studied the physiopathology of MDR Ab. We focused on the contribution
of
non-characterized outer membrane proteins (OMPs) associated to OMVs, with special
focus on
lipoproteins (LP).
Methods: We conducted a bioinformatic prediction using available datasets to
construct a list of OMV-associated OMPs putatively acting as VF in AB5075. Seven genes
were
selected and the corresponding mutants were obtained from Manoil Lab collection.
Physiological analyses of the mutants were performed, and the involvement of the selected
proteins in Ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity
assays on
human lung cells A549.
Results: Biochemical analysis indicated similar growth rates in rich media, as
well as similar levels of OMV production for all the mutants as compared to WT. Also,
no
differences in susceptibility to chaotropic agents were observed, indicating no alteration
of the OM function as a general permeability barrier. All mutants similarly reduced
A549
cell viability, but to a lesser extent than the WT. Moreover, three of them exhibited
less
adhesion and invasion compared to the WT, and OMV isolated from these mutants displayed
variable levels of cytotoxicity.
Summary/Conclusion: These results suggest roles for the mutant gene products
in Ab pathogenesis and contribute to the better understanding of Ab virulence mechanisms,
revealing novel possible targets for therapeutic development.
Funding: Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT
2017–3536) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET,
PIP-11220170100377CO), from Argentina.
Instituto de Salud Carlos III (PI12/02553); Plan Nacional de I + D + i 2013‐2016 and
Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades
(BFU2016-77297-P), and Spanish Network for Research in Infectious Diseases (REIPI
RD16/0016/0009) co‐financed by European Development Regional Fund, from Spain.
PS01.06
Talaromyces marneffei yeasts-derived Extracellular Vesicles
Mediate Inflammatory Response and Modulate Macrophage Functions
Biao Yanga
, XiuMei Hub,
Lei Zhengc and Qian Wangd
aNanfang Hospital, Southern Medical University, Guangzhou,
China, guangzhou, China (People’s Republic); bSouthern medical university,
guangzhou, China (People’s Republic); cDepartment of Laboratory Medicine, Nanfang
Hospital, Southern Medical University, Guangzhou, 510515, China, guangzhou, China
(People’s
Republic); dZhujiang Hospital, Southern Medical University, Guangzhou, China,
guangzhou, China (People’s Republic)
Introduction: Talaromyces marneffei (T. marneffei) grows as a mycelial form in
the environment but multiplies rapidly as a yeast form in the host and within macrophages.
The yeast can cause disseminated and progressive infections or lethal Talaromycosis.
But the
mechanisms of pathogenicity of T. marneffei are poorly understood. Fungal extracellular
vesicles (EVs) have previously been shown to transmit a proinflammatory message to
macrophages. However, the characteristics and effects of T. marneffei EVs on the progress
of
infection have not yet been investigated.
Methods: In this study, EVs of T. marneffei yeasts were isolated by
ultracentrifugation method. EVs were detected and confirmed by Electron microscopy
and
nanoparticle tracking analysis (NTA). The RAW 264.7 murine macrophages were incubated
with
the T. marneffei vesicles to observe the changes of macrophage morphology and function,
especially in inflammatory response. The proteins, DNAs, RNAs of T. marneffei vesicles
were
respectively removed with protease, DNase and RNase. All treated EVs were used to
incubate
with murine macrophages observe the effect on macrophages in inflammatory response.
Results: We observed that EVs secreted by T. marneffei have a typical
spherical shape with a diameter of 30 to 200 nm. T. marneffei EVs were internalized
by RAW
264.7 murine macrophages and promoted the production of NO and proinflammatory cytokine
by
macrophages in a dose-dependent manner. T. marneffei EVs stimulate macrophages to
generate
Reactive oxygen species (ROS). Addition of T. marneffei EVs to macrophages also promoted
transcription of the M1-polarization marker CD86 and diminish that of the M2 markers
CD206.
Incubation of T. marneffei vesicles with murine macrophages resulted in increased
levels of
extracellular interleukin-1β(IL-1β), interleukin-6 (IL-6) and interleukin-12 (IL-12).
The
proinflammatory effect of vesicles was weakened when the proteins of the vesicles
were
destroyed. In contrast, no similar changes were observed in degraded DNA and RNA.
Summary/Conclusion: Our results indicate that the extracellular vesicles of T.
marneffei can stimulate macrophage towards to M1 polarization phenotype and promote
proinflammatory function.
PS01.07
Plasma-derived extracellular vesicles as potential biomarkers in
chronic Chagas disease patients
Nuria Cortes-Serra
a, Maria Tays
Mendesb, Cameron C Ellisb, Joaquim Gascona, Igor C.
Almeidab, María Jesús Pinazoa and Carmen
Fernández-Becerrac
aISGlobal, Hospital Clínic – Universitat de Barcelona,
Barcelona, Spain., Barcelona, Spain; bDepartment of Biological Sciences, Border
Biomedical Research Center, The University of Texas at El Paso, El Paso, TX USA.,
El Paso,
USA; cISGlobal, Hospital Clínic – Universitat de Barcelona, Spain., Badalona,
Spain
Introduction: Chagas disease (CD), caused by the parasite Trypanosoma cruzi
(T. cruzi), is a neglected tropical disease affecting about 8 million people worldwide.
Currently, one of the main clinical problems is the lack of effective biomarkers for
therapeutic response and disease prognosis during chronic infections. In that context,
extracellular vesicles (EVs) are raising attention as novel, minimally invasive, and
inexpensive method for diagnostic and screening of diseases, as well as a new source
to
identify new biomarkers. The main objective of this study is to use EVs derived from
biological fluids of chronic CD patients for identifying novel biomarkers, specifically
in
the context of therapeutic response and disease prognosis.
Methods: Plasma, saliva and urine from a cohort of chronic CD patients are
being collected before and at the end of benznidazole treatment. As negative controls,
healthy donors have been also included. The purification and characterization of the
EVs was
performed by size exclusion chromatography, followed by nanoparticle tracking analysis,
bead-based flow cytometry assay and transmission electron microscopy. A proteomic
analysis
of the EVs was also performed.
Results: Proteins associated with EVs secreted by infective T. cruzi have been
previously identified in cell culture, but never in human samples. Our results, based
on the
analysis of a single heart-transplanted patient with chronic CD, showed the presence
of T.
cruzi and human proteins specifically associated with plasma-derived EVs. Noticeably,
several human and parasite proteins identified in EVs obtained from plasma samples,
were
present or upregulated before chemotherapy and were absent or downregulated following
treatment. Currently, proteomics analyses are being performed with higher numbers
of CD
plasma samples.
Summary/Conclusion: To the best of our knowledge, this is the first proteomic
profiling of plasma-derived EVs from a heart-transplanted patient with chronic CD.
These
results thus open the possibility of using EVs from biological fluids as a tool for
the
identification of new biomarker candidates in chronic CD. These biomarkers are essential
for
assessing disease prognosis, evaluating the recovery of chronic patients after treatment,
and for testing new drugs in clinical trials.
Funding: This work received funding from Fundació La Marató de TV3, Fundación
Mundo Sano and FEDER with the support of Secretaria d’Universitats i Recerca del Departament
d’Empresa i Coneixement de la Generalitat de Catalunya.
.
PS01.08
Eukaryotic extracellular vesicles and prokaryotes – a different
perspective
Joni R. White
a, Priscila
Dauros-Singorenkob, Joni Whitec, Jiwon Hongc, Anthony
Phillipsc and Simon Swiftc
aDepartment of Molecular Medicine and Pathology, 85 Park
Road, The University of Auckland, Auckland, New Zealand, Auckland, New Zealand;
bThe University of Auckland, Auckland, New Zealand; cUniversity of
Auckland, Auckland, New Zealand
Introduction: Eukaryotic cells communicate with one another through multiple
pathways. An established route of communication between eukaryotic cells is via the
production of a range of different membrane bound signalling “packages”, called
extracellular vesicles (EVs). EVs are produced by all domains of life and carry proteins,
nucleic acid (RNA and DNA), and other biological material, travelling between cells
and
around the body to deliver a range of chemical messages. Bacteria can also produce
EVs that
communicate with each other to co-ordinate population behaviour, as well as with eukaryotic
cells to stimulate host defence or induce tolerance. Here I investigate the poorly
explored
axis where EVs are the vehicle for communication between eukaryotic cells and bacteria.
Methods: As a first step, I have isolated EVs from tissue cultured eukaryotic
cells grown in Advanced RPMI media with minimal EV-depleted FBS. NanoEVs were isolated
from
spent culture media using sequential centrifugation (2,000 × g, 10,000 × g) and
concentration (100 kDa filter) before purifying using size exclusion chromatography
columns.
NanoEV-rich fractions were pooled based on particle (Nanoparticle Tracking Analysis)
and
protein quantity data. NanoEVs were characterised by electron microscopy and expression
of
exosomal markers. Eukaryotic nanoEVs were then characterised in their effect upon
the growth
of Escherichia coli as a model bacterium, also grown in tissue culture media to mimic
relevant in vivo conditions.
Results: Further experiments with increased dosages are required to determine
the effect of human EVs on bacteria.
Summary/Conclusion: Our work will investigate whether human EVs communicate
with the resident and pathogenic microbiota, while examining the mechanisms behind
this
communication.
Escherichia coli
pathogenic bacteria
commensal bacteria
PS01.09
Hydrogen sulphide (H2 S) derived extracellular vesicles: a
potential protective role in response to respiratory syncytial virus (RSV)
infection
Tiziana Corsello-Gorgun, Roberto P. Garofalo
and Antonella Casola
Department of Paediatrics, Division of Clinical and Experimental
Immunology and Infectious Diseases, University of Texas Medical Branch, Galveston,
TX,
Galveston, USA
Introduction: Hydrogen sulphide (H2 S) is as a critical endogenous
gasotransmitter with antiviral and anti-inflammatory activity against respiratory
syncytial
virus (RSV) infection. Extracellular vesicles (EVs), novel mediators of cell–cell
communication, have the potential to modulate cellular responses of recipient cells
following a variety of stimuli. In this study, we investigated the composition/function
of
EVs derived from airway epithelial cells treated with GYY4137, a slow-releasing H2 S
donor,
and whether GYY-derived EVs can alter airway epithelial cell response in recipient
cells
following RSV infection.
Methods: EVs were isolated from untreated (control EVs) and GYY4137 treated
(GYY-EVs) A549 cells, a human alveolar type II–like epithelial cell line. EVs were
purified
using a two-step enrichment procedure. EVs were characterized using particle sizing
(size
and concentration) and Western blot for the EV markers. Electron microscopy and
immunofluorescence staining were used to investigate presence of multivesicular bodies
(MVBs), EVs precursors, in both groups. Recipient A549 cells were cultured for 24 hours
in
the presence or absence of control- or GYY- EVs, then infected with RSV for 24 hours.
Viral
titres by plaque assay were measured in recipient infected A549 cells.
Results: We confirmed the presence and purity of our EVs. We found that
GYY4137 reduced the particles number of EVs, but did not change EV size. A549 cells
treated
with GYY4137 showed an accumulation of MVBs/lysosomes-like structures, as well as
an
increase in CD63 expression, a MVBs marker, compared to untreated cells. Recipient
A549
cells treated with GYY-EVs showed lower viral replication than control EV-treated
cells in
response to RSV infection. We are currently investigating the potential mechanism
for this
observation and characterizing the RNA cargo composition of GYY-EVs.
Summary/Conclusion: No vaccine or effective treatment is currently available
for RSV. Cellular pretreatment with GYY-EVs reduced the RSV replication in airway
epithelial
recipient cells, suggesting that H2 S could exert its antiviral activity in the context
of
RSV infection potentially through modulation of EV composition. Therefore, GYY-EVs
could
represent a future novel pharmacological approach for ameliorating virus-induced lung
disease.
PS01.10
Effects of extracellular vesicle-mediated transmission on
Reoviridae infection
Syndi Smith
a, Ariana von
Lersnerb, Andries Zijlstraa and Kristen Ogdena
aVanderbilt University Medical Center, Nashville, USA;
bVanderbilt University, Nashville, USA
Introduction: The Reoviridae family contains important viruses that infect
humans and other animals. Reovirus is in advanced clinical trials as an oncolytic
therapeutic and has been linked to onset of coeliac disease. Rotavirus is a leading
cause of
diarrhoeal gastroenteric mortality in children aged 5 years or younger in the developing
world. Traditionally, viruses have been thought to transit between cells as independent
particles. This viewpoint is being challenged with the recent discoveries that many
virus
families utilize extracellular vesicles (EVs) for non-lytic transport. EV-mediated
transmission potentially enables viral evasion of immune responses and collective
transmission to drive enhanced productive infection. Recently, rotavirus was shown
to egress
host cells in large EVs, and EV-mediated transmission enhances rotavirus virulence
in vivo.
Mechanisms of reovirus egress are incompletely defined, and effects of multiparticle
transmission on rotavirus and reovirus genetic complementation are unknown.
Methods: Using cultured cells, microflow cytometry, and genetically barcoded
viruses, we provide evidence that i) reovirus particles egress in large EVs in a virus
strain- and cell type-dependent manner, ii) rotavirus upregulates EV release from
infected
cells, and iii) EV-mediated transmission increases the frequency of reovirus genotype
mixing.
Results: Taken together, these data suggest that multiple particles of
reovirus and rotavirus egress in large, virus-modulated EVs, and that transmission
in EVs
increases segment complementation compared to transmission as free particles.
Summary/Conclusion: These discoveries may be broadly applicable to viruses
that travel in EVs and will contribute to general principles of virus transmission
and
diversification. Continued studies will illuminate the specific cellular pathways
reovirus
and rotavirus utilize for successful egress. These pathways may prove to be critical
targets
for the improvement of vaccines and oncolytic therapy.
PS01.11
Multiparameter flow cytometry analysis of the human spleen and
its interaction with plasma-derived EVs from Plasmodium vivax patients
Melisa Gualdrón-López, Miriam Diaz-Varela,
Haruka Toda, Iris Aparici-Herraiz, Laura Pedró-Cos, Ricardo Lauzurica, Marcus VG Lacerda,
Marco Fernández, Carmen Fernández-Becerra and Hernando A. del Portillo
ISGlobal, Hospital Clínic – Universitat de Barcelona, Spain
Introduction: The spleen is a secondary lymph organ that filters blood and
elicits immune responses against blood-borne pathogens, such as malaria parasites.
Extracellular vesicles (EVs) are membrane-bound particles involved in intercellular
communication. EVs play several roles in malaria ranging from modulation of immune
responses
to induction of vascular alterations. Here, we report the first integrated characterization
of human spleen cells using multiparameter flow cytometry (MFC) describing subpopulations
of
splenic leukocytes and red blood cells (RBCs), and studied their interaction with
plasma-derived EVs from P. vivax patients (PvEVs).
Methods: Human spleens were obtained from organ transplantation donors.
Myeloid, lymphoid, erythroid and haematopoietic stem cells (HSCs) were immunophenotyped
by
MFC. T cells, dendritic cells (DCs) and RBCs were enriched by density centrifugation
and
immunomagnetic isolation. PvEVs and healthy donors EVs (hEVs) were purified by
size-exclusion chromatography (SEC) and characterized by bead-based flow cytometry.
Enriched
EVs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes
or
enriched populations. EVs-cells interaction was assessed by flow cytometry.
Results: Human spleen immunophenotyping showed that CD45+ cells included B
(30%), CD4 + T (16%), CD8 + T (10%), NK (6%) and NKT (2%) lymphocytes. Myeloid cells
comprised neutrophils (16%), monocytes (2%) and DCs (0.3%). Erythrocytes represented
70%
whereas, unexpectedly, reticulocytes were 0.93% of total cells. In addition, we also
detected HSCs, which accounted for 0.02%. SEC separated EVs from the bulk of soluble
plasma
proteins as shown by the enrichment of CD63, CD5 L and CD71 markers. Interaction studies
showed an increased proportion of T cells (CD4 + 3-fold and CD8 + 4-fold), monocytes
(1.5-fold), B cells (2.3-fold) and erythrocytes (threefold) interacting with PvEVs
as
compared to hEVs.
Summary/Conclusion: The integrated cellular analysis of the human spleen and
the methodology employed here allowed in vitro interaction studies of human spleen
cells and
EVs. A larger proportion of monocytes, T and B lymphocytes as well as erythrocytes
was found
to interact with PvEVs compared to hEVs. Future functional studies of these interactions
can
unveil pathophysiological processes involving the spleen in vivax malaria.
PS02: EVs in Cancer Metastasis and Tumour Angiogenesis
Chair: Vincent Hyenne – Tumour Biomechanics INSERM UMR_S 1109 Institut d’hématologie
et d’immunologie, CNRS SNC5055, Strasbourg, France
Chair: Shiv Ram Krishn – Thomas Jefferson University, Philadelphia, USA
PS02.01
Activating protein-1 and extracellular vesicles in cancer
metastatic behaviour phenocopying
Sherif A. Ibrahima
and Michele
Fluckb
aXavier University Medical School – Istanbul, Istanbul,
Turkey; bMichigan State University, East Lansing, USA
Introduction: Activating protein 1 (AP-1) is a dimeric transcription factor
that is formed by one member of the Fos family and one member of the Jun family of
proteins.
The expression of Fra-1 and c-Jun, two members of the AP-1 family, is highly increased
in
triple-negative breast cancer. AP-1 regulates several soluble factors which play autocrine
and paracrine roles to enhance cancer progression and metastasis. My previously published
work showed that factors secreted by the metastatic MDA-231 cells and regulated by
AP-1
induced proliferation and migration of non-tumorigenic MCF10A cells and the non-metastatic
MDA-468 cells through the induction of Fra-1. Previous studies showed that exosomes
from
MDA-231 cells increased the migration and invasion of the less invasive MCF7 cells.
Proteomic comparison of the cargo of the exosomes from these two cell lines found
that
exosomes from MDA-231 tended to carry proteins that enhance the metastatic process
like
vimentin. So, I hypothesized that AP-1 controls the cargo of the MDA-231 released
exosomes
so it directs its oncogenic and metastatic phenocopying behaviour.
Methods: To study the role of AP-1, I utilized the dominant-negative version
of Fos protein named A-Fos. MDA-MB-231 cells were infected with the gene for a flag
tagged
A-Fos cloned to a retroviral vector under doxycycline (Dox) inducible promoter to
produce a
stable cell line (MDA231/Afos). Exosomes were extracted from conditioned medium acquired
from Dox(-) and Dox(+) MDA231/Afos cells using ultracentrifugation. The presence of
exosomes
was confirmed using TEM and nanoparticle tracking analysis. The exosomes were added
to
serum-starved MDA468 cells.
Results: Exosomes from Dox(-) MDA231/Afos cells increased the Fra-1 level in
serum-starved MDA-468 cells. This effect was reduced in the case of exosomes acquired
from
Dox(+) MDA231/Afos.
Summary/Conclusion: These results suggest a role of AP-1 both in controlling
and in mediating the action of exosomes from MDA-231 cells on non-metastatic cells.
My plan
is to explore the cargo of the exosomes regulated by AP-1 to transfer the malignant
and the
metastatic properties of MDA-231 cells. I will use RNAseq and proteomic analysis to
analyse
the content of MDA-231 exosomes in the presence and absence of A-Fos.
PS02.02
Neuroblastoma-secreted exosomes carrying miR‐375 promote
osteogenic differentiation of bone marrow mesenchymal stromal cells
Marta Collettia
, Luigi
Tomaoa, Angela Galardia, Alessandro Paolinib, Virginia Di
Paoloa, Cristiano De Stefanisc, Paolo Mascioa, Francesca
Nazioa, Stefania Petrinid, Aurora Castellanoa, Ida
Russoa, Roberta Carusoa, Simone Pigae, Rita De
Vitof, Andrea Masottib, Franco Locatellia and Angela Di
Giannatalea
aDepartment of Paediatric Haematology/Oncology and Cell and
Gene Therapy, IRCCS, Ospedale Pediatrico Bambino Gesù, Rome, Italy, Rome, Italy;
bResearch Laboratories, IRCCS, Ospedale Pediatrico Bambino Gesù, Rome, Italy,
Rome, Italy; cDepartment of laboratories – Pathology Unit, IRCCS, Ospedale
Pediatrico Bambino Gesù, Rome, Italy, Rome, Italy; dDepartment of Laboratories,
Confocal Microscopy Core Facility, IRCCS, Ospedale Pediatrico Bambino Gesù, Rome,
Italy,
Rome, Italy; eUnit of Clinical Epidemiology, IRCCS, Ospedale Pediatrico Bambino
Gesù, Rome, Italy, Rome, Italy; fDepartment of laboratories – Pathology Unit,
IRCCS, Ospedale Pediatrico Bambino Gesù, Rome, Italy
Introduction: Bone marrow (BM) is the major target organ for neuroblastoma
(NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism
by
which NB cells invade BM is largely unknown. Tumour microenvironment represents a
key
element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized
as a
fundamental part of the associated tumour stroma. Here, we explore the potential role
of
NB-derived exosomes in induction of a pro-osteogenic phenotype on BM-MSCs.
Methods: In this prospective study, we assessed the osteogenic differentiation
of BM-MSCs isolated from patients with and without BM metastasis. Expression profiling
of
exosomal microRNAs was performed on 8 NB cell lines by Q-PCR. MiR-375, represented
the main
candidate related to the osteogenic phenotype. Its over- and down-regulation was performed
in vitro. MiR-375 was then evaluated in plasma (peripheral and BM derived) and BM
biopsies
of NB patients.
Results: BM-MSCs isolated from NB patients with BM involvement exhibit a
greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM
metastasis-derived NB cell lines secrete higher levels of exosomal miR-375, which
promotes
osteogenic differentiation in MSCs. Clinical data demonstrate that high level of miR-375
correlate with BM metastasis in NB patients.
Summary/Conclusion: Our findings suggest a potential role for exosomal miR-375
in determining a favourable microenvironment in BM to promote metastatic progression.
MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients
with
BM involvement.
Funding: This work was supported by grants from the Ministero della Salute
(GR-2016-02364088 to ADG), the Associazione Italiana per la Ricerca sul Cancro
(AIRC)-Special Program Metastatic disease: the key unmet need in oncology 5 per mille
2018
Id. 21147 to F.L. and Ministero dell’Istruzione, Università e Ricerca (MIUR, grant
PRIN 2017
to F.L).
PS02.03
Effects of Small-Extracellular Vesicles on cell biomechanical
properties in metastatic breast cancer
Beatrice Senigagliesi, Pietro Parisse,
Loredana Casalis, Riccardo Sgarra, Sara Petrosino, Giuseppe Samperi, Federica Caponnetto
and
Daniela Cesselli
University of Trieste, Trieste, Italy
Introduction: Extracellular Vesicles (EVs) are nano-sized particles delimited
by a lipid bilayer which transfer functional molecular cargos from the cells of origin
to
target cells. This intercellular crosstalk controls both physiological and pathological
conditions. Given their presence in body fluids and their characteristics, these
nanocarriers might be potentially used in diagnostics and/or therapy. Breast Cancer
is the
most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality
in
women worldwide; the triple negative breast cancer, in particular, is the most aggressive
subtype with a poor prognosis. Since it is recognized that cell stiffness of cancer
cells
play a crucial role during the metastatic spreading, we set ourselves the goal of
clarify
the effects and the activity of small-EVs (i.e. with a diameter below 200 nm) in metastatic
breast cancer, with a special attention on their correlation with the biomechanical
properties of cells.
Methods: Functional assays were performed on the non-invasive MCF7 breast
cancer cell line, before and after the cellular uptake of small-EVs originating from
the
invasive MDA-MB-231 triple negative breast cancer cell line. The mechanical properties
(cell
stiffness, cytoskeleton organization and focal adhesions) of MCF7 cells were investigated
before and after the vesicle uptake.
Results: The uptake of small-EVs derived from MDA-MB-231 significantly reduces
the Young’s modulus values of MCF7 cell line making them more invasive. Moreover actin
and
focal adhesion variations were observed in MCF7 cells before and after small-EV’s
uptake,
suggesting a molecular rearrangement inside MCF7 cells upon uptake.
Summary/Conclusion: Our results evidence that small-EVs play a key role in
altering biomechanical properties of target cells and underline their relevance in
cell-cell
crosstalk. Our approach is very promising to identify new molecular mechanisms through
which
EVs perform their oncogenic function.
PS02.04
Stratification of angiogenic or non-angiogenic lesions in
colorectal cancer liver metastases patients using extracellular vesicle miRNA
Migmar Tsamchoea
, Audrey Kapelanski
Lamoureuxb, Jaclyn Chabotc, Stephanie Petrilloa, Janusz
Raka, Anthoula Lazarisc and Peter Metrakosb
aMcGill University, Montreal, Canada; bMcGill
University, Montreal, Canada; cResearch Institute-McGill University Health
Centre, Montreal, Canada
Introduction: Colorectal carcinoma (CRC) is the second leading cause of cancer
death in the western world. Over 50% of the CRC patients develop liver metastasis
(LM) and
90% will die from metastatic disease. In the current clinical setting, liver resection
provides the only possible cure, but only 20% of CRCLM patients are resectable. The
combination of angiogenic inhibitors with chemotherapy is used to downsize CRCLM with
the
goal of converting unresectable patients to resectable ones. However, only 10–15%
of these
patients can be successfully converted to a resectable state. We have no way of identifying
those CRCLM patients that would respond/benefit to the addition of anti-angiogenic
therapies
(e.g. Bevacizumab: Bev)). Proper stratification of patients into Angiogenic Inhibitor
responders and non-responders will permit a proper assessment of the efficacy of Angiogenic
Inhibitors. CRCLM forms 2 distinct histopathological growth patterns (HGP): angiogenic
(desmoplastic) and non-angiogenic (replacement) HGP. We demonstrated that CRCLM patients
with predominant angiogenic lesions receiving Bev plus chemotherapy have a more than
double
5-year overall survival compared to patients with non-angiogenic lesions. Therefore,
non-angiogenic lesions do not respond to angiogenic inhibitors. Our study focuses
on
stratifying angiogenic vs non-angiogenic lesions of CRCLM through extracellular vesicle
miRNAs. We are using two approaches in the selection of miRNAs to target: 1. Text
mining of
published EV miRNA from CRCLM patients; and 2. Differentially expressed miRNAs present
in
tumour tissue from both lesion types, we have obtained by sequencing 50–60 patients.
These
two strategies will generate a list of miRNAs that we will target using qPCR on
plasma-derived EV miRNA from the patients used in approach 2, where we have classified
the
lesions in the patients. Preliminary data on 10 patients will be presented.
Methods: EV isolation was performed using the gold standard centrifugation
method. RNAseq and qPCR are used to generate the expression profile for angiogenic
vs
non-angiogenic type of CRCLM.
Results: The research is under progress.
Summary/Conclusion: The research is under progress.
PS02.05
The role of extracellular vesicles from cancer cells in bone
metastasis
Akiko Kogurea
, Fumihiko
Urabeb, Kagenori Itob and Takahiro Ochiyac
aTokyo Medical University, Shinjuku-ku, Japan;
bThe Jikei University School of Medicine, Minato-ku, Japan; cTokyo
Medical University, Shinjuku-ku, Japan
Introduction: It is known that bone metastasis causes a reduction in the
quality of life of cancer patients due to fractures and nerve compression. Therefore,
it is
important to elucidate the mechanism of bone metastasis and develop new treatments.
Metastatic bone tumours occur at particularly high rates in cancers of the prostate,
breast,
and lung. In this study, we focused on extracellular vesicles (EVs) in bone metastasis,
and
investigated that the role of EVs derived from cancer cells in osteolysis.
Methods: The prostate, breast, and lung cancer cell-derived EVs were added to
osteoclast precursors with RANKLs. The osteoclast differentiation was evaluated by
Tartrate-resistant acid phosphatase (TRAP) stain and by measuring the expression level
of
osteoclast markers using by qRT-PCR. A proteome analysis (LC-MS/MS) and siRNA approaches
were used to identify molecules which are responsible for promotion of osteoclast
differentiation in the prostate cancer cell-derived EVs. To investigate whether the
molecules are suitable for the detection of bone metastasis in serum EVs, we isolated
EVs
from serum of prostate cancer patients, and analysed the protein level of the molecules
by
western blot analysis.
Results: We found that the prostate cancer and lung cancer-derived EVs
significantly promoted the RANKL-stimulated osteoclast differentiation. Our analysis
revealed that CUB domain-containing protein 1(CDCP1), which is a membrane protein
on the
prostate cancer cell-derived EVs, was responsible for promotion of osteoclast
differentiation. Moreover, CDCP1 was markedly detected in the EVs-derived from serum
of
prostate cancer patients who had bone metastasis than that of normal subjects. We
also found
that CDCP1 exits on the breast and lung cancer cell-derived EVs.
Summary/Conclusion: We showed that the EVs-derived from bone metastatic
tumours have a role in activation of osteoclastogenesis. Moreover, we revealed that
CDCP1 in
the EVs is responsible for promoting of osteoclast differentiation. These EVs could
be the
novel diagnostic and therapeutic target for bone metastasis.
PS02.06
Increased expression of chemokine receptor CXCR4 in non-invasive
colorectal cancer cells after incorporation of platelet-derived extracellular
vesicles.
Izabela Papiewska-Pająk, Hassan Kassassir,
Damian Krzyżanowski, Jakub Kryczka and M. Anna Kowalska
Institute of Medical Biology of Polish Academy of Sciences, Lodz,
Poland
Introduction: Blood platelets and platelet-derived extracellular vesicles
(P-EVs) play a crucial role in tumour growth and metastasis. P-EVs, also referred
to as
platelet microparticles, are recognized as a carrier for proteins and nucleic acids
that
control cell-to-cell communication, mediate the formation of metastatic niches and
affect
tumour invasion and metastasis. Among the other factors, P-EVs contain the chemokine
receptor CXCR4, known as a co-receptor for HIV entry but also regarded as important
in
cancer development due to the importance of CXCR4/CXCL12 signalling. Overexpression
of CXCR4
was reported in various, especially in invasive cancers, including colorectal cancer
(CRC).
CRC, the third most commonly diagnosed cancer, is usually diagnosed at the late stage
and
patient’s death is mainly related to metastasis. Increased levels of CXCR4 has been
reported
as a poor prognostic factor for survival of CRC patients and its blocking has been
suggested
as therapeutic approach. The aim of this study was to analyse the effect of P-EVs
on the
levels of CXCR4 in CRC cells on various epithelial-to-mesenchymal transition stage.
Methods: We used CRC cell lines HT29 and SW620, which represent distant
invasive potential and different phenotypes, epithelial and strongly mesenchymal,
respectively. P-EVs were isolated from outdated concentrates of human blood platelets
after
activation by thrombin in the presence of calcium ions, by subsequent centrifugation
and
ultracentrifugation. The P-EVs were labelled using PKH67 fluorescent dye to visualize
their
uptake into cell lines by confocal microscopy. We also quantified the levels of CXCR4
in
HT29 and SW620 by Western blot analysis. The effect of P-EVs uptake on the migration
of CRC
cells was studied by “wound healing” method.
Results: We found that the levels of CXCR4 in CRC lines used in the study were
correlated with their EMT stage. We show here that P-EVs released by activated platelets
were incorporated into both HT29 and SW620 cell lines. The expression of CXCR4 in
HT29 was
increased after the uptake of P-EVs. Additionally we observed that migration rate
of HT29
cells with incorporated P-EVs was elevated as compared to control cells.
Summary/Conclusion: We posit that circulating P-EVs can be incorporated into
yet not invasive CRC cells to significantly increase the level of CXCR4 receptors
and that
may lead to the their more invasive characteristics.
Funding: Supported by 2018/29/B/NZ5/01756 from the National Science Centre,
Poland.
PS02.07
Changes of miRNA profile in extracellular vesicles released by
colorectal cancer cells during early EMT
Izabela Papiewska-Pająk, Damian Krzyżanowski,
Patrycja Przygodzka, Joanna Boncela and M. Anna Kowalska
Institute of Medical Biology of Polish Academy of Sciences, Lodz,
Poland
Introduction: For cancer therapy it is important to identify markers and key
processes induced during cancer progression. One of them is epithelial-mesenchymal
transition (EMT) which is associated with cell acquisition of invasiveness, stem cell
characteristics and resistance to apoptosis and therapy. Also the extracellular vesicles
(EVs) released from tumour cells, which can be taken up by cells constituting pre-metastatic
niches, can alter cancer progression by promoting cells’ reprogramming. Our group
has
recently reported that Snail transcription factor, a key factor of EMT, when overexpressed
in CRC HT29 cells, drives their early EMT and alters the expression of microRNA (miRs).
In
the present study we analysed the miRs profile of EVs released from those cells.
Methods: EVs from three HT29 clones stably overexpressing Snail and from
control HT29-pcDNA were isolated by differential centrifugation and ultracentrifugation
of
conditioned media after 24 h of culturing in serum-free medium. Total RNA was isolated
and
next-generation sequencing (NGS) analysis of the miRNAs was performed followed by
Gene
Ontology (GO) Enrichment Analysis.
Results: MiRs profiling shows that overexpression of Snail in HT29, while does
not affect the global miRNAs content of EVs, significantly triggers changes in individual
miRs levels. Three miRs: miR-205, let-7i and miR-130b were upregulated and five miRs:
miR-1246, miR-3131, miR-375, miR-552-3p, miR-552-5p were decreased in HT29-Snail-EVs
in
comparison to control EVs. We also report the top Biological process and Molecular
functions
and cellular compartment that are GO terms associated with differentially expressed
microRNAs.
Summary/Conclusion: We show here that EVs from CRC cells undergoing partial
EMT driven by Snail, have different miR profile as compared to control cells. Particular
miRNAs are considered to be either tumour suppressor or oncogenic thus changes in
their
profile need to be taken into account during the cancer treatment. The EVs miRNA profile
may
also serve as a marker of CRC progression.
Funding: Supported by the project DEC-2011/02/A/NZ3/00068 from the National
Science Centre, Poland.
PS02.08
Extracellular vesicles from prostate cancer cells deliver
microRNAs to promote osteogenesis
Yu Lijuan, Weixiao Fan, Lin Lei and Xiaoke
Hao
Air Force Medical University, Xi’an, China (People’s Republic)
Introduction: Prostate cancer (PCa) is the most common malignant tumour in
male urinary system and osteoblastic bone metastasis is the most observed metastasis
in
prostate cancer patients. It has been demonstrated that circulating microRNAs contained
in
extracellular vesicles are potential early biomarkers and therapy targets for many
diseases.
However, the potential role of microRNAs in prostate cancer bone metastasis, is not
yet to
be fully explored.
Methods: After isolation and purification EVs using ultracentrifugation from
conditioned media of bone metastatic co-opting prostate cancer cells and normal cells,
total
RNA was extracted. Subsequent to library preparation and small RNA-Seq, differential
gene
expression analysis was performed. Data were filtered by mean miRNA expression of
≥ 50
reads, two fold up or down regulation between 2.5 − 7.5 and adjusted p-value ≤ 0.05.
The
uptake of PCa-sEVs was performed. Three candidate miRNAs (has-miR-200 c-3p; has-miR-1275;
has-miR-383-5p) were internalized and osteoblast differentiation were detected by
qPCR,
histochemical staining and protein activity detection.
Results: Total reads of miRNAs in bone metastatic co-opting PCa-EVs exceeded
significantly than that in normal EVs (p < 0.001), indicating that miRNAs delivered
by
PCa cells play critical role in PCa bone metastasis. PCa-CM enhanced osteoblast
differentiation and can be reversed by GW4869. The uptake of PCa-EVs by MC3T3-E1 was
efficient. The high expression of the three candidate miRNAs in PCa-EVs was verified
by
qPCR. All the three candidate miRNAs promoted osteogenesis, verified by mRNA expression
of
osteoblastic markers (ALP, OCN, RUNX2, OSX), ALP activity, ALP staining and Aliza
Red S
staining.
Summary/Conclusion: These findings suggest that miRNA cargos in PCa-EVs play a
pivotal role in the development of osteoblastic bone metastasis of PCa, which can
be
potential early biomarkers and therapy targets for prostate cancer bone metastasis.
Funding: This work was supported by grants from the National Natural Science
Foundation of China (81872347); Xijing Hospital Science and Technology Foundation
Project
(XJZT19PTK14).
PS02.09
Proteomic profiling of retinoblastoma-derived exosomes reveals
potential biomarkers of vitreous seeding
Angela Galardia
, Marta
Collettia, Chiara Lavarellob, Virginia Di Paoloa, Paolo
Mascioa, Ida Russoa, Raffaele Cozzaa, Antonino
Romanzoc, Paola Valentec, Rita De Vitod, Luisa
Pascuccie, Hector Peinadof, Angel Montero Carcabosog,
Andrea Petrettob, Franco Locatellia and Angela Di
Giannatalea
aDepartment of Paediatric Haematology/Oncology and Cell and
Gene Therapy, IRCCS, Ospedale Pediatrico Bambino Gesù, Rome, Italy, Rome, Italy;
bCore Facilities-Clinical Proteomics and Metabolomics, IRCCS, Istituto
Giannina, Gaslini, Genoa, Italy, Genoa, Italy; cOphtalmology Unit, IRCCS,
Ospedale Pediatrico Bambino Gesù, Rome, Italy, Rome, Italy; dDepartment of
laboratories – Pathology Unit, IRCCS, Ospedale Pediatrico Bambino Gesù, Rome, Italy;
eDepartment of Veterinary Medicine, University of Perugia, Perugia, Italy,
Perugia, Italy; fMicroenvironment and Metastasis Group, Molecular Oncology
Program, Spanish National Cancer Research Center (CNIO), Madrid, Spain, Madrid, Spain;
gInstitut de Recerca Sant Joan de Deu, Barcelona, Spain, Barcelona, Spain
Introduction: Retinoblastoma (RB) is the most common intraocular cancer of
childhood. Despite recent advances in conservative treatment have greatly improved
the
visual outcome, local tumour control remain difficult in presence of massive vitreous
seeding. Thus, the identification of new biomarkers is crucial to design more effective
therapeutic approaches. Traditional biopsy has long been considered unsafe in RB,
due to the
risk of extraocular spread. Exosomes, nano-sized vesicles containing nucleic acids
and
proteins, represent an interesting alternative to detect tumour-associated biomarkers.
The
aim of this study was to determine the protein signature of exosomes derived from
RB tumours
(RBT) and vitreous seeding (RBVS) primary cell lines.
Methods: Exosomes from 4 RBT (HSJD-RBT1, HSJD-RBT2, HSJD-RBT5, HSJD-RBT14) and
3 RBVS (HSJD-RBVS1, HSJD-RBVS3, HSJD-RBVS10) cell lines were isolated by high speed
ultracentrifugation. Vesicles number and size were confirmed by NanoSight and Scanning
Electron Microscopy. Protein content was analysed by bicinchonic-acid assay and high
resolution mass spectrometry.
Results: A total of 5666 proteins were identified. Among these, 5223 and 3637
were expressed in exosomes RBT and one RBVS group respectively. Gene enrichment analysis
of
exclusively and differentially expressed proteins and network analysis identified
identified
in RBVS exosomes upregulated proteins specifically related to invasion and metastasis
such
as proteins involved in Extracellular Matrix (ECM) remodelling and interaction, resistance
to anoikis and metabolism/catabolism of glucose and aminoacids.
Summary/Conclusion: In conclusion, in this study, we isolated exosomes from RB
primary tumour and vitreous seeding cell lines and characterized their content with
a
proteomic approach. This is the first evidence describing a proteomic exosome signature
specifically associated with vitreous seeding in RB. This characterization may represent
a
starting point for future analyses that allow defining exosomal markers as promising
diagnostic and potential prognostic markers in RB as well as therapeutic targets.
PS02.10
Activation of hepatic stellate cells by extracellular vesicles
released by uveal melanoma cells
Léo Piqueta
, Nathan
Schoonjansb, Kelly Coutanta, Julie Bérubéc, Francois
Bordeleau, Alain R. Brissond and Solange Landrevillea
aUniversité Laval, Quebec City, Canada;
bUniversité de Lille, Lille, France; cCentre de recherche du CHU de
Québec-Université Laval, Quebec City, Canada; dUniversité de Bordeaux, Bordeaux,
France
Introduction: Uveal melanoma (UM) is the main intraocular tumour in adults,
and is particularly resistant to treatments when disseminated to the liver. Our hypothesis
is that extracellular vesicles (EVs) released by the primary tumour are priming the
liver
stroma for metastatic cell colonization by activating hepatic stellate cells (HSteCs).
This
study aimed to characterize EVs from UM cells, and to determine their interactions
with
liver cells.
Methods: EVs were isolated from cell lines derived from ocular tumours and
liver metastases by differential centrifugation. Their concentration/diameter range
were
determined by high-sensitivity flow cytometry. Cryo-TEM combined with receptor-specific
gold
labelling was used to reveal the morphology/size of melanomic EVs. The presence of
melanoma
and EV markers was assessed by Western blotting. The internalization of fluorescent
melanomic EVs in HSteCs and their subsequent activation were assessed by confocal
imaging
using alpha-smooth muscle actin (alpha-SMA) and phalloidin stainings. EV impact on
invasion
was measured with a tumour spheroid model embedded in extracellular matrix. Melanomic
EVs
were inoculated into the retro-orbital sinus of immunodeficient mice to study their
selective organ distribution.
Results: Melanomic EVs were positive for Annexin-5, tetraspanins, as well as
some melanoma markers. Stellate cells with internalized melanomic EVs expressed more
alpha-SMA, reflecting their activation. Adding EVs on tumour spheroids increased the
invasion process. Melanomic EVs were localized into different murine organs, but mainly
into
the liver, as observed by in vivo fluorescent imaging.
Summary/Conclusion: Metastatic UM cells produce EVs that can reach the liver
and activate stellate cells. Analysis of plasmatic EVs collected from UM patients
should
help designing new strategies for monitoring the progression of this cancer and its
therapeutic response.
Funding: Vision Health Research Network; Fonds de recherche du Québec – Santé
(FRQS); Fondation du CHU de Québec; Canada Foundation for Innovation
PS02.11
Exosome of cancer-associated fibroblast induce anti-cancer
drug-resistance of NSCLC
So-young Kima
and Yeon-ju
Leeb
aChonnam National University Hwasun Hospital Biomedical
Research Institute, Gwangju, Republic of Korea; bChonnam National University
Hwasun Hospital Biomedical Research Institute, Gwangju, Republic of Korea
Introduction: The understanding of interaction mechanisms between cancer cells
and the tumour microenvironment (TME) is crucial for developing therapies that can
arrest
tumour progression and metastasis. CAFs are the major constituent of the TME in many
cancers. Recent studies indicate that exosomes harbour the potential to regulate
proliferation, survival and immune status in recipient cells. Most of the current
studies
are focused on cancer cell secreted exosomes; and little is known about CAF-derived
exosomes
and their influence on cancer cells.
Methods: NSCLC cell lines (PC9GR) and MRC5 (normal fibroblast cells) were
grown in culture with exosome-free FBS. Cutured media was filtrated by Tangential
Flow
Filtration Systems. Exosomes in supernatant were isolated with the ExoQuick-TC™ system.
Considering the important role of cell extrinsic factors on cell growth and survival,
we
assessed whether factors contained in the MPA exosome could affect proliferation and
survival of recipient cancer cells. Cells were then treated with 1 μM Osimertinib
or PBS for
3 days prior to cell quantification of live cells.To investigate mechanisms of resistance
to
osimertinib mediated by MA or MPA-Exosome in NSCLC cell lines, we test cell viability
by
crystal violet assay in Trametinib or Osimertinib treated after pre-treated MA or
MPA-exosome, PC9GR during 6 days. We will investigate how MPA-exsomes activate ERK
signalling pathway in PC9GR cells to induce antitumor effects by western blot.
Results: MPA exosome increased proliferation of PC9GR cells by more than 50%
compared to control PBS. PC9GR cells grown in MPA-exosome and subsequently treated
with
osimertinib showed a significant increase in cell survival compared to PC9GR cells
grown in
MA-exosome. Osimertinib is used to treat EGFR-mutant non-small cell lung cancer (NSCLC)
with
tyrosine kinase inhibitor resistance mediated by the EGFR T790 M mutation.These data
show
that “MRC5-PC9GR-crosstalk factors” affect proliferation and adaptive drug resistance
of
cancer cells. MPA-Exosome mediates ERK signalling activation and attenuated after
treatment
of 1uM osimertinib.
Summary/Conclusion: CAFs support cancer growth and invasion. Co-cultured NSCLC
with MRC5 lung fibroblast increased cell viability and exosomal miR-21 through the
TGF-ß
pathway in treatment Osimertinib. Exosomal miR-21 up-regulation in co-cultured NSCLC
with
MRC-5 induced drug resistance to drug-induced apoptosis. Thus, exosomal miR-21 expression
in
co-cultured NSCLC with MRC5 may support drug tolerance persister cells.
Funding: This work was supported by the National Research Foundation of
Korea(NRF) grant funded by the Korea government(MEST) (No. 2019R1A2C1005593)
PS03: EVs in Intrinsic Medicines
Chair: Ewa K. Zuba-Surma – Department of Cell Biology, Faculty of Biochemistry,
Biophysics and Biotechnology, Jagiellonian University
Chair: Cristina Grange – Deparment of Medical Sciences, University of
Torino
PS03.01
Proteomic analysis of amniotic fluid-derived exosome cargo
reveals a therapeutic potential for regenerative therapies.
Michael A. Bellioa
, Zanub
Abdullaha, Danique Stewarta, Aisha Khanb, Xiumin
Xub, George Shapiroa, Albert Mitrania and Maria Ines
Mitrania
aOrganicell Regenerative Medicine Inc., Miami, USA;
bAssureimmune, LLC, Miami, USA
Introduction: Exosomes are being tested for their use as therapeutic agents in
degenerative and chronic diseases. However, the optimal source of exosomes is currently
under investigation. Amniotic fluid (AF) is a naturally-rich source of exosomes that
is
easily obtained for use in regenerative medicine. Organicell Flow™ is a
minimally-manipulated, acellular product derived from human AF and consist of over
300
cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding
tissues. We characterized the exosome fraction of our product to elucidate the protein
cargo
of AF exosomes and demonstrate the therapeutic potential as a novel regenerative
therapy.
Methods: The exosome fraction of our product was analysed using Nanosight
nanoparticle imaging and MACsplex exosome surface marker array analysis. Exosomes
were
precipitated using size-exclusion filtration followed by ultracentrifugation from
3
independent products (in triplicate) and subjected to protein lysis and preparation
for mass
spectrometry analysis using the Easy nLC 1000 and Q Exactive instruments. Tune (version
2.9)
and Xcalibur (version 4.1) was used to collect data while Proteome Discoverer (version
2.2)
was used to analyse data. Protein expression lists were created by merging the 3 sample
replicates together and commonly expressed proteins were determined using Vinny 2.0
vin
diagram analysis. WebGestalt tool kit classification system was used to identify top
protein
function and pathway hits.
Results: Organicell Flow™ contain a mean concentration of 5.24x10^11
particles/mL (n = 12) with a mean mode size of 125.2nm (n = 12). Surface marker analysis
confirms the presence of exosome associated proteins CD63, CD81, and CD9 in addition
to a
high expression of CD133 (n = 3). The completed analysis revealed 1225 commonly detected
proteins across 3 products. The top molecular functions of identified proteins included
protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription
regulators, and transporter proteins representing the most abundant protein groups.
Pathway
enrichment analysis revealed top hits for Integrin, PDGF, and P53 pathways. A deeper
dive
into the enzyme category of the protein cargo further demonstrates the presence of
proteins
that promote DNA repair such as DNA polymerase (beta and lambda), telomerase reverse
transcriptase, and BRCA1.
Summary/Conclusion: Organicell Flow™ characterization demonstrates the
therapeutic potential of AF-derived exosomes. Proteomic analysis revealed protein
cargo that
may regulate various growth factor and cell-cycle associated pathways. Furthermore,
the
presence of DNA damage response proteins suggests a possible mechanism for induction
of
cellular repair.
PS03.02
Generation of CAR-T and γδT cell-derived exosomes for future
cell free immunotherapies
Vaques Georgea
, Ayesha
Khana, Jack Firtha, Mona Bajaj-Elliotta and Kenth
Gustafssonb
aUniversity College London, London, UK;
bUnviersity College London, London, UK
Introduction: T cell therapies have predominantly focused on treating cancers,
with the more recent example of CAR-T cells targeting CD19+ cells in lymphoma patients.
Despite recent success, challenges including demand for reduced off-target toxicities,
reduced donor-donor variabilities and the targeting of multiple malignant cell types,
still
remain.
γδT cells are a subset of T cells with dual innate and adaptive qualities. This duality
provides various advantages over their more studied and used counterpart, αβT cells.
In the
present study, we sought to compare the immunotherapeutic potential of CAR-T cell
and γδT
cell-derived exosomes as novel cell-free based alternatives.
Methods: CD19-targeting CAR-T cells were obtained following the isolation,
expansion and transduction of αβT cells using a lentiviral vector bearing the CAR
construct.
γδT cells were isolated and expanded from Peripheral Blood Mononuclear Cells (PBMCs)
following innate or adaptive stimulation.
Exosomes from both cell sources were isolated after a 3-day culture in serum-free
media
using ultracentrifugation-based methods. Exosomes were characterized by Nanoparticle
Tracking Analysis (determination of size) and western blot assays (detection of the
appropriate surface markers).
Nalm-6 (B cell precursor leukaemia) cells were used as target cells for assessment
of
exosome cytotoxic/killing function. CAR-T cell and γδT cell-derived exosomes were
incubated
at 1000 particles/target cell for 24-hours. Total viable cell counts were assessed
via
imaging-based cytometry (NC-3000) utilizing acridine orange and DAPI staining.
Results: Exosomes derived from γδT cells activated via innate mechanisms
showed significant killing of Nalm-6 as compared to exosomes from non-activated or
adaptively activated γδT cells. In comparison, CAR-T cell-derived exosomes showed
minor
killing capabilities of the target cells.
Summary/Conclusion: Here, we report for the first time that exosomes derived
from CD19 CAR-T cells and innately activated-γδT cells show/exert inhibitory action
on
Nalm-6 cells. Further studies are currently underway to identify the underlying mechanism(s)
responsible.
Funding: UCL EPSRC Centres for Doctoral Training
PS03.03
Characterization, composition, and biological properties of
extracellular vesicles generated from human induced pluripotent stem cell-derived
neural
stem cells
Raghavendra Upadhyaa
, Leelavathi N.
Madhub, Sahithi Attaluria, Daniel Gitaic, Marisa
Pinsond, Maheedhar Kodalib, Geetha Shettyd, Gabriele
Zaniratid, Smrithi Kumard, Bing Shuaia, Susan
Weintraube and Ashok K Shettya
aInstitute of Regenerative Medicine, College of Medicine,
Texas A&M University, College Station, USA; bInstitute for Regenerative
Medicine, College of Medicine, Texas A&M University, College Station, USA;
cInstitute for Regenerative Medicine, College of Medicine, Texas A&M
University, Maceio, Brazil; dInstitute of Regenerative Medicine, College of
medicine, Texas A & M university, College Station, USA; e4Department of
Biochemistry and Structural Biology, UT Health San Antonio, San Antonio, USA
Introduction: Neural stem cell (NSC) therapy has shown promise for brain
repair after injury or disease mostly through bystander effects. Nevertheless, the
translation of NSCs derived from human induced pluripotent stem cells (hiPSCs) to
the clinic
remain constrained due to safety issues, which include immunogenic risks, tumorigenesis
potential, and incomplete differentiation. A way to avoid these issues is by using
extracellular vesicles (EVs) generated from NSCs, as NSC-EVs likely have similar
neuroprotective properties as NSCs and are amenable for non-invasive administration
as an
autologous or allogeneic off-the-shelf product. However, this would require reliable
purification and characterization processes, and testing of EVs for composition and
biological properties.
Methods: We generated EVs from hiPSC-derived NSCs using a combination of
ion-exchange chromatography (IEX) and size-exclusion chromatography (SEC) and investigated
their composition through small RNA sequencing and proteomics. We also performed in
vitro
and in vivo experiments to determine their biological and functional properties.
Results: IEX and SEC facilitated purification of hiPSC-NSC EVs nearly to
homogeneity, which expressed EV markers such as CD9, CD63, CD81, and ALIX with a mean
size
of 145 nm. Small RNA sequencing revealed enrichment of miRNAs related to different
neuroprotective signalling pathways and diverse metabolic functions consistent with
their
role in cell-cell communication. The proteomic analysis identified >1,000 proteins,
including EV markers and many other proteins involved in central nervous system function
and
cellular processes. The EVs also displayed antiinflammatory activity in an in vitro
mouse
macrophage assay. Intranasal (IN) administration of NSC-EVs resulted in their rapid
incorporation by neurons, microglia, and astrocytes in virtually all regions of the
brain.
Functionally, IN administration of NSC-EVs reduced inflammatory activity in the brain
in a
model of status epilepticus, and increased hippocampal neurogenesis in the adult brain.
Summary/Conclusion: Biologically active EVs with antiinflammatory and
neurogenic properties could be purified and harvested from hiPSC-NSCs. Such EVs also
contain
many miRNAs and proteins that are of interest for brain repair after injury or disease.
Funding: Supported by a grant from the National Institute of Neurological
Disorders and Stroke (1R01NS106907-01 to A.K.S.)
PS03.04
Promise of neural stem cell-derived extracellular vesicles for
combating age-associated cognitive and mood impairments
Sahithi Attaluria
, Raghavendra
Upadhyaa, Andrew Vogela, Maheedhar Kodalib, Leelavathi N.
Madhub, Bing Shuaia and Ashok K. Shettya
aInstitute of Regenerative Medicine, College of Medicine,
Texas A&M University, College Station, USA; bInstitute for Regenerative
Medicine, College of Medicine, Texas A&M University, College Station, USA
Introduction: Age-related cognitive dysfunction is associated with increased
oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis
in
the brain. From this perspective, biologics capable of modulating oxidative stress
and
neuroinflammation, and stimulating neural stem cell activity in the brain might be
useful as
anti-ageing interventions.
Methods: We investigated the efficacy of intranasal administration of
extracellular vesicles (EVs) generated from cultures of rat subventricular zone neural
stem
cells (SVZ-NSCs) in the middle-aged mice to alleviate cognitive and mood dysfunction,
increased oxidative stress, neuroinflammation, and neurogenesis decline in old age.
Mice
were treated intranasally with NSC-EVs once weekly for three weeks (50 billion per
administration) starting from 18.5 months of age. A month later, the animals were
examined
for cognitive, memory, and mood function using multiple behavioural tests, and brain
tissues
were examined for oxidative stress, neuroinflammation, and neurogenesis.
Results: Object-based tests revealed that aged animals receiving vehicle
displayed cognitive impairments for discerning minor changes in the environment as
well as
for distinguishing similar but not identical experiences. These animals also exhibited
spatial memory dysfunction and anhedonia. In contrast, aged animals receiving NSC-EVs
showed
improved cognitive and mood function. Biochemical analyses of brain tissues revealed
that
NSC-EV treatment normalized elevated concentrations of oxidative stress markers
malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin-1
beta.
Moreover, NSC-EV treatment stimulated increased production of antiinflammatory protein
interleukin-10 and the antioxidant superoxide dismutase. Immunohistochemical analysis
revealed modulation of neuroinflammation typified by reduced activity of reactive
astrocytes
and activated microglia and improved hippocampal neurogenesis.
Summary/Conclusion: The results suggest that the intranasal administration of
NSC-EVs is a promising approach for maintaining better cognitive and mood function
in ageing
through modulation of oxidative stress, neuroinflammation, and neurogenesis.
Funding: Supported by a grant from the National Institute of Neurological
Disorders and Stroke (1R01NS106907-01 to A.K.S.)
PS03.05
Chemically modified myocytes-derived EVs for the treatment of
cardiac fibrosis.
Marta Prieto-Vilaa
, Asao
Muranakaa and Takahiro Ochiyab
aTokyo Medical University, Tokyo, Japan; bTokyo
Medical University, Shinjuku-ku, Japan
Introduction: Myocardial fibrosis is a disorder that may occur after cardiac
injure due to a malfunction of the cardiac remodelling. Fibroblasts resident in myocardium
are erroneously activated causing an excessive accumulation of extracellular matrix,
which
decreases cardiac function and eventually, leads to death. It is known that cardiomyocytes
communicate with the surrounding cells such as fibroblast and endothelial cells by
extracellular vesicles (EVs). The loss of this communication is thought to play a
central
role in cardiac fibrosis. Therefore, cardiomyocytes-derived EVs may be a promising
a
cell-free system for the treatment of fibrosis inhibition.
Methods: A novel culture medium was stablished to improve the expansion of
primary cardiac myocytes. This was tested using two commercially available primary
myocytes
cell lines. EVs were collected by serial ultracentrifuges, and their effect on fibrosis
was
tested. For that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast
were activated overnight with TGFβ.
Results: By the use of a defined conjunct of chemicals, mature cardiomyocytes
culture was highly improved to ensure a high collection of EVs. Terminal differentiation
markers, as well as senesce apparition was delayed in comparison to predetermined
culture
medium. Interestingly, those primary cells secreted a rather large amount of EVs,
which
expressed common EVs membrane marker. TGFβ-treated cardiac fibroblasts were co-cultured
with
myocytes showing a decrease of fibroblast activation markers both at mRNA and protein
levels. Similar results were found when activated fibroblast were treated with EVs.
Summary/Conclusion: Our findings indicate that the use of EVs derived from
chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis.
However, further molecular experiments have to be done to identify the molecules within
EVs
responsible for the inactivation of fibroblast.
PS03.06
Evaluation of osteoinductive and anti-inflammatory properties of
spine-derived exosomes
Renaud Sicarda
, Tania del
Riverob, Jonathan Messerc, Shabnam Naminc and Timothy
Ganeyc
aVivex, Biologics, Inc., Miami, USA; bVIVEX,
Biologics, Inc., Miami, USA; cVIVEX, Biologics, Inc., Miami, USA
Introduction: Over the last 2 decades, Mesenchymal Stem Cell-derived exosomes
have been shown to play a crucial role in a myriad of cell function such as extracellular
matrix synthesis, proliferation, differentiation or cell migration. Biological sources
of
exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct
influence on the content of their cargo and their therapeutic application and potential.
In
this study, we evaluated exosomes excreted from cadaveric spine-derived cells. We
hypothesized that exosomes derived from a bone source such as the spine, will drive
the
osteogenic differentiation of progenitor cells. We also investigated their effects
on
inflammation in Nucleus Pulposus cells using an in-vitro assay.
Methods: After their isolation and characterization, exosomes derived from
cadaveric human spines were assayed for osteoinductive properties. A C2C12 myoblast
cell
line was treated with different concentrations of exosomes and expression of alkaline
phosphatase was measured after 5 days incubation. Treatment with BMP-4 was used as
positive
control.
Anti-inflammatory properties were assessed by incubating TNF-treated Nucleus Pulposus
cells
with exosomes for 3 days. qPCR analysis of mRNA expression of inflammatory cytokines
(IL-6,
IL1-beta, IL-8) metalloproteinases (MMP3 and ADAMTS4), and apoptotic genes (BAX, BCL2)
was
used to determine the effects of exosomes on inflammation.
Results: Spine-derived exosomes positively expressed the exosome flow
cytometry markers tested (CD81, CD63 and CD9). The mean number of exosomes per microgram
of
protein was 3.31 ± 2.33 x 108 indicating a relatively high purity. Osteoinductive
(OI)
testing was performed using different concentrations of exosomes. The OI index of
treatment
of C2C12 cells with BMP-4, 2 x 108, 1 x 109, 2 x 109, 5 × 109 or 1 × 1010 exosomes
alone was
28.5, 1.0, 3.7, 7.4, 11.8 and 27.6 respectively. Anti-inflammatory properties of exosome
are
currently being assessed and will be presented at the time of the poster presentation.
Summary/Conclusion: Administering exosomes alone or in combination with an
exogenous scaffold has the potential to repair injured tissue and to restore bone
function.
The clinical significance of this application is aimed to promote the patients’ bone
healing
process and provide a cell-free therapeutic platform that is safe and effective.
PS03.07
Administration of human Mesenchymal Stem Cell derived
extracellular vesicles modulates the abnormal plasticity of newly born neurons and
neuroinflammation in a rat model of status epilepticus
Maheedhar Kodalia
, Daniel
Gitaib, Dong Ki Kima, Mariam Atobiloyea, Bing
Shuaic, Sahithi Attaluric, Raghavendra Upadhyac,
Leelavathi N Madhua, Olagide W. Castroa, Darwin J. Prockopa
and Ashok K. Shettyc
aInstitute for Regenerative Medicine, College of Medicine,
Texas A&M University, College Station, USA; bInstitute for Regenerative
Medicine, College of Medicine, Texas A&M University, Maceio, Brazil;
cInstitute of Regenerative Medicine, College of Medicine, Texas A&M
University, College Station, USA
Introduction: Extracellular vesicles (EVs) generated from human bone
marrow-derived mesenchymal stem cells (hMSCs) display anti-inflammatory and neuroprotective
properties. Our recent study has shown that intranasally (IN) administered hMSC-EVs
incorporate into significant percentages of neurons and microglia in virtually all
regions
of the intact as well as the injured forebrain within 6 hours (Kodali et al., Int
J Mol Sci,
2019). In this study, using a rat model, we investigated the efficacy of a low dose
of
hMSC-EVs administered intranasally for alleviating the abnormal plasticity of newly
born
neurons and the activation of microglia after SE.
Methods: Approximately 10 billion EVs were dispensed bilaterally into both
nostrils of young F344 rats that experienced two hours of Kainate-induced SE. Animals
were
euthanized seven days after SE, and brain tissue sections were processed for
immunohistochemical staining of NeuN (a neuronal marker), DCX (a marker of newly born
neurons), IBA-1 (a microglial marker), and parvalbumin (PV) and reelin (markers of
subclasses of interneurons). In addition, activated microglia were quantified using
IBA-1
and CD68 dual immunofluorescence.
Results: IN administration of EVs reduced the SE-induced loss of pyramidal
neurons in the hippocampal CA1 subfield. Also, EV administration after SE maintained
higher
levels of PV+ interneurons in the dentate gyrus. Furthermore, EV treatment after SE
modulated abnormal neurogenesis, which was evidenced by a decline in the percentage
of newly
born neurons displaying basal dendrites. Besides, EV treated animals displayed higher
percentages of resting microglia (ramified microglia), reduced percentages of activated
microglia (microglia expressing IBA-1 and CD68), in comparison to animals receiving
vehicle
after SE. Interestingly, diminished abnormal plasticity of newly born neurons was
accompanied by the preservation of interneurons positive for reelin; a protein believed
to
guide newly born neurons to their correct locations.
Summary/Conclusion: The results suggest that even a low dose IN administration
of MSC-derived EVs after SE can limit neurons loss, dampen the abnormal plasticity
of newly
born neurons, and modulate the activation of microglia.
Funding: Supported by a grant from the National Institute of Neurological
Disorders and Stroke (1R01NS106907-01 to A.K.S.)
PS03.08
Intranasal administration of exosomes improves the symptoms in
three mice model of Autism
Nisim Peretsa, Reut Horeva, Yona
Gefenb, Uri Danonb, Ehud Maromb, Dimitri
Yudinc, Oded Orond, Evan Elliotte and Daniel
Offena
aTel Aviv University, Tel Aviv, Israel; bStem Cell
Medicine LTD., Jerusalem, Israel; cStem Cell Medicine LTD, Israel, Jerusalem,
Israel; dBar Ilan University, Safed, Israel; eBar-Ilan University,
Safed, Israel
Introduction: Autism spectrum disorders (ASD) are neurodevelopmental disorders
characterized by three core symptoms that include social interaction deficits, cognitive
inflexibility, and communication disorders. They have been steadily increasing in
children
over the past several years, with no effective treatment. Two percent of all ASD patients
are suffering from a disorder caused by a mutation in the shank3 gene. Shank3 is an
important synaptic protein, disruption of this gene directly leads to cognitive and
motor
impairments. During the recent decade, exosomes that derived from mesenchymal stem
cells
(MSC-exo) have been spotlighted as a promising therapeutic target for various clinical
indications, including neurological disorders. Here we test three different autistic
mice
models. BTBR as a multifactorial mice model of autism and two different Shank3 mutated
mice.
The first is a complete deletion of exon 22 (22q13.3) and the second is a specific
insertion
mutation of Guanine to 3680 position in the gene (insG3680) that leads to stop codon.
Methods: Exosomes were isolated using differential centrifugation protocol and
characterized using the 2018MISEV guideline recommendations. Each animal received
an
intranasal administration of 20ul containing 107 exosomes/µl. For intravenous
administration, the same number of exosomes, were used, injected in 100µl.
Results: All three animal models showed significant improvement in their
autistic behavioural phenotypes following intranasal administration. The improvement
seems
to be dose-dependent and was better achieved via intranasal vs intravenous administration.
Biodistribution of MSC-exo showed accumulation in the brain within 72 hours, yet the
reduction of the signal was observed in the kidneys, heart and lungs.
Summary/Conclusion: Our data suggest that Exosomes derived from adipose MSC,
carry a therapeutic potential in ASD, via non-invasive intranasal administration in
three
different mice models. These data further emphasize our potential therapeutic strategy
to
reduce symptoms of autism in clinical trials.
Funding: Stem Cell Medicine LTD. Israel.
PS03.09
Equine tendon injury treatment by EVs: an in vitro
study
Robert Soukupa
, Hayeon
Baika, Iris Ribitscha, Johannes Grillarib and Florien
Jennera
aUniversity of Veterinary Medicine, Vienna, Vienna, Austria;
bChristian Doppler Laboratory for Biotechnology of Skin Ageing, Department of
Biotechnology, BOKU – University of Natural Resources and Life Sciences, Vienna, Austria
Evercyte GmbH Austrian Cluster for Tissue Regeneration Ludwig Boltzmann Institute
for
Experimental and Clinical Traumatology, Vienna, Austria, Vienna, Austria
Introduction: Current treatment options for tendinopathies (chronic, painful
tendon disorders), are not able to restore the functional properties of native tendons.
Hence, new treatment options are sought.
The efficacy of mesenchymal stem cells (MSCs) therapies, which combined with a
rehabilitation programme including controlled exercise is the current gold standard
in
equine tendon treatment, has been shown to be largely due to the cells´ paracrine
activity.
The aim of this study was therefore to evaluate the effect of bone marrow MSC derived
autologous and allogeneic conditioned medium (CM, full secretome) and their extracellular
vesicles (EVs) on “tendon healing” in vitro.
Methods: To compare the “therapeutic” effect of MSC derived EVs and CM, a
standardized scratch assay (wound healing assay) was performed. CM from equine tenocytes,
EV
depleted medium and medium with or without FCS served as controls.
Tendons and bone marrow aspirates were obtained from three horses (6, 19 and 23 years)
which were euthanized for reasons unrelated to this study. MSCs were isolated by Ficoll
density gradient centrifugation and tenocytes were obtained by migration from tendon
explants. For CM and EV production, cells were cultured in EV depleted medium.
EVs were harvested by a stepwise ultracentrifugation approach and characterized by
Nanoparticle Tracking analysis (NTA), Western Blot (CD9, CD63) and Transmission-Electron
Microscopy (TEM).
Results: Western blot, NTA and TEM confirmed successful isolation of EVs from
equine MSCs. The strongest positive effect on wound healing (fastest gap closure)
was
achieved by MSC-CM (p < 0.0071). The gap closure achieved with MSC-EVs was slower
than
with MSC-CM (p < 0.7985) but faster than with CM of tenocytes (p < 0.8289). Donor
specific differences in wound healing capability were shown for both autologous and
allogeneic application.
Summary/Conclusion: Treatment with MSC-CM resulted in significantly faster
wound healing of adult tenocytes in vitro than MSC- EVs or tenocyte-CM.
MSCs donor age shows a significant effect on gap closure following autologous but
not
allogeneic administration.
PS03.10
EV-enriched secretome fraction from GMP-compatible, scalable,
human iPSC-derived cardiac progenitors improve heart function in chronic heart failure
mice
Michele Hamricka
, Jacquelyn
Wonga, Valerie Bellamyb, Philippe Menaschec and Nisa
Renaulta
aFUJIFILM Cellular Dynamics, Inc (FCDI), Madison, USA;
bINSERM U 970, Paris, France; cDepartment of Cardiovascular Surgery
and INSERM U 970, Hôpital Européen Georges Pompidoiu, Paris, France
Introduction: We have shown that research-use-only grade (res) human
iPSC-derived cardiac progenitors (CPCres) can produce a secretome whose small-EV-enriched
fraction (sVF) can treat chronic heart failure (CHF) in mice. GMP-compatible, scalable
processes for a CPC-derived sVF suitable for human therapeutic use is needed.
Methods: iPSC-derived CPC were produced and cultured using GMP-compatible,
scalable processes (CPCtx). Media without cells were “cultured” in parallel for “virgin
media” controls (MV). CPCres were cultured as previously described. As a proof of
concept,
sVFs were isolated from conditioned media by ultracentrifugation: CPCtx-EV, CPCres-EV
and
MV. Particle size distributions/concentrations (nanoparticle tracking analysis), protein
levels (BSA), and the presence of CD-63 (ELISA) were determined. In vitro activity
was
assessed by HUVEC scratch wound healing assay, and by rat and human cardiomyocyte
(CM)
survival assays. C57BL/6 mice in CHF received echo-guided myocardial injection of
PBS
vehicle control (60uL, n = 11), CPCtx-EV (60uL, n = 11), or CPCres-EV (45uL, n = 11).
Change
in cardiac function was assessed by echocardiography.
Results: CPCtx-EV particle sizes were polydisperse (mode ~72 nm) at a
concentration of ~1.6e11 particles/mL (~2,600 particles/cell) and ~0.975mU CD63/ug
protein.
CPCtx-EV increased wound healing, human CM survival, and rat CM survival in vitro
by 4.75x,
1.4x, and 2x, respectively over MV controls. In CHF mice, significantly less CPCtx-EV
mice,
and less CPCres-EV mice had severely progressive heart failure (Left Ventricular End
Systolic Volume, LVESV, increased >14%) than PBS control mice (PBS vs CPCtx-EV,
p < 0.05; PBS vs CPCres-EV, p < 0.056), and the Average Ejection Fraction of the PBS
group deteriorated 2.5x more than the CPCtx-EV group (−4% vs −1.6%, respectively;
ns).
Summary/Conclusion: We have a process for CPC differentiation and production
of conditioned media suitable for use in human clinical trials from which can be made
an sVF
with the potential to treat CHF, possibly through re-vascularization or preservation
of CM
viability.
Funding: FUJIFILM, Fondation pour la Recherche Médicale, Labex REVIVE
PS03.11
Electrospun collagen coupled with spine bone marrow-derived
exosomes
Renaud Sicarda
, Timothy
Ganeyb, Javorina Milosevicc, Connie Chungd, Shabnam
Naminb and Hans Meiselc
aVivex, Biologics, Inc., Miami, USA; bVIVEX,
Biologics, Inc., Miami, USA; cSpin Plant GmbH, Leipzig, Germany;
dVIVEX, Miami, USA
Introduction: Exosomes are nanoscale vesicles that mediate cell-to-cell
communication via exchanging molecular cargo. Mesenchymal stem cell (MSCs) modification
towards an osteogenic path can occur by uptake of exosomes from other cells. It is
less
clear whether vesicle placement in the absence of cells will facilitate site-specific
delivery through acellular transfer of osteogenic activity. An electrospun fleece
was
combined with bone marrow-derived exosomes in the absence of cells to evaluate
osteoinductive potential that might be thermo-stable and be used in a biologically
neutral
collagen carrier. Comparisons were made of standard laboratory assay of osteoinductivity
(OI), and in vivo expression in a mouse calvarial defect model.
Methods: Electrospun Type-I collagen was prepared with and without
hydroxyapatite (HA) (SpinPlant GmbH, Leipzig) as a foundation base for application
of the
bone marrow-derived exosomes. Individual discs of the collagen enhanced scaffolds
(3-mm)
were prepared and placed in a mouse calvarial skull defect. Animals were followed
for 4 and
8 weeks. Exosomes were isolated from qualified cadaveric human spines by differential
ultracentrifugation. Microscopic observation, quantitative assessment of OI with an
alkaline
phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid
nature of the addition to the nano-collagen fibres. A fluorescent protein reporter
transgenic mouse model expressing osteocalcin, Type-I collagen, Phex, and SP7 (Osterix)
was
evaluated at 4 and 8 weeks to determine bone formation across the defect.
Results: ALP activity on the scaffold with HA demonstrated an approximate
tenfold increase to that of the collagen scaffold alone. While a dose-dependent effect,
with
higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression,
expression that exceeded that of the 50ng BMP-4 control. Dose escalation from 1.5,
2, and
4E9 resulted in similar increases in expression that was statistically greater with
the
combination of the fleece with the exosome component. Bone formation in the mouse
calvaium
did not demonstrate gap closure at 4 or at 8 weeks, but did demonstrate enhanced
osteoclastivity and robust bone remodelling at the margins of the defect.
Summary/Conclusion: Bone marrow-derived exosomes dried into an electrospun
fibrillar collagen demonstrated in vitro osteoinductive potential that might provide
site-specific placement that could enhance biologic potential. With the capacity for
ambient
temperature storage, the provision of site-specific placement becomes a technical
consideration. Placement of the human tissue derived exosomes in a transgenic mouse
calvarial defect model did not demonstrate bridging bone across the defect.
PS03.12
Exosomes loaded with PTEN-Interfering RNA enables functional
recovery in rats after complete spinal cord transection
Daniel Offena
, Nisim
Peretsa, Shaowei Guob, Oshra Betzerc, Rachela
Popovtzerc and Shulamit Levenbergb
aTel Aviv University, Tel Aviv, Israel; bTechnion,
Haifa, Israel, Haifa, Israel; cBar Ilan University, Israel, Ramt Gan, Israel
Introduction: Complete spinal cord transection is a debilitating disease that
usually leads to permanent functional impairments, with various complications and
limited
spontaneous recovery. The current investigation of molecular mechanisms controlling
axon
regeneration, (e.g., signalling networks and environmental cues), led to new strategies
to
enhance axonal regeneration. We have previously shown that intranasal administration
of
mesenchymal stem cells derived exosomes (MSC-exo), cross the blood-brain barrier and
significantly ameliorate motor and behavioural phenotype in several animal models
of
neurotrauma and neuropsychiatric disorders.
Methods: MSC-exo were isolated from human bone marrow and were loaded with
phosphatase and tensin homolog small interfering RNA (PTEN-siRNA). The exosomes were
given
intranasally to rats two hours after complete spinal cord transaction. Eight weeks
later we
followed the motor function and histology and electrophysiology study was performed
in order
to reveal the connectivity and the biochemical changes in the treated rats.
Results: We demonstrate that Intranasal (IN) administrations of MSC-derived
exosomes could penetrate the blood-brain barrier, home selectively to spinal cord
lesion via
chemotaxis, and integrated in neurons within the lesion. Furthermore, in rats with
complete
spinal cord transection, MSC-exo loaded with PTEN-siRNA silenced PTEN protein expression
in
the lesion and promoted robust axonal regeneration and angiogenesis, companied with
decreased astrogliosis and microgliosis. Moreover, the intranasal treatment partially
restored electrophysiological and structural integrity, and most importantly, enabled
the
remarkable functional recovery and significant improvement in their movements.
Summary/Conclusion: This rapid, non-invasive, approach, using cell-free
nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel
strategy for clinical translation to spinal cord injury and beyond.
PS03.13
A novel umbilical cord derived wharton’s jelly formulation for
regenerative medicine applications
Ashim Guptaa
, Sobrasua
Ibimb, Howard Levyc, Rebecca Sze Tud and Saadiq
El-Aminc
aBioIntegrate LLC, New York, New York, USA, Lawrenceville,
USA; bMorris Brown College, Atlanta, Georgia, USA, Atlanta, USA;
cBioIntegrate LLC, New York, New York, USA, New York, USA; dColumbia
University, New York, New York, USA, New York, USA
Introduction: Musculoskeletal injuries have traditionally been treated with
activity-modification, physical therapy, pharmacological agents and surgical procedures.
These modalities have limitations, as well as potential side-effects. Over the last
decade,
there has been an increased interest in the use of biologics for regenerative medicine
applications (RMA), including umbilical cord (UC) derived Wharton’s Jelly (WJ). Despite
this
increase, there is insufficient literature assessing the amount of growth factors,
cytokines, hyaluronic acid (HA) and extracellular vesicles (EV) including exosomes
in these
products. The purpose of this study was to develop a novel WJ formulation and evaluate
the
presence of growth factors, cytokines, HA and EV including exosomes.
Methods: WJ was isolated from human-UC obtained from consenting C-section
donors and formulated into an injectable form. Randomly selected samples from different
batches were analysed for sterility testing and quantified for presence of growth
factors,
cytokines, HA and particles in EV size range.
Results: The results showed all samples passed the sterility test. Growth
factors including IGFBP 1, 2, 3, 4 and 6, TGF-α, PDGF-AA were detected. Expression
of
several immunomodulatory cytokines, RANTES, IL-6 R, IL-16, were also detected. Expression
of
pro-inflammatory cytokines MCSFR, MIP-1a; anti-inflammatory cytokines TNF-RI, TNF-RII,
IL-1 RA; and homoeostatic cytokines TIMP-1 and TIMP-2 were observed. Cytokines associated
with wound-healing, ICAM-1, G-CSF, GDF-15, and regenerative properties, GH were also
expressed. High concentrations of HA were observed. Particles in the EV size range
(30–150 nm) were detected and were enclosed by the membrane, indicative of true EV.
Summary/Conclusion: Our results confirmed the presence of numerous growth
factors, cytokines, HA and EV in the WJ formulation. More studies are underway to
confirm
the presence of exosomes in detected EV using exosome-specific markers. We believe
the
presence of multiple factors within one WJ formulation may play a role in reducing
inflammation, pain and augment healing of musculoskeletal injuries. This offers a
potential
expanded use for RMA.
Funding: This study was funded by BioIntegrate LLC, New York, NY, USA.
PS03.14
Collagen sponge loaded with mesenchymal stem cell-derived small
extracellular vesicles promote robust bone regeneration
Shang Jiunn Chuah
a, Chee Weng
Yonga, Jacob Ren Jie Chewa, Ruenn Chai Laib, Yi Ann
Cheowa, Raymond Chung Wen Wonga, Asher Ah Tong Lima, Sai
Kiang Limc and Wei seong Tohd
aFaculty of Dentistry, National University of Singapore,
Singapore, Singapore, Singapore; bInstitute of Medical Biology, Agency for
Science, Technology and Research, Singapore, Singapore, Singapore; cInstitute of
Medical Biology, Agency for Science, Technology and Research, Singapore. Department
of
Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore,
Singapore, Singapore; dFaculty of Dentistry, National University of Singapore,
Singapore. Department of Biomedical Engineering, Faculty of Engineering, National
University
of Singapore, Singapore. Tissue Engineering Program, Life Sciences Institute, National
University of Singapore, Singapore. Graduate School for Integrative Sciences and
Engineering, National University of Singapore, Singapore, Singapore, Singapore
Introduction: Mesenchymal stem cell (MSC) therapy has demonstrated effective
bone regeneration in clinical studies. However, the therapeutic efficacy of MSCs have
been
attributed to the secretion of extracellular vesicles (EVs), particularly 50–200 nm
small
EVs (sEVs). Here, we investigate the efficacy of MSC-sEVs loaded in collagen sponge
in the
regeneration of critical-sized calvarial defects in immunocompetent rats.
Methods: sEVs were isolated from conditioned medium of human MSCs and stored
at −20 C. Calvarial defects of 8-mm diameter were surgically created on thirty-two
10-week-old male Sprague-Dawley rats. These rats were then randomly assigned to 2
groups
(n = 16 rats/group): defects treated with collagen sponge containing 100 μg of sEVs
in
100 μl saline (CS/sEVs) and defects treated with control collagen sponge containing
an
equivalent volume of saline (CS/Control). At 1 and 8-week post-surgery, the calvarial
bone
samples was harvested for analyses by micro-computed tomography (micro-CT), histology,
immunohistochemistry and histomorphometry.
Results: At 1-week post-surgery, micro-CT analysis showed little bone
formation at the defect site in both CS/sEVs and CS/Control groups. No statistical
differences were observed in micro-CT and histology scores in both groups. Interestingly,
CS/sEVs group showed significantly higher osteocalcin (OCN)+ area of 5.7 ± 2.4% than
that of
CS/Control group (2.1 ± 1.3%; P = 0.003). CD31+ microvessels at sizes ≤50 µm and >50 µm
in CS/sEVs group (34.4 ± 2.9 and 8.6 ± 2.1 microvessels/HPF) were also significantly
higher
than that of CS/Control (18.8 ± 1.1 and 5.2 ± 1.4 microvessels/HPF; P = 0.002 and
P = 0.038
respectively). By 8 weeks, CS/sEVs group displayed enhanced new bone formation that
completely bridged the calvaria defect. In contrast, rats in CS/Control showed limited
bone
formation. Consequently, CS/sEVs group displayed a micro-CT score of 3.9 ± 0.2 which
was
significantly better than that of CS/Control group (2.5 ± 0.8; P = 0.001). CS/sEVs
group
also exhibited >twofold increase in bone volume, and improved bone quality with higher
trabecular thickness and number, and smaller separation (P < 0.01), compared to
CS/Control group. Consistently, CS/sEVs group displayed a significantly better histology
score of 5.4 ± 1.0 than that of CS/Control (2.6 ± 1.8; P = 0.001). Moreover, CS/sEVs
group
showed significantly higher OCN+ area of 48.9 ± 7.9% than that of CS/Control group
(20.4 ± 4.4%; P = 0.004).
Summary/Conclusion: This study demonstrates that single-stage implantation of
collagen sponge loaded with ready-to-use MSC sEVs can promote robust bone regeneration
in a
rat calvarial defect model.
Funding: National University of Singapore, R221000114114, National Medical
Research Council Singapore, R221000123213.
PS03.15
Immunomodulatory potential of extracellular vesicles derived
from Mesenchymal stromal cells
Fabiola Nardi Bauera, Michel Bremerb, Verena
Börgerc, Peter A. Hornc and Bernd
Giebel
d
aUniversity Hospital Essen, Essen, Germany;
bUniversity of Heidelberg, Heidelberg, Germany; cInstitute for
Transfusion Medicine, University Hospital Essen, 45147 Essen, Germany, Essen, Germany;
dHeidelberg, Heidelberg, Germany
Introduction: Extracellular vesicles (EVs) derived from mesenchymal
stem/stromal cells (MSCs) are promising new agents in regenerative medicine and
immunotherapy. Considering that independent MSC-EV preparations might differ in their
therapeutic function, we have set up a functional assay allowing testing for the potential
immunomodulatory properties of independent MSC-EV preparations.
Methods: Human peripheral blood-derived mononuclear cells (PBMCs) were pooled
from up to 12 different healthy donors warranting high allogeneic cross-reactivity,
even
following an optimized freezing and thawing procedure. After thawing, mixed PBMCs
were
cultured for 5 days in the absence or presence of MSC-EVs. Thereafter, cell morphologies
were documented, supernatants were harvested for cytokines quantification and cells
were
phenotypically characterized by flow cytometry. By analysing the expression of a collection
of different lineage and activation markers, we selected a panel of antigens apparently
being regulated by MSC-EV preparations considered to be therapeutically active.
Results: We observed that in the presence of active MSC-EV preparations more
CD14+ (monocytes) are recovered from the MLR assay than in corresponding control samples.
Focusing on T cells, we learned that active MSC-EV preparations reduced the content
of CD4
and CD8 T cells expressing activation markers like CD54 and CD25.
Summary/Conclusion: The MLR assay allows elaborated functional testing of
immunomodulatory activities of given MSC-EV preparations. Currently, we are comparing
the
immune modulatory capabilities of EVs derived from distinct sources and optimize the
marker
panel to distinguish discrete immune cell subtypes such as different CD4 cell types,
i.e.
TH1, TH2, TH17 and TRegs.
PS03.16
Extracellular vesicles in platelet-rich plasma: dependency on
sample processing
Zala Jan
a, Saba
Battelinob, Darja Božičc, Matej Hočevard, Ales
Igliče, Marko Jeranc, Manca Pajniča, Ljubiša
Pađena, Domen Vozelf and Veronika Kralj-Igliča
aLaboratory of Clinical Biophysics, Faculty of Health
Sciences, University of Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia;
bFaculty of Medicine, University of Ljubljana, Slovenia, Ljubljana, Slovenia;
cLaboratory of Clinical Biophysics, Faculty of Health Sciences, University of
Ljubljana, Ljubljana, Slovenia; Laboratory of Physics, Faculty of Electrical Engineering,
University of Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia; dDepartment of
Physics and Chemistry of Materials, Institute of Metals and Technology, Ljubljana,
Slovenia,
Ljubljana, Slovenia; eUniversity of Ljubljana, Ljubljana, Slovenia;
fDepartment of Otorhinolaryngology and Cervicofacial Surgery, University Medical
Centre Ljubljana, Slovenia; Faculty of Medicine, University of Ljubljana, Slovenia,
Ljubljana, Slovenia
Introduction: Platelet-rich plasma (PRP) proved effective in regenerative
medicine. Numerous protocols for its preparation and application are available in
the
published literature. PRP possesses important immune, haemostasis and regenerative
factors,
however, the mechanisms of their action are yet poorly understood. Extracellular vesicles
(EVs) could be one of the important factors that would contribute to the beneficial
effects
of preparations.
This study was performed as a part of a registered randomised controlled clinical
trial
(Nr: NCT04281901). PRP was used to treat chronic middle ear inflammations. Here we
present
the results of PRP analyses from 29 blood samples of volunteers with no record of
disease.
Methods: Plasma obtained from 20 ml of blood was depleted of erythrocytes and
enriched with other particles by repetitive centrifugation of samples. Flow cytometry
(FCM)
was employed to monitor particle contents (cells and smaller particles) throughout
the
sample processing. The platelet gate was divided into two parts: intact platelets
and
smaller particles. Identity and morphology of particles in the preparations were examined
by
scanning electron microscopy (SEM). Standard laboratory tests of blood were performed.
Results: SEM images revealed the presence of heterogeneous population of
particles in the preparation of PRP, most of which were activated and partially fragmented
platelets. The population of smaller particles measured with FCM, was identified as
EVs. The
erythrocyte sedimentation rate was statistically significantly correlated to the volume
of
plasma obtained in the initial centrifugation step (R = 0,70, p < 0,001) and to the
concentration of EVs (R = 0,82; p < 0,0001). Time from sample collection to the
preparation of PRP was negatively correlated with the concentration of platelets in
PRP and
positively with the concentration of EVs (R = 0,75, p < 0,001). Platelet concentration
in
preparation samples was found to depend on the concentration of platelets in the blood
and
parameters of sample processing connected with larger centrifugal and shear forces
on the
samples during centrifugation. These include: sample volume, the size and shape of
the
centrifuge tube and the distance of the sample from the rotor axis.
Summary/Conclusion: EVs are gradually forming upon activation and degradation
of cells in the sample throughout the sample processing. Optimal processing may importantly
contribute to the healing properties of preparation.
Funding: Authors acknowledge support from the European Union’s Horizon 2020
research and innovation program under grant agreement No. 801338 (VES4US project)
and
Slovenian Research Agency (ARRS, grants P2-0232, P3-0388, J1-9162).
PS03.17
Satellite cell-derived extracellular vesicles as a therapeutic
for mitochondrial dysfunction in duchenne muscular dystrophy
Kyle T. Shuler
a, Brittany E.
Wilsona, Eric R. Muñoza, Andrew D. Mitchella, Joshua T.
Selsbyb and Matthew B. Hudsona
aUniversity of Delaware, Newark, USA; bIowa State
University, Ames, USA
Introduction: Satellite cells (SCs) are muscle stem cells that play a central
role in muscle remodelling. Despite their therapeutic potential, SC-based therapies
have met
numerous logistical challenges in the treatment of systemic muscle diseases, such
as
Duchenne muscular dystrophy (DMD). SC-derived extracellular vesicles (SC-EVs) may
unlock the
therapeutic potential of SCs by overcoming these limitations. To investigate their
therapeutic potential, we assessed the ability of SC-EVs to reverse mitochondrial
dysfunction, a key pathological feature of DMD, in oxidatively-damaged C2C12 and primary
DMD
myotubes.
Methods: SCs from C57 mice were isolated and cultured. EVs were isolated from
the supernatant of SCs via polyethylene glycol precipitation and characterized using
nanoparticle tracking analysis. The ability of SC-EVs to deliver protein cargo to
C2C12
myotubes, and the localization of the cargo once delivered, were analysed using fluorescence
microscopy. To examine SC-EV potential to restore the function of damaged mitochondria,
C2C12 myotubes were treated with 50 µM H2O2 for 24 h followed by treatment with 3.12x108
SC-EVs for 24 h. Separately, cultured dystrophic myotubes were treated with 3.12 × 108 EVs
every 24 h for 72 h. In both sets of experiments, maximal oxygen consumption rate
(max OCR)
was measured via Seahorse XF Cell Mito Stress Test. Where appropriate, a t-test was
performed to test for statistical significance (p < 0.05).
Results: Based on estimated cell number and EV quantification, each SC
released approximately 2.35 × 105 ± 3.10x104 EVs/day. EVs delivered protein cargo
into
myotubes within 2 h. Fluorescent labelling of intracellular mitochondria showed
co-localization of delivered protein and mitochondria. Incubation of myotubes with
H2O2
resulted in a 42% decline in max OCR relative to untreated myotubes. Subsequent treatment
with SC-EVs resulted in a 76% increase in max OCR. Treatment of undamaged myotubes
with
SC-EVs had no effect on max OCR. Primary DMD myotubes treated with SC-EVs showed a
78%
increase in max OCR relative to untreated DMD myotubes.
Summary/Conclusion: SC-EVs rapidly deliver proteins into myotubes, much of
which co-localizes with mitochondria, and reverses mitochondria dysfunction in
oxidatively-damaged and dystrophic myotubes.
Funding: Supported by NIH R01 NS102157, NIH P20 GM113125, NIH P20 GM103446,
NIH R03 HD094594 to MBH
PS04: Advances in EV Quantification
Chair: Edwin van der Pol – Department of Clinical Chemistry, Amsterdam UMC, University
of Amsterdam; Vesicle Observation Center, Amsterdam UMC, University of Amsterdam;
Department of Biomedical Engineering and Physics, Amsterdam UMC, University of
Amsterdam
PS04.01
Quantitation of extra-cellular vesicle surface markers with MESF
liposomes
Oleg Guryev, Elena Afonina and Majid
Mehrpouyan
BD Biosciences, San Jose, USA
Introduction: Flow cytometry has been used extensively for analysis of EV
particles stained with fluorescent antibodies directed to the known cell surface markers.
Quantitation of the surface markers in terms of the number of molecules or the number
of
antibodies bound per specific marker has remained one of the largest challenges in
the EV
research field. Changes in instrument setup as well as changes in fluorescent antibodies
from different vendors, all impact the relative MFI values for the same EV sample.
In this
work we report a standardization method of quantitating extra-cellular vesicle surface
markers with MESF liposomes.
Methods: Liposomes labelled with FITC fluorescent dye were prepared with a BD
proprietary technology. Dynamic light scattering analysis was used for size determination
of
the liposomes. BD FACSAria™ Fusion system, modified with a small particle side scatter
module (SP SSC), was used for analysis of the labelled liposomes by flow cytometry.
Results: We created a set of 180 nm FITC-modified liposomes of various
fluorescent intensities with a known number of FITC molecules incorporated in each
liposome
intensity. The MFI values of each liposome population (intensity) had a linear relationship
to the amount of FITC used for labelling the liposome nanoparticles, suggesting that
no
self-quenching of FITC fluorescence had occurred. The number for the FITC fluorophores
for
each liposome intensity was expressed in the units of Molecules of Equivalents Soluble
Fluorochrome (MESF). A plot of MESF vs. the fluorescent intensity of the liposomes
(MFI
values) obtained from flow cytometry analysis provided a calibration curve, from which
the
fluorescent intensity (MFI value) of a stained EV sample can be converted to the number
of
fluorophores bound (MESF value) to the surface of the EV particles.
Summary/Conclusion: By this approach, the MFI values of stained EV particles
are converted to standardized MESF values that are independent of instrument variation,
resulting in further improvement of inter-laboratory standardization. Furthermore,
utilization of liposomes with similar size and refractive index to EV particles simplifies
the data evaluation and improves the accuracy of EV surface marker quantitation by
flow
cytometry. Currently, other fluorescent dyes are being explored to expand the utility
of
MESF liposomes with other fluorescent colours.
PS04.02
Measuring cholesterol as a high-throughput method for
quantifying extracellular vesicles
Aaron Sulentica, Shelly
Martina
, Beth DiBiaseb, Jorge
Sanchez-Salazarb, Charlotte Piardb, Joon Chong Yeeb,
Kevin Dooleya and Jonathan Finna
aCodiak BioSciences, Cambridge, USA; bCodiak
Biosciences, Cambridge, MA, USA
Introduction: The extracellular vesicle (EV) field currently lacks a
high-throughput method for accurately quantifying EVs in solution. EV quantification
has
traditionally relied on nanoparticle tracking analysis (NTA), which is time intensive
and
indiscriminately counts non-EV particles, such as membrane fragments and protein aggregates.
We have rigorously assessed two commercially available methods for measuring cholesterol,
a
major lipid component of the EV lipid bilayer, and evaluated the utility of these
assays to
quantify EVs in minimally processed samples.
Methods: The Amplex® Red Cholesterol Assay and Cedex Bio HT were used to
quantify cholesterol in EV samples via enzymatic oxidation, with dynamic ranges of
80–8,000 ng/mL and 4–800 µL/mL, respectively. Samples throughout various stages of
purification were analysed, from clarified cell culture medium to highly purified
EVs
separated on an iodixanol gradient. We evaluated several pre-processing methods, to
remove
non-EV cholesterol content prior to analysis.
Results: The Amplex® and Cedex Bio HT assays were found to perform comparably
for quantifying cholesterol in purified EVs (R2 = 0.92). Importantly, cholesterol
quantification on purified EV samples, ranging from 1e11 to 4e13 particles/mL, correlated
well with NTA measurements (R2 = 0.96). Both 45 µM filtration or an additional 16,000
RCF
centrifugation step following clarification removed cholesterol associated with cellular
debris or other non-EV sources, allowing for accurate quantification of conditioned
medium
samples or ultracentrifugation pellets (UCP) instead of needing to rigorously purify
samples
with an iodixanol density gradient.
Summary/Conclusion: Cholesterol quantitation can be used to accurately
estimate EV concentration, allowing for rapid characterization of samples from clarified
cell culture supernatant to highly purified EVs. This high-throughput analytical capability
may enable more comprehensive assessment of methods to boost EV yield through mass
screening
of cell culture conditions.
PS04.03
Optimization of nanoparticle tracking analysis of extracellular
vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small
cell lung cancer
Magdalena Dlugoleckaa
, Joanna
Domagala-Kulawikb, Malgorzata Polubiec-Kownackac and Malgorzata
Czystowska-Kuzmiczd
aDoctoral School, Medical University of Warsaw, Warsaw,
Poland; Chair and Department of Biochemistry, Medical University of Warsaw, Warsaw,
Poland,
Warsaw, Poland; bDepartment of Internal Diseases, Pneumonology and Allergology,
Medical University of Warsaw, Warsaw, Poland, Warsaw, Poland; cDepartment of
Surgery, Institute of Tuberculosis and Lung Diseases, Warsaw, Poland, Warsaw, Poland;
dChair and Department of Biochemistry, Medical University of Warsaw, Warsaw,
Poland, Warsaw, Poland
Introduction: Recent studies show that tumour-derived extracellular vesicles
(EVs) greatly influence the tumour microenvironment and impact the therapy. In non-small
cell lung cancer (NSCLC), bronchopulmonary lavage fluid (BALF) appears to be a good
source
of tumour-derived EVs, providing more accurate information about the tumour microenvironment
than EVs from plasma. So far there is a lack of accurate and standardized methods
for EV
quantification. Fluorescence nanoparticle tracking analysis (FL-NTA) is an emerging
method
of EV-analysis, allowing discrimination of EVs and exosomes from impurities. Here
we perform
an optimization of the FL-NTA method to compare EVs from plasma and BALF of NSCLC
patients
and healthy controls (NC).
Methods: EVs were isolated using homemade size-exclusion chromatography (SEC)
columns (plasma) and ultrafiltration or differential ultracentrifugation (BALF). NTA
was
performed using ZetaView PMX220 (Particle Metrix) after EV-staining with membrane
dyes or
fluorescence-labelled antibodies against typical EV-marker (CD9, CD81, CD63).
Results: NTA scatter measurements showed a higher total particle concentration
in plasma than in BALF. However, membrane-specific staining showed a much greater
purity of
EV-preparations from BALF, where nearly 100% of the particles detected in scatter
mode
showed positive membrane-staining. In contrast, only around 40–50% of particles in
the
plasma EV-preparations were positive for the membrane dyes. Fluorescence-staining
for EV
surface marker requires further optimization to obtain reproducible results.
Summary/Conclusion: Classical NTA using only the scatter mode fails to
discriminate between EVs, lipoproteins and protein aggregates. For EV-analysis from
complex
biofluids like plasma, FLA-NTA and staining for specific EV marker is necessary to
receive
reliable data. BALF seems to be a better source of tumour-derived EVs than plasma,
since the
obtained EV-preparations show a higher purity. Improving conditions for
fluorescence-staining and NTA measurement of EVs from plasma and BALF of NSCLC patients
will
provide an additional method for quantifying and phenotyping of EVs.
Funding: National Science Centre – OPUS 14 2017/27/B/NZ6/01990
PS04.04
Introducing a universal fluorescence extracellular vesicle stain
for NanoView Bioscience’s Exoviewer platform
Dennis Zimmermann and George Daaboul
NanoView Biosciences, Boston, USA
Introduction: The ExoViewer platform currently enables the user to capture
extracellular vesicles (EV) by means of surface antigen-specific antibodies (e.g.
targeting
tetraspanins), making possible the enumeration of individual particles using single-particle
interferometric reflectance imaging sensor (SP-IRIS, interferometric) imaging as well
as
fluorescence. Currently, through interferometric imaging particles smaller than 50 nm
cannot
be detected, while fluorescently stained EV smaller than 50 nm can be well resolved.
Further, it is conceivable that small EV contain antigen numbers in the single digits,
making antigen-specific immunostaining a challenge. To further characterize EV populations
of different sizes and surface marker composition, it would be highly advantageous
to target
the vesicular nature of the detected particles linked to a fluorescence readout.
Methods: The goal of this project is to detect EV with a probe that is
ubiquitously distributed across the surface (or lumen) of the vesicle. Small (30–150 nm)
EV
present fairly distinctive lipid membrane features in the extracellular environment,
turning
the EV membrane into a “universal” marker, and as such may serve as an alternative
marker
that is complementary to canonical EV surface markers.
Results: Here we present data on successfully staining EV with the membrane
dye Di-8-ANEPPS (Di-8) and the luminal dye Calcein-AM. We demonstrate that EV from
different
sources can be efficiently stained with either dye, allowing the quantitative
characterization of EV in an unbiased manner using Exoviewer’s fluorescence mode.
While both
dyes certainly have their own unique strengths, they exhibit the wanted linear correlation
of EV staining versus concentration. Further, both dyes are compatible with subsequent
immuno-staining applications, allowing the user to target specific surface or luminal
markers (Di-8).
Summary/Conclusion: While a large-panel screening featuring other powerful
dyes is continuously ongoing, the current data support the notion of providing the
experimenter with a reference for total particle count and at the same time fully
exploring
the larger dynamic range of the fluorescence mode. Moreover, the universal probe will
enable
the user to correlate intensity and particle size measurements, thereby significantly
improving the ExoViewer platform and its applications.
PS04.05
Membrane labelling is essential for the identification and
quantification of extracellular vesicles via FACS
Fanny Ender, Frank Gieseler, Piet Zamzow and
Nikolas von Bubnoff
Experimental Oncology, University Hospital and Medical School (UKSH),
University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany, Luebeck, Germany
Introduction: Extracellular vesicle (EV) research is challenged by the lack of
standard protocols to identify and distinguish between exosomes and ectosomes being
released
via exocytosis or plasma membrane shedding, respectively. Analysis of small EV populations
requires high-resolution technology and can be further improved using fluorescent
labels
such as carboxyfluorescein diacetate succinimidyl ester (CFSE). At the inner leaflet
of the
plasma membrane, CFSE is cleaved enzymatically resulting in covalent binding of the
dye. In
this study we optimized the conditions for membrane labelling of EVs and their subsequent
detection by flow cytometry to obtain a maximum yield of intact EVs.
Methods: Using sequential centrifugation, we separated EV subpopulations from
supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000x
g
centrifugation, we reconstituted EVs from the pellet. We used CFSE for EV detection
and
analysed the expression of tetraspanins by FACS to confirm the lipid bilayer structure.
Furthermore, we determined size distribution of EVs by nanoparticle tracking analysis
(NTA)
and electron microscopy. Detecting EVs as CFSE+ events, we quantified our samples
and
investigated the impact of threshold adjustment on EV quantification.
Results: After high speed centrifugation of cell free supernatants, we
identified CFSE+ events as EVs, which appeared as round structures under the microscope,
and
ranged from 80 to 40 nm in size. Interestingly, tetraspanin markers CD9 and CD81 were
detectable only on a subpopulation of purified EVs, suggesting heterogeneity of our
preparations. For sufficient labelling of EVs, minimal temperature variations and
short
incubation times correlated with EV stability. Of note, threshold adjustment significantly
improved the sensitivity of the flow cytometer for the detection of labelled EVs and
hence,
is central for data comparability.
Summary/Conclusion: Protocol standardization is of major importance for the
use of EVs as diagnostic markers in liquid biopsies.
Funding: This project has been supported in part by Annelise-Asmussen
foundation, Luebeck (grant 180802), LEO Pharma Germany (grant 180208).
PS04.06
Surface plasmon field-enhanced fluorescence spectroscopy (SPFS)
system for quantitative and qualitative extracellular vesicles total evaluation without
any sample pretreatment
Takatoshi Kayaa
, Yoichi
Aokib and Hiroaki Okac
aKONICAMINOLTA, Inc. Corporate R&D Headquarters,
Hino-shi, Japan; bKONICAMINOLTA Inc. Corporate R&D Headquarters, Hino-shi,
Japan; cKONICAMINOLTA, Inc. Corporate R&D Headquarters, Hino-shi, Japan
Introduction: The function of extracellular vesicle (EV) is interested in the
immunology and oncology fields as a key transmitter for cellular communication. However,
the
conventional EV evaluation methods are required complicated EVs preconcentration from
the
sample, its leads EV analysis uncertainty. In this study, we applied the SPFS highly
sensitive automated system for quantitative and qualitative EV evaluation without
any sample
pre-concentration and preparation step.
Methods: SPFS automated system and plastic disposable sensor had been
developed by Konica Minolta corporation in house. Anti-membrane protein (CD9, CD63,
CD83)
antibody was chemically bonded on hydrophilic polymer which was immobilized through
the gold
thin film on the SPFS sensor. The concentration of standard EV materials was evaluated
by
the Q-nano system before using. EV detection without pre-concentrating was achieved
by
sandwich immunoassay step in microchannel round-trip flow reaction (TAT 120 min) with
the
SPFS system, and ELISA was adapted as a conventional standard method. After SPFS highly
sensitive fluorescent measurements step, extracted and detected EV were effectively
recovered by using the recovery buffer reaction.
Results: The EV sensitivity performance between SPFS and ELISA clearly showed
a significant difference, and the LOD of SPFS (8.3 particles/μl) method was estimated
3000
times superior to the LOD of conventional ELISA (26,000 particles/μl). The SPFS calibration
curve showed a wide dynamic range at least over 5 logs as an additional specificity.
SPFS
method also showed fine results in the dilution linearity test with high reproducibility
under the serum/plasma sample condition. The data for recovery test of EV expected
us that
highly accurate measurement can be guaranteed under the condition of dilution about
10 times
or less even in the whole blood sample. After the SPFS measurement, extracted EV on
the SPFS
sensor chip could be effectively recovered and could be analysed nucleic acid which
contains
micro RNA.
Summary/Conclusion: SPFS system might have great potential for quantitative
and qualitative EV evaluation. Our strategy with SPFS system for EV proteomic and
genomic
profiling will be possible for applying to EV quality control as well as a novel biomarker
development.
PS04.07
Identification of a novel compound that inhibits small EV
secretion and tumour progression by a sensitive ELISA screening.
Yunfei Maa, Takeshi Yoshidaa, Duc Tuan
Nguyena, Kazutaka Matobab, Katsuhiko Kidab, Taito
Nishinob and Rikinari Hanayamac
aKanazawa University, Kanazawa, Japan; bNissan
Chemical Corporation, Tokyo, Japan; cWPI Nano Life Science Institute, Kanazawa
University, Kanazawa, Japan
Introduction: Small EVs from tumour cells are known to promote tumour
progression, therefore, it is expected to develop drugs that regulate small EV secretion,
which can be used in clinical applications.
Methods: To identify such regulators, we first developed a sensitive ELISA
system for the quantification of small EV secretion using a high-affinity EV binding
protein
Tim4. By using this ELISA system, we screened for small compounds that promote or
inhibit
small EV secretion using a drug-repositioning compound library (about 1,600 compounds).
Results: As a result, we identified eight promoters and two inhibitors,
including compound A, which significantly reduced small EV secretion from various
cell types
without affecting cell growth. We further investigated the effects of compound A on
a mouse
model of osteosarcoma and found that compound A suppressed tumour progression
efficiently.
Summary/Conclusion: These data suggest that compound A would be useful not
only for the characterization of small EV function but also for the clinical therapy
against
tumour progression, by inhibiting small EV secretion.
Funding: JST CREST
PS04.08
Quantitative proteomics identifies proteins enriched in
microvesicles and exosomes respectively
Anna Lischniga, Takahiro Ochiyab and
Cecilia Lässerc
aKrefting Research Centre, Institute of Medicine, Sahlgrenska
Academy at University of Gothenburg, Göteborg, Sweden; bTokyo Medical University,
Shinjuku-ku, Japan; cKrefting Research Centre, Institute of Medicine, Sahlgrenska
Academy at University of Gothenburg, Gothenburg, Sweden
Introduction: For many years it was believed that several proteins such as
CD63, CD9 and Flotillin-1 were unique for exosomes, however recent studies have shown
that
several of these markers also can be present in other subpopulations of EVs (Kowal
et al
PNAS 2016). Furthermore, few markers have been identified as uniquely present in
microvesicles. The aim of this study was to in depth compare the proteome of microvesicles
and exosomes.
Methods: MDA-MB-231-luc-D3H1, -D3 H2LN and -BMD2a were cultured in EV-depleted
media. Microvesicles (16,500 x g, 20 min) and exosomes (118,000 x g 2.5 h) were isolated
using a combination of differential ultracentrifugation and a density cushion (~1.1 g/ml).
Purity and yield of EVs were determined by nanoparticle tracking analysis (NTA), Western
blot, and electron microscopy (EM). Quantitative mass spectrometry (TMT-LC-MS/MS)
was used
to identify differently enriched proteins in microvesicles and exosomes (n = 3 x 3
cell
lines).
Results: In total 6493 proteins were quantified, with 4851 being quantified in
all samples. In total 818 and 1567 proteins were significantly upregulated in exosomes
and
microvesicles, respectively. GO Terms associated with the proteins significantly upregulated
in exosomes were “Extracellular Exosome” and “Plasma membrane”, while the microvesicle
proteome was associated with “Membrane” and Mitochondrion”. In exosomes tetraspanins,
annexins, ESCRT and rab proteins were significantly upregulated. In contrast, proteins
that
were upregulated in microvesicles were involved in protein translocation into the
mitochondrial membrane (TIMM and TOMM proteins), in cytokinesis, and in MICOS complex.
However, Flotillin-1 was not differently expressed in the EV subtypes.
Summary/Conclusion: This study identifies several proteins to be differently
enriched in exosomes and microvesicles. Several of the proteins suggest recently by
Kowal
and colleagues, such as ADAM10 and Mitofilin could be validated. Additionally several
novel
proteins could be identified. Identifying markers separating microvesicles and exosomes
is
of high importance for the EV field and future studies will have to validate them
also in
other cells to determine if they are generic.
PS05: EVs in the Central and Peripheral Nervous Systems
Chair: Norman Haughey – Department of Neurology, Johns Hopkins University School of
Medicine
Chair: Sowmya V. Yelamanchili, PhD – Department of Anaesthesiology
PS05.01
Vesicle secretion by ependymal cilia in brain
ventricle
Aline dos Santos Santana, Rafael Pedro Madeira, Andréa Aurélio
Borges, Rita Sinigglia and Ana Claudia Trocoli Torrecilhas
Universidade Federal de Sao Paulo, Sao Paulo, Brazil
Introduction: The cellular elements composing the lining of brain ventricles
have drawn much attention from neuroscientists, especially the role of subependymal
cells in
neurogenesis, but the role of ependymal cells in brain function and disease is still
neglected.
Our objective is to study the morphological aspects of rat brain ventricles and the
ependymal cells as analysed by transmission and field emission scanning microscopy
in normal
or ischaemic rats.
Methods: For this purpose, male Wistar rats were submitted to 10 minutes of
global brain ischaemia and divided into two groups: a) sham-operated animals and b)
saline-treated ischaemic animals. All animals were allowed to survive for seven days.
All
procedures were approved by the ethics committee of the Federal University of São
Paulo
(2018/7633081117). Transmission and scanning electron microscopic analysis of lateral
brain
ventricles were done in buffered 2,5% glutaraldehyde/2%formaldehyde perfused brains.
Cerebrospinal fluid was collected for NTA analysis.
Results: The morphological characterization of brain ventricle revealed a
slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared
to
normal animals. Field emission electron microscopy revealed the secretion of vesicles
by the
ependymal cilia in the lateral ventricle. Size and concentration of particles in the
cerebrospinal fluid was confirmed by NTA and transmission electron microscopy.
Summary/Conclusion: Our results are unprecedented and bring innovative
potential regarding the role of extracellular vesicles in both the physiology and
pathogenesis of the nervous system. These data may also contribute to the development
of new
technologies for diagnosis and therapy of chronic degenerative diseases.
Funding: FAPESP and CAPES
PS05.02
Mitochondrial vesicles in neurons
Pamela J. Yaoa
, Ronald
Petraliab, Erden Erenc, Jeffrey Gua, Ya-Xian
Wangb and Dimitrios Kapogiannisd
aNIA/NIH, Baltimore, USA; bNIDCD/NIH, Bethesda,
USA; cNational Institute on Ageing, NIH, Baltimore, USA; dLaboratory
of Clinical Investigation, National Institutes of Ageing, Baltimore, USA
Introduction: The function of mitochondria relies on precise and effective
quality controls. Neurons have high metabolic demands and employ multiple mechanisms
to
ensure functional mitochondria. We investigated mitochondrial vesicles – a less understood
quality control mechanism for mitochondria – and assessed the effect of cellular stress.
Methods: We surveyed mitochondrial vesicles in rat and planaria brains with
electron microscopy. We quantified these vesicles with serial-section electron microscopy
(FIB-SEM). We also conducted confocal microscopy with Airyscan analysis of cultured
neurons
expressing fluorescently tagged mitochondrial markers.
Results: Electron microscopy showed the ultrastructure of various types of
mitochondrial vesicles. Serial-section electron microscopy revealed the 3D ultrastructure
of
mitochondrial vesicles and their prevalence in neurons. Confocal microscopic analysis
showed
increased numbers of mitochondrial vesicles in neurons under mild stress.
Summary/Conclusion: Our findings provide direct structural evidence for
mitochondrial vesicles in neurons and their abundance in response to neuronal stress.
Their
detection in the extracellular compartment (evidence for which is expected to be presented
by the time of ISEV) may allow for development of biomarkers for mitochondrial health,
with
relevance to numerous pathologic conditions.
PS05.03
The role of small extracellular vesicles in chronic neuropathic
pain
Zhucheng Lina
, Renee
Jean-Toussaintb, Yuzhen Tianb, Ahmet Sacana and Seena
Ajitb
aDrexel University, Philadelphia, USA; bDrexel
University College of Medicine, Philadelphia, USA
Introduction: Chronic pain is the most prevalent, disabling, and expensive
public health condition in the USA. Exosomes are 30–150 nm extracellular vesicles
that can
transport RNAs, proteins, and lipid mediators to recipient cells via circulation.
Exosomes
can be beneficial or harmful depending on their source and contents. We hypothesized
that
the composition of small extracellular vesicles (sEVs) can be altered following nerve
injury
and these alterations can provide insight into how the body responds to neuropathic
pain.
Methods: To characterize changes following nerve injury, small extracellular
vesicles (sEVs) were purified by ultracentrifugation from mouse serum four weeks after
spared nerve injury (SNI) or sham surgery. miRNA profiling and proteomics analysis
using
tandem mass spectrometry were performed to determine differential expression of miRNAs
and
protein cargo respectively. For in vivo studies, sEVs were administrated intrathecally
into
the mouse lumbar region. Animals were evaluated for mechanical and thermal hypersensitivity
over 21 days after injection.
Results: Our miRNA profiling showed a distinct miRNA signature in SNI model
compared to sham control. Proteomics analysis detected 274 gene products. Of these,
24 were
unique to SNI model. Neuropathic pain can induce the activation of the complement
cascade
and we observed significant upregulation of complement component 5a (C5a) in sEVs
from SNI
model. Intercellular Adhesion Molecule 1 (ICAM-1), required for the leukocyte recruitment,
adhesion and homing of exosomes was also upregulated in sEVs from SNI model compared
to sham
control. Administration of sEVs from SNI model increased paw withdraw threshold in
naïve
recipient mice and inflammatory pain model, indicating a protective role for sEVs
in
attenuating chronic pain.
Summary/Conclusion: Our preliminary studies suggest a critical role for sEVs
cargo in regulating pain. Additional studies are ongoing to determine the functional
significance of alterations in sEVs composition using mouse models of pain.
Funding: NIH NINDS R01NS102836
PS05.04
Large and small extracellular vesicles in ALS: friends or
foes?
Daisy Sprovieroa
, Stella
Gagliardia, Carlo Morassob, Maria Chiara Mimmia, Matteo
Bordonia, Luca Diamantic, Maria Garofaloa, Orietta
Pansarasaa, Fabio Corsib and Cristina Ceredaa
aGenomic and post-Genomic Centre, IRCCS Mondino Foundation,
Pavia, Italy; bIstituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy;
cDivision of General Neurology, IRCCS Mondino Foundation, Pavia, Italy
Introduction: Amyotrophic Lateral Sclerosis (ALS) is a progressive adult-onset
neurodegenerative disease caused by selective motor neurons (MNs) death. The rapid
disease
progression strongly suggests that cell-to-cell spreading of noxious factors could
take
place in ALS pathogenesis. Extracellular vesicles could potentially spread the disease.
In
this study, we characterized large (lEVs) and small extracellular vesicles (sEVs)
isolated
from plasma of sporadic ALS patients and healthy controls and determined their different
composition in order to understand their neuroprotective or neurotoxic role in ALS
pathogenesis.
Methods: lEVs and sEVs were isolated from plasma of 40 ALS patients and 30
healthy volunteers by differential centrifugation and characterized by Nanosight NS300.
CD45, CD31, CD61, CD235a and Annexin V were used for flow cytometry. SOD1, TDP43,
FUS
protein level was investigated by Western Blot. For Raman Spectroscopy, EVs were dried
on
top of a CaF2 slide and Raman spectra were acquired using a 633 nm laser line. miRNA
libraries were prepared by TruSeq Small RNA Library kit (Illumina).
Results: The mean size both for lEVs and for sEVs resulted increased in ALS
patients compared to controls. lEVs derived from ALS patients were enriched in SOD-1,
TDP-43
and FUS proteins compared to CTRLs. sEVs showed a distinct spectral pattern from lEVs.
In
addition, lEVs of ALS patients were richer in lipids and had less intense bands relative
to
aromatic aminoacids compared to healthy controls. We also found a great presence of
leukocyte derived lEVs (LMVs) in ALS patients compared to AD patients and healthy
donors and
significant correlation with the Progression Rate of the disease. On the other hand,
miRNA
and RNA whole transcriptome sequencing identified a specific signature of miRNAs in
plasma
derived sEVs of ALS patients compared to a group of healthy controls and three neurological
groups of control.
Summary/Conclusion: These data may suggest that lEVs derived from ALS
patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation
and immunity of ALS disease, while sEVs, deriving from endosomes, might be involved
in the
impairment of RNA, specific feature of ALS disease.
Funding: This work was supported by Italian Ministry of Health (Grant No.
RC2017-2019); Fondazione Regionale per la Ricerca Biomedica for TRANS–ALS (Translating
Molecular Mechanisms into ALS risk and patient’s well-being: FRRB 2015–0023) and by
Italian
Ministry of Health (GR-2016-02361552).
PS05.05
Combining high-resolution flow cytometry and surface marker
analysis using an automated platform to study extracellular vesicle in cerebrospinal
fluid
Raphael Schneider
Unity Health Toronto, Toronto, Canada
Introduction: There is growing enthusiasm that extracellular vesicles (EVs)
carry the potential for a variety of applications in medicine. As biomarkers, EVs
may aid
clinicians in the evaluation of diagnoses, disease progression, or even response to
therapy.
However, proper characterization of the amount, size, and phenotype of EVs in a given
sample
remains challenging due to their sub-micrometre size and heterogeneity. Over the last
years,
technologies, including high-sensitivity flow cytometry and automated platforms that
simultaneously assess EV amount, size, and phenotype, have matured, providing new
opportunities to study EVs for future clinical applications. Using such technologies
to
analyse cerebrospinal fluid (CSF), which is in direct contact with the brain and spinal
cord, may yield valuable insights into neurological disease processes. While there
is often
uncertainty about the exact source of EVs in a biological sample, CD171 has emerged
as a
surface marker that suggests a neuronal origin.
Methods: CSF samples that had been stored at – 80 degrees Celsius for advanced
biomarker studies were analysed using two distinct approaches. A Becton, Dickinson
and
Company (BD) Aria III flow cytometer was converted into using violet side scatter
(SSC) for
improved detection of EVs with 405 instead of 488 nm SSC. For the combined analysis
of
amount, size, and phenotype, samples were analysed with the NanoView Bio R100 platform.
Phenotype analysis included probing for the classic tetraspanins associated with exosomes
(CD9, CD63, CD81) and the neural cell adhesion molecule L1 (CD171).
Results: Flow of CSF samples showed similar vesicle counts in control vs.
disease and an increase of counts in later disease stages when neurodegeneration is
thought
to be more prominent. All CSF samples showed some binding to classic exosomal markers
(CD9,
CD63, CD81). The sample taken at the latest time point showed relatively high vesicle
counts, overall larger vesicle size, and abundant CD171 binding. Interestingly, the
CD171
positive EVs were not positive for any of the classic exosomal markers (CD9, CD63,
and
CD81).
Summary/Conclusion: This data supports the notion that analysing the amount,
size, and surface markers of EVs in CSF can reveal intriguing dynamics in such basic
EV
characteristics over time and suggests important differences between EV populations
in
different disease stages. While previous studies indicated that CD171 could identify
an EV
to be of neuronal origin, it remains to be determined whether such specific surface
markers
will emerge as clinically relevant tools to support the evaluation of people affected
by
neurological diseases.
PS05.06
A distinct microRNA signature in plasma derived small
extracellular vesicles of different neurodegenerative diseases
Stella Gagliardia, Daisy
Sprovieroa
, Maddalena Arigonib, Cecilia
Pandinia, Orietta Pansarasaa, Raffaele A. Calogeroc and
Cristina Ceredaa
aGenomic and post-Genomic Centre, IRCCS Mondino Foundation,
Pavia, Italy; bDepartment of Molecular Biotechnology and Health Sciences,
Bioinformatic and Genomic Unit, University of Turin, Torino, Italy; cDepartment
of Molecular Biotechnology and Health Sciences, Bioinformatics and Genomics Unit,
University
of Turin, Torino, Italy
Introduction: Exploring Identifying robust biomarkers is essential for early
diagnosis of neurodegenerative diseases. Blood stream transports large (lEVs) and
small
extracellular vesicles (sEVs), which are extracellular vesicles of different sizes
and
biological functions that are transported in blood. Aim of our study was to investigate
mRNA/miRNA signatures in plasma derived lEVs and sEVs of Amyotrophic Lateral Sclerosis
(ALS), Alzheimer’s Disease (AD), Parkinson’s disease (PDPD), Fronto-temporal Dementia
(FTD)
and Alzheimer’s Disease (AD) patients.
Methods: lEVs and sEVs were isolated from plasma of patients and healthy
volunteers (CTR) by ultracentrifugation and RNA was extracted. Whole transcriptome
and miRNA
libraries were prepared with TruSeq Stranded Total RNA kit and TruSeq Small RNA Library
kit
(Illumina).
Results: Our data suggested that the RNA cargo in lEVs and sEVs varies among
different diseases. miRNA analysis in sEVs provided the most informative disease specific
signatures, while whole transcriptome analysis did not show any specific signature.
ALS was
characterized by a small but specific group of circulating miRNAs. miRNAs profiling
revealed
that PD and FTD can be subgrouped in two classes while AD appears to be a homogeneous
disease population. Furthermore, miRNAs profiling show the presence of overlaps in
the
signatures between the analysed diseases. miRNA profiling in lEVs is similar to that
observed in sEVs, although in lEVs the overall differences between diseases are less
marked.
Summary/Conclusion: In this study we have demonstrated that miRNAs are the
most interesting subpopulation of transcripts transported by plasma derived sEVs since
they
discriminate a disease from the other and they can provide a signature for each
neurodegenerative diseases.
Funding: This work was supported by Italian Ministry of Health (Grant No.
RC2017-2019); Fondazione Regionale per la Ricerca Biomedica for TRANS–ALS (Translating
Molecular Mechanisms into ALS risk and patient’s well-being: FRRB 2015–0023) and by
Fondazione Cariplo 2017 (Extracellular vesicles in the pathogenesis of Frontotemporal
Dementia 2017–0747).
PS05.07
Role of exosomes in the development of dendritic filopodia,
spines and synapses
Mikin Patela
, Mingjian
Shib and Alissa Weaverc
aDepartment of Biological Sciences, Vanderbilt University,
Nashville, USA; bDepartment of Biomedical Informatics, Vanderbilt University
Medical Center, Nashville, USA; cDepartment of Cell and Developmental Biology,
Vanderbilt University School of Medicine, Nashville, USA
Introduction: Dendritic spines are actin-rich structures at the postsynaptic
sites of most excitatory synapses in the central nervous system. They are highly important
structures for higher brain functions such as learning and memory. Several live imaging
studies have shown that long, thin, actin-rich protrusions called dendritic filopodia
are
precursors of dendritic spines in hippocampal and cortical neurons. So far, many
intracellular factors that regulate filopodia formation have been identified. However,
extracellular mechanisms of filopodia formation are largely unknown. Also, detailed
molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are
not
well understood. Small extracellular vesicles (SEVs)/exosomes have potential to regulate
filopodia, spine and synapse formation in autocrine or paracrine manner due to their
unique
cargo composition. Here, we examine role of exosomes in filopodia, spine and synapse
formation.
Methods: Primary rat hippocampal and cortical neurons were transiently
transfected with the multivesicular body (MVB) docking regulator GFP-Rab27b or with
shRNAs
against the exosome secretion and biogenesis regulators Rab27b and Hrs. Transfected
neurons
were immunostained for synaptic proteins and analysed for filopodia at day in vitro
(DIV) 6
or spines at DIV12. For rescue experiments, exosomes were isolated using differential
ultracentrifugation method from conditioned media of DIV9 cortical neurons or primary
astrocytes and characterized for their size, common protein markers and morphology.
Results: Here, we find that MVB docking factor GFP-Rab27b localizes to both
the tips and bases of actin-rich filopodia and spines in primary neurons. Furthermore,
genetic regulation of exosome secretion by overexpression or knockdown of Rab27b or
Hrs
leads to respective increases or decreases in the number of filopodia, spines and
synapses.
The defects of exosome-inhibited neurons in filopodia density are rescued by add-back
of
neuronal exosomes. Additionally, treatment of primary neurons with exosomes isolated
from
primary astrocyte cultures leads to enhanced spine and synapse formation.
Summary/Conclusion: These results indicate that autocrine and paracrine
communication via exosomes are a key part of the process of neuronal filopodia, spine
and
synapse formation.
PS05.08
Effects of apolipoprotein E genotype on protein and small RNA
profiles of brain tissue-derived extracellular vesicles of Alzheimer’s disease
patients
Yiyao Huanga
, Lesley
Chengb, Andrew F. Hillb, Andrey Turchinovichc, Vasiliki
Machairakid, Juan Troncosod, Olga Pletnikováe, Norman
Haugheyd, Lei Zhengf and Kenneth Witwerg
aDepartment of Molecular and Comparative Pathobiology
Neurology, The John Hopkins University School of Medicine, Baltimore, USA;
bDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular
Science, La Trobe University, Melbourne, Australia; cSciBerg, mannheim, Germany;
dDepartment of Neurology, Johns Hopkins University School of Medicine,
Baltimore, USA; eDepartment of Pathology, Johns Hopkins University School of
Medicine, Baltimore, USA; fDepartment of Laboratory Medicine, Nanfang Hospital,
Southern Medical University, Guangzhou, 510515, China, Guangzhou, China (People’s
Republic);
gDepartment of Molecular and Comparative Pathobiology and Department of
Neurology, The Johns Hopkins University School of Medicine, Baltimore, USA
Introduction: In Alzheimer’s disease (AD), three Apolipoprotein E (APOE)
polymorphic alleles contribute to risk: ε4 represents higher risk, ε2 is protective,
and ε3
is neutral. To determine if extracellular vesicles in brain may be linked with APOE
genotype, we investigated the possible effect of APOE genotype on brain-derived EVs
(bdEVs)
and their protein and RNA molecular cargo.
Methods: Cortical brain tissues of AD patients with different APOE genotypes
[ε2/ε3 (n = 5), ε3/ε3 (5), ε3/ε4 (6), ε4/ε4 (6)] and non-AD controls (n = 7) were
obtained.
bdEVs were separated by size exclusion chromatography plus ultracentrifugation (UC)
and
characterized per MISEV2018. Proteins were analysed by mass spectrometry. After protein
identification, data were normalized using the Cyclicloess method and analysed by
principal
component analysis (PCA). Nested Factorial design highlighted differentially expressed
proteins. RNA from bdEVs was extracted by miRNeasy Mini kit. Small RNA libraries were
constructed using the Ion Total RNA-Seq kit and sequenced on the Ion Torrent S5™ using
Ion™
540 chips. Reads were aligned to human reference transcriptomes using bowtie. Differential
gene expression was quantified by edgeR and limma.
Results: Among 28 proteins dysregulated in AD bd-sEVs, several have reported
roles in AD, e.g., microtubule-associated protein tau and peroxiredoxin-6. Regarding
APOE
genotypes, 16 proteins were differentially expressed between ε4 carriers (ε3/ε4 and
ε4/ε4)
with non ε4 carriers (ε2/ε3 and ε3/ε3). However, EV markers did not differ by APOE
genotype.
In contrast to protein cargo of bdEVs, the overall small RNA expression pattern was
similar
among AD patients with different APOE alleles and non-AD patients. Only a few miRNAs
showed
different abundance level between ε2/ε3 and ε4/ε4 groups, or between AD and non-AD
groups.
Summary/Conclusion: bdEVs carry proteins and miRNAs related to AD development
and APOE genotypes. Further verification of protein and RNA expression in brain and
plasma
derived EVs may reveal mechanisms of EV function in neuroinflammation and develop
biomarkers
for AD disease.
Funding: This project was funded by MH118164.
PS05.09
Efficient pathology spread by extracellular vesicles from human
brain tissues in mouse brain and tissue cultured neurons: Transmission and propagation
to
GABAergic neurons
Tsuneya Ikezua
, Zhi
Ruana, Dhruba Pathaka, Satoshi Muraokaa, Annina
Deleoa, Srinidhi Venkatesan Kalavaia, Rakez Kayedb,
Santhi Gorantlac, Howard Gendelmanc, Jennifer Luebkea and
Seiko Ikezua
aBoston University, Boston, USA; bUniversity of
Texas Medical Branch, Galveston, USA; cUniversity of Nebraska Medical Center,
Omaha, USA
Introduction: Tau-containing extracellular vesicles (EVs) are transmissible
and induce tau phosphorylation and conformational change in the recipient neurons.
However,
whether human brain-derived EVs induce tau pathology has not yet been characterized
in the
mouse brain. Here, we assess the mechanisms of disease spread after intrahippocampal
injection of human brain-derived EVs into the aged mouse model.
Methods: EV-enriched fractions were isolated from unfixed frozen human brain
samples from AD, prodromal AD (pAD), control (CTRL) cases, and tau knockout (TKO)
mouse
brains. Isolated EVs containing 300 pg of human total tau were sterotaxically injected
into
the right outer molecular layer of the dentate gyrus of 18 months-old C57BL/6 female
mice.
4.5 months after the injection, hippocampal slices were prepared for whole-cell patch
clamp
recordings of CA1 pyramidal neurons were undertakent. Hippocampi were analysed with
immunohistochemistry using phosphorylated-tau (p-tau) epitopes including AT8. EVs
were
examined for protein composition by protein mass-spectroscopy, the neuronal uptake
in vitro,
and structural analysis by the atomic force microscopy (AFM).
Results: Semiquantitative brain-wide immunohistochemistry of p-tau revealed
that inoculation of AD or pAD-EVs induced tau propagation throughout the hippocampus,
including the dentate gyrus, CA3 and CA1 subregions. AT8 was localized primarily in
GAD67+ GABAergic neurons in pAD and AD EVs groups, accompanied with reduced amplitude
of
inhibitory postsynaptic currents and Excitatory-Inhibitory ratio in amplitube of
postsynaptic currents in CA1 pyramidal neurons in pAD EVs. AFM analysis showed higher
density of tau oligomers in both AD and pAD EVs while only AD EVs showed significantly
higher neuronal uptake compared to CTRL EVs. Finally, proteomic analysis showed that
AD EVs
are enriched in disease and glia-related molecules compared to CTRL EVs, which may
contribute to their enhanced neuronal uptake.
Summary/Conclusion: Intracranial injection of AD or pAD EVs induced p-tau
accumulation primarily in GABAergic neurons throughout the hippocampus, resulted in
higher
uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency
as
seeding factors. GABAergic neuronal dysfunction in the hippocampal neuronal circuitry
reported in early AD brains could be attributed to specific EV mediated tau propagation
in
this cell type, a phenomenon meriting further investigation and validation.
Funding: NIH RF1AG054199, NIH R01AG054672, NIH R56AG057469, Cure Alzheimer’s
Fund, BrightFocus Foundation, CurePSP, Coins for Alzheimer’s Research Trust
PS05.10
Isolation of extracellular vesicles from cerebrospinal fluid and
characterization of their bioactive compounds to investigate multiple sclerosis
pathogenesis
Ludovic D’auria, Zakia Nasr and Vincent van
Pesch
Catholic University of Louvain, Brussels, Belgium
Introduction: Multiple sclerosis (MS) is the most frequent chronic
inflammatory disease of the young adult central nervous system. Nevertheless, the
pathogenesis remains largely unknown. It is therefore relevant to better characterise
in
cerebrospinal fluid (CSF), which irrigates the brain, novel bioactive compounds whose
dysregulation could be involved in MS pathology. The concentration of extracellular
vesicles
(EVs) has been already found affected in MS patient fluids but the content in bioactive
molecules, particularly the microRNAs (miRNAs), remains barely investigated. The miRNA
are
short oligonucleotides that are major posttranscriptional regulators and we previously
showed the dysregulation of specific miRNAs in CSF of MS patients. EVs can potentiate
miRNA
effects by allowing remote action through the shuttling within biological fluids such
as CSF
while providing a protection from circulating RNAse. Nevertheless, CSF remains a challenging
fluid to analyse due to limited access, low volume and presence of lipoproteins (other
putative miRNA carrier) that can be co-isolated with EVs.
Methods: We performed a comparative analysis of EV isolation from CSF by
size-exclusion chromatography (SEC), density-gradient ultracentrifugation, ultrafiltration
or chemical precipitation (ChemP) to determine the optimal technique(s) to enrich
EV.
Results: SEC applied on CSF of control patients showed optimal EV purification
with sufficient EVs from 0.5 ml of CSF for downstream EV characterization. Furthermore,
we
were able to isolate miRNAs from CSF and determined their enrichment in EVs by
RNAse-sensitivity treatments. Finally, we have combined ChemP and SEC to enable a
fast and
large-scale isolation of EVs from > 5 ml of CSF, which successfully provided an increase
in particles detected by nanoparticle tracking analysis. We are currently characterising
the
particles to confirm that they are purified EVs, cleared from contaminants.
Summary/Conclusion: This work opens perspective to analyse EVs from MS
patients and to determine whether miRNAs participates in MS pathogenesis through their
transit in EVs.
Funding: Fondation Louvain, Charcot Foundation.
PS05.11
Differences in circulating number of extracellular vesicles
between contact sport athletes with and without acute mTBI: a pilot study
Meghan Rath
a, Jacqueline
Sayoca, Soo-Young Choia, Karlee Burnsb, Aja
Corchadoc, Jane McDevittb, Jingwie Wud, Ryan
Tierneyb, Michael Selzere, Xiaoxuan Fanf and Joon-Young
Parka
aDepartment of Kinesiology, College of Public Health &
Cardiovascular Research Center, Lewis Katz School of Medicine, Temple University,
Philadelphia, USA; bDepartment of Health and Rehab Sciences, College of Public
Health, Temple University, Philadelphia, USA; cNovaCare Rehabilitation,
Philadelphia, USA; dDepartment of Epidemiology and Biostatistics, College of
Public Health, Temple University, Philadelphia, USA; eShriners Hospitals
Pediatric Research Center (Center for Neural Repair and Rehabilitation); Department
of
Neurology, the Lewis Katz School of Medicine at Temple University, Philadelphia, USA;
fMarlene and Stewart Greenebaum Comprehensive Cancer Center, University of
Maryland School of Medicine, Baltimore, USA
Introduction: Extracellular vesicles (EVs) are released by cells of the
central nervous system as a result of injury, including mild traumatic brain injury
(mTBI).
Since mTBI may alter circulating levels of EVs, this study aimed to investigate differences
in circulating EV numbers between contact sport athletes with and without acute mTBI.
Methods: Circulating EVs containing CD63 (CD63+ EV), CD81 (CD81+ EV), and
neural cell adhesion molecule (L1CAM+EV) were analysed in young, male athletes with
or
without mTBI (18–29 yo, n = 6 per group). Sodium citrate-treated blood samples were
obtained
from athletes with mTBI within 48-hours of injury and from control athletes free of
mTBI for
one year. Athletes were best matched for age and history of prior mTBI. Samples were
double-centrifuged to obtain platelet-poor plasma and stored at −80°C until analysed.
Quantification of EVs was performed using a spectral flow cytometer. The study was
approved
by Temple University’s IRB, and all athletes provided written informed consent.
Results: Mann-Whitney U tests showed that population percentages of small size
(179–304 nm) CD63+ EV, CD81+ EV and L1CAM+EVs were significantly higher in mTBI athletes
(mean rank: 8.0, 9.5, 9.3) than controls (mean rank: 3.6, 3.5, 3.7) (U = 3.0, p = 0.03;
U = 0.0, p > 0.01; U = 1.0, p > 0.01, respectively). Population percentages of large
size (500–800 nm) CD63+ EV, CD81+ EV and L1CAM+EVs were also significantly higher
in mTBI
athletes (mean rank: 8.2, 9.2, 9.5) than controls (mean rank: 3.4, 3.8, 3.5) (U = 2.0,
p = 0.02; U = 2.0, p > 0.01; U = 0.0, p > 0.01, respectively). There were no
significant differences between percentages of EVs associated with blood brain barrier
function (CD144+ EV) or platelets (CD42a+EV) among mTBI athletes or controls.
Summary/Conclusion: Athletes with acute mTBI demonstrate different EV profiles
than contact sport athlete controls. Further investigation of EV biomarkers is necessary
to
determine their potential for future, diagnostic usage.
Funding: NIH R01NS102157
PS05.12
Plasma-derived Extracellular Vesicles profiling as a biomarker
for Parkinson’s Disease
Elena Vacchi
a, Jacopo
Burrellob, Sara Bolisb, Alessio Burrelloc, Cinthia
Farinad, Lucio Barilee, Alain Kaelin-Langf and Giorgia
Mellia
aLaboratory for Biomedical Neurosciences, LBN-EOC,
Taverne-Torricella, Switzerland; bCellular and Molecular Cardiology Laboratory
and Laboratory for Cardiovascular Theranostics, Cardiocentro Ticino Foundation, Lugano,
Switzerland, Lugano, Switzerland; cDepartment of Electrical, Electronic and
Information Engineering, University of Bologna, Bologna, Italy, Bologna, Italy;
dImmunobiology of Neurological Disorders Laboratory, Institute of Experimental
Neurology (INSpe) and Division of Neuroscience, San Raffaele Scientific Institute,
Milan,
Italy, Milano, Italy; eCardiocentro Ticino, Lugano, Switzerland;
fNeurology Department, Neurocenter of Southern Switzerland, Ente Ospedaliero
Cantonale, Lugano, Switzerland, Lugano, Switzerland
Introduction: Parkinson’s disease (PD) is characterized by clinical
heterogeneity, different rates of progression and absence of definitive biomarkers.
Extracellular vesicles (EVs) are easily isolated from plasma and play a central role
in
intercellular communication which is highly relevant for inflammatory processes implicated
in protein misfolding-related neurodegenerative disorders. Thus, we characterized
distinctive plasmatic EV subpopulations of PD and atypical parkinsonisms (AP) patients,
with
the aim to identify candidate biomarkers among EVs surface membrane-proteins.
Methods: Plasmatic EVs were collected from 27 PD, 19 matched healthy controls
(HC), 9 AP with multiple system atrophy (MSA) and 9 AP with tauopathies (AP-Tau).
EVs were
quantified by Nanoparticle Tracking Analysis. The expression of 37 EV-surface markers,
related to inflammatory and immune cells, were measured by MACSPlex and correlated
to
clinical scales. A diagnostic model based on EV markers expression was built via supervised
machine learning algorithms and validated in an external cohort (10 PD, 20 HC, 5 MSA,
5
AP-Tau). The Cantonal Ethics committee approved the study protocol. All enrolled subjects
gave written informed consent.
Results: PD showed the highest EV concentration compared to others groups. PD
and MSA displayed a greater pool of overexpressed immune markers compared to AP-Tau.
EV
antigens correlate to cognitive impairment and disease gravity in PD and MSA. The
ROC curve
analysis of a compound EV marker showed optimal diagnostic performance for PD (AUC
0.908;
sensitivity 96.3%, specificity 78.9%) and MSA (AUC 0.974; sensitivity 100%, specificity
94.7%) and good accuracy for AP-Tau (AUC 0.718; sensitivity 77.8%, specificity 89.5%).
A
diagnostic model based on EV markers expression, correctly classified 88.9% of patients
with
reliable diagnostic performance after validation in an external cohort (77% of
accuracy).
Summary/Conclusion: This analysis of multiple immune surface markers of
circulating EVs in PD and AP well captured the clinical heterogeneity of PD and showed
optimal diagnostic performance. Furtherly it suggests a different immune dysregulation
in PD
and MSA vs. AP-Tau, to be confirmed by functional analysis in experimental models
of
disease.
Funding: Supported by ABREOC.
PS05.13
Separation and characterization of extracellular vesicles from
human cerebrospinal fluid
Julia Costa
a, Ana
Pronto-Laborinhob, Susana Pintob, Marta Gromichob, Sara
Bonuccic, Erin Tranfieldc, Catarina Correiad, Bruno
Alexandred and Mamede de Carvalhoe
aITQB NOVA, Oeiras, Portugal; bInstituto de
Fisiologia, Instituto de Medicina Molecular- Faculdade de Medicina, Universidade de
Lisboa,
Lisbon, Portugal; cElectron Microscopy Facility, Instituto Gulbenkian de Ciência,
Oeiras, Portugal; dUniMS – Mass Spectrometry Unit, IBET – Instituto de Biologia
Experimental e Tecnologica; ITQB NOVA, Oeiras, Portugal; eInstituto de
Fisiologia, Instituto de Medicina Molecular- Faculdade de Medicina, Universidade de
Lisboa,
Lisboa, Portugal
Introduction: Extracellular vesicles (EV) are released from cells to the
surroundings and are found in human biofluids, where they constitute promising targets
for
novel biomarker identification. EV have been found in cerebrospinal fluid (CSF) where
they
may provide with markers for neurological diseases.
Here, we aimed at purifying and characterizing EV from human CSF.
Methods: CSF was collected by lumbar puncture from patients with amyotrophic
lateral sclerosis. Patients gave written consent and studies were agreed by the local
ethics
committee. CSF was fractionated by ultrafiltration (Vivaspin, cut-off 3,000), and
size-exclusion chromatography (SEC; qEVsingle Izon Science). Eluted fractions were
analysed
by dynamic light scattering (DLS) and electron microscopy. Proteins were analysed
by
immunoblotting and nano-liquid chromatography-tandem mass spectrometry.
Results: EV eluted in early fractions (3 + 4) after the SEC void volume as
evaluated by detection of CD63 and CD9 markers (immunoblotting) and annexin A2 (peptide
mapping by nanoLC-MS/MS). There, nanoparticles around 150 nm were identified by DLS.
In
agreement, electron microscopy showed EV with characteristic shape and sizes typically
between 55 and 165 nm, with average diameter 94 ± 31 nm. CD63 was visualized by
immunocytochemistry at the surface of EV around 80 nm. On the other hand soluble proteins
IgG and albumin eluted in later fractions. Curiously, galectin-3 binding protein (LGALS3BP
or 90 K) was also partially detected in early-eluting fractions as nanoparticles of
irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those
nanoparticles had ring-like appearance. Occasionally 90 K also appeared on EV of variable
dimensions.
Summary/Conclusion: In conclusion, EV from the CSF may be separated from
soluble proteins and small molecules by a combination of ultrafiltration with SEC
fractionation. However, using this strategy a population of 90 K-containing nanoparticles
co-eluted with EV from the CSF. Further separation techniques need to be applied to
separate
EV from 90 K nanoparticles to investigate their individual physiological relevance
and
biomarker potential.
Funding: Euronanomed 2 ERA-NET project GlioEx (ENMed/0001/2013), FCT,
Portugal; iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344.
PS05.14
Separation and characterization of extracellular vesicles from
human cerebrospinal fluid
Julia Costa
a, Ana
Pronto-Laborinhob, Susana Pintob, Marta Gromichob, Sara
Bonuccic, Erin Tranfieldc, Catarina Correiad, Bruno
Alexandred and Mamede de Carvalhoe
aITQB NOVA, Oeiras, Portugal; bInstituto de
Fisiologia, Instituto de Medicina Molecular- Faculdade de Medicina, Universidade de
Lisboa,
Lisbon, Portugal; cElectron Microscopy Facility, Instituto Gulbenkian de Ciência,
Oeiras, Portugal; dUniMS – Mass Spectrometry Unit, IBET – Instituto de Biologia
Experimental e Tecnologica; ITQB NOVA, Oeiras, Portugal; eInstituto de
Fisiologia, Instituto de Medicina Molecular- Faculdade de Medicina, Universidade de
Lisboa,
Lisboa, Portugal
Introduction: Extracellular vesicles (EV) are released from cells to the
surroundings and are found in human biofluids, where they constitute promising targets
for
novel biomarker identification. EV have been found in cerebrospinal fluid (CSF) where
they
may provide with markers for neurological diseases.
Here, we aimed at purifying and characterizing EV from human CSF.
Methods: CSF was collected by lumbar puncture from patients with amyotrophic
lateral sclerosis. Patients gave written consent and studies were agreed by the local
ethics
committee. CSF was fractionated by ultrafiltration (Vivaspin, cut-off 3,000), and
size-exclusion chromatography (SEC; qEVsingle Izon Science). Eluted fractions were
analysed
by dynamic light scattering (DLS) and electron microscopy. Proteins were analysed
by
immunoblotting and nano-liquid chromatography-tandem mass spectrometry.
Results: EV eluted in early fractions (3 + 4) after the SEC void volume as
evaluated by detection of CD63 and CD9 markers (immunoblotting) and annexin A2 (peptide
mapping by nanoLC-MS/MS). There, nanoparticles around 150 nm were identified by DLS.
In
agreement, electron microscopy showed EV with characteristic shape and sizes typically
between 55 and 165 nm, with average diameter 94 ± 31 nm. CD63 was visualized by
immunocytochemistry at the surface of EV around 80 nm. On the other hand soluble proteins
IgG and albumin eluted in later fractions. Curiously, galectin-3 binding protein (LGALS3BP
or 90 K) was also partially detected in early-eluting fractions as nanoparticles of
irregular shapes and heterogeneous sizes typically between 15 and 60 nm; some of those
nanoparticles had ring-like appearance. Occasionally 90 K also appeared on EV of variable
dimensions.
Summary/Conclusion: In conclusion, EV from the CSF may be separated from
soluble proteins and small molecules by a combination of ultrafiltration with SEC
fractionation. However, using this strategy a population of 90 K-containing nanoparticles
co-eluted with EV from the CSF. Further separation techniques need to be applied to
separate
EV from 90 K nanoparticles to investigate their individual physiological relevance
and
biomarker potential.
Funding: Euronanomed 2 ERA-NET project GlioEx (ENMed/0001/2013), FCT,
Portugal; iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344.
PS06: EVs in Cardiovascular Diseases and Vascular
Disorders
Chair: Ahmed Ibrahim, Ph.D., MPH – Smidt Heart Institute, Cedars-Sinai Medical
Centre
PS06.01
Release of extracellular vesicles from platelets requires
platelet-platelet interaction
Aleksandra Gąseckaa
, Naomi C.
Buntsmab, Sytske Talsmac, Krzysztof J. Filipiakd, Rienk
Nieuwlande and Edwin van der Polf
a1st Chair and Department of Cardiology, Medical University
of Warsaw, Warsaw, Poland; bDepartment of Neurology, Amsterdam UMC, University of
Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands; cLaboratory of
Experimental Clinical Chemistry and Vesicle Observation Center, Amsterdam University
Medical
Centre, University of Amsterdam, Amsterdam, USA; d1st Chair and Department of
Cardiology, Medical University of Warsaw, Warszawa, Poland; eDepartment of
Clinical Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands,
Vesicle Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the
Netherlands, Amsterdam, Netherlands; fDepartment of Clinical Chemistry, Amsterdam
UMC, University of Amsterdam, Amsterdam, the Netherlands; Vesicle Observation Center,
Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands; Department of
Biomedical
Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands,
Amsterdam, Netherlands
Introduction: Arterial thrombosis is a major and global cause of human death
and disability, but a biomarker for early-diagnosis of thrombosis is absent. Platelet
activation and aggregation are the first steps of platelet-rich thrombus formation,
but
their relative contribution to platelet extracellular vesicles (PEVs) release is
unknown.
Methods: To study the relation between PEV release and platelet interaction
(aggregation), citrate-anticoagulated whole blood (WB) from healthy donors was diluted
2, 4,
8, 16 and 32-fold and activated by 30 μM thrombin-receptor activating peptide (TRAP).
In
addition, undiluted WB and 10-fold diluted WB, which totally blocked PEV release,
were
activated with various TRAP concentrations. Concentrations of PEVs (CD61+ and CD61+,
CD62p +
>1000 nm) and activated platelets (CD61+, CD62p+ >1000 nm) were measured by flow
cytometry (Apogee A60-Micro). Platelet aggregation was assessed using impedance
aggregometry.
Results: A 10-fold dilution of WB blocked both aggregation and the release of
PEVs. Compared to baseline, activation of undiluted WB with TRAP increased the
concentrations of CD61 + 2.2-fold and CD61+-CD62p+ PEVs 7.2-fold. The concentration
of CD61+
(R2 = 0.71) and CD61+-CD62p+ (R2 = 0.78) PEVs as well as platelet aggregation (R2 = 0.8)
scaled inversely (reciprocal) with the dilution of WB. Further, we found a linear
correlation between the % of activated platelets and the concentration of CD61+ (R2 = 0.80)
and CD61+, CD62p+ (R2 = 0.64) PEVs in undiluted WB, which was absent in 10-fold diluted
blood (R2 < 0.05).
Summary/Conclusion: The absence of aggregation and PEV release upon platelet
activation in 10-fold diluted blood shows that aggregation directly depends on the
distance
between platelets, which is confirmed by the reciprocal relationship between PEV release
and
blood dilution. Because PEVs are only released when platelet activation is followed
by
aggregation, PEVs are a potential early biomarker of thrombosis.
Funding: AG is supported by the National Science Centre, research programme
PRELUDIUM 2018/31/N/NZ7/02260.
EvdP is supported by the Netherlands Organisation for Scientific Research – Domain
Applied
and Engineering Sciences (NWO-TTW), research programmes VENI 15924.
PS06.02
Age-dependent alteration in concentration and size distribution
of extracellular vesicles in plasma of normotensive and hypertensive rats
Kosuke Otani, Muneyoshi Okada and Hideyuki
Yamawaki
Laboratory of Veterinary Pharmacology, School of Veterinary Medicine,
Kitasato University, Towada, Japan
Introduction: Spontaneously hypertensive rats (SHR) are the most widely used
animal model of human essential hypertension. We previously reported that plasma small
extracellular vesicles (sEVs) in SHR regulate systolic blood pressure, however, the
mechanism has not been clarified. In the present study, we compared the concentration
and
size distribution of plasma EVs (sEVs and large EVs) from young and aged normotensive
Wistar
Kyoto rats (WKY) and SHR.
Methods: Heparin-anticoagulated plasma was collected from male WKY and SHR at
5 ~ 7- (young) and 15- (aged) week-old. Large EVs were isolated from the plasma by
centrifugation (10000 x g). sEVs were isolated by ultracentrifugation (164,071 x g)
following precipitation with polyethylene-glycol. The concentration and size distribution
of
sEVs and large EVs were measured by a tunable resistive pulse sensing analysis.
Results: There was no significant difference in the total concentration of
plasma sEVs between WKY and SHR or between young and aged rats. The mean diameter
of plasma
sEVs from aged rats was larger than that from young rats in both WKY and SHR. Also,
the
number of particles with a diameter of smaller than 150 nm in plasma sEVs from aged
rats was
lower than that from young rats. The concentration of plasma large EVs from aged rats
was
higher than that from young rats in both WKY and SHR. There was no significant difference
in
the size distribution of plasma large EVs between WKY and SHR or between young and
aged
rats.
Summary/Conclusion: The present results for the first time demonstrate that
the concentration of plasma large-sized EVs is increased by ageing, while there is
no
difference in the concertation and size distribution of EVs between WKY and SHR. Further
research is required to clarify the cause of age-dependent alternation in plasma EV
size
distribution and its physiological meaning.
PS06.03
microRNA profiling of circulating extracellular vesicles is
involved with susceptibility to age-related diseases: relevance to cardiovascular
signalling in ageing process
Ionara Rodrigues Siqueiraa
, Laura
Cechinelb, Rachael Batabyalc and Robert Freishtatc
aUniversidade Federal do Rio Grande do Sul (UFRGS), Porto
Alegre, Brazil; bUniversidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;
cChildren’s National Hospital, Washington, USA
Introduction: Ageing represents a central risk factor for several diseases,
such as cardiovascular diseases. Our hypothesis is that extracellular vesicles (EVs)
can be
potential mechanism of spreading molecules, such as microRNAs, involved with susceptibility
to chronic age-related diseases and geriatric syndromes. In this context, the role
of
microRNAs in age-induced detrimental changes in the cardiovascular system has been
suggested. Although EVs can protect microRNAs from endogenous RNAses and internalization
of
these vesicles into cells is involved with cell communication, delivering microRNAs
even to
distant tissues, the relationships between EVs microRNAs profile and chronic age-related
diseases has not been evaluated. Our aim was to investigate the microRNA profile of
circulating EVs during ageing process and their downstream signalling pathways.
Methods: The Ethics Committee (CEUA – Comissão de Ética no Uso de Animais –
UFRGS; nr. 29,818) approved all animal procedures and experimental conditions. Male
Wistar
rats of 3- and 21-month-old were used, and plasma was obtained from the trunk blood.
EVs
were isolated with ExoQuick following the manufacturer’s instructions. microRNA was
isolated
from EVs and then amplified. microRNA was labelled using the FlashTag Biotin HSR RNA
Labelling Kit and profiled on Affymetrix GeneChip microRNA 4.0 Arrays. Ingenuity Pathway
Analysis (IPA) was used to identify pathways regulated by significantly altered
microRNAs.
Results: Microarray analysis revealed 728 microRNAs. Of these microRNAs, 48
were differentially expressed between aged and young-adult animals, 18 microRNAs were
significantly upregulated and 30 were downregulated in aged animals compared to young
adult
(p < 0.05; fold change of |1.1|). A conservative filter was applied on IPA and only
experimentally validated and highly conserved predicted mRNA targets for each microRNA
was
used. IPA analysis showed that cardiac hypertrophic signalling is ranked as highly
predicted
targets for these differentially expressed microRNAs (p < 0.0001). Moreover, IPA
demonstrated that this canonical pathway is upregulated in aged animals when compared
to
young adult. In addition to cardiac hypertrophic signalling, other relevant cardiovascular
canonical pathways, such as endothelin-1 signalling and intrinsic prothrombin activation
pathway have predicted targets.
Summary/Conclusion: Our results showed for the first time that microRNAs
profile in circulating EVs has a potential role to drive heart senescence and consequent
cardiac diseases which represents the leading cause of death.
Funding: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Finance Code 001–88881.189257/2018-01; Conselho Nacional de Desenvolvimento Científico
e
Tecnológico (CNPq) – 307980/2018-9
PS06.04
Endothelial cell-derived extracellular vesicles induce a smooth
muscle cell pro-inflammatory phenotype via HMGB1
Michael Boyera, Yayoi Kimurab, Tomoko
Akiyamab, Grant Knollc, Ariele Baggettc, Kyle
Prestonc, Rosario Scaliac, Satoru Eguchic and
Victor Rizzoc
aLewis Katz School of Medicine Temple University,
Phialdelphia, USA; bAdvanced Medical Research Center Yokohama, Yokohama, Japan;
cLewis Katz School of Medicine Temple University, Philadelphia, USA
Introduction: Introduction: The vascular endothelium and smooth muscle form
adjacent cellular layers that comprise part of the vascular wall. Here, we examined
the
extent to which extracellular vesicles (EVs) vesicles participate in endothelial-vascular
smooth muscle cell communication.
Methods: Methods: EVs were collected from rat aortic endothelial and smooth
muscle cell serum-free media by ultracentrifugation. Vesicle morphology, size and
concentration were evaluated by transmission electron microscopy and nanoparticle
tracking
analysis. Endothelial cell and vascular smooth muscle cell cultures were subjected
to
various concentrations of EVs for various times. Functional assays were performed.
Results: Results: Western blot as well as shot gun proteomic analyses revealed
sets of proteins common to both endothelial- and smooth muscle-derived EV as well
as
proteins unique to each vascular cell type. Functionally, endothelial-derived EVs
stimulated
vascular cell adhesion molecule-1 (VCAM-1) expression and enhanced leukocyte adhesion
in
vascular smooth muscle cells while smooth muscle EVs did not elicit similar effects
in
endothelial cells. EVs from endothelial cells also induced protein synthesis and
senescence-associated β galactosidase activity in vascular smooth muscle cells. Proteomic
analysis of vascular smooth muscle cells following exposure to endothelial cell-derived
EVs
revealed upregulation of several proteins including pro-inflammatory molecules,
high-mobility group box (HMGB) 1 and HMGB2. Pharmacological blockade of HMGB1 and
HMGB2 and
siRNA depletion of HMGB1 in smooth muscle cells attenuated NF-kb (p65) phosphorylation
and
nuclear translocation, VCAM-1 expression and leukocyte adhesion induced by endothelial
cell
EVs.
Summary/Conclusion: Conclusions: These data suggest that endothelial
cell-derived EVs can enhance signalling pathways that induce a pro inflammatory in
vascular
smooth muscle cells.
Funding: This work was supported by National Institute of Health grants,
HL128324 (V.R. and S.E.), HL133248 (S.E.), DK111042 (R.S. and S.E.) and F31HL146081
(M.J.B.), and American Heart Association grants, 16GRNT30130013 (V.R.), 16 GRNT30410007
(S.E.), and 19PRE34430038 (M.J.B.).
PS06.05
Double value of Microvesicles in CABG: patency predictive
capacity and tool for personalized antiplatelet therapy
Marina Cameraa
, Marta
Brambillab, Paola Canzanob, Laura Cavallottib, Calogero
Tedescob and Elena Tremolib
aDepartment of Pharmaceutical Sciences, Università degli
Studi di Milano, Milan, Italy; bCentro Cardiologico Monzino IRCCS, Milan,
Italy
Introduction: Graft patency is one of the major determinants of long-term
outcome following coronary artery bypass graft surgery (CABG). Biomarkers, if indicative
of
the underlying pathophysiological mechanisms, would suggest strategies to limit graft
failure. Many studies have generated compelling data on the sensitivity of MVs as
biomarkers
of cardiovascular disease progression and events. The MV usefulness in CABG has been
tested
only in a study that highlighted their importance in surgical haemostasis. No information
is
so far available on the association between the amount or pattern of circulating MVs
and
CABG outcome. We aimed to evaluate whether MV pre-operative signature could predict
mid-term
graft failure.
Methods: This was a nested case-control substudy of the CoronAry bypass
grafting: factors related to late events and Graft patency (CAGE) study that enrolled
330
patients undergoing elective CABG. Of these, 179 underwent coronary computed tomography
angiography 18 months post-surgery showing 24% graft occlusion. Flow cytometry MV
analysis
was performed in 60 patients (30/group with occluded [cases] and patent [controls]
grafts)
on plasma samples collected the day before surgery and at follow-up.
Results: Before surgery, cases had two-fold (p = 0.020) and four-fold
(p = 0.042) more activated platelet-derived and TF+ MVs, respectively than controls.
The MV
thrombin generation capacity was also significantly greater (p < 0.05). This MV signature
predicted graft occlusion (AUC of 0.897 [95%CI: 0,77–0,96], p = 0.02). By using a
MV-score
(0–6), the OR for re-occlusion for a score above 3 was 16.3 (95% CI 4.1–65.3,
p < 0.001).
Summary/Conclusion: The pre-operative signature of MVs is an independent
predictor of mid-term graft occlusion in CABG patients and a cumulative MV-score stratifies
patient’s risk. Since the MV signature mirrors platelet activation, patients with
a high
MV-score would benefit from a personalized antiplatelet therapy.
PS06.06
Exosomes from engineered immortalized human heart cells improve
ventricular function and attenuate fibrosis in mice with arrhythmogenic
cardiomyopathy
Yen-Nien Lin, Lizbeth Sanchez, Rui Zhang,
Thassio Ricardo Ribeiro Mesquita, Chang Li, Ahmed Ibrahim, Eduardo Marbán and Eugenio
Cingolani
Heart Institude, Cedars Sinai Medical Center, Los Angeles, USA
Introduction: Arrhythmogenic cardiomyopathy (AC) is characterized by
progressive loss of cardiomyocytes and fibrofatty tissue replacement. Currently, there
is no
effective treatment for this disease. Exosomes (imEXOs) secreted by heart stromal
cells,
engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and
anti-fibrotic effects and improve ventricular function in models of ischaemic injury
(Ibrahim et al., Nature BME 2019).
Methods: To investigate the effectiveness of imEXOs in a murine model of AC,
four-week old homozygous DSG2 knockout (DSGKO) mice and wild type (WT, age- and
strain-matched) mice were compared. DSGKO mice were randomized to receive weekly imEXOs
or
vehicle via intravenous injection for 4 weeks. Neonatal rat ventricular myocyte (NRVM)
proliferation and apoptotic assays were performed to explore potential effects of
exosomes.
Results: Biodistribution studies of DiR-labelled imEXOs revealed some cardiac
uptake, along with strong signals in spleen. At 4 weeks, DSGKO mice which had received
intravenous imEXOs showed improved cardiac function (echocardiographic ejection fraction
73 ± 2 VS 59 ± 5% in vehicle mice, P = 0.03), with an underlying attenuation in myocardial
fibrosis by histology. Electrophysiology test showed shorter QRS duration (4.0 ± 2.0 ms
imEXO VS 6.5 ± 2.9 ms vehicle, P = 0.02) and effective refractory period. Programmed
ventricular stimulation showed DSGKO mice which had received imEXOs were remarkably
less
prone to ventricular tachycardia induction (10.0 ± 32% VS 55.0 ± 52% in vehicle, P = 0.031).
In vitro study showed NRVM exposed to imEXOs for 2 days exhibited higher BrDU expression
relative to vehicle group, and less Annexin-V expression after oxidative stress induced
by
10-minute illumination with 254 nm UV.
Summary/Conclusion: Intravenous administration of imEXOs improved cardiac
function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in AC. Our findings
motivate clinical testing of imEXOs in AC, an orphan disease with great unmet medical
need.
Funding: NIH R01 HL124074 (to EM)
PS06.07
Cardiac-derived extracellular vesicles contribute to
communication between heart and brain in chronic heart failure (CHF) and target Nrf2/ARE
signalling
Changhai Tiana
, Lie Gaob
and Irving Zuckerb
aDepartment of Cellular and Integrative Physiology,
University of Nebraska Medical Center, Omaha, USA; bDepartment of Cellular and
Integrative Physiology, University of Nebraska Medical Center, Omaha, USA
Introduction: miRNAs regulate the translation of proteins that are involved in
redox homoeostasis in the heart and brain. Intra- and/or inter-organ communication
takes
place by multiple mechanisms including extracellular vesicular (EV) transport. Our
previous
studies suggested that cardiac derived miRNA-enriched EVs contribute to the dysregulation
of
Nrf2/antioxidant enzyme (ARE) signalling in the myocardium via intercellular cross-talk,
and
result in the decreased Nrf2/ARE signalling in the sympatho-regulatory areas of the
brain in
CHF. However, it is unclear if cardiac derived EVs circulate to the central nervous
system
evoking sympatho-excitation by disrupting central redox homoeostasis.
Methods: Cardiac-specific membrane GFP+ mice were generated to track the brain
distribution of cardiac EVs in rats with CHF (coronary ligation). The isolation and
characterization of EVs were carried out by differential ultracentrifugation, TEM,
NanoSight, western blotting, and qRT-PCR. Transfection, labelling, and microinjection
of EVs
into the rostral ventrolateral medulla (RVLM) were performed.
Results: Nrf2 protein was reduced in the RVLM of CHF rats consistent with an
upregulation of Nrf2-targeting miRNAs. Nrf2-targeting miRNAs were enriched in cardiac
and
circulating EVs of CHF rats. Nrf2-targeting and cardiac-specific miRNAs were abundant
in
brain-derived EVs. Circulating EVs were taken up by neurons in sympatho-regulatory
areas of
the brain. miRNA-enriched EVs from CHF animals increased sympathetic tone which was
prevented by a cocktail of Nrf2-targeting miRNA inhibitors.
Summary/Conclusion: Myocardial infarction-induced miRNA-enriched EVs mediate
the inter-organ cross-talk between heart and brain in the oxidative regulation of
sympathetic outflow through targeting the Nrf2/ARE signalling pathway. These findings
suggest that cardiac-derived EV miRNAs targeting Nrf2/ARE signalling may act as an
endocrine
signalling mediator of CHF that has potential as a novel therapeutic target.
Funding: This work was supported by the National Institution of Health Grant
P01 HL62222 to IHZ, and by the American Heart Association (AHA) Career Development
Award
(19CDA34520004) to CT.
PS06.08
Ischaemia impairs the ubiquitin-mediated secretion of Cx43 into
extracellular vesicles
Tania Martins-Marques
a, Saskia
Jagerb, Teresa M. Ribeiro-Rodriguesc, Monica Zuzartea,
Lino Gonçalvesa, Joost Sluijterd and Henrique Giraoe
aFaculty of Medicine, University of Coimbra, Coimbra,
Portugal; bLaboratory of Experimental Cardiology, UMC Utrecht Regenerative
Medicine Centre, Circulatory Health Laboratory, University Medical Centre Utrecht,
University Utrecht, Utrecht, the Netherlands, Utrecht, Netherlands; cInstitute
for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University
of
Coimbra, Coimbra, Portugal, Coimbra, Portugal; dDepartment of Experimental
Cardiology, University Medical Centre Utrecht, Utrecht University, The Netherlands,
Utrecht,
Netherlands; eFaculty of Medicine, University of COimbra, Coimbra, Portugal
Introduction: A fine-tuned communication between cardiac cells is vital to
maintain myocardial integrity and contractility. Not only an impairment of gap junction
(GJ)-mediated intercellular communication, but also defects in EV-mediated communication
have been associated with ischaemic heart disease, a major causative factor of heart
failure. We have previously shown that Cx43, the main ventricular GJ protein, assembles
into
channels at the EVs surface, mediating the release of vesicle content into target
cells.The
main objective of this work was to characterize the signals underlying protein sorting
into
extracellular vesicles (EVs) in a human pathophysiological context, using connexin43
(Cx43)
as a model substrate.
Methods: Animal models of ischaemia/reperfusion (I/R) injury by ligation of
the left anterior descending coronary artery, ex vivo and in vitro ischaemia models
and
human patients were used to investigate the secretion of EV-Cx43.
Results: Release of Cx43 was downregulated in circulating vesicles from
I/R-injured mice and patients with ST-segment elevation myocardial infarction, as
well as in
intracardiac and cardiomyocyte-derived EVs. Additionally, we show that ubiquitin signalled
the release of Cx43 in basal conditions but appeared to be dispensable during ischaemia.
Depletion of the autophagy adaptor p62 partially restored the secretion of Cx43, suggesting
an interplay between ischaemia-induced Cx43 degradation and secretion.
Summary/Conclusion: Overall, we demonstrated that ischaemia impairs the
sorting of Cx43 into EVs, which may ultimately affect long-distance communication.
Through
the identification of the underlying molecular mechanisms and players, these results
pave
the way towards the development of innovative diagnostic and therapeutic strategies
for
cardiovascular disorders.
Funding: European Regional Development Fund (ERDF) through the Operational
Program for Competitiveness Factors (COMPETE) [under the projects PAC “NETDIAMOND”
POCI‐01‐0145‐FEDER‐016385; HealthyAging2020 CENTRO‐01‐0145‐FEDER‐000012‐N2323;
POCI‐01‐0145‐FEDER‐007440, CENTRO‐01‐0145‐FEDER‐032179, CENTRO‐01‐0145‐FEDER‐032414,
POCI-01-0145-FEDER-022122, FCTUID/NEU/04539/2013, UID/NEU/04539/2019, UIDB/04539/2020
and
UIDP/04539/2020]. This work was supported by the Project EVICARE (No. 725229) of the
European Research Council (ERC) to JPGS. TMM was supported by PD/BD/106043/2015 and
TRR by
PD/BD/52294/2013 from Fundação para a Ciência e a Tecnologia (FCT).
PS06.09
Cardioprotection mediated by calcium-ionophore induced
extracellular vesicles
Szabolcs Hambalkó
a, Peter
Pečanb, Csilla Pelyhea, Van Thai Hac, Csilla Terezia.
Nagyd, Duško Lainščekc, Bence Kenyeresa, Anikó
Görbea, Ágnes Kittele, Monika Bartekovaf, Péter
Ferdinandyd, Mateja Manček Keberc and Zoltán Giriczd
aSemmelweis University, Department of Pharmacology and
Pharmacotherapy, 1085 Budapest, Hungary, Budapest, Hungary; bNational Institute
of Chemistry, SI-1000 Ljubljana, Slovenia, Ljubljana, Slovenia; cNational
Institute of Chemistry, Ljubljana, Slovenia; dDepartment of Pharmacology,
Semmelweis University, Budapest, Hungary; eInstitute of Experimental Medicine,
Hungarian Academy of Sciences, Budapest, Hungary; fSlovak Academy of Sciences,
Centre of Experimental Medicine, Institute for Heart Research, 84104 Bratislava, Slovakia,
Bratislava, Slovakia
Introduction: Remote ischaemic conditioning is a cardioprotective intervention
which protects the heart against ischaemia/reperfusion injury. Transient activation
of
Toll-like receptor 4 (TLR4) and its downstream regulators (TNFα and IL-6) have been
implicated in cardioprotective interventions. Extracellular vesicles (EVs) play a
role in
cardioprotection through the activation of the TLRs. However, since isolation of EVs
in high
amounts with suitable purity from blood is a challenge, our aim was to develop a cellular
model system from which TLR-inducing, cardioprotective EVs can be isolated in a reproducible
manner.
Methods: EV release from HEK293 cells was induced by calcium-ionophore A23187.
EVs were characterized, cytoprotection by EVs against simulated ischaemia/reperfusion
injury
and its mechanism were investigated in H9c2 and AC16 cell lines.
Results: A23187 induction of HEK293 cell induced EV release and the isolates
contained mostly large EVs. EVs decreased cytotoxicity and apoptosis due to 16 h ischaemia
followed by 2 h reperfusion in H9c2 and AC16 cells in a dose-dependent manner. EVs
activated
TLR4 and its downstream signalling pathway in H9c2 and AC16 cells as well as the expression
of cytoprotective haem oxigenase 1 (HO-1) in H9c2 cells.
Summary/Conclusion: A23187-induced EVs exert cytoprotection in H9c2 and AC16
cells by inducing TLR4 signalling and HO1 expression. Therefore, EVs released via
calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective
therapy.
PS07: EV Nucleic Acid Biomarkers
Chair: Kendall Van Keuren-Jensen – Translational Genomics Research
Institute
PS07.01
Extracellular-Vesicle microRNAs for detecting Pancreaticobiliary
Cancers
Daniel Liua
, Flora
Uptonb, Mireia Mato Pradoa, Jonathan Krella, Long
Jiaoc and Adam Framptona
aDivision of Cancer, Department of Surgery & Cancer,
Imperial College London, London, UK; bDivision of Cancer, Department of Surgery
and Cancer, Imperial College London, London, UK; cDepartment of Surgery and
Cancer, Imperial College London, London, UK
Introduction: Biliary strictures may be benign or malignant. The major
malignant causes of biliary stricture are a primary cholangiocarcinoma (CCA) or pancreatic
ductal adenocarcinoma (PDAC). There is ongoing debate about adequate diagnostics in
biliary
strictures of unknown aetiology. MicroRNAs (miRNAs) are small non-coding RNAs important
in
tumourigenesis. MiRNA have been found to be enriched in exosomes, small membrane-bound
extracellular vesicles (EV) of endocytic origin, which is a novel pathway for intercellular
signalling within the tumour microenvironment and have been implicated in loco-regional
pre-metastatic niche formation. This project aims to investigate circulating-free
and EV
miRNAs as biomarkers that can aid diagnosis in patients with a biliary stricture.
We will
(1) isolate and characterise EVs in plasma and bile from patients with benign and
malignant
biliary strictures (i.e. pancreaticobiliary cancers); and (2) identify differentially
expressed circulating-free and EV miRNAs in plasma and bile suitable for detecting
malignancy.
Methods: Sample size (n = 126) was calculated for a study power of 90% and α
error of 5% for the ability of extracellular miRNAs to discriminate benign from malignant
biliary strictures. Prospective matched plasma and bile samples will be collected
from
patients with benign (n = 63) and malignant (n = 63) biliary strictures undergoing
endoscopic retrograde cholangio-pancreatography (ERCP). EVs will be isolated from
the
biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised
(TEM, NTA and immunoblotting). Circulating-free and EV-associated miRNAs will be profiled
using small RNA sequencing. Extracellular miRNA “signatures” will then be validated
by
RT-qPCR, and diagnostic accuracy confirmed (sensitivity, specificity, AUC).
Results: EVs derived from patient samples have been characterised using NTA,
Western blotting and TEM. SEC derived EVs appear to be more well-defined than UC EVs
with
marker positivity for CD63, CD81 and CD9. Ongoing work will be focused on RNA profiles
of
EVs from both malignant and benign cohorts.
Summary/Conclusion: There is currently no effective method to differentiate
benign from malignant biliary strictures. Novel plasma and bile circulating-free and
EV-associated miRNA biomarkers may improve the speed and accuracy of diagnosis, resulting
in
considerable patient benefits. Furthermore, as little is known about the EV-associated
function of these tumours, candidate EV-miRNAs could be taken from “bedside to bench”
and
their function further investigated using in vivo, vitro and silico models.
PS07.02
Optimization of urinary extracellular mRNA profiling during
pregnancy
Priyadarshini Pantham
a,
Srimeenakshi Srinivasanb, Rob Moreyc, Peter DeHoffd, Mana
Paraste, Derek Wildmanf and Louise Laurentb
aDepartment of Obstetrics, Gynaecology, and Reproductive
Sciences, University of California San Diego, La Jolla, USA; bDepartment of
Obstetrics, Gynaecology, and Reproductive Sciences and Sanford Consortium for Regenerative
Medicine, University of California, San Diego, La Jolla, USA; cUniversity of
California San Diego, La Jolla, USA; dDepartment of Obstetrics, Gynaecology &
Reproductive Sciences, University of California San Diego, La Jolla, USA;
eDepartment of Pathology, University of California San Diego, La Jolla, USA;
fCollege of Public Health, University of South Florida, Tampa, USA
Introduction: Urine is a source of extracellular RNA (exRNA) biomarkers that
can be obtained non-invasively throughout pregnancy. Several studies have profiled
extracellular miRNAs in biofluids during pregnancy, but few have profiled extracellular
mRNAs (ex-mRNAs) in urine. Objective: To optimize methods for ex-mRNA isolation and
RNA-Seq
library preparation from urine of healthy pregnant and non-pregnant females.
Methods: RNA was isolated from pooled non-pregnant urine using kits based on
EV precipitation (miRCURY Exosome kit for CSF/urine, SeraMir), EV affinity purification
(ExoRNeasy), and protein precipitation (miRNeasy Serum/Plasma Advanced). Next, long
(>200nt) and short RNAs were isolated from EV enriched urine of pregnant (n = 5) and
non-pregnant (n = 5) individuals using the mIRCURY kit followed by the miRNeasy Micro
kit.
RNA-Seq libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA (Oligo(dT)
priming) and the SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input (random priming)
methods
(Takara). Preliminary data were obtained using the Illumina MiSeq, and aligned using
STAR
v.2.7.3.a.
Results: Overall, RNA isolation using miRCURY followed by the SMART-Seq v4
library preparation kit yielded the highest % of mapped reads: 42% in pooled non-pregnant,
27% in individual non-pregnant, and 31% in individual pregnant urine. For RNA extracted
using the miRCURY kit, the SMART-Seq v4 libraries had higher % of mapped mRNA reads
compared
to Pico libraries (P < 0.05, t-test). In contrast for miRNeasy Advanced it was reversed
(38% vs 21%).
Summary/Conclusion: Early results from low-depth sequencing show the highest
mRNA mapping rates for miRCURY followed by the SMART-Seq v4 kit. High-depth sequencing
data
are now being generated, which will enable us to perform detailed comparisons of different
RNA species from the RNA profiles obtained using different library preparations and
RNA
isolation methods from urine of pregnant and non-pregnant subjects.
Funding: This study was funded by NIH 7 K99 HD096125-02, NIH U01 HL126494, and
a UCSD IGM-Illumina Mini-Grant.
PS07.03
IL-2 mutein-induced changes of exosomal miRNA cargo in a
humanized mouse model
Emily Lurier, Erik Sampson, Patrick Halvey,
Mike Cianci and Katalin Kis-Toth
Pandion Therapeutics, Cambridge, USA
Introduction: Regulatory T cells (Tregs) are key contributors to immune
homoeostasis. Decreased number and/or function of these cells are frequent features
of many
autoimmune diseases linked to the development of tissue inflammation. While interleukin-2
(IL-2) is essential for pan T cell proliferation and performance, low dose IL-2 treatment
has been shown to preferentially affect Tregs and is being evaluated as an intervention
in
autoimmune diseases. PT101 is a novel IL-2 mutein Fc fusion molecule (IL-2 M) designed
to
selectively engage with Tregs. Using a humanized NOD-scid IL2R☓-null (NSG) mouse model
we
have shown that PT101 expanded Tregs without significant effects on other immune cells.
We
have also shown that Tregs from PT101-dosed humanized mice exhibit increased expression
of
FOXP3 and CD25, and demethylation of FOXP3 and CTLA-4 genes, suggesting enhanced function
and stability. In the current study we investigated the miRNA content of plasma exosomes
isolated from PT101- or vehicle-treated mice in order to identify Treg specific miRNAs
from
the IL-2 M treated animals.
Methods: CD34+ haematopoietic stem cell humanized NSG mice were dosed once
subcutaneously with PT101 or vehicle. Plasma samples from 8 mice were collected at
Day 7 and
exosome isolation was conducted using the ExoQuick method. Small RNA was extracted
and
quantified using the Bioanalyzer Small RNA assay. An Illumina NextSeq instrument was
used
for library preparation and sequencing with 75bp single end reads at an approximate
depth of
10–15 million reads per sample. Raw sequences were mapped to human genome GRCh37 and
analysed via a pipeline provided by the University of California Santa Cruz.
Results: RNA within the exosomes from vehicle and IL-2 M-treated groups was
mostly comprised of miRNA and tRNA. Plasma was pooled from 8 animals per treatment
group and
differential expression was determined using a twofold change cut-off. We found that
PT101
treatment actively altered the miRNA content of plasma exosomes, compared to exosomes
from
vehicle-treated mice. Many of the differentially expressed miRNAs are involved in
immunoregulation.
Summary/Conclusion: Plasma exosomes from PT101-treated humanized mice
encapsulated treatment-specific miRNAs which can potentially be used as systemic biomarkers
of Treg expansion and function.
PS07.04
Identification of potential biomarkers in microglial specific
exosomes isolated from prion-infected serum
Lise Lamoureux, Sarah Medina, Kathy Frost,
Kristyn Burak and Stephanie Booth
Public Health Agency of Canada, Winnipeg, Canada
Introduction: Transmissible spongiform encephalopathies (TSE) are
neurodegenerative disorders caused by the misfolding of the cellular prion protein
(PrPc) to
the beta-sheet rich abnormal prion protein (PrPsc). PrPsc aggregates in the brain
and causes
amyloid plaques, neuronal loss, spongiform degeneration and microglial activation.
Currently, definitive diagnosis of TSE diseases is only confirmed post-mortem thus
a
diagnostic test in accessible body fluid is of interest. Exosomes are a good resource
for
biomarker discovery since they cross the blood-brain barrier easily and contain protein,
lipids and nucleic acids from the cells of origin. The goal of this study was to look
at
biomarkers from brain-originating exosomes (specifically microglia) isolated in the
serum of
prion-infected animals.
Methods: Westerns and nanoparticle tracking analysis (NTA) were used to look
at the composition of microglial-specific exosomes. As proof of principle, exosomes
were
isolated from a microglial cell line (BV2 cells). A CD63 antibody was labelled with
a
fluorophore and binding to exosomes was visualized via NTA. Exosomes were isolated
from
serum of both prion-infected and mock-infected mice throughout disease course. A macrophage
specific antibody (F4/80) was bound to beads which were used to isolate exosomes which
includes those of microglial origin. microRNA was extracted from these exosomes and
Next-Generation Sequencing (NGS) was performed using the Illumina platform. CLC Genomics
Workbench was used for bioinformatics analysis.
Results: Microglial and macrophage proteins (TMEM119 and Iba1) were identified
in exosomes isolated from BV2 cells and prion-infected mouse serum. Macrophage exosomes
were
isolated via a novel antibody-bead based system. Results of the NGS analysis of the
microRNA
isolated from these exosomes indicated a series of miRNA that could differentiate
between
control and infected samples as well as age-specific markers.
Summary/Conclusion: To our knowledge, this is the first time
microglial-specific exosomes have been isolated from prion-infected serum from early
and end
stage disease. The results of this analysis could facilitate the diagnosis of prion
disease
in easily-accessible biofluids pre-mortem.
PS07.05
Comparison of urinary extracellular vesicle isolation methods
for transcriptomic biomarker research in diabetic kidney disease
Karina A. Barreiroa
, Om
Dwivedia, German Leparcb, Marcel Rolserb, Denis
Delicb, Carol Forsblomc, Leif Groopa, Tobias
Huberd, Maija Puhkaa and Harry Holthofera
aInstitute for Molecular Medicine Finland FIMM, University of
Helsinki, Finland, Helsinki, Finland; bBoehringer Ingelheim Pharma GmbH & Co.
KG, Biberach, Germany, Biberach an der Riss, Germany; cFolkhälsan Institute of
Genetics, Folkhälsan Research Centre, Helsinki, Finland, Helsinki, Finland; dIII
Department of Medicine, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany,
Hamburg, Germany
Introduction: Urinary Extracellular Vesicles (uEVs) are emerging as a source
for early biomarkers of kidney damage, holding the potential to replace the conventional
invasive techniques including kidney biopsy.
Several methods are available for uEV isolation. Our aim was to compare different
workflows
and isolation by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC) and
a kit
based isolation method for their subsequent use in miRNA-seq and RNA-seq for biomarker
discovery in diabetic kidney disease.
Methods: Type 1 diabetic patients (T1D) with macroalbuminuria and
normoalbuminuric healthy controls were included in the study. Sample collection and
all
experiments were performed in accordance with the declaration of Helsinki. EVs were
isolated
from 10–50 ml of 24 h urine collections by UC, HFD, or a commercially available kit
(Purification based on spin column chromatography, Urine exosome purification and
RNA
isolation Midi kit, Norgen Biotech, Canada) each with different established urine
clarification steps.
Quality control of the EVs was performed with negative staining EM, NTA and Western
blotting. Isolated RNAs were profiled with Bioanalyzer Pico kit and subjected to miRNA
and
mRNA sequencing.
For RNA-seq, cDNA library was prepared using SMART-seq v4 Ultra Low Input RNA Kit
for
Sequencing (Takara Bio, Japan). RNA-seq was performed using HiSeq 2000 (Illumina).
miRNA-seq library was prepared using QIAseq miRNA Library Kit (Qiagen, Germany). miRNA-seq
was performed on the Illumina HiSeq 4000 platform (Illumina).
Results: Our data showed that uEV yield, morphology and size distribution were
closely similar in HFD and UC preparations, while lower yields were obtained using
the
kit.
By Western blot, EV markers were detectable in samples isolated by HFD and UC but
not
readily in samples isolated with the kit. Tamm-Horsfall Protein was detected in all
the
samples and albumin levels appeared higher in HFD and Kit isolated samples relative
to UC
samples.
The number of paired-end reads for RNA-seq in HFD and UC samples (in both > 5 M) were
closely similar. Instead, RNA reads were lower than 2 M for the kit samples. For miRNA-seq,
the number of reads as well as the molecular biotype distribution were similar for
the three
methods. By principal component analysis of the RNA-seq data, we observed that HFD
and UC
grouped together showing similarities. However, for miRNA-seq data such similarities
were
not obvious. This suggests that the three different workflows and isolation principles
may
enrich different miRNA-rich uEV preparation components.
Summary/Conclusion: Our transcriptomics data shows that HFD and UC are
suitable methods to isolate uEVs for miRNA-seq and RNA-seq. The kit based method appears
better suited for miRNA-seq.
Funding: This work was supported by the H2020-IMI2 consortium BEAt-DKD
PS07.06
cfDNA distribution in bioliquids: exosome-associated vs. free
circulating form
Andrei Malykha
, Vladimir
Evtushenkob and Anastasia Malekc
aCapital Biosciences, Inc., Gaithersburg, USA;
bA.M. Granov Russian State Centre of Radiology and Surgery Technologies, St.
Petersburg, Russia; cN.N. Petrov Institute of Oncology, St. Petersburg,
Russia
Introduction: Exosomes contain a variety of biomolecules including DNA.
Knowledge of cfDNA distribution and localization in bioliquid is important for understanding
both biological function of cfDNA and exosomes. Some publications state that a large
proportion of plasma cfDNA is localized in exosomes. To quantify cfDNA content in
free vs.
exosomal form in human plasma, urine, and saliva, we employed SubX technology, which
allows
affinity capture DNA via phosphates groups of the polynucleotide chain and exosomes
via
membrane surface phosphate moiety clusters. SubX is a proprietary compound that can
simultaneously bind to both cfDNA and exosomes in bioliquids, thus allowing precipitation
of
the [SubX-DNA/SubX-Exosomes] complexes without ultracentrifugation.
Methods: Detection of SubX-DNA and exosomes binding was done by measurement of
particle sizes using Zetasizer Nano ZS and Nanosight NS300. The samples were processed
with
the SubX Exo-DNA isolation kit following the standard protocols. DNA, protein and
lipid
concentrations were measured by fluorescent assays using Qubit Fluorometer.
Results: SubX efficiently and selectively captures and co-precipitates cfDNA
and exosomes directly from bioliquids. Exosomes are easily extracted from the pellet
in
exosome reconstitution buffer (ERB), followed by subsequent isolation of tightly bound
cfDNA
from the SubX pellet. ERB does not extract DNA form the [SubX -DNA] pellet and thus
does not
contaminate reconstituted exosomes with cfDNA. Thus, we separate two distinct types
of
extracellular material – intact exosomes and purified cfDNA in a single protocol from
the
same sample. Over 90% of DNA in plasma and urine exist as a free circulating pool,
while in
saliva up to 30% is associated with exosomes. Thus, cfDNA distribution is probably
bioliquid-specific and must be evaluated by methods that eliminate cfDNA-outer exosomal
membrane aggregation.
Summary/Conclusion: SubX technology is suitable for simultaneous isolation of
both cfDNA and exosomes from the same bioliquid sample. SubX separates cfDNA fragments
non-specifically attached to the outer lipid layers of the exosome membrane from the
true
intra-exosomal cfDNA. In contrast, salting-out PEG technique is associated with aggregation
of macromolecules and vesicles and thus leads to overestimation of exosome-associated
polymers content, including cfDNA.
PS07.07
Tracing extrachromosomal DNA inheritance patterns in
glioblastoma using CRISPR
Eunhee Yi, amit Gujar, Hoon Kim, Albert Cheng
and Roel Verhaak
Jackson Laboratory for Genomic Medicine, Farmington, USA
Introduction: Glioblastoma multiforme (GBM) is the most lethal brain tumour;
it is characterized by poor response to standard post-resection radiation and cytotoxic
therapy, resulting in a dismal prognosis with a five-year survival rate of 10%. Recurrence
after therapy for GBM is unavoidable. There are substantial differences among the
cells of
GBM tumours in the abundance and types of genetic material. This heterogeneity likely
is the
major cause of therapy failure, the development of treatment resistance, and ultimately
recurrence. A recent study has suggested that the amount of a particular type of DNA
–
extrachromosomal DNA (ecDNA) – differs substantially among different GBM tumours,
and
differs within a given GBM tumour over time. Despite the speculation that ecDNA is
a key
factor of tumour heterogeneity, how ecDNA is propagated and distributed among – and
how it
behaves within – cancer cells is completely unknown.
Methods: To address this gap in knowledge, this study focused on developing a
novel cytogenetic CRISPR-based tool that enables visualization and tracking ecDNA
behaviour
in live GBM cells.
Results: We found breakpoint sequences resulting from genome rearrangements
during ecDNA formation by performing computational analysis from Whole Genome Sequencing
data. And each breakpoint was regarded as a unique target sequence for ecDNA-specific
labelling. The uniqueness of each breakpoint was validated by breakpoint-PCR (BP-PCR).
Furthermore, the location and the amount of each breakpoint were observed by breakpoint-FISH
(BP-FISH) analysis in GBM cells.
Summary/Conclusion: This results will be strong evidence to make
ecDNA-specific CRISPR system in further research. Tracing ecDNA dynamics will provide
new
insight into the impact of ecDNA on cancer evolution.
Funding: Basic Research Fellowship from American Brain Tumour Association
PS07.08
RNA signatures of mouse primary neurons, astrocytes and their
small extracellular vesicles
Xuan Luoa
, Renée
Jean-Toussainta, Ahmet Sacanb and Seena Ajita
aDrexel University College of Medicine, Philadelphia, USA;
bDrexel University, Philadelphia, USA
Introduction: Small extracellular vesicles (sEVs) are 30–150 nm vesicles that
mediate intercellular communication by transferring RNA and proteins to the recipient
cells.
These cargo molecules are selectively sorted into sEVs and mirror the physiological
state of
the donor cells. Given that sEVs can cross the blood-brain barrier and their composition
can
change in neurological disorders, there is an increasing interest in elucidating the
molecular signatures of sEVs in circulation as disease biomarkers. However, circulating
sEVs
are derived from multiple cellular sources and determining their source is challenging.
Information on sEV composition can be beneficial in predicting whether these sEVs
are
released predominantly from central nervous system cells. We hypothesized that
differentially expressed miRNAs between neuronal sEVs and astrocytic sEVs could be
used as
cell-type-specific signatures.
Methods: Small extracellular vesicles were isolated from cell culture media of
postnatal mouse primary neurons and astrocytes using differential centrifugation and
characterized using nanoparticle tracking analysis, transmission electron microscopy
and
western blotting. RNA from neurons, astrocytes, and their respective sEVs were used
for
transcriptome and small RNA sequencing.
Results: We observed that only a subset of cellular miRNAs was packaged into
sEVs; differential expression of specific miRNAs between sEVs and their corresponding
cells
suggest that cells employ special mechanisms to sort miRNAs into sEVs. These mechanisms
could be cell-type specific since neuronal sEVs showed a different miRNA profile compared
to
astrocytic sEVs. EXOmotifs, the short sequence motifs that control the loading of
RNA into
sEVs, were present in differentially expressed miRNAs. We also observed that five
RNA-binding proteins, which are associated with passive or active RNA sorting into
sEVs,
were differentially expressed between neuronal and astrocytic cells.
Summary/Conclusion: miRNA signatures of sEVs from neurons and astrocytes could
be beneficial in determining if these cell types contribute to the alterations of
sEV
composition in circulation in neurological disorders. Cell-type-specific selectivity
in RNA
loading might be attributed to the differential expression of RNA-binding proteins.
Funding: NIH NINDS R01NS102836
PS07.09
Isolation and characterization of Extracellular vehicles (EVs)
and EV RNA in human urine samples
Amit Aroraa
, Jaclyn
Chabotb, Gagandeep Narulaa, evgueni Doukhaninea, Anthoula
Lazarisb and Rafal Iwasiowa
aDNA Genotek, Ottawa, Canada; bResearch
Institute-McGill University Health Centre, Montreal, Canada
Introduction: Analytes present in the extracellular fraction of bodily fluids
(ex. blood, urine) have utility as a tool for uncovering the molecular landscape of
tumours
and hold great potential for discovery of individualized cancer medicine. Urine, being
non-invasive as a sample type, has an obvious advantage over blood when used for liquid
biopsy purposes. However, potential for microbial proliferation and the labile nature
of
host cells and extracellular vesicles (EVs) at the point of sample collection/transport
to
the lab drives the need for stabilization of urine samples. Development of such sample
stabilization opens up capability for the detection of various biomarkers present
in the
extracellular fraction to be used in liquid biopsy. This is of particular concern
as studies
around urinary analytes for cancer diagnosis, progression and therapeutic effect are
rapidly
expanding in cohort sizes. Multi-site collections and at-clinic collections are increasingly
prohibitive for large scale recruitment and also lead to variability in the time between
collection and processing.
Methods: In this study, we have analysed two commercially available EV
extraction kits and compared them with ultracentrifugation technique for size, concentration
and specificity of the isolated EVs from human urine samples with and without our
proprietary preservation solution using nanoparticle tracking analysis and western
blot
analysis for exosomal membrane markers. EV RNA contents in various urine fractions
(first
morning first void, random first void and midstream) were compared using RT-qPCR assay
to
provide better understanding of the collection techniques and fractionations that
are ideal
for EV research work.
Results: In our current work, we have bench-marked human urine collection and
EV extraction in order to provide recommendations in standardization of sample acquisition
and processing for urinary EV studies. We have utilized these standardization in order
to
develop a novel and efficient sample stabilization principle for preservation of EVs
and EV
RNA in urine samples during an ambient temperature hold.
Summary/Conclusion: Taken together, we have established a framework for
evaluating technologies and techniques in the EV sample processing space, which can
be
utilized by other research groups.
PS07.10
Vn96-isolated plasma extracellular vesicles improve tumour
mutation detection by next-generation sequencing compared to cell-free DNA and correlate
with tissue biopsy of NSCLC patients
Catherine R. Taylor, Simi Chacko, Jacynthe
Lacroix, Sebastien R. Fournier, Nicolas Crapoulet, Gabriel Wajnberg, Anirban Ghosh,
Stephen
M. Lewis and Rodney J. Ouellette
Atlantic Cancer Research Institute, Moncton, Canada
Introduction: Liquid biopsy is a minimally-invasive diagnostic method that
detects circulating biomarkers and has the potential to improve access to molecular
profiling for NSCLC patients when tissue biopsy material is unavailable or insufficient.
Although isolation of cell-free DNA (cfDNA) from plasma is the standard liquid biopsy
method
for detecting DNA mutations in cancer patients, the sensitivity can be highly variable.
Vn96
is a 27 amino acid peptide with an affinity for heat shock proteins that are exposed
on the
surface of extracellular vesicles (EVs); peptide-EV aggregates readily sediment using
a
benchtop centrifuge and therefore the Vn96 peptide provides a rapid, clinically-amenable
procedure for EV isolation. In this study, we determine whether isolation of EVs from
NSCLC
patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection
compared to cfDNA and correlate genetic changes observed by liquid biopsy with tumour
FFPE
tissue biopsy.
Methods: Blood was collected from stage III/IV NSCLC patients with informed
consent in either EDTA or Cell-Free DNA BCT® collection tubes and plasma was harvested
within 30 minutes. Total nucleic acid (TNA) was extracted from either Vn96-isolated
EVs from
EDTA plasma or directly from plasma collected in EDTA or Cell-Free DNA BCT® tubes
(cfDNA).
SNVs were detected by next-generation sequencing (NGS)
Results: Vn96 isolation of EVs from plasma resulted in higher recovery of DNA
than cfDNA isolation. The SNVs detected in both EV-DNA and cfDNA correlated well with
those
reported in matched FFPE tumour tissue using NGS, including 100% specificity for EGFR
mutations. No improvement in SNV detection was observed using Cell-Free DNA BCT® collection
tubes compared to EDTA tubes. Isolation of EVs with the Vn96 peptide prior to sequencing
improved a number of NGS parameters including library yield, total reads, median read
coverage and molecular coverage, resulting in improved sensitivity of SNV detection.
Summary/Conclusion: In summary, our research demonstrates that Vn96-based EV
isolation is useful for molecular profiling of NSCLC patients for whom tissue biopsy
is not
an option, thereby improving access to molecular profiling and targeted therapies.
Funding: Atlantic Canada Opportunities Agency
PS07.11
Novel markers for neuroendocrine prostate cancer
Divya Bhagiratha, Michael Listonb, Theresa
Akotoa and Sharanjot Sainia
aAugusta University, Augusta, USA; bVeteran
Affairs, San Francisco, USA
Introduction: Prostate cancer (PCa) is fuelled by androgens and androgen
receptor (AR) signalling. Therefore, ablation of AR signalling by Androgen deprivation
therapy (ADT) is the goal of first-line therapy that results in cancer regression
initially.
However, two to three years post-ADT, the disease develops into castration-resistant
prostate cancer (CRPC). As a second-line of therapy, next generation of AR pathway
inhibitors (API) such as Enzalutamide (ENZ) are used that are effective initially
followed
by emergence of drug resistance. A subset of API-resistant tumours emerges to an AR
independent state via undergoing a trans-differentiation to neuroendocrine lineage,
a
process referred to as neuroendocrine differentiation (NED). Due to lack of AR signalling,
these PCa variants, referred to as neuroendocrine prostate cancer (NEPC), are impervious
to
anti-androgen therapy and constitute an aggressive variant of advanced CRPC with poor
prognosis. Currently, there is a lack of effective molecular biomarkers for predicting
API
therapy resistance and emergence of therapy-induced NED.
Methods: Exosomes/EVs were isolated from sera of a patient cohort with/without
NED. The study was conducted in accordance with ethical guidelines of US Common Rule
and was
approved by the institutional committee on human research. Written informed consent
was
obtained from all patients. Following extensive characterization of EVs by electron
microscopy, nanosight tracking analyses and Western blotting of exosomal markers,
small RNA
sequencing was carried out on Illumina HiSeq platform to identify differentially expressed
transcripts. Machine learning algorithms were applied to clinical sequencing data
to train a
“miRNA classifier”. Further, we probed the proteomic profile of exosomes isolated
from NEPC
cellular model NCI-H660 and enzalutamide resistant CRPC cell lines by mass spectrometry.
Results: We identified that transition from CRPC-Adenocarcinomas to
neuroendocrine states is associated with significant EV-miRNA dysregulation, with
a specific
dysregulation in certain miRNA families. With the application of machine learning
algorithm,
we identified an EV-based “molecular classifier” that can robustly stratify CRPC-NE
tumours
from CRPC-Adenocarcinomas. Proteomic analyses identified novel NEPC-specific, glycosylated
proteins that can be exploited for NEPC diagnosis.
Summary/Conclusion: Our data suggest that EV miRNA and protein profile can
predict neuroendocrine differentiation in advanced castration-resistant prostate cancer
patients.
Funding: This work is supported by the US Army Medical Research Acquisition
Activity (USAMRAA) through the Idea Development Award under Award No. W81XWH-18-1-0303
and
the National Cancer Institute at the National Institutes of Health (Grant Number
RO1CA177984).
PS07.12
Exosomal mRNA in diagnosis strategy for hepatocellular
carcinoma
Aleksandr Abramov, Alisa Petkevich, Vadim
Pospelov and Pavel Ogurtsov
Peoples’ Friendship University of Russia (RUDN University), Moscow,
Russia
Introduction: Exosomal cargo is informative source illustrating the genetic
events happening in cells, what can be especially advantageous in case of cancer development
for disease progression or treatment effectiveness monitoring.
Methods: 10 plasma samples of hepatocellular carcinoma (HCC) patients, 10
plasma samples of patients with liver cirrhosis 3–4 on the hepatitis C virus (HCV)
background, 5 healthy donors’ plasma samples. Exosomes were isolated with
ultracentrifugation, western blot (CD63, CD9) was performed. Total mRNA was isolated
with
exosomal RNA isolation kit, Norgen Biotec Corp. Sequencing was carried out on a MinIon
sequencer. Housekeeping genes (GAPDH, B2 M, ACTB, TUBA1A). Detected mutations were
confirmed
by real-time PCR with specific highly sensitive LNA probes.
Results: Significant changes in expression levels were identified for 50 genes
in HCC and liver cirrhosis groups (increasing up to X200 compared to control samples
and
decreasing up to no detected expression). In 6 out of 10 patients with HCC mutant
burden was
significant increased compared to mutant burden in groups with cirrhotic samples.
In 8 out
of 10 patients with HCC increased expression for mRNA LINE-1 was identified compared
to
cirrhotic patients.
Summary/Conclusion: Exosomal mRNA expression levels may serve as a prognostic
and diagnostic marker for patients with liver cirrhosis caused by HCV for HCC risk
development.
Funding: Research is supported with federal funds “5–100”
PS07.13
Circulating extracellular vesicle signatures in small cell lung
cancer
Michela Savianaa
, Giulia
Romanoa, Giovanni Nigitab, Robin Tofta, Patricia
Lea, Kai Wangc, Mario Acunzoa and Patrick
Nana-Sinkama
aVirginia Commonwealth University, Richmond, USA;
bThe Ohio State University, Columbus, USA; cInstitute for Systems
Biology, Seattle, USA
Introduction: Lung cancer is the leading cause of cancer deaths worldwide and
classified primarily as either non-small cell lung cancer (NSCLC) or small cell lung
cancer
(SCLC). Compared to NSCLC, SCLC has a faster growth rate, earlier widespread metastasis,
and
shorter overall survival. The early diagnosis of SCLC and the development of novel
therapeutics have proven challenging. Thus, progression and recurrence rates remain
high.
Non-invasive methods for cancer detection are increasingly being used to inform clinical
decision making. Extracellular vesicles (EVs) have recently emerged as potential carriers
of
genetic contents such as microRNAs (miRs) to induce reprogramming of components of
the
microenvironment in cancer initiation and progression. Moreover, extracellular miRs
expression profiles have been shown to have signatures related to tumour classification,
diagnosis, and progression.
Methods: We selected a cohort of patients divided into 4 groups: high-risk
smokers, adenocarcinomas, squamous carcinomas, and SCLC. We extracted total circulating
EV
and plasma RNA from plasma (38 patients in total) and RNA from plasma in a separate
group
(24 patients in total). Utilizing both next-generation sequencing (NGS) and nanostring
platforms, we analysed for global microRNA (miRs) expression patterns. Candidate miRs
were
then validated by qRT-PCR.
Results: We identified several deregulated miRs in both EVs and plasma of SCLC
patients compared to the other groups. For EVs, we validated miR-1285-5p as a significant
biomarker for the late stage of SCLC compared to controls. In the case of plasma,
we
validated the upregulation of miR-375 in SCLC compared to controls.
Summary/Conclusion: Our results indicate that a potential combination of
plasma (miR-375) and EV-based (miR-1285-5p) miRs be valuable biomarkers for SCLC detection
and serve as a basis for a non-invasive SCLC classifier.
Funding: Virginia Commonwealth University, DOIM – NIH/NCI
PS08: Separation and Concentration
Chair: Annalisa Radeghieri – Department of Molecular and Translational Medicine,
University of Brescia, Brescia, Italy and Consorzio Sistemi a Grande Interfase, Department
of Chemistry, University of Florence
Chair: Aleksandra Gąsecka – 1 st Chair and Department of Cardiology, Medical
University of Warsaw
PS08.01
A fast and reliable in vitro cell culture system reveals that
acidification of bovine milk extracellular vesicles impairs their functionality
Martijn J. C. van Herwijnen, Marije Kleinjan
and Marca H. M. Wauben
Department of Biomolecular Health Sciences, Faculty of Veterinary
Medicine, Utrecht University, Utrecht, Netherlands, Utrecht, Netherlands
Introduction: The isolation of EVs from milk is technically challenging due to
the complexity of milk. Currently used separation procedures allow for the removal
of milk
fat globules and cells (by low speed centrifugation of fresh milk), removal (by
acidification), or disruption (by addition of EDTA) of casein micelles. Using these
protocols the integrity, composition and targeting of bovine milk EVs has been evaluated
and
has led to believe that milk EVs might withstand these conditions. However, the effects
on
functionality of milk EVs (i.e. immunomodulatory properties) after processing and
isolation
have not been studied. Therefore, we have set up an in vitro culture system using
a human T
cell line that allows for the rapid screening of milk EV functionality.
Methods: Fresh bovine milk was defatted and cells were removed after 2x3,000 g
centrifugation, followed by differential centrifugation at 5,000 g and 10,000 g. This
milk
was either subjected to acidification with HCL, or EDTA was added, or the milk supernatant
remained untouched. Top down Optiprep density gradient separation followed by SEC
was used
to further purify EVs. These highly purified milk EVs were added to human Jurkat T
cells,
which were simultaneously stimulated using anti-CD3 and anti-CD28 antibodies. After
24 h T
cell activation was measured by IL-2 cytokine production.
Results: Precipitation or disruption of casein micelles allowed for the
substantial removal of proteins during isolation compared to directly isolated EVs,
which
aids in the purification of milk EVs. In vitro analysis revealed that in the presence
of
directly isolated, or EDTA isolated milk EVs, Jurkat cells were suppressed in their
activation as measured by IL-2 production. Remarkably, EVs isolated from HCL-acidified
milk
were impaired in their suppressive capacity to inhibit IL-2 production.
Summary/Conclusion: Although casein removal from bovine milk greatly improves
purity of isolated milk EVs, the detrimental effects on EV functionality should be
considered. Interestingly, EVs exposed to acidic conditions lost their ability to
modulate T
cell activation, which is in contrast with the general believe that milk EVs could
withstand
the gastro-intestinal tract.
Funding: This work is funded by the European Union’s Horizon 2020 Framework
Programme under the grant FETOPEN-801367 evFOUNDRY.
PS08.02
Optimising methods for separation and characterisation of
extracellular vesicles from skim milk and infant milk formula
Anindya Mukhopadhya, Jessie Santoro, Barry
Moran, Zivile Useckaite and Lorraine O’ Driscoll
Trinity College Dublin, Dublin, Ireland
Introduction: Infant milk formula (IMF) is intended to impart nutrition to
infants, similar to breast milk. However, although industrial IMF production involves
harsh
treatment, potential consequences on extracellular vesicles (EV) in IMF are not yet
established. This study aimed to optimise methods for separating EVs from IMF and
skim milk
(SM) and to characterise the EVs in accordance with MISEV2018.
Methods: SM and IMF were either not treated (NT) or treated with acetic (AA)
or HCl acid (isoelectric precipitation, IP), to remove caseins. Samples were then
subjected
to differential ultracentrifugation (DUC) or gradient ultracentrifugation using iodixanol
solution (GUC). For DUC, 38 mL samples were centrifuged at 12 K g, 35 K g, 75 K g,
100 K g
and 200K g sequentially for 75 min each and pellets re-suspended in 1 mL PBS. For
bottom-up
GUC, increasing iodixanol gradients with 2.3 mL of samples were centrifuged at 186 K
g for
18 h. Fractions were then pooled based on densities (1.1–1.2 g/mL). BCA and SDS-PAGE
were
used to analyse total protein; nanoparticle tracking (NTA) and transmission electron
microscopy (TEM) for EV presence; and immunoblotting and imaging flow cytometry (IFCM)
to
evaluate EV specific markers. (EV-TRACK ID: EV190096).
Results: Immunoblotting showed absence of Actinin4 from all samples, while
CD63 and TSG101 were detected for all samples; apart from IMF_IP. NT_samples were
not
analysed reliably by NTA and IFCM, due to the high concentration of casein micelles
present
(~10^14/mL in milk) that otherwise would be co-counted with EVs. As expected, following
IP,
which most efficiently removed casein micelles, BCA showed that samples had lowest
total
protein. This was confirmed by SDS-PAGE. Thus, most effects were then focused on the
IP
casein-depleted samples. IFCM indicated that, post-GUC, SM_IP EVs had significantly
(P < 0.05) more CD81-positive particles/mL of milk vs all other GUC and 200 KDUC samples.
While there were no significant differences in sizes of EV separated from SM or IMF,
directly comparing the IP pre-treated samples, SM had significantly (P < 0.01) higher
quantities of EVs when compared to IMF. Additionally, TEM indicated that EVs separated
from
SM by GUC were intact with limited background debris, whereas those separated from
SM by DUC
and all IMF EVs were not.
Summary/Conclusion: In conclusion, regardless of the method used, IMF has
fewer intact EVs compared to SM. Also, to obtain purest SM EVs, IP followed by GUC
separation is optimal.
Funding: Dept. Agriculture, Food & Marine, Ireland [17/F/234]
PS08.03
A novel platform to isolate extracellular vesicles based on
surface molecule expression uncovers functional differences between EV
subpopulations
Rowan Frunta, Olivier G. de Jonga, Ioanna
Paspalia, Mark Tielemansa, Raymond M. Schiffelersa,
Pieter Vaderb and Sander A.A. Kooijmansa
aLaboratory of Clinical Chemistry and Haematology, UMC
Utrecht, The Netherlands, Utrecht, Netherlands; bLaboratory of Clinical Chemistry
and Haematology, UMC Utrecht; Department of Experimental Cardiology, UMC Utrecht,
The
Netherlands, Utrecht, Netherlands
Introduction: Extracellular vesicles (EVs) exist as subpopulations with
heterogeneous content. The surface heterogeneity of EVs may reflect differences in
functionality between EV subpopulations, as interactions with recipient cells may
differ
between EV subpopulations with different surface profiles. However, it is currently
challenging to study functional differences between EV subpopulations due to the lack
of
suitable techniques to purify intact EVs based on their surface signature. Here, we
showcase
a novel capture-and-release platform to enrich intact EV subpopulations by their surface
profile and compare their characteristics.
Methods: MDA-MB-231 and SKOV-3 cell-derived EVs were isolated using size
exclusion chromatography. EV subpopulations were enriched based on surface markers
CD9,
CD63, CD81 or phosphatidylserine (PS) using a novel magnetic bead-based capture-and-release
platform. Obtained EVs were characterized by transmission electron microscopy (TEM),
Nanoparticle Tracking Analysis (NTA) and western blotting. EVs were fluorescently
labelled
using PKH67 and CellTracker Deep Red (CTDR) and their uptake by recipient cells was
examined
using flow cytometry.
Results: Western blot analysis showed that EV subpopulations enriched for the
selected tetraspanins and PS were successfully isolated using a novel capture-and-release
platform. Interestingly, EVs isolated based on PS exposure (PS+) lacked most canonical
EV
markers. All EV subpopulations showed intact, cup-shaped morphology when analysed
by TEM,
but contained less protein contaminants compared to the initial EV isolate. PS+ EVs
were
slightly larger than other EV subpopulations when analysed by TEM and NTA. To test
the
capacity of EV subpopulations to interact with recipient cells, EVs were labelled
with PKH67
and CTDR prior to subpopulation fractionation. After fractionation, PS+ EVs showed
a
significantly higher CTDR/PKH67 ratio than other EV subpopulations as determined by
fluorescence spectroscopy, suggesting higher esterase activity of PS+ EVs compared
to other
tested subpopulations. Furthermore, MDA-MB-231-derived EVs isolated based on CD9 and
CD81
expression were taken up more efficiently by HMEC-1 and MDA-MB-231 cells than EVs
isolated
based on presence of CD63 or PS.
Summary/Conclusion: Using a novel technology to isolate EV subpopulations
based on their surface profile, we here show that composition and cellular uptake
efficiency
differs between EV subpopulations. Theoretically, this technology is applicable to
any
surface marker of interest, allowing its use to further establish EV surface-functionality
relationships and enrich EVs with desirable characteristics for therapeutic purposes.
Funding: This work was supported by a VENI grant (no. 17296) of the Dutch
Research Council (NWO).
PS08.04
Preparation of agarose microspheres for high-efficient
separation of extracellular vesicles
Cheng-Tai Chen, Chien-An Chen, Carolyn Yen and
Nien-Tzu Chou
Industrial Technology Research Institute, Chutung, Taiwan (Republic of
China)
Introduction: Size exclusion chromatography (SEC) is becoming a widely used
technique for separating of extracellular vesicles. Various commercially available
products
were launched on the market, however, their separation efficiencies were not fully
disclosed. Herein, novel porous agarose microspheres with the tunable diameter and
pore size
were synthesized by emulsion reaction. The performance was evaluated and compared
with
commercial products. The modified SEC column packing materials were shown to exhibit
advantages for rapid, high-recovery and high-purity separation of extracellular vesicles
from cell culture-conditioned medium and human plasma.
Methods: The homemade SEC column was packed by gravity flow. 100 μL of the
sample was loaded and the PBS buffer was used as eluent. 24 factions were collected
and
analysed by CD9/CD9 sandwich ELISA assay and by micro BCA assay for determining respectively
extracellular vesicles and total protein content.
Results: Agarose microspheres were prepared by emulsification. The particle
size can be controlled by the types and concentrations of surfactants. The product
was
collected by desired screen meshes and used as packing materials of the SEC column.
Our
results showed that the extracellular vesicles were clearly separated from proteins.
More
than 99.8% of proteins were removed while the recovery of extracellular vesicles was
close
to 70%, which is much higher than 32% of the commercial product. The total separation
time
was less than 15 min.
Summary/Conclusion: We have established an approach for generating spherical
agarose microspheres as packing materials of homemade SEC columns, which are capable
of
separating extracellular vesicles from complex samples with high efficiency. Further
validations with additional samples are currently ongoing.
PS08.05
Immunomagnetic Sequential Ultrafiltration (iSUF) platform for
enrichment and purification of extracellular vesicles from large and small volumes
of
biofluid
Eduardo Reategui, Jingjing Zhang, Luong T. H.
Nguyen, Richard Hickey, Nicole Walters and Andre F. Palmer
The Ohio State University, Columbus, USA
Introduction: EVs derived from tumour cells have the potential to provide a
much-needed source of non-invasive molecular biomarkers for liquid biopsies. However,
compromises have to be made when using a particular technology/methodology for the
isolation
of EVs. Currently, there is a trade-off between sample volume and specificity in EV
isolation technologies that limits quantitative molecular analysis of EV contents,
ultimately impacting the utility of EVs in cancer diagnostics. Here, we present an
approach
called immunomagnetic sequential ultrafiltration (iSUF). Our platform combines
ultrafiltration and immunoaffinity separation. Using iSUF, we demonstrate that small
or
large volumes of biofluid can be processed (~ 100 µL or > 100 mL) while concomitantly
removing 99.9 % contaminating proteins. We also processed serum from breast cancer
patients
enabling the characterization of different tumour and immune biomarkers on the isolated
EVs.
Methods: Human samples were collected under an approved IRB. Size distribution
and concentration of EVs were measured using a tunable resistive pulse sensing (TRPS)
method. EV proteins and RNAs were extracted and quantified using a BCA protein assay
and UV
spectroscopy. iSUF and other EV isolation methods were compared for EV concentration,
protein, and RNA quantity.
Results: 50 mL of cell culture media (CCM), 0.5 mL serum, and 100 mL urine
samples were processed with the iSUF platform and recovered in 100 µL. For all cases,
EVs
were enriched with recovery efficiency greater than 95%. The processing time for a
100 mL
sample was 120 min with over 99% of purity. We compared EV concentration and purity
isolate
from 0.5 mL serum using iSUF and other commercially available methods, iSUF demonstrated
superior performance on isolating EVs at high concentrations and purities. Analysis
of total
RNA amounts in the isolated EVs using different methods was corresponding to higher
EV
recovery efficiency of iSUF. We also compared protein and RNA levels of EVs enriched
with
iSUF present in urine and serum samples from the same donors (n = 10), and we found
that for
the same number of EVs, the EV RNA concentration from both biofluids showed no significant
difference. Finally, we have processed serum samples from 10 metastatic breast cancer
patients and demonstrated that their isolated EVs have expression levels of HER2,
CD24 and
miR21 biomarkers at significantly higher levels than healthy controls.
Summary/Conclusion: The iSUF platform can be scale-down or -up to work with
small or large volumes of biofluids for the isolation of EVs. Using the iSUF platform
with
clinical samples shows the potential of our platform to be used for cancer diagnosis
or
monitoring treatment response.
Funding: National Institutes of Health (NIH) grants UG3TR002884 (E.R.); R01HL
126945, R01HL138116, and R01EB021926 (AFP).
PS08.06
Challenges in exosomes isolation from primary biological samples
derived from multiple myeloma patients
Antonia Realea
, Andrew
Spencerb, Tiffany Khongc, Rong Xud, Maoshan
Chene, Richard Simpsond, Ioanna Savvidoue, Malarmathy
Ramachandrane, Sridurga Mithraprabhue, Nicholas Binghame
and David Greeningf
aMonash University, Coburg North, Australia;
bMonash University – Alfred Hospital, Melbourne, Australia; cMonash
University, Melbourne, Australia; dLa Trobe University, Melbourne, Australia;
eMonash University, Melbourne, Australia; fBaker Heart and Diabetes
Institute, Melbourne, Australia
Introduction: Multiple myeloma (MM) remains incurable despite advances in its
treatment and research progress on the crosstalk between MM and surrounding host cells.
Exosomes are important regulators of the cellular niche. Their importance for diagnostic
and
therapeutic applications has been proven in many cancers. In this context we hypothesized
that a better understanding of the molecular role and features of MM-derived exosomes
would
provide a basis for their use for both risk stratification and as predictive biomarkers
of
response to anti-MM drugs already in use in clinical settings, given the optimization
and
validity of their isolation/purification method.
Methods: Exosomes were isolated from human MM cell lines (HMCLs) supernatants
and peripheral blood plasma (PBPL) isolated from healthy donors, MM and MGUS (monoclonal
gammopathy of undetermined significance) patients. Both fresh and frozen samples were
tested. We evaluated 3 commercially-available kits, density-based separation and
ultracentrifugation.
Results: Higher purity and recovery, evaluated by western blotting,
nanoparticle tracking analysis and electron microscopy, were observed for supernatant
density-based purification and for PBPL resin-based isolation.
Exploring the function of MM-derived exosomes, we observed an increase in proliferation
of
the immortalized stromal cell (SC) line HS5 treated with exosomes when compared to
untreated
cells, and a higher increase in proliferation of SCs treated with MM-exosomes when
compared
to exosomes derived from normal and MGUS PBPL samples.
Summary/Conclusion: The method of isolation represents a critical step in the
study of exosomes as many factors can affect the purity, yield and downstream application.
Our data demonstrated that density and resin-based isolation methods provided functional
MM-derived exosomes with proliferative effects on SCs. Altogether our findings may
serve as
a guide to choose exosome isolation methods for MM studies. Further optimization steps,
including albumin-depletion from plasma samples and use/type of serum in cell cultures,
should be taken into consideration when planning proteomics and genomics as downstream
applications.
Funding: Australian Government RTP and Monash Departmental Scholarship.
PS08.07
A rigorous method for exosome isolation from post-mortem
eyes
Mikael Klingeborn, Belinda Hernandez, Nikolai
P. Skiba, Martha A. Cady, Una Kelly, W. Daniel Stamer and Catherine Bowes Rickman
Duke University, Dept. of Ophthalmology, Durham, USA
Introduction: In order to determine and validate the tissue-specific content
of extracellular vesicles (EVs) in biofluids, robust EV isolation methods from tissues
must
be developed. However, to date very few rigorous methods to isolate or enrich for
intact EVs
from tissues have been reported. We present a comprehensive exosome isolation method
with a
sufficient level of characterization to unequivocally demonstrate true EV identity
from ex
vivo eyes.
Methods: Iodixanol (OptiPrep) buoyant density gradient ultracentrifugation
(DGUC), cushioned DGUC (C-DGUC), and our newly developed C-DGUC immunocapture (C-DGUC-IP)
method were used to compare yield and enrichment of exosomes isolated from porcine
eyes
between 3 to 5 hours post-mortem. Yield was assessed by Nanoparticle Tracking Analysis
(NTA)
and immunoblotting for exosomal markers along with total protein quantitation. Enrichment
was assessed by comparison of exosomal markers, ocular-specific markers and known
contaminant markers, plus in-depth proteomic mass spectrometry analyses.
Results: High enrichment of posterior eyecup small EVs (sEV) were achieved by
DGUC and C-DGUC, with C-DGUC resulting in an eightfold increase in yield by NTA and
two to
fivefold increases of exosomal protein markers such as Syntenin-1 and CD81 by
immuno-blotting compared to DGUC. Interestingly, in-depth proteomic analyses revealed
that a
majority of these sEVs with densities of 1.07–1.11 g/ml isolated by DGUC and C-DGUC
were
likely of endoplasmic reticulum (ER) and Golgi origin, suggesting ER-to-Golgi transport
vesicles resulting from post-mortem tissue cell rupture. In order to enrich further
for sEVs
(including exosomes) we subjected sEVs isolated by C-DGUC to anti-CD81 immunocapture.
The
resulting sEV proteome was enriched 1.5- to 4-fold for bona fide sEV and exosome markers
compared to C-DGUC.
Summary/Conclusion: The C-DGUC method provides an enhanced yield and purity of
sEVs and exosomes from ex vivo eye tissue. However, to avoid significant contamination
with
ER and Golgi-derived vesicles from post-mortem eyes, a final EV-specific immunocapture
step
is required to achieve sufficient purity for subsequent analyses. Our highly rigorous
method
paves the way for identification and validation of ocular-derived exosomes in blood
and
their potential use as eye disease biomarkers.
Funding: This work was supported by NIH grants EY026161 (CBR), EY023287 (WDS),
EY022359 (WDS), EY019696 (WDS), a grant from Foundation Fighting Blindness (CBR),
a Research
to Prevent Blindness (RPB)/International Retinal Research Foundation Catalyst Award
(CBR). A
Core Grant for Vision Research (P30; EY005722) from NEI (to Duke University) supported
mass
spectrometric analyses carried out by NPS. In addition, Duke University Department
of
Ophthalmology is supported by an unrestricted grant to the Duke Eye Centre from RPB.
PS08.08
Characterization of the extraction of extracellular vesicles
using a lab-on-a-disc filtration system
Lucile F. Alexandre
a, Philippe
Decorwin-Martinb, Rosalie martelb, Molly L. Shenb, Johan
Renaultb, Lorenna Oliveirab, Andy Ngb and David
Junckerb
aMcGill University, Montréal, Canada; bMcGill
University, Montreal, Canada
Introduction: Personalized treatment for cancer is a promising way to face the
multiplicity of the disease, to increase the efficacy of drugs and to decrease their
toxicity. As part of this strategy, liquid biopsy explores a new non-invasive approach
to
diagnose cancer, guide treatment and monitor its efficacy. Extracellular vesicles
(EVs) are
nanometric lipid bilayers micelles with high potential as biomarkers. They are involved
in
the transfer of information (proteins, RNA and DNA) between cells. EVs include a broad
spectrum of particle sizes, from the tens to thousands of nanometres.
The isolation of EVs from complex matrices is the first step of any protocol and is
particularly important for the reproducibility and fidelity of the results presented,
as it
could bring bias in further analysis. In order to explore the heterogeneity of EVs,
a full
characterization (physical and biological) of the extracted EVs is needed. We evaluate
and
compare 3 EVs purification methods, including ultracentrifugation, size-exclusion
chromatography (SEC) column and an emerging microfluidic technology: LabSpinner filtration
lab-on-a-disc device isolating EVs between two filters of 600 and 20nm.
Methods: A431 cell supernatant was used as a model matrix. We compared three
methods of extraction of EVs: ultracentrifugation with two cycles of 1 h10 at 110,000 g
at 4
degrees Celsius (rotor type 70Ti, Beckman Floor Ultracentrifuge Optima L90 K), qEV
size
exclusion chromatography columns from Izon (qEVoriginal/70 nm) and lab-on-a-disc filtration
system (LabSpinner, Exodisc C). EVs characterization was conducted with NTA
(NanoSightNS500), TRPS (Izon), nanodrop (spectrometerND1000), TEM (FEI Tecnai 12 120
kV) and
custom micro- immuno-assay.
Results: In this study, we characterize a filtration system made of two serial
filters of 600 nm and 20 nm pores for isolation of EVs. Compared to ultracentrifugation
and
chromatography columns, yield of extraction is up to 10 times higher and the size
of the
extracted particles is smaller. TEM imaging was used for assessment of the quality
of the
extracted EVs. However, albumin concentration measurement tends to show that the purity
of
the solution is decreased. The immuno-labelling analysis shows that the proteomic
signature
of the extracted EVs differs according to the extraction methods. The new filtration
technology seems to give us access to a broader range of EVs compared to standard
methods.
Summary/Conclusion: In this study, we characterized 3 purification methods
including lab-on-a-disc filtration, and were able to demonstrate an increase of the
concentration of EVs by a factor of 10, a decrease of the size of the accessible extracted
particles and access to new proteomic signatures.
Funding: We acknowledge the support of Génome Québec and Action Marie
Skłodowska-Curie.
PS08.09
Effects of sample processing on isolation of extracellular
vesicles from blood plasma by centrifugation
Darja Božič
a, Matej
Hočevarb, Veno Kononenkoc, Marko Jerana, Urška
Štiblerd, Immacolata Fiumee, Manca Pajničf, Ljubiša
Pađenf, Ksenija Kogejg, Damjana Drobnec, Ales
Igličh, Gabriella Pocsfalvii, Veronika Kralj-Igličf and
Darja Bozicj
aLaboratory of Clinical Biophysics, Faculty of Health
Sciences, University of Ljubljana, Ljubljana, Slovenia; Laboratory of Physics, Faculty
of
Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia;
bDepartment of Physics and Chemistry of Materials, Institute of Metals and
Technology, Ljubljana, Slovenia, Ljubljana, Slovenia; cDepartment of Biology,
Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia;
dLaboratory of Physics, Faculty of Electrical Engineering, University of
Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia; eExtracellular Vesicles and
Mass Spetrometry Group, Institute of Bioscience and BioResosrces (IBBR) – CNR, Naples,
Italy; fLaboratory of Clinical Biophysics, Faculty of Health Sciences, University
of Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia; gDepartment of Chemistry
and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana,
Ljubljana, Slovenia, Ljubljana, Slovenia; hUniversity of Ljubljana, Ljubljana,
Slovenia; iInstitute of Biosciences and BioResources (IBBR) – National Research
Council (CNR), Naples, Italy; jUniversity of Ljubljana, Faculty of Health
Sciences; University of Ljubljana, Faculty of Electrical Engineering, Ljubljana,
Slovenia
Introduction: The isolation of extracellular vesicles (EV) from body fluids is
still controversial and the poor understanding of vesicle stability and effects of
sample
processing is probably one of the core issues preventing the breakthrough of this
field into
applicative practices.
Methods: We performed an in-depth study of sample changes in blood, blood
plasma and samples throughout the increasing speed of centrifugation, considering
the
number, size, contents and shape of particles in the isolates. Flow cytometry, light
scattering, mass spectrometry and scanning electron microscopy were employed to reveal
the
properties of material in the samples.
Results: The particles of size about 100–500 nm with characteristic topology
of membrane vesicles without internal structure were observed by the scanning electron
microscope only in EV isolates prepared from fresh blood sample. Inspection of the
tube
surface in which the isolation took place suggests that those particles are likely
formed
from activated platelets tearing at the tube wall due to the centrifugal pull.
The isolates prepared from frozen blood plasma prepared by centrifugation with different
forces contained different amounts of particles with similar protein contents, predominated
by highly abundant human plasma proteins, including albumins and immunoglobulins.
Some
lipoprotein clearance and fibronectin precipitation were however observed through
increased
speed and time of centrifugation.
Summary/Conclusion: The results of this study [1] contribute to the
understanding of stability and dynamics of membrane particles. The reported evidence
provides the support for viewing EV isolates as a product, shaped by uniqueness of
the
starting samples and the thermal and mechanical stress applied upon processing. We
believe
this kind of insights strengthen our ability of reading the story of EVs.
Reference:
[1] Božič D., Hočevar M., Kononenko V., et al.: Pursuing mechanisms of extracellular
vesicle formation. Effects of sample processing, Ch 17 in: Biological Membrane Vesicles:
Scientific, Biotechnological and Clinical Considerations 32, Elsevier 2020, in press.
Funding: Authors acknowledge support from the European Union’s Horizon 2020
research and innovation program under grant agreement No. 801338 (VES4US project)
and
Slovenian Research Agency (ARRS. Research core fundings No. P2-0132, P2-0232, P3-0388
and
projects No. Z2-9215, J2-8166, J1-9162).
PS09: EVs in Cancer Pathogenesis
Chair: Liang Dong – The Brady Urological Institute, Johns Hopkins University School
of
Medicine
Chair: Jeffrey Franklin – Vanderbilt University Medical Centre
PS09.01
Imaging extracellular vesicles arising from apoptotic tumour
cells for cancer diagnosis and monitoring
Maria S. Panagopouloua
, Margaret
Patersona, Fabio Nudelmana, Alastair Warkb, David
Birchb and Christopher D. Gregorya
aThe University of Edinburgh, Edinburgh, UK;
bUniversity of Strathclyde, Glasgow, UK
Introduction: Apoptosis is a form of programmed cell death with diverse roles
in the tumour microenvironment and emerging data show that, besides its role in tumour
suppression, it can also promote oncogenic proliferation. Highly aggressive tumours
such as
Burkitt Lymphoma (BL) show high levels of apoptosis, which has a diagnostic and prognostic
value for classifying and staging the disease. We hypothesize that amongst other elements,
extracellular vesicles (EV) are key mediators of apoptotic cell-derived tumour
microenvironment signals. Here, we report on EV released in vitro by apoptotic BL
cells
(Apo-EV) in relation to their potential use as cancer biomarkers.
Methods: Basic physical properties of Apo-EV such as structure, size
distribution, surface charge and membrane fluidity are discussed using Cryo Electron
Microscopy (EM) and Tomography, Nanoparticle Tracking Analysis, Dynamic Light Scattering
and
fluorescence anisotropy respectively. For phenotypic analysis we apply immunocapture
and
flow cytometry, immunogold labelling on transmission EM, fluorescence microscopy and
quantitative PCR. In addition, we study the interaction of Apo-EV with blood components
such
as platelets, leucocytes and red cells, in order to understand their effects in the
circulation and therefore their potential for analysis in blood samples.
Results: Looking at the differences between Apo- and non-Apo-EV, Apo-EV have
larger diameter, while structurally are not different. However, we have identified
distinct
Apo-EV markers such as active caspase 3 and histones, or DNA and small non-coding
RNA-Y.
There is also strong interaction of EV with platelets and leucocytes but not with
red cells,
indicating potential routes of transfer of EV cargo in the circulation.
Summary/Conclusion: It is concluded that for the characterization of the
heterogenous EV populations, combination of multiple techniques is often required,
and also,
understanding the strengths and limitations of each method is essential for choosing
the
appropriate set of analytical tools. Finally, we consider that monitoring free circulating
Apo-EV or blood cells with which they have interacted is a promising approach to improve
cancer diagnosis, prognosis and evaluation of therapeutic response.
Funding: Engineering and Physical Sciences Research Council & Medical
Research Council [grant number EP/L016559/1].
PS09.02
Casting a small netrin: functional roles of a novel surface
factor on stroma-derived extracellular vesicles in pancreatic cancer
Kristopher S. Raghavana
, Ralph
Francesconeb, Janusz Franco-Barrazab and Edna
Cuckiermanb
aDrexel University; Fox Chase Cancer Center, Philadelphia,
USA; bFox Chase Cancer Center, Philadelphia, USA
Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) is a devastating disease
driven and supported by changes in its microenvironment, or stroma. Here we dissect
the
intercellular communication that exists between the primary stromal component,
cancer-associated fibroblasts (CAFs) and PDAC. PDAC communicates with its microenvironment,
in part, through the exchange of specific types of extracellular vesicles (EVs).
Specifically, we focus on the mechanism by which CAF-secreted EVs support PDAC survival,
with an additional goal to identify biomarkers suitable to generate a future “liquid
biopsy”
test for early PDAC detection and prognosis.
Methods: EVs are isolated from Patient-Derived PDAC-associated fibroblasts via
differential ultracentrifugation and validated by ISEV standards. Human PDAC cell
lines used
as recipient cells are treated with CAF-EVs to assess their role in supporting PDAC
survival. Recombinant proteins, neutralizing peptides, and non-functional mutant proteins
are used to block EV interaction with target cells.
Results: We observe sub-types of CAF-EVs containing unique surface receptors.
One EV sub-population of interest contains a novel surface protein (NSP) expressed
on the
plasma membrane of pancreatic CAFs, but not their healthy counterparts. Further, PDAC
cells
up-regulate NSP’s lone binding partner, suggesting a role for these factors in
PDAC-selective EV uptake. Functional assays designed to test PDAC viability suggest
these
NSP(+)-EVs protect PDAC cells from programmed cell death as a result of physiological
stress. This EV-mediated survival benefit can also be inhibited by blocking the interaction
of NSP and its binding partner, suggesting the engagement of these two factors is
necessary
for CAFs to support PDAC via EVs. Pursuing our biomarker goal we confirm stromal NSP
expression increases during early PanIn stages prior to tumour development, and we
are
currently seeking to validate NSP(+)-EVs in blood of PDAC patients.
Summary/Conclusion: This research shines light on a novel mechanism of
tumour-stroma communication that may be crucial for cancer progression during early
disease
stages and a potential target for disrupting the supportive role of the tumour
microenvironment. Additionally, we describe a sub-population of NSP(+)-EVs that have
the
potential to serve as biomarkers for identifying PDAC development.
PS09.03
Exosomes carry distinct miRNAs that drive medulloblastoma
progression
Hannah K. Jacksona
, Franziska
Linkea, Ian Kerrb and Beth Coylec
aChildren’s Brain Tumour Research Centre, School of Medicine,
University of Nottingham Biodiscovery Institute, Nottingham, UK; bSchool of Life
Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK;
cChildren’s Brain Tumour Research Centre, School of Medicine, University of
Nottingham Biodiscovery Institute, Nottingham, UK
Introduction: Extracellular vesicles (EVs) represent an ideal source of
functional biomarkers due to their role in intercellular communication and their ability
to
protect cargo, including RNA, from degradation. The most investigated EV’s are exosomes,
nanovesicles secreted by all cell types and able to cross the blood-brain-barrier.
Here we
characterised the RNA of exosomes isolated from medulloblastoma cell lines, with the
aim of
investigating exosomal RNA cargo as potential functional biomarkers for medulloblastoma.
Methods: Exosomes derived from a panel of matched (original tumour and
metastasis) medulloblastoma cell lines were isolated and characterised by NanoSight,
electron microscopy, western blotting and Nanoscale flow cytometry. Exosomal miRNA
and mRNA
from our matched cell lines and foetal neuronal stem cells, which were used as a normal
control, were analysed by RNA-sequencing technology.
Results: Based on hierarchical clustering, malignant derived exosomes were
distinctly separated from normal control exosomes. miRNA profiling revealed several
established oncomiRs identified in our malignant derived exosomes compared to control
samples. Using interaction pathway analysis, we identified that our malignant exosomes
carry
numerous miRNAs implicated in migration, proliferation, cellular adhesion and tumour
growth.
Several previously identified oncomiRs were also identified to be present at higher
levels
in metastatic exosomes compared to primary and normal, including hsa-miR-455-3p and
hsa-miR-92a-3p.
Summary/Conclusion: This study shows that exosomes from MB cells carry a
distinct miRNA cargo which could enhance medulloblastoma progression. The use of circulating
exosomes as markers of metastatic disease could be an innovative and powerful non-invasive
tool.
Funding: James Tudor Foundation, Children’s Brain Tumour Research Centre, The
University of Nottingham Life Sciences
PS09.04
Leukaemic extracellular vesicles induce inflammatory regulators
and suppress haematopoietic stem and progenitor cell function
Ding-Wen Chena
, Theresa
Mennaa, Jian-Meng Fana, Seul Junga, Angelica
Ortizb, Serge Fuchsb and Peter Kurrea
aComprehensive Bone Marrow Failure Center, Children’s
Hospital of Philadelphia; Perelman School of Medicine, University of Pennsylvania,
Philadelphia, USA; bDepartment of Biomedical Sciences, School of Veterinary
Medicine, University of Pennsylvania, Philadelphia, USA
Introduction: Inflammatory changes in the bone marrow (BM) and suppression of
haematopoietic stem and progenitor cell (HSPC) function during acute myeloid leukaemia
(AML)
significantly contribute to patient morbidity and mortality. Our laboratory has previously
shown that AML-derived extracellular vesicle (EV-AML) trafficking confers a state
of
enforced quiescence and leads to lasting DNA damage in HSPCs. Here we explore the
underlying
cause. Specifically, we hypothesize that EV-AML incite inflammatory regulators as
potential
mediators of DNA damage.
Methods: As a validated model of AML, we utilized the murine TiB49 cell line
as a source of EV-AML. EV-AML were harvested from TiB49 cells cultured in EV-free
medium
using serial ultracentrifugation. HSPC (KSL; Lin-Sca1+ cKit+) clonogenicity and inflammatory
responses were assessed using colony-forming unit (CFU) assay and real-time polymerase-chain
reaction, respectively. IFN-alpha receptor 1 (IFNAR1) expression and intracellular
reactive
oxygen species (ROS) levels were assessed by flow cytometry. DNA damage were assessed
by
quantifying nuclear γ-h2ax using immunofluorescent microscopy.
Results: Similar to EVs derived from AML patients, TiB49 EV-AML elicited
double-stranded breaks in HSPCs, and actively suppressed HSPC clonogenicity. Transcriptional
profiling revealed that exposure to EV-AML induced the upregulation of several inflammatory
mediators in HSPCs, including ISG15, IL-6, IFNα, CH25 H. Inflammatory signalling triggered
by EV-AML did not depend on IFNα signalling as evident from suppression of clonogenicity
in
IFNAR1-null HSPCs as well as the lack of EVs-induced STAT1 phosphorylation or IFNAR1
downregulation. Instead, we found increased levels of ROS following EV-AML exposure.
Summary/Conclusion: Our findings support a model whereby EV-AML inflammatory
signalling and oxidative stress lead to DNA damage in HSPCs.
PS09.05
SARI prevents glioblastoma multiforme progression by inhibiting
the recruitment of myeloid-derived suppressor cells via SARI-bearing extracellular
vesicles
Xin Zhanga
and Lei
Zhengb
aLaboratory Medicine Center, Nanfang Hospital, Southern
Medical University, Guangzhou, Guangdong, 510515, P. R. China, Guangzhou, China (People’s
Republic); bDepartment of Laboratory Medicine, Nanfang Hospital, Southern Medical
University, Guangzhou, 510,515, China, Guangzhou, China (People’s Republic)
Introduction: Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2) is
implicated in inflammatory response and anti-tumour effects. Although the tumour suppressive
function of BATF2 has been reported, its extracellular role in maintaining a non-supportive
cancer microenvironment has not been explored.
Methods: In this study, we established GBM orthotopic and subcutaneous tumour
models in nude and balb/c mice and Flow cytometry analysis determined the BATF2 inhibitory
effects of MDSCs recruiting. We used transwell assay to determine BATF2-positive EVs
(EVs-BATF2) inhibitory of the chemotaxis of myeloid-derived suppressor cells (MDSCs)
in
vitro. In addition, exo-counter detection during the development of the GBM-BATF2
model to
demonstrate EVs-BATF2 crosstalk with distant tissues. AMD3100 blocking in tumour model
confirms that EVs-BATF2 dominated by the SDF-1a/CXCR4 signalling pathway. In addition,
Exo-counter detection of EVs in 32 pairs of gliomas in different stages proposes
plasma-EVsBATF2(plEVs-BATF2) as a prognostic marker.
Results: We found that tumour-derived EVs-BATF2 regulate crosstalk between
glioma cells and tumour microenvironment by inhibiting MDSCs recruitment. EVs-BATF2
can be
detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence
that glioma-derived EVs can communicate with distant site by crossing blood-brain
barrier.
Besides, EVs-BATF2 injection significantly reduced SDF-1α expression in the tumour
tissues.
After blocking SDF-1α signalling by AMD3100, the inhibitory effects of BATF2 overexpression
on MDSCs recruitment were rescued. EVs-BATF2 inhibit MDSCs recruiting and secreting
MMP2,
MMP9, and VEGFA which promote GBM progression. Strikingly, Exo-counter detection of
EVs in
32 pairs of gliomas in different stages reveals that the number of plEVs-BATF2 can
distinguish stage III–IV glioma from stage I–II glioma and healthy donors.
Summary/Conclusion: Our results suggest that EVs-BATF2 may be an effective
circulating biomarker associated with glioma progression. Of note, we are the first
to
determine the regulatory role of EVs-BATF2 in regulating tumour microenvironment and
propose
plEVs-BATF2 as a prognostic marker predicting glioma progression and candidate target
for
GBM therapy.
Funding: National Natural Science Foundation of China (National Science
Foundation of China) – 81,772,939 [Deng]
National Natural Science Foundation of China (National Science Foundation of China)
–
81,902,147 [Zhang]
PS09.06
Desmoglein 2 modulates extracellular vesicle microRNA content in
squamous cell carcinoma
Brianna L. Hilla
, Joseph P.
Flemmingb, Mohammed Haquec, Kenneth Tsaid, Andrew
Overmillere and My Mahoneyf
aThomas Jefferson University, Mohrsville, USA;
bThomas Jefferson University, haddonfield, USA; cThomas Jefferson
University, Lansdale, USA; dMoffitt Cancer Center, Tampa, USA; eThomas
Jefferson University, Bethesda, USA; fThomas Jefferson University, Philadelphia,
USA
Introduction: miRNAs are short, non-coding RNAs that regulate gene expression
post-transcriptionally. Previous work has indicated that miRNAs, notably miR-146a
and
miR-155, play a critical role in SCC tumour development. EVs are membrane-bound vesicles
involved in cell-cell communication carrying actively sorted cargo, protected from
degradation. The potential pathways these vesicular miRNAs modulate and the implication
they
have on cancer biology is under active investigation. We have previously shown that
the
cadherin Dsg2, a stem cell marker, modulates EV release. Dsg2 is upregulated in a
number of
cancers, including SCC, and correlates with poor prognosis. Here we aim to elucidate
the
impact of EV-associated miRNAs in SCCs by bioinformatic analysis.
Methods: SCC cells stably expressing Dsg2 were generated and EVs isolated by
sequential ultracentrifugation. Total cellular and EV RNA was isolated by miRNeasy,
analysed
using RNAseq and identified by GRCh37 alignment. Results were confirmed by qPCR. Altered
pathways based on targets were identified using miRNet and KEGG pathway analysis.
Potential
cancer-associated cytokine targets were confirmed by antibody array.
Results: RNAseq revealed 87 cellular and 15 EV miRNAs that were differentially
expressed in response to Dsg2 with 7 overlapping. The highest altered miRNAs were
validated
by qPCR. KEGG pathway analysis determined that these miRNAs have the highest number
of
shared targets in Cancer, Cell Cycle, and p53 Signalling pathways. Interestingly,
miR-155
was upregulated while miR-146a was dramatically downregulated in EVs. Targets of miR-146a,
ICAM-1, IL-6, and IL-8, cytokines critical for cancer progression were upregulated.
Summary/Conclusion: These results suggest that the miRNA content of EVs is
tightly regulated. By altering the miRNA profile, Dsg2 contributes to the pathogenicity
of
these EVs by increasing levels of cytokines important for cancer stem cell renewal
and
metastasis. In addition, these miRNAs may serve as non-invasive diagnostic markers
for
SCCs.
Funding: NIH R01
PS09.07
Cancer cells grown in 3D release distinct extracellular vesicles
during tumour growth and invasion
Jens C. Luoto, Sara Bengs, Leila Coelho Rato,
Lea Sistonen and Eva Henriksson
Åbo Akademi University, Turku, Finland
Introduction: Cancer cells secrete extracellular vesicles (EVs) that affect
tumour progression. The characteristics of EVs produced during tumour growth and invasion
are however poorly understood. In this study, we identify the composition and
characteristics of EVs produced by non-invasive and invasive tumours and correlate
these
characteristics with the invasive status of the tumour. For that purpose, we established
a
protocol for isolating EVs from extracellular matrix (ECM)-based three-dimensional
(3D)
cancer cell cultures.
Methods: Human prostate cancer PC3 cells were grown in 3D cultures using
ECM-based hydrogel, in standard 2D culture conditions and in bioreactor. EVs were
isolated
from these cultures with differential and density gradient centrifugation. The isolated
EVs
were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting
and mass spectrometry (MS).
Results: Our results demonstrate that 3D ECM-based hydrogel cell cultures
secrete EVs that can be isolated from both the conditioned media and the hydrogel.
The
invasive 3D cultivated PC3 organoids were found to secrete large amounts of EVs compared
to
the non-invasive organoids. Interestingly, our MS results revealed that non-invasive
and
invasive organoids secrete EVs with partially distinct protein cargo.
Summary/Conclusion: We have established a novel protocol for EV production in
a 3D cell culture system utilizing ECM-based hydrogel, in which invasive tumour growth
can
be mimicked. Our method allows the specific isolation and characterization of EVs
derived
from different stages of 3D culture, such as non-invasive and invasive organoids.
Importantly, we found that tumour-derived EVs change in composition during the tumour
progression. Taken together, our method can be used to define the distinct EV
characteristics involved in cancer invasion.
Funding: Åbo Akademi University
Svenska Kulturfonden
K. Albin Johanssons Stiftelse
Magnus Ehrnrooths Stiftelse
PS09.08
Exploring Extracellular Vesicles release inhibition in Prostate
Cancer
Mariadelva Catalanoa
, Niamh
McNameeb, Anindya Mukhopadhyaa and Lorraine O’
Driscolla
aTrinity College Dublin, Dublin, Ireland; bTrinity
College Dublin, Dundalk, Ireland
Introduction: Although pharmacological treatment options are available for
prostate cancer, drug resistance can occur leading to limited survival for patients.
We
previously showed extracellular vesicles (EVs) to be causally involved in transmitting
drug
resistance. This study aimed to evaluate compounds proposed to reduce/block EV release.
Specifically, we selected calpeptin and Y2763 (proposed to inhibit EVs budding from
the cell
membrane) and manumycin A and GW4869 (proposed to inhibit EVs deriving from MVBs).
Associated effects on -and consequences of- EV release were then investigated.
Methods: Suitable compounds concentrations that were non-toxic to cells were
first selected by performing cytotoxicity assay and flow cytometry (FC). Conditioned
medium
(CM) was collected from docetaxel-resistant PC3 (PC3 RD) cells after 48 h incubation
in
dFBS-medium with or without the 4 compounds. EVs were separated from tangential flow
filtration concentrated CM using Optiprep density gradient. 1.03–1.16 g/mL fractions
were
then pooled and washed. EVs were characterised using NTA, immunoblot, TEM and lipid
assay
and FC. Influences on growth and migration, of EVs continuing to be released (at
1x105EVs/3x103cells, 1x107EVs/3x103cells), were evaluated on recipient DU145 and 22Rv1
cells.
EVTRACK ID EV190113, score 88%
Results: Calpeptin and Y27632, alone and in combination, did not significantly
affect quantities of EVs released. However, GW4869 significantly (p < 0.004) increased
quantities of released EVs, of a larger size; very high protein to lipid ratio; and
carrying
GRP94 compared to control EVs (p < 0.006). This effect was reverted when GW4869 was
combined with manumycin A (p < 0.004).
Following all compounds treatments, 1x105EVs/3x103cells inhibited 22 RV1 proliferation
(p < 0.0014), while at 1x107EVs/3x103cells only EVs from manumycin A (p < 0.05) and
Y27632 (p < 0.00036) treated cells reduce 22 RV1 proliferation. EVs following GW4869
treatment significantly (p < 0.001) inhibited DU145 migration compared to bulk
non-treated control and compared to the effect obtained using the entire pool of EVs
(p < 0.001).
Summary/Conclusion: While none of the 4 proposed inhibitors significantly
reduced EV release, the resulting EVs were less potent in transmitting aggressive
behaviour,
such as proliferation and migration, to receiving cell lines.
Funding: H2020-MSCA-ITN-TRAIN-EV grant [722148]
PS09.09
Patient-derived organoids represent a novel tool to study the
effect of intra-tumoral heterogeneity on EV release in non-small cell lung
cancer
Gyöngyvér Orsolya Sándor1; Anikó Zeöld2; Zoltan
Wiener2
1Semmelweis University, Department of Genetics, Cell and
Immunobiology, Molecular Cancer Research Group, Budapest, Hungary; 2Semmelweis
University, Department of Genetics, Cell and Immunobiology, Molecular Cancer Research
Group,
Budapest, Hungary, Budapest, Hungary
Introduction: Lung adenocarcinoma (LUAD) is the leading cause of
cancer-related death with a low 5-year survival. Although the importance of intra-tumoral
cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging,
proper models to study its effects on EV release and cargo in human tissues still
lack. The
3D organoid technology maintains the cellular and genetic heterogeneity of in vivo
tissues
and has proved to be so far the best ex vivo model of human cancers. By using
patient-derived and mouse organoids we set out i) to compare the EV release from normal
and
tumor tissues and ii) to follow changes in EV secretion when the relative ratio of
tumor
cell subpopulations is shifted.
Methods: We used mouse and LUAD patient-derived normal and tumor organoids.
The Medical Research Council of Hungary approved our experiments with human samples
and
informed consent was obtained from patients. EVs were detected by antibody-coated
beads, NTA
and TEM. Intra-organoid heterogeneity was proved by immunostaining and RT-qPCR.
Results: We provide evidence that both mouse and human normal organoids
contain all the bronchiolar cell types. Interestingly, LUAD organoids selected for
TP53
mutation contained not only Ki67+ proliferating cells, but differentiated cell types
as
well. Furthermore, all the lung organoid cultures produced EVs and this was shifted
to the
smaller size range. Interestingly however, when modifying the proportion of organoid
cell
types, we observed an increased EV release when more Ki67+ proliferating cells were
present
both in normal and in LUAD samples.
Summary/Conclusion: Our data show that patient-derived lung organoids
represent a novel model to study the role of intra-tissue heterogeneity in EV functions
in
the humans, leading to improved diagnosis.
Funding: This work was funded by the National Competitiveness and Excellence
program NVKP_16-0007 (National Research, Development and Innovation Office, Hungary)
and by
the National Excellence Program in Higher Education (Ministry of Human Resources,
Hungary).
PS09.10
Exosome mediate heart-adipocyte communication after myocardial
Ischaemia/Reperfusion and impairs adipocyte endocrine function
Yajing Wang, Lu Gan, Dina Xie, Wayne Lau,
Theodore Christopher, Bernard Lopez and Xinliang Ma
Thomas Jefferson University, Philadelphia, USA
Introduction: By incompletely understood mechanisms, MI patients sustain
systemic metabolic disorder. Adipocytes are an important cellular type regulating
energy
homoeostasis. The impact of MI upon adipocyte function remains unknown. Exosomes (Exo)
are
critical vehicles mediating organ-organ communication. However, whether and how Exo
may
mediate post-MI cardiomyocyte/adipocyte communication have not been previously
investigated.
Methods: Adult male mice were subjected to MI/R. Serum Exo were isolated
3 hours after R and incubated with 3T3 L cells for 24 hours. The effects of Exo upon
adipocyte function were determined.
Results: Compared to control, MI/R Exo significantly altered the expression of
17 genes known to be important in adipocyte function. GO analysis revealed that genes
associated with endoplasmic reticulum (ER) function and adipocyte endocrine function
are the
primary two pathways altered by MI/R Exo. Venn analysis identified 11 mi-RNAs as
cardiac-enriched, adipocyte-poor, and ER function-related miRNAs. RT-qPCR confirmed
the
miR-23a/27a/24-2 family members are the most markedly increased mi-RNAs in MI/R Exo.
Incubation of 3T3 L cells with mi-R27a mimic significantly downregulated EDEM3, DsBA-L,
and
PPAR☓, and upregulated PERK and CHOP. Conversely, mi-R27a inhibitor significantly
decreased
the impact of MI/R Exo upon ER function genes. Additional studies demonstrated EDEM3
and
PPARγ (two critical molecules maintaining ER function and adipocyte endocrine function)
to
be direct targets of mi-R27a. One of the most significant endocrine molecules of adipocyte
origin, adiponectin is regulated by PPAR☓ at the transcriptional level and by DsBA-L
at the
post-translational level. We next determined whether MI/R Exo may affect adiponectin
expression/assembly. Incubation of 3T3 L cells with MI/R Exo significantly inhibited
total
and high molecular weight adiponectin expression, an effect blocked by miR27a mimic.
Finally, in vivo administration of GW4869 (Exo biogenesis inhibitor) or miR27a inhibitor
attenuated adipocyte ER dysfunction and restored plasma adiponectin level in MI/R
animals.
Summary/Conclusion: We demonstrate for the first time that MI/R causes
significant adipocyte ER and endocrine dysfunction by Exo mediated cardiomyocyte/adipocyte
communication via miR-23a/27a/24-2.
Funding: NIH and American Diabetes Association
PS09.11
Pancreatic cancer cell extracellular vesicles drastically alter
the behaviour of recipient normal pancreas cells
Charles P. Hinzmana
, Yaoxiang
Lia, Meth Jayatilakea, Jose Trevinob, Partha
Banerjeea and Amrita Cheemaa
aGeorgetown University Medical Center, Washington, USA;
bUniversity of Florida Health Science Center, Gainesville, USA
Introduction: Pancreatic cancer (PaCa) is predicted to become the 3rd leading
cause of cancer-related deaths by 2030. Patients diagnosed with pancreatic ductal
adenocarcinoma (PDAC) have a 5-year survival rate ~9%. Detection of pre-neoplastic
lesions
can potentially improve survival. However, there is currently no screening test for
early
stage detection. Importantly, PaCa tumours are 90% non-tumorigenic cells. A better
understanding of early PaCa oncogenesis is needed. Cancer cells shed extracellular
vesicles
(EVs) that are internalized by neighbouring and distant cells to induce a myriad of
cancer
progression events. We hypothesize that in early PaCa oncogenesis, EVs mediate a behavioural
change in surrounding normal cells, leading to the formation of this unique stroma.
The
purpose of this study was to develop a model to examine the phenotypic changes undergone
by
normal human pancreas cells when they are exposed to PaCa cell EVs.
Methods: EVs were isolated using differential ultracentrifugation with
filtration from established (PANC-1, SW-1990, Capan-1 and MiaPaCa-2) and patient-derived
xenograft (PPCL-46 and PPCL-68) PaCa cell lines. Cells were grown using EV-depleted
FBS. EV
isolations were validated and quantified using transmission electron microscopy,
quantitative ELISA, immunoblot and nanoparticle tracking analysis. Normal pancreas
cells
(hTERT-HPNE and HPDE-H6c7) were co-cultured with cancer cell EVs for 24–48 hours.
Metabolic
activity was measured using a Mito Stress Test on a Seahorse XFe96 Extracellular Flux
Analyser.
Results: We discovered that normal cells undergo vast behavioural
transformations, including significant morphological changes, increased proliferation
and an
uncharacteristic invasive capability, when co-cultured with PaCa cell EVs. These responses
were EV dose dependent. Further, PaCa cell EVs metabolically reprogrammed normal cells,
causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic
and
glycolytic profile.
Summary/Conclusion: Our results indicate that PaCa cell EVs confer enormous
transformational properties to normal human pancreas cells in vitro. We hypothesize
that EVs
impart distinct transformational properties to normal cells in vivo and this influence
could
unveil novel mechanisms regulating cancer onset and progression. These signals may
be
detectable before progression of early-stage PaCa to PDAC, leading to the development
of
assays for earlier diagnosis in patients. Further studies are underway to identify
the
biochemical mediators of these changes.
Funding: Research supported by the National Center For Advancing Translational
Sciences of the National Institutes of Health under Award Number TL1TR001431. Content
is
solely the responsibility of the authors and does not necessarily represent the official
views of the National Institutes of Health.
PS09.12
Plasma extracellular vesicles-miRNAs released by hypoxic cells
are associated to pro-tumorigenic and immunosuppressive microenvironment in lung
cancer
Orazio D. Fortunatoa
, Luca
Rozb, Mavis Mensahb, Massimo Morob, Anna Maria
Ferrettic, Ugo Pastorinob and Gabriella Sozzib
aFondazione IRCCS Istituto Nazionale dei Tumori, Milano,
Italy; bFondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;
cConsiglio Nazionale delle Ricerche, Milan, Italy
Introduction: Extracellular vesicles (EV) containing specific subset of
functional biomolecules, such as microRNAs (miRNAs) are released by all cell types.
It has
been widely demonstrated that EV-miRNAs from cancer cells can manipulate the tumour
microenvironment modulating the gene expression of recipient cells. We previously
identified
a three levels risk classifier (MSC) based on 24 plasma-miRNAs associated with lung
cancer
development and prognosis. The aim of this study was to investigate the potential
role of
EV-24 miRNAs as mediators of pro-tumorigenic features.
Methods: EVs were isolated from plasma of heavy-smoker individuals with high
(MSCpos) or low (MSCneg) risk of lung cancer by differential centrifugation method.
Purity
of EVs isolated was confirmed by sizing using NTA and TEM analysis and expression
of
EV-enriched proteins. miRNA levels were analysed by dPCR. In vitro and in vivo analyses
were
used to assess the biological effect of plasma EVs on different recipient cells.
Results: Levels of miRNAs in EVs correlated with determination of whole plasma
(80% of correlation with MSC classifier). MSCpos-EVs stimulated 2D and 3D proliferation
of
non-tumorigenic epithelial cells through c-myc transfer into recipient cells. Furthermore,
MSCpos-EVs increased the ability of HUVEC to form tubular structures compared to MSCneg-EVs.
In vivo co-injection of MSCpos-EVs-treated HUVEC with A549 lung cancer cells resulted
in an
increase of tumour growth compared to MSCneg-EVs-treated HUVEC. miR-126 modulation
in EVs
with miRNA mimics or in recipient cells using miRNA inhibitors demonstrated that this
miRNA
is implicated in the autocrine proangiogenic modulation of HUVEC phenotype. MSCpos-EVs
induced M2 polarization of macrophages both in vitro and in vivo. We demonstrated
using
synthetic oligonucleotides that miR-320 is responsible for the immunosuppressive modulation
of these cells. Regarding the potential origin of EVs in MSCpos individuals, we observed
that hypoxia stimulated the secretion of EVs containing c-myc from fibroblasts, miR-126-EVs
from endothelial cells and miR-320-EVs from granulocytes.
Summary/Conclusion: These data show that plasma EVs of high-risk individuals
display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic
and immunosuppressive microenvironment suggesting an involvement of EVs in lung cancer
development.
PS09.13
Exploration of EV-associated metastatic targets on melanoma
cells
Isadora F. Semionatto, Jessica M. Toscaro,
Romenia R. Domingues, Bianca A. Pauletti, Adriana P. Leme and Marcio C. Bajgelman
Brazilian Biosciences National Laboratory (LNBio/CNPEM), Brazil,
Campinas, Brazil
Introduction: Cancer cells secrete EVs that may harbour metastatic proteins.
Previous studies have demonstrated the decrease of CD63 tetraspanin expression in
melanoma
cells are correlated with enhancing its metastatic potential. However, other proteins,
such
as CD44, CD47, MET and NRP2 which are overexpressed in melanoma cells, are also associated
with the spread of cancer. In this study, we sought to investigate the expression
of
metastatic proteins in EVs derived from murine melanoma B16F10 lineage.
Methods: B16F10 cells were cultured in DMEM supplemented with 10% FBS and
antibiotics. The cells supernatant were harvested each 24 hours, filtered through
0.22 µm
and ultracentrifuged at 110000 x g for 90 min at 4ºC to pellet EVs. Next, size and
concentration was determined using nanoparticle tracking analyses technique, and the
morphology of EVs were analysed by negative-staining transmission electron microscopy
(TEM).
The EV’s surface protein were characterized by flow cytometry and protein content
was
profiled by mass spectrometry.
Results: Our flow cytometry results have shown the presence of tetraspanins
markers CD63, CD9 and CD81 on vesicle´s surface. In addition, our assay demonstrated
a
diminished expression of CD63 molecule in comparison to CD9 and CD81. We have profiled
melanoma-EVs by mass spectrometry, identifying the presence of proteins that may be
associated to metastasis, such as CD44, CD47, MET and NRP2.
Summary/Conclusion: These preliminary results are consistent with the
literature and suggest that melanoma-derived EVs harbour proteins, which may contribute
to
promote tumour metastasis. In our next step, we plan to generate B16F10 lineages harbouring
shRNA vectors, in order to knockdown gene expression of CD63, CD44, CD47, MET and
NRP2 to
investigate the metastatic potential in vitro, in comparison to parental cells. We
also may
use syngeneic mice models to explore metastatic potential of genetically modified
B16 F10-derived cells.
Funding: Grant #2018/16449-0, São Paulo Research Foundation (FAPESP).
PS09.14
Analysing extracellular vesicles from drug-resistant and
drug-sensitive cancer cells as potential predictive biomarkers in the liquid
biopsy
Sarai Martinez Pacheco and Lorraine O’
Driscoll
Trinity College Dublin, Dublin, Ireland
Introduction: Overexpression of HER2 occurs in ~ 20% of breast cancers and
confers aggressive behaviour and poorer prognosis. Thankfully, a number of drugs such
as
neratinib have been developed to target HER2, potentially providing substantial benefit
for
many patients. Nevertheless, it is estimated that up to 70% of patients with
HER2-overexpressing tumours do not gain benefit, as a result of innate or acquired
drug-resistance. This study aimed to investigate if nano-sized membrane-surrounded
extracellular vesicles (EVs) released from drug-sensitive and drug-resistant cells
reflect
the HER2 status of their cells of origin and thus have potential as minimally-invasive
biomarkers.
Methods: EVs were isolated from conditioned media (CM) of HER2-positive cell
lines (HCC1954 and SKBR3) and their neratinib-resistant counterparts (HCC1954-NR and
SKBR3-NR) that we developed in our laboratory. EVs from CM of a triple-negative breast
cancer (TNBC) cell line variant, Hs578 Ts(i)8, were evaluated as negative control
for HER2.
In brief, CM was centrifuged at 300 g, for 10 min x3 to get rid of any cells. The
supernatant was then centrifuged at 200,000 g for 6 h at 4ºC to collect EVs. The resulting
EVs were washed in PBS, centrifuged as before, and re-suspended in 100 μL PBS. EV
quantities
were estimated by nanoparticle tracking analysis (NTS). EV lysates were characterised
by
immunoblots, for established positive and negative EV markers. Particle concentration
as
well as size distribution of EVs were measured using the ZetaView (Particle Metrix,
Germany). Surface proteins on single EVs were analysed by high-resolution flow cytometry
(FC), using an AMNIS ImageStreamX Mark II. All data was submitted to EV-TRACK (EV-TRACK
ID:
EV190112).
Results: Neratinib-resistant cell line variants were found to have reduced
HER2 protein expression compared to their respective drug-sensitive counterparts.
Neratinib-resistant cell line variants released fewer EVs, when normalised per number
of
secreting cells, compared to their-drug sensitive counterparts. Furthermore, EVs from
drug-sensitive cells carried HER2, while those from drug-resistant cells lacked HER2
(similar to the EVs from the TNBC cells); reflecting the HER2 status of their cells
of
origin.
Summary/Conclusion: This study indicates that a reduction in HER2 protein
expression is a mechanism by which cancer cells manifest resistance to HER2-targeted
drugs
(i.e. by making fewer HER2 receptors available on the cells surface to accommodate
the drugs
activity). Furthermore, EV-carried HER2 seems to reflect the HER2 status of their
cells of
origin. This suggest that analysis of HER2 on EVs in the peripheral circulation may
help
predict response to HER2-targeted drugs. Thus, analysis of EVs in appropriate cohorts
of
patients’ specimens is warranted.
Funding: From H2020-MSCA-ITN TRAIN-EV (722148)
PS09.15
Downregulation of Rab27a expression correlates with replication
restriction of oncolytic herpes virus in tumour cells
Xusha Zhou
a, Lei Wanga,
Weixuan Zoua, Xiaoqing Chenb and Grace Guoying Zhoub
aGuangzhou Medical University, Guangzhou, China (People’s
Republic); bShenzhen International Institute for Biomedical Research, Shenzhen,
China (People’s Republic)
Introduction: Rab27a, a small GTPase involved in exosome biogenesis by
regulating MVE docking at the plasma membrane, and its expression level highly correlated
with oHSV replication ability in vitro. Oncolytic viruses is a newly promising therapeutic
agent for cancer treatment. However, more than 50% of tumours naturally showed highly
resistant to oncolytic viruses for unknown reasons. Uncovering the underlying mechanisms
of
resistance to oHSV can offer potential therapeutic targets to enhance oHSV activity
to kill
tumour cells. In addition, it will give new insights into the identification of therapeutic
biomarkers, which can be used to predict patient response to oHSV in clinical.
Methods: Deep-sequencing data showed that lower expression level of Rab27a is
present in oHSV resistant tumour cells compared to that in sensitive tumour cells.
Then an
oHSV resistant MC38 tumour cell line was established by repeated injections with oHSV
in
MC38 tumour-bear mouse model. Lastly, it was verified that oHSV resistance is associated
with a downregulation of Rab27a and overexpression of Rab27a can rescue oHSV
replication.
Results: 1) The lower expression level of Rab27a is shown in oHSV resistant
tumour cells compared to that in sensitive tumour cells shows in Deep-sequencing data.
2)
Furthermore, we established an oHSV resistant MC38 tumour cell line by repeated injections
with oHSV in MC38 tumour-bear mouse model. Similarly, in oHSV resistant MC38 cell
line,
Rab27a expression was lower than parental MC38 cells. And the release of exosomes
and virus
was decreased in oHSV resistant MC38 cell line. These results were confirmed by Rab27a
siRNA
knockdown. 3) We verified that in oHSV naturally resistant human cancer cell lines,
oHSV
resistance is associated with a downregulation of Rab27a and overexpression of Rab27a
can
rescue oHSV replication.
Summary/Conclusion: Downregulation of Rab27a expression restricts the
efficiency of oHSV replication and the spreading ability of the released progeny virus
which
also provide Rab27a as a potential target and biomarker for oHSV cancer therapy.
Funding: These studies were supported by grants from Shenzhen Overseas
High-Calibre Peacock Foundation KQTD2015071414385495, Shenzhen Science and Innovation
Commission Project Grants JCYJ20170411094933148, JCYJ20180306173333907 to Shenzhen
International Institute for Biomedical Research.
PS09.16
Inactivation of EMILIN-1 by proteolysis and secretion in
extracellular vesicles favours melanoma progression and metastasis
Ana Amor Lopeza, Marina S. Mazariegosa, Marta
Hergueta-Redondob, Alessandra Capuanoc, Pilar
Ximénez-Embúnd, Fatima Al-Shahroura, Javier Muñoze, Eva
Muñozf, Juan Angel Reciog, Diego Megiash, Roberto
Dolianai, Paola Spessotoi and Hector
Peinado
j
aCNIO, Madrid, Spain; bMicroenvironment and
Metastasis Group, Molecular Oncology Programme, Spanish National Cancer Research Centre
(CNIO), Madrid, Spain, Madrid, Spain; cCentro di Riferimento Oncologico di
Aviano, Aviano, Italy; dProteomics Unit – ProteoRed-ISCIII, Spanish National
Cancer Research Centre (CNIO), Madrid 28029, Spain, Madrid, USA; eSpanish
National Cancer Research Centre, Madrid, South Georgia & South Sandwich Islands;
fVall D’Hebron Institute of Oncology (VHIO), Barcelona, Spain; gVall
d’Hebron Research Institute, Barcelona, Spain; hConfocal Microscopy Unit,
Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain;
iCentro di Riferimento Oncologico di Aviano (CRO), Aviano, Italy;
jMicroenvironment and Metastasis Group, Molecular Oncology Program, Spanish
National Cancer Research Center (CNIO), Madrid, Spain, Madrid, Spain
Introduction: Studies have demonstrated that melanoma-derived extracellular
vesicles (EVs) home in distal organs and sentinel lymph nodes favouring metastasis.
Although
lymph node metastases are themselves rarely life threatening, they could be considered
as
one of the first step of metastasis in many cancer types. Therefore, defining the
mechanisms
involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes
could
bring the clue to block the metastatic process from the beginning.
Methods: We have characterized secreted exosomes from a panel of mouse
melanoma models representative of low metastatic potential (B16-F1), high metastatic
potential (B16-F10) and lymph node metastasis (B16-F1 R2). We analysed the RNA expression
in
cells and protein content of exosomes derived from mouse melanoma lymph node metastatic
models by RNA sequencing and mass spectrometry respectively. We validated expression
by
Western-Blot, qPCR and immunofluorescence. To define the mechanism of EMILIN-1 secretion
cells were treated with the EV secretion inhibitor (non-competitive inhibitor of
sphingomyelinase), GW4869. Cell viability and cell cycle assays with an overexpression
model
(B16-F1-EMILIN-1) were also performed. In vivo experiments based on subcutaneous and
intrafootpad injection for studying the role of this protein during melanoma progression
were performed to define the relevance of our findings in vivo. Human paraffin samples
were
analysed for EMILIN-1 expression by immunohistochemistry.
Results: We found a signature of over-expressed genes and hyper-secreted
proteins in exosomes related to lymph node metastasis in the B16 mouse melanoma model.
Among
them, we found that EMILIN-1, a protein with an important function in lymph node physiology,
was hyper-secreted in exosomes. Interestingly, we found that EMILIN-1 is degraded
and
secreted in exosomes as a mechanism favouring metastasis. Further, we found that EMILIN-1
has a tumour suppressive-like role regulating negatively cell migration. Importantly,
our in
vivo studies demonstrate that EMILIN-1 overexpression reduced primary tumour growth
and
metastasis in mouse melanoma models. Analysis in human melanoma samples showed that
cells
expressing high levels of EMILIN-1 are reduced in metastatic lesions.
Summary/Conclusion: Overall, our analysis suggests that the inactivation of
EMILIN-1 by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive
activities in melanoma favouring tumour progression and metastasis.
Funding: This work was supported by grants from MINECO (SAF2014 54541 R),
Asociación Española Contra el Cáncer, Fundación de Investigación Oncológica FERO and
MINECO-Severo Ochoa predoctoral program.
PS10: Diverse EV Biomarkers Chair: Pia Siljander – Faculty of
Biological and Environmental Sciences, University of Helsinki, Finland Chair: Angela
Zivkovic – Department of Nutrition, UC Davis
PS10.01
Raman spectral signature of urinary extracellular vesicles as a
diagnostic biomarker for diabetic kidney disease
Agnieszka Kamińskaa
, Maciej
Romanb, Andrzej Wróbela, Agnieszka Gala-Błądzińskac,
Marek Kuźniewskid, Wojciech Kwiatekb, Czesława
Paluszkiewiczb and Ewa Stępieńa
aDepartment of Medical Physics, M. Smoluchowski Institute of
Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian
University, Kraków, Poland, Kraków, Poland; bInstitute of Nuclear Physics Polish
Academy of Sciences, Kraków, Poland, Kraków, Poland; cDepartment of Internal
Medicine, Nephrology and Endocrinology, St. Queen Jadwiga Clinical District Hospital
No2 in
Rzeszów, Rzeszów, Poland, Rzeszów, Poland; dDepartment of Nephrology,
Jagiellonian University Medical College, Kraków, Poland, Kraków, Poland
Introduction: Diabetic kidney disease (DKD) is a leading cause of chronic
kidney disease. This complication of diabetes develops in about 10% of type 2 diabetes
mellitus (T2DM) patients as a result of chronic hyperglycaemia. Urine is a reach reservoir
of extracellular vesicles (EVs) released by glomerular epithelial cells present in
the
urinary tract. The alterations in molecular content of EVs related to the disease
state make
them promising biomarkers for DKD.
Methods: T2DM patients with different stage of diabetic kidney disease
(n = 24) and healthy subjects (n = 7) were enrolled to the study. Patients were classified
into 4 groups according to the estimated glomerular filtration rate (eGFR CKD-EPI)
level: G2
eGFR = 60–89, G3 eGFR = 30–59, G4 eGFR = 15–29, G5 eGFR <15 ml/min/1.73cm2. The study
protocol was approved by the Jagiellonian University Bioethics Committee (permission
no.
1072.6120.268.2018 from 25 October 2018). Urinary EVs were isolated using low vacuum
filtration method followed by ultracentrifugation. Raman spectra of urinary EVs were
recorded using a Renishaw InVia Raman spectrometer. Data analysis was performed using
Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA). The size
distribution and morphology of EVs were analysed by Transmission Electron Microscopy
and
Nanoparticle Tracking Analysis methods.
Results: Average Raman spectra obtained for urinary EVs from studied groups
showed differences in intensities of specific bands in the region of 400–1800 cm-1.
We found
significant correlations between mean area under curve (AUC) calculated for Raman
bands
(phenylalanine, DNA, Proteins, Lipids and Amide I) and selected clinical parameters
such as:
eGFR, serum creatinine, glucose, urine creatinine. Chemometric methods showed spectral
pattern responsible for separation between studied groups. NTA measurements visualized
EVs
with size of 204.7 ± 96.5 nm.
Summary/Conclusion: Our results showed that characteristic Raman spectra of
urinary EVs are promising candidates for new, non-invasive biomarkers for DKD. Raman
spectra
of urinary EVs can be used to differentiate DKD patients with different stage of kidney
damage.
Funding: This work was supported by the National Centre for Research and
Development (Grant No. LIDER/9/0031/L-9/NCBR/2018 to A. Kamińska).
PS10.02
Isolation of circulating extracellular vesicles and cfDNA allows
for ERBB2 detection in a single aliquot of breast cancer patients plasma
Vera Mugonia
, Caterina
Nardellaa, Yari Ciania, Orsetta Quainia, Michela
Notarangeloa, Mattia Barbareschib, Antonella Ferroc,
Orazio Caffoc, Vito D’Agostinoa and Francesca
Demichelisd
aDepartment of Cellular, Computational and Integrative
Biology (CIBIO), University of Trento, Trento, Italy, Trento, Italy; bDepartment
of Pathology, Santa Chiara Hospital, Trento, Italy, Trento, Italy; cDepartment of
Medical Oncology, Santa Chiara Hospital, Trento, Italy, Trento, Italy;
dUniversity of Trento, Weill Cornell Medical College-New York Presbyterian
Hospital, Trento, Italy
Introduction: The amplification of ERBB2 gene and the consequent
overexpression of the encoded protein HER2 have an important role in breast cancer
classification at diagnosis and subsequent treatment decision with the anti-HER2 monoclonal
antibody Trastuzumab. FISH and IHC have been used so far to detect ERBB2 gene amplification
and HER2 protein overexpression respectively in tissue biopsies. In this context,
a major
goal for liquid biopsies is to take advantage of the information carried by circulating
tumour – derived materials (such as extracellular vesicles (EVs) and cell free DNA
(cfDNA))
to non-invasively detect ERBB2 gene status in the blood. However, the isolation of
diverse
tumour–derived materials from a single aliquot of patients’ plasma and the accurate
detection of cancer biomarkers is still challenging.
Methods: By adopting a recently published nickel – based EVs isolation (NBI)
protocol1 that allows for recovery of cfDNA after EVs isolation, we generated a
high-sensitivity molecular assay to accurately detect ERBB2 amplification and consequent
HER2 overexpression on a limited volume of plasma (1.5 ml) collected from breast cancer
patients (stage I, II and III) at diagnosis.
Results: 1) we detected ERBB2 amplifications by droplet digital PCR (ddPCR) on
the cfDNA isolated from the plasma of ERBB2 positive patients; 2) we confirmed HER2
overexpression on a subset of patients by setting up an antibody–based affinity reaction
designed to detect HER2 protein on the surface of the isolated EVs; 3) we succeeded
in the
quantification of HER2 transcripts enclosed within EVs by performing ddPCR in samples
of
patients showing a range of circulating tumour – derived material. The specificity
and
sensitivity of these novel methodological assays were tested on a cohort of healthy
individuals (n = 20) and on a cohort of HER2 positive (n = 20) or HER2 negative breast
cancer patients (TNBC; n = 20).
Summary/Conclusion: Here we report a pilot study on a novel multimodal method
for ERBB2 detection from a minimal amount of plasma. This approach integrates information
from cfDNA with EVs-derived RNA and proteins analysis. This proof of concept may ultimately
translate into relevant clinical applications for disease diagnosis as well as for
therapy
selection and monitoring of disease progression.
Reference: [1] Notarangelo, M. et al. EBioMedicine 43, 114–126 (2019).
PS11: Single EV Analysis Chair: M. Selim Ünlü – Boston
University, Department of Electrical Engineering Chair: Randy Carney, PhD – Department
of
Biomedical Engineering, University of California
PS11.01
Fluorescence-based strategies for EVs quantification,
characterization and assay development
Mattia Criscuolia
, Diogo
Fortunatob, Francesco Carpic, Laura Bianciardid, Danilo
Mladenoviće, Riccardo Vagof, Stefania Zupponef, Marta
Venturellag, Davide Zoccoc and Natasa Zarovnid
aUniversity of Siena, Via Aldo Moro 2, 53100 Siena, Italy,
Siena, Italy; bUniversity of Siena, Siena, Italy; cExosomics SpA,
Siena, Italy; dExosomics S.p.A., Siena, Italy, Siena, Italy; eTallin
University, Narva Rd 25, 10120 Tallinn, Estonia, Tallin, Estonia; fIRCCS San
Raffaele Scientific Institute, Milan, Italy; gExosomics Spa, 53048 Siena, Italy,
Siena, Italy
Introduction: The biological and medical importance of exosomes recognized
over the last decade has given rise to a crucial need for the discrimination between
true
EVs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris.
Additionally, it is imperative to develop methods to identify and characterize EV
sub-populations. Considering EV biology and the reliability of labelled biomolecules,
we
developed both exogenous and endogenous labelling protocols, taking advantage of different
dye properties which can target a multitude of compartments. This approach reveals
key
aspects of EV structure and integrity.
Methods: Nanosized EVs/exosomes were purified by size exclusion chromatography
(SEC) from model cell lines and human plasma. Diverse dyes were orthogonally evaluated
through different single particle and bulk analysis technologies, such as high-resolution
cytometry, Nanoparticle Tracking Analysis and plate fluorimeter. Concomitant profiling
of
specific EV subpopulations was optimized using antibodies (Abs) against tetraspanins
and
cell type specific targets and assessed by single particle analysis and ELISA. To
assess
specificity of labelling protocols we used specific controls such as recombinant RFP
expressing vesicles and purified lipoproteins.
Results: Our EV staining protocols allowed for high labelling efficiency and
unprecedent EV discrimination, quantification and characterization by combining single
particle analysis and bulk measurements in simple matrices (saline buffers) and in
complex
biofluids (i.e. plasma). Different approaches have diverse and complementary advantages
(costs, capacity, sensitivity, informative readout) for implementation in research
and
diagnostic development flows, directly feeding in-house R&D and QC pipeline.
Summary/Conclusion: Overall, fluorescent EVs are versatile and valuable
tracers that can be applied in the optimization of pre-analytical and purification
protocols, selection of target biomarkers and diagnostic assay calibration and
validation.
Funding: EndeVor (POR-FSE 2014–2020) Region Tuscany and Exosomics R&D
Program.
PS11.02
Subpopulations of tissue-derived extracellular vesicles –
methodological evaluation for vesicle size measurement
Rossella Crescitellia
, Cecilia
Lässerb, Nasibeh Karimic, Aleksander Cvjektkovicc, Roger
Olofsson Bagged and Jan Lötvallc
aKrefting Research Centre, Institute of Medicine, Sahlgrenska
Academy at University of Gothenburg, Götenborg, Sweden; bKrefting Research
Centre, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg,
Sweden; cKrefting Research Centre, Institute of Medicine, Sahlgrenska Academy at
University of Gothenburg, Göteborg, Sweden; dSahlgrenska Cancer Centre,
Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University
of
Gothenburg, Göteborg, Sweden
Introduction: Introduction: Subpopulations of extracellular vesicle (EVs) are
commonly classified by their different size, however, the EV size cut off is still
under
discussion. The aim of the study was to evaluate size range of six EV subpopulations
using
three methods: electron microscopy (EM), nanoparticle tracking analysis (NTA) and
ExoView™.
Methods: Methods: EV subpopulations were isolated from melanoma tissues by a
centrifugation based protocol recently established in our lab. Large and small EVs
(lEVs and
sEVs) were isolated with differential ultracentrifugation and these were further separated
into low and high-density fractions (LD and HD). Size of EV subpopulations was then
evaluated by: EM (N = 8, large EVs and small EVs; N = 2, large and small EVs LD and
HD), NTA
(N = 7) and ExoView™ (N = 1).
Results: Results: Tissue-derived large and small EVs showed difference in size
(mean 142 nm vs 75 nm) when examined by EM, whereas NTA and ExoView™ were unable to
show a
clear difference between the populations (NTA: mean 125.7 nm vs 122 nm, ExoView™:
mean
81.7 nm vs 69.2 nm). After iodixanol density gradients, lEVs-LD were significantly
larger in
size than the sEVs-LD as determined by EM (mean 128.1 nm vs. 67.3 nm) but no difference
was
observed by NTA and ExoView™ (NTA: mean 134.1 nm vs 124.5 nm, ExoView™: mean 71.1 nm
vs
63 nm). HD vesicles from both large and small EVs were the smallest in size when analysed
by
EM (mean size 41 nm vs 34 nm) but no significant difference was observed by NTA and
ExoView™
(NTA: mean 127.8 nm vs 116 nm, ExoView™: mean 98 nm vs 66.4 nm).
Summary/Conclusion: Conclusions: Different methods have different limitations:
EM preparations can shrink vesicles, NTA can only detected vesicles above 70 nm and
ExoView™
only measures vesicles between 50–200 nm. Of the three different methods, EM analysis
of
single vesicles visualized in a significant number of micrographs was the only one
able to
distinguish EV subpopulations by size.
Funding: Funding: Swedish Research Council (K2014-85X-22504-01-3), Swedish
Heart and Lung Foundation (20120528), Swedish Cancer Foundation (CAN2014/844), Knut
och
Alice Wallenberg Foundation.
PS11.03
Imaging of human plasma-derived small-extracellular vesicles
using Transmission Electron Microscopy and Structured Illumination Microscopy
Soraya Moradi Bachillera
, Stefano
Fumagallia, Nicolò Baranzinib, Annalisa Grimaldib,
Pierluigi Quadric, Mauro Tettamantic, Gianluigi Forlonia
and Diego Albania
aDepartment of Neurosciences, Mario Negri Institute for
Pharmacological Research IRCCS, Milan, Italy; bDepartment of Biotechnology and
Life Science, University of Insubria, Varese, Italy; cGeriatric Division,
Mendrisio and Lugano Regional Hospital, Mendrisio, Switzerland
Introduction: Small-extracellular vesicles have an important role in cell
metabolism and cell-to-cell communication. Moreover, sEVs when secreted from cells
are
capable to act as mediators of various neurological diseases. However, sEVs show
heterogeneity and this may impact their functions. Therefore, to characterize sEVs
at a
single-particle level is important to better detail the associated biological activity.
In
this scenario, we innovatively propose the Structured Illumination Microscopy (SIM)
as a
technique able to complement the non-optical methods (Transmission Electron Microscopy,
TEM)
to analyse single sEVs and their markers.
Methods: Human plasma sEVs were separated from healthy cognitive control
(ctrl), Mild Cognitive Impairment (MCI) and demented subjects. The sEVs-containing
pellet
was resuspended in 4% paraformaldehyde or 2% glutaraldehyde solutions. For SIM,
sEVs-enriched preparations were washed with the blocking solution and incubated with
the
primary antibody (L1CAM). The secondary fluolabelled antibody was then added. Between
the
steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge
steps were performed. The image acquisition was done on a Nikon SIM system with a
100x oil
immersion objective. sEVs were imaged with a 3D-SIM acquisition protocol. TEM was
performed
on 300 mesh formvar copper-coated grids. sEVs-enriched preparations were incubated
first
with the blocking solution and then, immunogold-labelled for CD63. Samples were
counterstained with uranyl acetate and observed under a Jeol 1010 EX electron microscope.
Data were recorded with a MORADA digital camera system. Participation to the present
study
was approved by relevant local ethics committee of Mendrisio and Lugano Hospital and
written
informed consents were obtained from subjects.
Results: For SIM methodology, only vesicles in the range from 180 to 190 nm
were detected, as the final resolution achieved was 160 nm. Instead, for TEM, sEVs
under
100 nm were identified. None of these methods provided relevant information about
possible
difference in morphology of the ctrl-, MCI or demented subjects-derived sEVs.
Summary/Conclusion: Even if both methods identified CD63 or L1CAM-positive
vesicles, SIM resolution and the complexity of the protocol represent some disadvantages
respect to TEM, that may be the first choice screening technique for EVs analysis
to be then
completed by SIM for particular tasks.
Funding: Horizon 2020 MSCA-ITN-2015-ETN: Marie Sklodowska-Curie Innovative
Training Networks (ITN-ETN) – “Blood Biomarker-based Diagnostic Tools for Early Stage
Alzheimer’s Disease [BBDIAG – 721281]”.
PS11.04
Fabrication of nanopore structures via conformal metal-film
deposition for EV sensing
kwanjung Kima
, Seung-Min
Hanb, Soo-Hyun Kimc and Sung-Wook Namd
aSchool of Medicine, Kyungpook National University, Daegu,
Republic of Korea, Daegu, Republic of Korea; bSchool of Materials Science and
Engineering, Yeungnam University, Gyengsanbuk-do, Republic of Korea, Daegu, Republic
of
Korea; cSchool of Materials Science and Engineering, Yeungnam University,
Gyengsanbuk-do, Republic of Korea, Dae-gu, Republic of Korea; dDepartment of
Molecular Medicine, School of Medicine, Kyungpook National University, Repulic of
Korea,
Daegu, Republic of Korea
Introduction: Electrofluidics is an emerging technology of combining
electronics and nanofluidics. One important device in electrofluidics is an ion transistor
in which the ionic current through a nanopore is regulated by gate voltage bias. Here,
we
suggest a fabrication method of nanopore by introducing focused ion beam (FIB) and
atomic
layer deposition (ALD) to sense extracellular vesicle (EV) via metal electrode
structures.
Methods: We deposited 100 nm-thick silicon-nitrite layers on both sides of
silicon wafer by low-pressure chemical vapour deposition (LPCVD). We fabricated rectangular
patterns by photolithography followed by reactive ion etching (RIE) on the backside
of the
wafer. Anisotropic silicon etching by KOH was performed. The front side of the chip
was
patterned by photolithography followed by Ti/Au deposition for the fabrication of
electrode
structures. We drilled 50 ~ 100 nm pores in the Si3N4 membrane by FIB. By the ALD
process,
we deposited highly-conformal metal film, either Platinum (Pt) or Ruthenium (Ru) to
shrink
nanopores by a self limiting process.
Results: We expect that the ion current through the nanopore is efficiently
controlled by the gate-surrounding structures. The nanopore ion transistor can be
used to
count the number of EVs.
Summary/Conclusion: we suggest a fabrication method of nanopore ion
transistors by introducing focused ion beam (FIB) and atomic layer deposition (ALD).
This
device will be applicable for single EV sensing.
Funding: This research was supported by the Bio & Medical Technology
Development Program of the National Research Foundation (NRF) funded by the Ministry
of
Science & ICT (2017M3A9G8083382).
PS11.05
Dimensional reduction of multi-parametric flow cytometry data
reveals existence of distinct extracellular vesicle subpopulations
Ariana von Lersnera
, Fabiane
Fernandesb, Bong Hwan Sungc and Andries Zijlstrad
aVanderbilt University, Nashville, USA;
bHogeschool Arnhem en Nijmegen University of Applied Sciences, San Francisco,
USA; cDepartment of Cell and Developmental Biology, Vanderbilt University,
Nashville, USA; dVanderbilt University Medical Center, Nashville, USA
Introduction: Extracellular vesicles (EVs) are key players in cell-cell
communication and increasing evidence has shown that EVs function in cancer by promoting
cancer cell motility and metastasis. Analysing tumour-derived EVs in biofluids is
attractive
because it would be a novel approach to a non-invasive liquid biopsy. Unfortunately,
EVs are
highly heterogeneous. They vary greatly in size, lipid composition, and cargo and
are
difficult to distinguish from other small particles in complex biofluids. We have
developed
a novel flow cytometry method to generate a distinct EV fingerprint to profile biological
specimens.
Methods: EVs from cell culture media (purified and unpurified) and biological
fluids (plasma and urine) were detected by flow cytometry using features on individual
EVs
produced by intrinsic (CD63-pHLuorin) and extrinsic (lipophilic dye, Di-8-ANEPPS,
and
antibodies) fluorescent labels. EV subpopulations were visualized with dimensional
reduction
(t-SNE and UMAP) of 20–150 features that defined the vesicle size, shape, and fluorescent
emission spectra associated with the fluorescent marker. Unsupervised density based
clustering (HDBSCAN) in conjunction with supervised machine learning (XGBoost) was
subsequently used to define subpopulations. We refer to this method as “EV
Fingerprinting”.
Results: EV Fingerprinting was successfully used to detect EVs in complex
biological specimens and trace their differential enrichment through conventional
purification methods. EVs were readily distinguished from protein complexes, lipoproteins
and non-lipid particles. Calibration with externally validated purified EV, as well
as size,
lipid, and fluorescence standards enabled EV Fingerprinting as a rigorous and reproducible
method for resolving heterogenous EV samples. EV Fingerprinting applied to conditioned
medium from tumour cells and biological fluids from cancer patients reveals unique
EV
profiles generated by cancer, further supporting the potential of EV fingerprinting
as a
liquid biopsy.
Summary/Conclusion: Our single-EV analysis approach characterizes whole EV
populations in complex biological fluids without the need for purification, reducing
time
intensive purification protocols and subsequent sample loss, permitting efficient
analysis
of liquid biopsy samples.
PS11.06
Detection and quantification of extracellular vesicles with
cargo protein and RNA Using the Amnis® CellStream® Flow Cytometer
Haley R. Pugsley and Raymond Kong
Luminex Corporation, Seattle, USA
Introduction: Extracellular vesicles (EVs) are membrane-derived structures
that include exosomes, microvesicles, and apoptotic bodies. These EVs – released under
normal physiological conditions as well as in the pathogenesis of neurological, vascular,
haematological, and autoimmune diseases – have been shown to transfer biological molecules
such as protein and RNA between cells, potentially transmitting signals. To understand
more
about these signalling mechanisms, there is a need for detecting and quantifying EVs
with
cargo protein and RNA in a reproducible and reliable manner. However, this has been
challenging due to the small size of EVs (ranging from 30 to 100 nm in diameter),
and the
lack of specific staining reagents.
Methods: Here, we utilize the Amnis® CellStream® Flow Cytometer, which enables
high-throughput flow cytometry with increased sensitivity for detecting small particles.
We
demonstrate that a charge-coupled device (CCD)-based, time-delay-integration image
capturing
system can be used to detect and quantify EVs and their cargo labelled with ExoGlow™-Protein
or ExoGlow™-RNA.
Results: In this study, we show flow cytometry data quantifying EV samples
that have been labelled with cargo markers for proteins and RNA. The EV cargo contents
along
with the appropriate control samples will be shown.
Summary/Conclusion: The CCD based detection of the CellStream Flow Cytometer
has the sensitivity to quantify EVs and their cargo.
PS11.07
Single EV imaging reveals novel EV biomarkers and DNA
cargo
Siobhan M. King
a, Ricardo
Bastosa and Andras Miklosib
aONI, Oxford, UK; bONI (Oxford Nanoimaging Ltd),
Oxford, UK
Introduction: Extracellular Vesicles (EVs) are cell-derived membrane-bound
particles that range in size from 30–1000 nm and carry active molecules such as DNA,
RNA and
proteins. Upon secretion, EVs can execute many biological functions such as initiating
intracellular communication or regulating immune responses. Depending on their origin
EVs
have different characteristics and cargo, making them attractive candidates for early
diagnostic and therapeutic applications. However due to their small size and heterogeneity,
direct visualization and characterization of the surface markers expressed remains
a
challenge since these vesicles are below the resolution limit of standard light
microscopy.
Methods: Here, we describe a method that provides size analysis of single-EVs,
which falls below the diffraction limit of light. This was done with purified EVs,
immunostained using fluorescently labelled primary antibodies raised against EV surface
markers (CD63, CD81, CD9), specific cargo such as DNA and probed for tissue specific
cargo.
Characterization of the molecular content and structural properties of surface-immobilized
EVs was performed using single-molecule localisation microscopy (SMLM) on the Nanoimager
platform.
Results: Multicolour SMLM was used to detect up to three EV biomarkers showing
successful characterization of the molecular signature for different EV subpopulations.
The
distribution of novel components on urinary EVs were visualized for the first time
using
this approach. In addition, SMLM revealed the presence of DNA on both the surface
and also
as a cargo inside EVs isolated from tumour cell culture media, which was validated
using
complementary biochemical characterization.
Summary/Conclusion: SMLM is a powerful technique for single-EV analysis and
characterization. Visualization of single-urinary EVs enabled accurate sizing and
further
insights into novel components expressed on the subpopulation’s membrane surface.
Together,
the data demonstrates that the quantitative abilities of SMLM can significantly enhance
our
understanding of EVs, as structure, phenotypes, and cargoes can now be successfully
resolved.
PS12: EVs in Musculoskeletal System- Bone, Muscle, Tendon Chair:
Aaron James
PS12.01
Extracellular vesicles, microRNA and muscle damage
Jason Lovetta
, Peter
Durcanb and Kathryn Myburghb
aStellenbosch University, Matieland, South Africa;
bStellenbosch Univeristy, Matieland, South Africa
Introduction: Working skeletal muscle is a common site for injury due to
unaccustomed exercise with or without underlying pathology. Direct analysis of SkM
injury
requires invasive tissue biopsies. Circulating extracellular vesicles (EVs) are abundant
in
blood and have been shown to be enriched in microRNA; profiles of which may reflect
the
state of tissues. EVs may therefore serve as a non-invasive indicator of muscle injury
and
regenerative processes in vivo.
Methods: Two consecutive bouts of muscle-damaging exercise (plyometric jumping
and downhill running) were performed by 9 healthy male volunteers. Serum creatine
kinase
(CK) and plasma EVs were analysed at baseline, 2 and 24 hr post-exercise. Perceived
muscle
pain (PMP) was assessed at 2 and 24 hr post-exercise. Large EVs were isolated using
a 10
000 G centrifugation step, and small EVs were isolated using qEV columns. EV-enriched
isolates were visualized using TEM, and size and numbers were quantified using NTA.
Based on
NTA results the highest particle fractions (7–10) were pooled for RNA analysis. qPCR
was
done on plasma, large EVs and small EVs. A group of muscle and immune cell-important
miRs
were analysed by means of normalization to an exogenous control.
Results: CK and PMP increased post-exercise, providing evidence for muscle
damage. TEM revealed an abundant and heterogeneous pool of EVs. A concomitant abundance
of
EVs was seen with NTA (mean = 1.17 x 1010 particles/ml plasma). Mean EV diameters
were
194 ± 22 nm across all timepoints. No change in EV size nor number was seen over time,
however, miR-31 decreased at 24 hr when compared to 2 hr in the small EV isolate only.
Plasma displayed an immediate increase in myomiRs-1 and −206 at 2 hr, which returned
to
baseline at 24 hr. In contrast, myomiRs-208b and 486 remained elevated over the 24 hr
period. MyomiR-133b and 486, as well as immune-miRs, did not change in EVs or plasma
as a
result of the intervention.
Summary/Conclusion: The decrease in miR-31 in small EVs at 24 hr is consistent
with previous data. No decrease in miR-31 in large EVs suggests specific packaging
and hence
a specific response to the muscle damage in small EVs. More changes occurred in plasma
myomiRs suggesting less specific passive leakage into circulation from damaged cell
membranes.
Funding: South African National Research Foundation
PS12.02
Pulsed electromagnetic fields potentiate the paracrine function
of mesenchymal stem cells for cartilage regeneration
Yingnan Wua
, Dinesh
Paratea, Eng Hin Leea, Zheng Yanga and Alfredo
Franco-Obregónb
aNational University of Singpaore Tissue Engineering Program,
YLL School of Medicine., National University of Singapore, Singapore, Singapore;
bBiolonic Currents Electromagnetic Pulsing Systems Laboratory, BICEPS, National
11 University of Singapore, Singapore, Singapore
Introduction: The mesenchymal stem cell (MSC) secretome, via the combined
actions of its plethora of biologically active factors, is capable of orchestrating
the
regenerative responses of numerous tissues by both eliciting and amplifying biological
responses within recipient cells. MSCs are “environmentally-responsive” to local
microenvironmental cues and biophysical perturbations, influencing their differentiation
as
well as secretion of bioactive factors. We have previously shown that exposures of
MSCs to
pulsed electromagnetic fields (PEMFs) enhanced MSC chondrogenesis. Here, we investigate
the
influence of PEMF exposure over the paracrine activity of MSCs and its significance
to
cartilage regeneration. Also, the subsequent extracellular vesicles analysis and isolation
are processed for the understanding of how the PEMFs affect stem cell EVs and consequent
differentiation induction.
Methods: Conditioned medium (CM) was generated from MSCs subjected to either
3D or 2D culturing platforms, with or without PEMF exposure. The paracrine effects
of CM
over chondrocytes and MSC chondrogenesis, migration and proliferation, as well as
the
inflammatory status and induced apoptosis in chondrocytes and MSCs was assessed. The
CMs
which have significant effects during chondrogenesis will be analysed by protein and
miRNA
studies.
Results: We show that the benefits of magnetic field stimulation over
MSC-derived chondrogenesis can be partly ascribed to its ability to modulate the MSC
secretome. MSCs cultured on either 2D or 3D platforms displayed distinct magnetic
sensitivities, whereby MSCs grown in 2D or 3D platforms responded most favourably
to PEMF
exposure at 2 mT and 3 mT amplitudes, respectively. Ten minutes of PEMF exposure was
sufficient to substantially augment the chondrogenic potential of MSC-derived CM generated
from either platform. Furthermore, PEMF-induced CM was capable of enhancing the migration
of
chondrocytes and MSCs as well as mitigating cellular inflammation and apoptosis.
The CMs protein results in the significant promotion chondrogenesis condition showed
an
increase in proliferation and anti-inflammatory cytokines.
Summary/Conclusion: The findings reported here demonstrate that
PEMF-stimulation is capable of modulating the paracrine function of MSCs for the enhancement
and re-establishment of cartilage regeneration in states of cellular stress. The
PEMF-induced modulation of the MSC-derived paracrine function for directed biological
responses 1 in recipient cells or tissues has broad clinical and practical ramifications
with high translational value across numerous clinical application.
PS12.03
Effects of extracellular vesicles from blood derivatives on
osteoarthritic chondrocytes within an inflammation model
Andrea De Lunaa
, Alexander
Otahala, Karina Kramera, Olga Kutenb, Zsombor
Laczab and Stefan Nehrera
aCenter for Regenerative Medicine; Danube University Krems,
Krems, Austria; bOrthosera GmbH, Krems, Austria
Introduction: The degenerative disease osteoarthritis (OA) is one of the
leading causes of disability especially of elderly people. Besides various treatment
options
depending on the severity of the cartilage degradation, the application of blood derived
products such as platelet rich plasma (PRP) are getting more and more popular in clinical
practice due to its high concentration of platelets and the perceived high growth
factor
levels. Drawbacks of using PRP include high donor variability, discrepancies among
preparation protocols and the presence of cells (platelets, leukocytes) which can
evoke
cellular processes, especially inflammation, when injected into the diseased tissue.
One
possibility is to isolate only extracellular vesicles (EVs) from blood derivatives
to
overcome these problems. In the current study the effects of EVs isolated from blood
derivatives on OA chondrocytes within an inflammation model was investigated.
Methods: CD14 positive primary monocytes were isolated from citrate
anticoagulated whole blood by magnetic bead sorting. Monocytes were differentiated
into
resting M0 macrophages and activated into M1 macrophages according to published protocols.
ELISA measurements verified successful differentiation and activation as IL1β and
TNFα
levels increased. As control, THP1 monocytes were used. Patient-derived OA chondrocytes
were
grown in 6 well plates and co-cultivated with activated M1 macrophages which were
seeded
into thincerts and added to the 6 wells representing the inflammation model. Furthermore,
cells were treated for 48 hours with media containing FCS, EV depleted FCS or EVs
isolated
from PRP or hypACT serum.
Results: Successful differentiation and activation of monocytes (THP1 and
primary monocytes) into M1 macrophages was demonstrated by elevated levels of the
inflammatory cytokines IL1β and TNFα. Within the inflammation model (co-culture of
OA
chondrocytes with M1 macrophages), addition of EVs isolated from PRP or hypACT serum
resulted in decreased secretion levels of IL1β and TNFα compared to media supplemented
with
either FCS or EV depleted FCS.
Summary/Conclusion: Taken together, EVs from blood derived products might be
chondroprotective and anti-inflammatory mediators which protect cartilage from being
degraded during OA.
Funding: The work was jointly supported by the European Fund for Regional
Development (EFRE) and the Fund for Economy and Tourism of Lower Austria, grant number
WST3-F-5030664/003-2017.
PS12.04
1α,25(OH)2D3 regulates growth cartilage matrix vesicle
microRNAs
Niels Asmussen, Michael McClure, Zhao Lin, Zvi
Schwartz and Barbara Boyan
Virginia Commonwealth University, Richmond, USA
Introduction: Matrix vesicles (MVs) are small (50–150 nm in diameter) lipid
bound extracellular organelles isolated from calcifying tissues including the growth
zone
(GC) of growth plate cartilage. 1α,25(OH)2 vitamin D3 (1α,25) is a regulator of GC
chondrocytes and the MVs they produce. These MVs are key players in the mineralization
process and are selectively enriched with enzymes and growth factors. We found that
MVs are
also selectively enriched with microRNAs (miR), including miR-22, miR-122 and miR-451.
The
aim of this study was to determine the regulatory role of 1α,25 in the packaging of
miRNA in
MVs by GC cells.
Methods: GC cells were isolated by enzymatic digestion from costochondral GC
cartilage harvested from 5 wk-old male Sprague Dawley rats (IACUC approved). Confluent
fourth passage GC cell cultures were treated with 10–8 M 1α,25 or vehicle for 24 h.
Media
were removed, cell monolayers digested with trypsin and cells and MVs isolated by
differential ultracentrifugation. RNA was precipitated from cells and MVs. Small RNAseq
data
were trimmed, aligned and counted before undergoing differential expression analysis.
Experimental groups had an n = 3 per variable. Significant differences (p < 0.05)
were
determined using R v 3.6.2.
Results: 1α,25 treatment altered expression of 30 MV miRs compared to control
MVs, whereas 5 cell miRNAs were differentially expressed. 62.1% of significantly up
or down
regulated miR found in MVs overlapped between 1α,25 and vehicle groups with the remaining
being uniquely differentially expressed. 1α,25 increased MV miR-150 and decreased
miR-384-3p
two miRs known to regulate osteoblast proliferation (150 increases, 384 decreases).
Summary/Conclusion: 1α,25 regulates GC chondrocyte and MV behaviour and this
study demonstrates that it also impacts the miR packaging within MVs. MiR discovered
in MVs
have been demonstrated to impact chondrocyte behaviour and the present study indicates
that
1α,25 regulates the growth plate through miR delivered by MVs.
Funding: NIH
PS13: Advances in Characterization of EV-Associated Molecules
Chair: John Nolan – Scintillon Institute
PS13.01
Evaluating the impact of culture conditions on human mesenchymal
stromal cell-derived extracellular vesicles molecular fingerprint through FTIR
spectroscopy
Ana Fernandes-Platzgummera
, Maria
Pereirab, Luís Ramalhetec, Sandra Aleixod, Cláudia
Silvaa, Joaquim Cabrala and Cecília Caladoc
aDepartment of Bioengineering and iBB – Institute for
Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa,
Lisboa,
Portugal, Lisboa, Portugal; bDepartment of Bioengineering and iBB – Institute for
Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa,
Lisboa,
Portugal, Llisboa, Portugal; cInstituto Superior de Engenharia de Lisboa, Lisboa,
Portugal; dInstituto Superior de Engenharia de lisboa, Lisboa, Portugal
Introduction: Increasing evidence has proposed extracellular vesicles (EVs) as
mediators of many of the therapeutic features of mesenchymal stromal cells (MSC) that
have
been widely studied in clinical trials over the last years. These EVs have been recognized
as nanocarriers of important biological information, which play a central role in
cell-to-cell communication. In this context, EVs can be used as an alternative to
a
cell-based therapy, with reduced risks. The present work aimed to evaluate the impact
of
different culture conditions on the MSC-derived EVs molecular composition through
Fourier-Transform InfraRed (FTIR) spectroscopy.
Methods: EVs derived from MSC from different sources, expanded in two
different culture media ((xenogeneic -free (XF) vs serum-containing medium (FBS))
were
characterized by FTIR spectroscopy, a highly sensitive, fast and high throughput technique.
Moreover, principal component analysis (PCA) of pre-processed FTIR spectra of purified
EVs
was conducted, enabling the evaluation of the replica variance of the EVs chemical
fingerprint in a reduced dimensionality space. For that, different pre-processing
methods
were studied as baseline correction, standard normal variation and first and second
derivative.
Results: EVs secreted by MSCs cultured with serum-containing medium presented
a more homogenous chemical fingerprint than EVs obtained with XF medium. The regression
vector of the PCA enabled to identified relevant spectral bands that enabled the separation
of samples in the score-plot of the previous analysis. Ratios between these spectral
bands
were determined, since these attenuate artefacts due to cell quantity and baseline
distortions underneath each band. Statistically inference analysis of the ratios of
spectral
bands were conducted, by comparing the equality of the means of the populations using
appropriate hypothesis tests and considering the significance level of 5%. It was
possible
to define ratios of spectral bands, that can be used as biomarkers, enabling the
discrimination of EVs chemical fingerprint in function of the culture medium used
for MSC
expansion and the MSC donor.
Summary/Conclusion: This work is a step forward into understanding how
different culture conditions affect MSC-derived EVs characteristics.
Funding: Fundação para a Ciência e Tecnologia (PTDC/EQU-EQU/31651/2017,
UIDB/04565/2020).
PS13.02
Performance qualification for MicroFlow Cytometers:
understanding technical limitations to improve your research
Desmond Pinka
, Michael
Wonga, Diana Phama, Renjith Pillaia, Leanne
Stifanykb, Sylvia Kochb, Rebecca Hieberta, Oliver
Kenyonc and John lewisa
aNanostics, Edmonton, Canada; bDynalife, Edmonton,
Canada; cApogeeFlow Systems, Hemel hempstead, UK
Introduction: As microflow cytometry and other techniques mature as validated
modalities for analysing extracellular vesicles (EV), there has been a concerted effort
to
improve reproducibility . In order for this reproducibility to occur there has to
be a
critical understanding of advantages and limitations for each technology. For microflow
cytometry, several instruments are available to analyse EVs. Each platform has different
limitations as well as advantages over other platforms. To provide the optimal data
for your
specific research, it is critical to understand the limitations of your platform.
To
accurately define these limitations, a performance qualification (PQ) of your instrument
should be undertaken.
Methods: An Apogee A60 platform was used in these experiments. Experiments
were designed with expected ranges and cut-offs for acceptance criteria.Initial tests
included autosampling of a 96 well plate with either single or double aspiration,
single
sample reproducibility and linearity proportional to flow rate. Other experiments
designed
to show machine performance included minimal time to achieve valid data, sample volume
required for double aspiration, determination of coincidence; detection sensitivity
using a
spiked sample; flow rate stability for extended periods (5–30 minutes). Tests should
also be
performed to determine carryover at a range of sample concentrations. If present,
the means
to remove contaminating samples should be determined. Any performance tests should
be
applicable to any instrument in the field.
Results: Auto-sampling helped demonstrate consistent data; reproducibility of
total events and biomarkers was 2–8% C.V. Detected bead concentrations were linear
with flow
rates between 0.75 and 10.5 uL/min. Double well aspirations provided similar data
with
aspirations between 60–80uL. Valid data was achieved for a low abundant target (~200-400
events/uL) after only 30 s, <10%C.V. Detection sensitivity was determined to be
~1/400,000. Carryover ranges were determined in the presence of nominal unstained
serum. An
optimal number of machine washes was determined. Some membrane stains, such as cell
mask and
CFSE require much more rigorous cleaning to remove stain carryover.
Summary/Conclusion: To improve data reproducibility, performance qualification
of any instrument is key. Operational limitations help define optimal performance
parameters
of any technology. Understanding the types of experiments to perform for your particular
type of characterization technology depends on the requirements you set for your research.
A
good performance test should be applicable to any related instrument in the field.
Funding: Funding provided by Nanostics, the Alberta Cancer Foundation, and
Alberta Innovates.
PS13.03
Operating guidelines to determine the size and concentration of
extracellular vesicles with microfluidic resistive pulse sensing
Michael J. Cimorellia
, Rienk
Nieuwlandb, Edwin van der Polc and Zoltan Vargad
aDepartment of Chemical Engineering, Drexel University,
Philadelphia, USA; Department of Clinical Chemistry, Amsterdam UMC, University of
Amsterdam,
Amsterdam, the Netherlands; Vesicle Observation Center, Amsterdam UMC, University
of
Amsterdam, Amsterdam, the Netherlands, Turnersville, USA; bDepartment of Clinical
Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Vesicle
Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands,
Amsterdam, Netherlands; cDepartment of Clinical Chemistry, Amsterdam UMC,
University of Amsterdam, Amsterdam, the Netherlands; Vesicle Observation Center, Amsterdam
UMC, University of Amsterdam, Amsterdam, the Netherlands; Department of Biomedical
Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands,
Amsterdam, Netherlands; dBiological Nanochemistry Research Group, Institute of
Materials and Environmental Chemistry, Research Centre for Natural Sciences, Budapest,
Hungary, Budapest, Hungary
Introduction: The particle size distribution (PSD) of extracellular vesicles
(EVs) is commonly measured by tunable resistive pulse sensing (TRPS) and nanoparticle
tracking analysis (NTA). Both TRPS and NTA have limitations that hamper the accurate
measurement of the PSD of EVs, specifically in the size range from 50 to 100 nm. An
alternative technique for measuring the PSD of EVs is micro-fluidic resistive pulse
sensing
(MRPS). Because a standard operating procedure (SOP) for characterizing EVs by MRPS
is
absent, we aim to establish a reliable SOP to ensure reproducible PSD measurements
of EVs by
MRPS.
Methods: Measurements (n = 3) of red-blood cell, prostate cancer cell line
supernatant, and human urine and plasma EVs were acquired in 10 × 10 s acquisitions.
Two
microfluidic cartridges were used to study a dynamic range of 50–450 nm. Samples were
diluted into phosphate buffered saline with different concentrations of Tween 20 or
BSA.
Because the excess of particles affects the detection limit, serial dilutions were
performed
to find the optimal dilution for each sample. Data were evaluated using Data Viewer
software.
Results: The optimal dilution was determined for each sample by maximizing the
particle rate and minimizing the measurement time while preserving a robust detection
limit
of 50 or 65 nm. Moreover, we developed a procedure to optimize the peak filter settings
of
Data Viewer by fitting data to normal distributions and identifying threshold values
for
signal-to-noise ratio, symmetry, and transit time within 99% confidence.
Summary/Conclusion: We recommend to use 0.1% w/v BSA in DPBS as sample
diluent, because Tween 20 affects EVs as confirmed by flow cytometry. By using orthogonal
techniques and well-characterized biological test samples, we developed and validated
a SOP
for EV detection by MRPS, thereby making MRPS a valuable tool for EV researchers.
PS13.04
Real-time measurements of extracellular vesicles binding
kinetics achieved through interferometric imaging in a multiplexed microarray
modality
Elisa Chiodia
, George
Daaboulb and M. Selim Ünlüa
aBoston University, Department of Electrical Engineering,
Boston, USA; bNanoView Biosciences, Boston, USA
Introduction: Extracellular vesicles are very promising diagnostic biomarkers.
As a matter of fact, the properties of these biological nanoparticles depend on the
health
conditions of each individual. However, experiments that involve EVs phenotyping are
time
consuming, due to 12 h- or overnight incubations. In order to get accurate results,
maximizing binding efficiency is a necessity; that normally involves ensuring the
saturation
of the capture reaction, which can result in an unnecessarily long incubation time.
With the
ability of label-free kinetic binding measurements using interferometric reflectance
sensing
in a microfluidic chamber, we perform an optimization of the incubation time in different
flow conditions, while demonstrating a new way of multiplexing for real-time EVs specific
capture and detection.
Methods: All the real-time binding measurements were performed with the
Interferometric Reflectance Imaging Sensor (IRIS). IRIS chips were first coated with
an
organic polymer (MCP-2), which provides an active surface for probe immobilization.
Then,
antibodies against CD9, CD81, CD63 markers were spotted at different densities in
a
microarray modality. The chips were then encapsulated with a glass window to form
a
microfluidic chamber that allows for imaging the sensor surface.
Samples of HEK-derived extracellular vesicles were flowed across the sensor surface
in the
IRIS system and real-time images were acquired. Incubation was performed at different
flow
rates, and in static and stop-flow modalities.
Results: In this work, we focus on the specific capture of EVs under different
flow conditions to achieve an optimization of the incubation time. Indeed, through
the
acquisition of real time binding data, we are able to precisely monitor the equilibrium
point of the capture reaction. In this configuration of IRIS, low magnification optics
allow
for simultaneous detection of binding on hundreds of capture ligand spots. Therefore,
surface probes (surface density and specificity) as well as assay conditions can be
optimized. We report on the optimization of antibodies against CD9, CD81, and CD63
markers.
Since the sensor chips are identical to the single-particle detection assays developed
by
Nanoview Biosciences, the optimization of binding assays will directly impact the
phenotyping of individual exosomes.
Summary/Conclusion: Our method proved to be very efficient in optimizing the
most crucial aspects concerning EVs capture – flow conditions, incubation time, surface
density and sample concentration.
Funding: This work was partially funded by European Union’s Horizon 2020
program (grant n° 766466 – INDEX).
PS13.05
Spectral methods for EV detection in diabetic renal
complications
Ewa Stępieńa
, Agnieszka
Kamińskaa, Andrzej Wróbela and Agnieszka
Gala-Błądzińskab
aDepartment of Medical Physics, M. Smoluchowski Institute of
Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian
University, Kraków, Poland, Kraków, Poland; bDepartment of Internal Medicine,
Nephrology and Endocrinology, St. Queen Jadwiga Clinical District Hospital No2 in
Rzeszów,
Rzeszów, Poland, Rzeszów, Poland
Introduction: Diabetes is a life treating diseases extending its impairing
influence on more than 500 billion of people around the world within upcoming 20 years.
The
most harmful complication generating high treatment and social costs is diabetic
nephropathy, which develops in about 10% of patients suffering diabetes. Still we
do not
have an effective and direct prognostic biomarker to diagnose renal complications
in the
primary stage of renal disease.
Methods: Extracellular vesicles were concentrated from diabetic patients’
urine and washed to perform spectral analysis: Fourier Transform Infrared Spectroscopy
(FTIR), based on the molecular absorption of electromagnetic radiation in the infrared
region of the spectrum in a range from 400 cm-1 to 4000 cm-1 and Raman spectroscopy
(RS) as
a technique based on inelastic scattering of monochromatic light. Both techniques
provide
information on the chemical structure of compounds by identifying functional groups
with
high molecular specificity.
Results: Average spectral signature obtained for EVs from urine samples of
patients in the different stage of kidney damage allowed distinguishing specific bands,
representative for Amide (I/II), lipids, cholesterol and nucleic acids. Spectral parameters
correlated with a clinical stage and a commonly used indicator of renal function
(creatinine) in diabetic patients.
Summary/Conclusion: Infrared and Raman spectroscopy are promising tools to
diagnose and monitor renal function in diabetes.
Funding: grant No: 2019/33/B/NZ3/01004
PS13.06
Charge detection mass spectrometry measurements of exosomes and
other extracellular particles enriched from Bovine Milk
Brooke Brown, Xuyao Zeng, Aaron Todd, Lauren
Barnes, Jonathan Trinidad, Milos Novotny, Martin Jarrold and David Clemmer
Indiana University, Bloomington, USA
Introduction: Several existing bioanalytical strategies for purifying and
characterizing exosomes have allowed for fundamental progress to be made. Mixtures
of EVs
can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion
chromatography. But, these processes require large amounts of material that are often
difficult to obtain and many different types of particles have similar sizes and densities.
It is likely that unique subfractions within enriched samples exist, particularly
in complex
biological matrices such as blood, urine or milk which remain difficult to characterize
and
isolate with existing analytical technologies.
Methods: Bovine milk exosomes were isolated via differential
ultracentrifugation and resolubilized in 100 mM ammonium acetate. These data were
recorded
using charge detection mass spectrometry (CDMS). In CDMS, individual particles are
reflected
back and forth through an electrostatic ion trap where they pass through a sensitive
charge
detector. Each time a trapped particle enters and exits the detector, its charge (z)
and
mass-to-charge (m/z) ratio is measured. Mass distributions are generated by multiplying
the
m/z values by the charge measured for each ion and binning the resulting masses.
Results: The masses of particles in a bovine milk extracellular vesicle (EV)
preparation enriched for exosomes were directly determined for the first time by CDMS.
Particle masses and charges span a wide range from m ~ 2 to ~90 MDa and z ~ 50 to
~1300 e
and are highly dependent upon the conditions used to extract and isolate the EVs.
In total,
57,350 particles were detected from eight CDMS measurements. A simple two-dimensional
Gaussian model suggests that eight unique subpopulations of particles may be resolvable
based on charge and mass. Complementary EM and proteomics analyses confirm that samples
are
enriched for exosomes. Particles associated with the S1, S2, and S3 families that
are
centred at ~ 3.5, ~5.9, and ~8.3 MDa, respectively, appear too small to be ascribed
to
exosomes. The remaining 45,229 (79%) particles detected by CDMS are within the mass
range
expected for exosomes. While CDMS measurements are at an early stage of development,
this
approach appears to provide a new physical basis for separating and characterizing
EV
particles.
Summary/Conclusion: This work describes a novel biophysical approach for
measuring and characterizing the masses and charges of the extremely heterogenous
population
of exosomes and other extracellular particles enriched in bovine milk. As new sample
preparation methods, aimed at purifying specific types of exosomes from different
cell
lines, tissues, and other body fluids continue to evolve, rapid and sensitive CDMS
measurements of the physical properties of mass and charge may become an important
means of
assessing the efficacy of different protocols.
Funding: NIH (R01 GM131100-01). BAB is supported by Indiana University
Quantitative Chemical Biology Fellowship (T32GM131994).
PS13.07
In situ detection of exosomal microRNA-10b by fusion with
liposome-encapsulated nanomotor
Bo Lia
, Weilun Pana and
Lei Zhengb
aDepartment of Laboratory Medicine, Nanfang Hospital,
Southern Medical University, Guangzhou, China (People’s Republic); bDepartment of
Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515,
China, guangzhou, China (People’s Republic)
Introduction: Breast cancer is the most common cause of cancer-associated
death in women and has raised global health concerns. Early diagnosis and treatment
are
crucial to improve the prognosis and survival rate of breast cancer patients. Liquid
biopsy
is expected to provide a strategy for early diagnosis of breast cancer. Exosomes have
been
regarded as novel liquid biopsy biomarkers due to their stable cargo of RNAs, lipids,
and
proteins from their origin cells. Exosomal micro(mi)RNAs have recently been recognized
as
promising indicators of cancer occurrence and progression. However, most of the reported
exosomal miRNA detection methods require the lysis or extraction process, which increases
the possibility of sample loss. In situ detection strategies avoid interference from
body
fluid. In this study, we developed a gold nanomotor fluorescence platform based on
liposome
fusion for breast cancer exosomal miRNA in situ detection.
Methods: The exosomal miRNA detection platform was constructed using a gold
nanomotor (detector) and liposomes (carrier). The DNAzyme amplification sequences
which
could be especially triggered by miRNA-10b were identified by SDS-PAGE before modified
on
gold nano-motor and the capacity of the nanomotor was assessed using synthetic target
sequence, breast cancer cell MDA-MB-231, miRNA-10b-encapsulated anionic liposomes,
and
miRNA-10b-expressing exosomes. Three kinds of liposomes were synthesized, characterized,
and
assessed for loading ability. Membrane fusion effect was evaluated by confocal laser
scanning microscopy (CLSM) and nano-flow cytometry. The performance of this method
to
discriminate between breast cancer patients and healthy individuals was investigated.
Results: The chosen DNAzyme amplification sequences transformed “locked”
status to “cleavable” status on target addition, releasing a fluorescence signal.
The
modified gold nanomotor showed a ten times higher fluorescence signal in the presence
of
miRNA-10b than the background and no noticeable fluorescence changes from a
single-base-mismatch sequence. Moreover, among the three different liposomes, cationic
liposomes exhibited great stability, high loading efficiency, and excellent membrane
fusion
effect. Furthermore, the fluorescent experiments confirmed that cationic liposomes
could
load and transfer the nanomotors into exosomes for miRNA-10b detection. Finally, we
were
able to distinguish breast cancer patients and healthy individuals by sensing exosomal
miRNA-10b directly from plasma samples without exosome isolation.
Summary/Conclusion: A separation-free and sensitive assay based on DNAzyme
amplification technique and membrane fusion effect was established for breast cancer-derived
exosomal miRNA-10b detection, which could be a promising tool for the liquid biopsy
of
breast cancer.
PS13.08
Isolation of exosomes by membrane affinity column increases
non-exosomal RNA recovery in comparison to differential ultracentrifugation
Maria M. Aguilar-Hernandez, Jessica Zalapa
Soto, Gabriela Galicia García, Mariana Rotzinger Rodriguez, Antonio Casas Aguilar,
Gilberto
Gutierrez García, Andrea Izquierdo Medina and Alvaro Aguayo González
Instituto Nacional de Nutricion Salvador Zubiran, Mexico City,
Mexico
Introduction: Exosome-based liquid biopsy is a potential aid in the diagnosis
and prognosis of cancer patients. However, in order to incorporate exosomes into clinical
routine, there is a need to compare different isolation methods. Here we analysed
the
impact, in exosomal RNA yield, of two intermediate recovery/intermediate specificity
methods: differential ultracentrifugation (UCD) and a membrane-affinity column (MAC)
kit.
Although MAC has a faster performance which is more suitable to the clinic, we found
that
UCD results in a higher recovery of exosomes and less contaminating non-exosomal RNA.
Methods: Exosomes were enriched by MAC and UCD from identical volumes of human
plasma (12,000xg, 34 min/0.22 )m filtration/110,000xg, 5 h)(n = 7) and lymphoma conditioned
medium(300xg, 10 min/200xg, 20 min/1000xg, 30 min/120,000xg 1.5 h/120,000xg, 1 h)
(n = 3).
All exosomes were characterized by nanoparticle tracking analysis (NTA), immunoblotting
of
CD63/CD9/Flotilin/Alix and electron microscopy (TEM). Exosome pellets were pre-treated
with
proteinase K (1 mg/ml/56°C/10 min) and RNAse A (2 mg/ml/37°C/20 min) before
phenol-chloroform/glycogen RNA extraction. RNA yield was measured by both fluorometer
and
bioanalyzer.
Results: Isolation of exosomes by UCD, in both plasma and medium, resulted in
a higher yield in comparison to MAC. This was shown by an augmented intensity of marker
bands in the UCD samples (p = 0.005, n = 4) as well as by an increased number of exosomes
in
TEM. In contrast, MAC final exosomal fraction (from both plasma and medium), resulted
in a
13-fold and 200-fold increase in RNA, respectively, in comparison to UCD when measured
by
fluorometer. This was confirmed by bioanalyzer. To further investigate if the isolated
RNA
was inside exosomes, MAC and UCD final exosomal fractions were treated with enzymes
before
RNA extraction. Importantly, no significant effect was observed in RNA yield.
Summary/Conclusion: Together this data shows that MAC isolates fewer exosomal
structures than UCD; however, it shows a greater recovery of total RNA thus suggesting
that
non-exosomal RNA is getting trapped by MAC. The fact that RNA showed resistance to
RNAse A
cleavage might result in the inclusion of contaminating RNA into the sample.
Funding: FOSISS CONACYT 2017
PS13.09
Fluorescent Extracellular Vesicle characterisation using a long
lasting cell tracker probe
Cass Whelana
, Keith
Morrisb, Aled Reesc and Philip Jamesb
aCardiff Metropolitan University, Ely, UK;
bCardiff Metropolitan University, Cardiff, UK; cUniversity Hospital of
South Wales, Cardiff, UK
Introduction: The visualisation of extracellular vesicles (EV) via fluorescent
means presents a promising method of characterisation. There is potential to specifically
label individual EV families in order to distinguish them in samples of multiple EV
subgroups. Nevertheless, fluorescent EV labelling has proven difficult. Nanosight
NTA© is
the standard for visualising EV and does possess means of fluorescence capture.
Methods: This method utilised a cell division probe consisting of 5-(and
6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) and anhydrous DMSO, forming
a
non-specific long-lasting fluorescent probe typically used for in vivo cell tracking
that is
internalised by cells. Human vascular endothelial cells (HECV) at confluence were
introduced
to the cell tracker at a concentration of 10uM, before cells were incubated in serum
free
media at 21% or 1% oxygen, respectively. This fluorescence was visualised using a
488 nm
fluorescent Nanosight© NTA laser under light scatter and fluorescent means of capture.
Results: The use of a CFSE Cell Tracker Probe proved that EVs were able to
take up the probe from their parent cells. EVs from the cells with the tracker probe
internalised, also contained the probe and exhibited fluorescence.The probe was long
enough
lasting to be identified on Nanosight and has shown promise with no significant difference
in EV number when compared to light scatter.
Summary/Conclusion: Identifying a potentially cost effective method for
visualising EV and combating photo-bleaching, the cell tracker proves that the technique
works on a functional level. Further investigation is needed to determine if this
can be
used to specifically label and identify individual EV families in a population. This
could
allow for the numbers of individual EV families to be shown in a given sample.
Funding: This PhD is solely funded by Cardiff Metropolitan University.
PS13.10
Multiplex electrochemiluminescence immunoassays for phenotyping
of intact extracellular vesicles (EVs)
David A. Routenberga
, Sigal
Shachara, Siddhartha Paulb, Alex Tucker-Schwartza, Abir
Malika and Jacob Wohlstadtera
aMeso Scale Diagnostics, LLC., Rockville, USA;
bAmerican Type Culture Collection, Gaithersburg, USA
Introduction: There is a need for better techniques for characterizing EV
populations. We developed a sensitive multiplexed electrochemiluminescence (ECL)-based
assay
format to characterize EVs in cell-conditioned medium (CCM) and human biofluids. Here
we use
the format to analyse EV samples for the presence of 66 EV surface proteins, and to
identify
changes in EV phenotype associated with different cell lines, purification methods
and
growth conditions.
Methods: Multiplex plates were prepared on MSD’s U-PLEX® platform with
antibodies for 66 putative EV-surface proteins. Each well displayed an array of nine
specific capture antibodies and a negative control antibody. EVs from samples were
captured
on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (CD9,
CD63
and CD81) conjugated to an ECL label. Three distinct cell types were grown at two
sites, MSD
and ATCC. Resulting CCM were each purified by four common methods: tangential flow
filtration, PEG-based precipitation, size-exclusion chromatography and centrifugal
ultrafiltration. All samples were also assayed without purification.
Results: Fifty-five of the surface markers were detected on intact EVs from at
least one evaluated cell type. Datasets were analysed using correlation matrices,
hierarchical clustering, and machine learning. For each cell type, when comparing
unpurified
CCM grown at different sites or EVs prepared by different purification methods, we
typically
observed correlations above 0.9, indicating that the purification methods did not
introduce
bias to EV phenotypes, and that the assay format can provide robust phenotypic information
without any purification of EVs. Two unsupervised clustering analyses – hierarchical
clustering and t-distributed stochastic neighbour embedding – both generated well-separated
clusters for each of the cell types, regardless of purification method or source.
Summary/Conclusion: We developed multiplex EV surface marker assays and
demonstrated their use for multimarker EV phenotyping. This flexible format enables
rapid
assay development for new EV subpopulations with or without sample purification. These
results also demonstrate EV surface marker phenotyping via multiplex ECL assays may
be used
to distinguish EV populations from various cell types, and characterize bias introduced
by
purification.
PS13.11
Detection of MISEV recommended EV protein-markers using
automated western blotting
Mikkel Noerholma, Lisa
Meyerb
, Daniel Enderleb, Carsten Lueckc, Anne
Bickela, Yasef Kahnd, Chris Hegere, Martin
Schlumpbergerf, Markus Sprenger-Hausselg and Johan
Skogh
aExosome Diagnostics GmbH, Martinsried, Germany;
bExosome Diagnostics, Martinsried, Germany; cProteinSimple, a
Bio-Techne brand, Abingdon, UK; dBio–Techne, San Jose, USA;
eProteinSimple, a Bio-techne brand, San Jose, USA; fQiagen GmbH,
Qiagen Str.1, Hilden, Germany, Hilden, Germany; gQiagen, Hilden, Germany;
hExosome Diagnostics, Inc, Waltham, USA
Introduction: The limited amount of material and the diverse methods for
isolation of extracellular vesicles (EV) pose unique challenges to proper characterization
of experimental EV preparations. The “Minimal Information for Studies of Extracellular
Vesicles” (MISEV) guidelines recommend characterizing preparations for both trans-membrane-,
cytosolic- and contaminating non-EV proteins. However, compliance with these guidelines
can
be a considerable effort due to lack of easy and robust analytical protocols and the
time
consuming and user variable nature of standard western blotting protocols. Here we
present a
simple method for isolation of EVs and a simple western blotting platform for automated
protein separation and immunodetection of MISEV-recommended proteins.
Methods: Total EVs were isolated by affinity-membrane spin columns from
pre-filtered 0.5–4 mL plasma or 2–20 mL urine, respectively. Intact vesicles were
eluted and
the EV-depleted biofluid fraction was collected from the flow-through. A small fraction
(4 μL) was analysed by a simple western blot workflow providing automated capillary
electrophoresis-based protein separation and immunodetection, characterizing each
fraction
for presence or absence of MISEV-recommended proteins.
Results: A range of specific antibodies were identified and the EV fractions
were shown to be enriched in EV-proteins, whereas contaminating non-EV proteins were
significantly reduced. Isolation of EVs was necessary to allow detection of the low
abundant
EV protein markers, whereas non-EV proteins were readily detectable both in the neat
biofluids and in the EV-depleted flow-through. We characterized the effect of washing
on the
purity of EV isolates and defined the dynamic range of the workflow using titrations
of
input volume of both plasma and urine EV isolations.
Summary/Conclusion: Simple western blotting protocols were established for
quality control of isolated EVs in accordance with MISEV-guidelines. EVs isolated
using
affinity-membrane spin columns were shown to be enriched in EV markers and depleted
for
non-EV proteins.
PS13.12
AL-PHA beads: a library of extracellular vesicle-associated
metalloproteinase biosensors
Richard Kelwick
a, Alexander
Webba, Yizhou Wanga, Amelie Heliota and Paul
Freemontb
aImperial College London, London, UK; bThe London
DNA Foundry, Imperial College London, London, UK
Introduction: Lung cancer is a leading cause of cancer-associated deaths and
early detection could lead to improved patient outcomes. Extracellular vesicles (EVs)
are
readily accessible from patient bio-fluids and could be a source of early-stage lung
cancer
biomarkers. Recent studies indicate that EVs contain metalloproteinases. The matrix
metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAMs) and a disintegrin
and
metalloproteinase with thrombospondin motifs (ADAMTSs) are highly promising cancer
biomarker
candidates that have complex roles in cancer pathogenesis and metastasis. Importantly,
within the context of lung cancer, the detection of ADAM10 proteolytic activity might
be
more informative than the level of ADAM10 protein. Therefore, the development of low-cost
metalloproteinase biosensors could serve as useful biomarker research tools.
Methods: To this end, we developed Advanced proteoLytic detector
PolyHydroxyAlkanoates (AL-PHA) beads – a library of biodegradable, biopolymer-based
protease
biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are
bound
to microbially manufactured bioplastic beads. These PhaC-fusions also incorporate
specific
protease cleavage sites. In the presence of a specific protease, reporter proteins
are
cleaved off of the AL-PHA beads – resulting in a loss of bead fluorescence that can
be
measured using flow cytometry. These biosensors were assayed using either
metalloproteinases, conditioned media or EVs from in vitro cancer models.
Results: Human metalloproteinase recognition motifs were identified in the
literature and a total of 70 different AL-PHA bead biosensors were designed. A control,
TEV-specific biosensor detected 0.5 U of tobacco etch virus protease activity and
the MMP14
biosensor successfully detected 0.033 mU of recombinant MMP14 activity. A panel of
AL-PHA
biosensors also detected an array of MMP, ADAM and ADAMTS proteases within cell conditioned
media and isolated EV samples from in vitro cancer models.
Summary/Conclusion: AL-PHA biosensors successfully detected EV-associated
metalloproteinases and in the longer-term, we envision that AL-PHA beads may lead
to the
development of low-cost cancer diagnostics for use in resource-limited settings.
Funding: RK is supported by the Cancer Research UK Imperial Centre Development
Fund and until recently a BBSRC-funded RSE Enterprise Fellowship. AW and PF are supported
by
the UK Government’s Global Challenges Research Fund (GCRF) through the EPSRC grant
[EP/P028519/1], as part of the WISER project. We also acknowledge the support of Imperial
Confidence in Concept (MRC/EPSRC), Imperial College London EPSRC Impact Acceleration
Account
(EP/R511547/1), EPSRC grant (EP/L011573/1), and BBSRC grant (BB/L027852/1).
PS14: Scholarship and Funding Opportunities Chair: Lucia
Languino – Professor of Cancer Biology, Thomas Jefferson University Chair: Alissa
Weaver –
Professor, Department of Cell and Developmental Biology, Vanderbilt University School
of
Medicine
PS14.01
Program to assess the rigour and reproducibility of
extracellular vesicle-derived analytes for cancer detection
Matthew Young and Sudhir Srivastava
National Cancer Institute, Rockville, USA
Introduction: Cancer cells release more EVs than normal cells and EVs secreted
from tumour cells can promote tumour progression, survival, invasion and angiogenesis.
The
EV cargo may mirror the altered molecular state of the cell of origin. Therefore,
EVs have
potential for the development of non-invasive markers for early detection of cancers.
EVs
and their cargo also have the potential to be multiplexed with other molecular markers
or
screening modalities (e.g., imaging) to develop integrated molecular-based computational
tools for the early detection of cancer.
One challenge with using EVs as a biomarker is the lack of robust and reproducible
methods
for the isolation of a pure vesicular population. There is a lack of clear consensus
for an
optimal method of isolation of a pure EV population that is devoid of contamination
with
similar-sized vesicles of different origins. There is also a lack of standards to
ensure
rigour reproducibility.
Methods: The current funding opportunity announcement (FOA), PAR20-053, is
promoting research on the isolation and characterization of extracellular vesicles
(EVs) and
their cargo for the discovery of biomarkers to predict cancer and cancer risk.
Results: The previous cycle of this FOA, PAR16-267/277, successfully funded 7
R01 and 4 R21 grants. These awards are focused on proteomics profiling of EVs, effect
of
methodological and biological variability, asymmetric-flow field-flow technology,
therapeutic monitoring, LSS and SERS lab on a chip optical spectroscopic, EVs in
obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular
heterogeneity, urinary EV DNA, and EV markers in paediatric cancers.
Progress from these awardees have shown separation of two discernible exosome
subpopulations and identified a distinct nanoparticle, the exomere (Nature Cell Biology,
2018); and have shown that large-EVs contain the entire genome of the cell of origin,
including cancer-specific genomic alterations (Journal of Extracellular Vesicles,
2019).
Protocols that critically evaluate and refine the existing methodologies to improve
utilization of EVs in clinical use have been shared (Nature Protocols, 2019).
Summary/Conclusion: Drs. Sudhir Srivastava and Matthew Young are the programme
directors for the PAR which began accepting applications on 5 January 2020. This and
other
EV funding opportunities will be discussed.
Funding: This is a Funding Opportunity Announcement offered by the National
Cancer Institute.
PS15 = OP3 Oral with Poster Session 3: Neurological & ID
Chair: Jereme Spiers – La Trobe University Chair: Sophie Rome – INRAE, department
of Human
Nutrition
PS15.01 = OP3.01
Mitovesicles: a new extracellular vesicle of mitochondrial
origin altered in ageing and neurodegeneration
Pasquale D’Acunzoa
, Tal
Hargashb, Yohan Kimb, Rocío Pérez-Gonzálezc, Chelsea
Millerb, Melissa J. Alldredb, Chris Goulbourneb, Hediye
Erdjument-Bromaged, Monika Pawlikb, Mitsuo Saitoe, Mariko
Saitof, Stephen D. Ginsbergb, Thomas Neubertg and Efrat
Levyb
aCenter for Dementia Research, Nathan S. Kline Institute, New
York, USA; bCenter for Dementia Research, Nathan S. Kline Institute, Orangeburg,
USA; cCIBERNED, Hospital de la Santa Creu i Sant Pau (Barcelona, Spain),
Orangeburg, USA; dDept. Cell Biology, New York University School of Medicine, New
York, USA; eDivision of Analytical Psychopharmacology, Nathan S. Kline Institute,
Orangeburg, USA; fDivision of Neurochemistry, Nathan S. Kline Institute,
Orangeburg, USA; gCell Biology, New York University School of Medicine, New York,
USA
Introduction: Brain extracellular vesicles (EVs) are heterogenous and include
previously described microvesicles and exosomes. Herein we characterized a formerly
unappreciated population of mitochondria-derived EVs that we term “mitovesicles”.
Mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative
disorders as Down syndrome (DS). Hence, we examined mitovesicle levels and cargo under
these
conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial
stressors.
Methods: Employing a high-resolution density gradient, distinct and novel
populations of EVs were isolated from murine and human DS and diploid control post-mortem
brains or from cell media. Morphometric EV features were analysed by nanoparticle
tracking
analysis and cryogenic electron microscopy, while EV constituents were characterized
by
Western blotting, mass spectrometry, lipid profiling and mitochondrial RNA qPCR.
Results: We identified a population of double-membrane, electron-dense brain
EVs containing multiple mitochondrial markers (“mitovesicles”) that are highly distinct
from
microvesicles and exosomes. Proteomic data show that mitovesicles contain a unique
subset of
mitochondrial proteins while lacking others, such as Tom20. Mitovesicles have a lipid
composition that is unlike that of previously described EVs and is consistent with
mitochondrial origin. Functionally, the complex-III inhibitor antimycin-A stimulated
in
vitro mitovesicle release into the cell media, suggesting an interrelationship between
mitochondrial dysfunction and mitovesicle biology. In mouse brains, mitovesicle levels
increased with age and were found to be higher in DS compared to diploid controls.
Mitochondrial RNA and protein levels were also altered in DS compared to diploid
controls.
Summary/Conclusion: We describe a previously unidentified type of
metabolically competent EVs of mitochondrial origin that we designate mitovesicles.
Our data
demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal
conditions and are modified during pathophysiological processes in which mitochondrial
dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player
in
mitochondria quality control and may have a role in the trans-cellular tissue response
to
oxidative stress.
Funding: AG017617
AG056732
PS15.02 = OP3.02
Reducing extracellular vesicle release with a novel neutral
sphingomyelinase 2 inhibitor for the treatment of Alzheimer’s disease
Carolyn Tallona
, Kristen
Hollingerb, Benjamin Bellb, Michal Salac, Ranjeet
Dashb, Ajit Thomasb, Amrita Datta Chaudhurib, Asit
Kumarb, Lyndah Lovellb, Ying Wub, Rana Raisb,
Norman Haugheyd, Radim Nenckae, Camilo Rojasb and Barbara
Slusherb
aJohns Hopkins University, Baltimore, USA; bJohns
Hopkins University School of Medicine, Baltimore, USA; cAcademy of Sciences of
the Czech Republic, Prague, Czech Republic; dDepartment of Neurology, Johns
Hopkins University School of Medicine, Baltimore, USA; eJohns Hopkins University
School of Medicine, Prague, Czech Republic
Introduction: Alzheimer’s disease (AD) is a devastating neurodegenerative
disease leading to progressive memory loss and ultimately death with limited therapeutic
options. Growing evidence supports the theory that toxic proteins, like tau and amyloid,
may
propagate from diseased cells by packaging toxic proteins into extracellular vesicles
(EVs)
and releasing them to infect other cells. One enzyme involved in the biogenesis of
EVs is
neutral sphingomyelinase 2 (nSMase2), which catalyzes the hydrolysis of sphingomyelin
to
produce phosphorylcholine and ceramide. Several groups have reported improved cognition
and
reduced tau propagation when nSMase2 is pharmacologically inhibited or genetically
knocked
down in AD mouse models. Unfortunately, current nSMase2 inhibitors are not suitable
for
clinical development due to poor solubility and inadequate pharmacokinetic profiles.
Methods: Our group carried out a high-throughput screening campaign followed
by extensive medicinal chemistry efforts leading to the discovery of phenyl
(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b] pyridazin-8-yl) pyrrolidin-3-yl)
carbamate (PDDC), an orally active, nM potent inhibitor with excellent selectivity
and brain
penetration. We tested PDDC’s ability to inhibit exosome release in cultured primary
glial
cells as well as an in vivo model of acute EV release. We then treated 5XFAD mice
with
10 mg/kg of PDDC daily for six months and monitored their behaviour in the fear conditioning
assay.
Results: PDDC dose dependently reduced EV release from cultured primary glial
cells and significantly reduced plasma EV numbers in an in vivo model. Following chronic
treatment with PDDC, 5XFAD mice demonstrated significantly improved cognitive function
in
the fear conditioning assay.
Summary/Conclusion: These promising findings are currently being expanded
using mouse models of tau propagation. If successful, these data would support PDDC
as a
novel compound for targeting the pathological spread of tau as a therapeutic for AD.
PS15.03 = OP3.03
Profiling EVs in the anterior cingulate cortex of individuals
with major depressive disorder
Pascal Ibrahima
, Zahia
Aouabedb, Corina Nagyb and Gustavo Tureckic
aIntegrated Program in Neuroscience, McGill University,
Montreal, Canada; bDouglas Hospital Research Centre, Montreal, Canada;
cDepartment of Psychiatry, McGill University, Montreal, Canada
Introduction: Major Depressive Disorder (MDD) is one of the leading causes of
disability worldwide, affecting 20% of the population. The environment has been thought
to
play a role in the disease development, resulting in biological changes mediated by
epigenetic mechanisms. MicroRNA’s (miRNA) are well known epigenetic regulators that
are
disrupted in the depressed brain, and they are packaged into extracellular vesicles
(EVs).
EVs have emerged as means of intercellular communication, a process that is also disrupted
in MDD. They are thought to transfer miRNA between cells, which can alter gene expression
in
recipient cells. Therefore, we hypothesize that EV cargo is altered in MDD subjects
compared
to healthy controls (HC). The aim is to extract EVs from human post-mortem anterior
cingulate cortex, a region previously associated with depression, and profile the
miRNA
cargo and compare it between MDD subjects and HC.
Methods: Post-mortem human brain tissue from the anterior cingulate cortex of
20 MDD subjects and 20 HC was mildly dissociated in the presence of collagenase type
III.
Residual tissue, cells, and large vesicles were eliminated, and EVs were isolated
using size
exclusion chromatography. The quality was assessed by western blots and transmission
electron microscopy (TEM). RNA was extracted and a small-RNA library was constructed
and
sequenced using the Illumina Platform. Differential expression analysis was then
performed.
Results: Western blots showed little to no Endoplasmic Reticulum (Calnexin),
Golgi (BiP), or mitochondrial (VDAC) contamination, along with enrichment of the exosomal
marker CD9. TEM images showed the typical cup-shaped morphology with sizes mostly
between 30
and 200 nm. Preliminary sequencing results revealed that miR-33a-5p, which is predicted
to
target glutamate receptors, is downregulated in EVs from MDD subjects.
Summary/Conclusion: High quality EV extractions can be obtained from
post-mortem brain tissue using our method. This will be the first study to profile
brain-derived EV miRNA in the context of depression. Future studies will be needed
to
determine the effect of the different levels of miR-33a-5p. This could provide novel
mechanistic insights into the pathophysiology of MDD and will serve as a starting
point to
examine the potential role of EVs in MDD pathology.
Funding: Réseau québécois sur le suicide, les troubles de l’humeur et les
troubles associés (RQSHA) Student Award
McGill Faculty of Medicine Internal Studentship Award (Max E. Binz Fellowship)
PS15.04 = OP3.04
Combining nanomagnetic isolation and artificial intelligence to
guide the treatment of traumatic brain injury
Zijian Yanga
, Kryshawna
Beardb, David Meaneyb, Danielle Sandsmarkb, Ramon
Diaz-Arrastiaa and David Issadorec
aUniversity of Pennsylvania, Philadelphia, USA;
bUniversity of Pennsylvania, philadelphia, USA; cDepartment of
Bioengineering, University of Pennsylvania, Philadelphia, USA
Introduction: Traumatic brain injury (TBI) is characterized by diverse primary
mechanisms of injury that lead to the development of secondary pathological cascades
that
drive neurological deficit post-TBI. Inability to separate patients based on the presence
of
these different endophenotypes represents a major challenge for diagnosis and treatment
of
TBI.
Extracellular vesicles including exosomes isolated from patient plasma have emerged
as
promising potential biomarkers for TBI due to their ability to cross the BBB into
systemic
circulation with molecular cargo intact for analysis. We have developed a novel microfluidic
platform for rapid isolation of brain-derived EVs providing a tool with which the
biochemical state of neurons and glia can be directly assessed post-TBI. We used the
ultra-sensitive, single molecule array (SIMOA) to quantify concentrations of 7 protein
biomarkers from the plasma and brain derived EVs from mild TBI patients and controls.
By
combining multiple protein biomarkers, we could discriminate mTBI patients from controls
in
both the training and the blinded test set.
Building on this work, we are also characterizing single EV heterogeneity of neuron
derived
EVs by developing novel droplet based digital assay for single EV quantification at
ultra-low concentration. Droplet based assay for single EV analysis would potentially
be
very informative for early disease diagnosis and therapy decision.
Methods: Our microfluidic platform for EV isolation consists of tracked-etched
membranes with millions of nanopores (600 nm), coated with a magnetic film (NiFe)
to
precisely capture immunomagnetically labelled brain-specific EVs from plasma. Single
molecule array (SIMOA) was used to quantify concentrations of the 7 protein biomarkers
(Tau,
UCHL-1, NFL, GFAP, IL6, IL10, and TNF) in the plasma and brain-derived exosomes of
mild TBI
(mTBI) patients and controls. To identify single EV, we applied droplet based enzyme-linked
immunosorbent assay and encoded the fluorescent signal for single EV quantification
within
parallelized microfluidic platform.
Results: We report that concentrations of plasma and exosome GFAP, NFL, and
UCHL1 were elevated in mTBI patients compared to controls (p < 0.05), and that each
of
these biomarkers are uncorrelated with one another. Discrimination of mTBI patients
from
controls was most accurate when machine learning algorithms on the panel of biomarkers.
Specifically, combining plasma NFL, GFAP, IL6 and TNF- with Tau from GluR2+ EVs showed
88%
accuracy with 80% sensitivity and 100% specificity.
Summary/Conclusion: This data suggests that neuron-derived exosomes contain
information that characterizes the injured and recovering brain. It also suggests
that
analysis of a panel of biomarkers from a combination of both blood and exosomal compartments
could lead to more accurate diagnosis of mTBIs.
PS15.05 = OP3.05
L1CAM is not associated with extracellular vesicles in
cerebrospinal fluid or plasma
Maia Norman, Dmitry Ter-Ovanesyan, Wendy Trieu
and David Walt
Wyss Institute, Boston, USA
Introduction: Neurons in living psychiatric and neurological patients are
inaccessible for cell type specific analysis of RNA and protein. Our understanding
of these
diseases instead relies upon imperfect sources of biochemical information such as
post-mortem brain tissue analysis and animal models. Furthermore, there is a paucity
of
biochemical assays available to diagnose and manage brain diseases. Extracellular
vesicles
(EVs) present an opportunity to noninvasively sample the contents of neurons in
cerebrospinal fluid (CSF) and plasma. In order to isolate neuron-derived EVs (NDEVs),
a cell
type specific transmembrane protein is necessary for immunocapture. L1CAM, a protein
abundant on the surface of neurons, has been used extensively in the literature for
NDEV
isolation. However, L1CAM exists in humans in several isoforms without a transmembrane
domain, and as such it can be secreted as a free protein. Additionally, the ectodomain
of
L1CAM can be cleaved off of the cell surface in physiological processes. It remains
to be
demonstrated whether the L1CAM found in CSF and plasma is EV associated, or if it
is instead
a spliced or cleaved isoform behaving as a free protein.
Methods: Using Single Molecule Arrays (Simoa), a digital form of ELISA, as
well as Western blotting, we quantify EV markers (CD9, CD63 and CD81) as well as L1CAM
and
Albumin. We use these assays to determine in which fractions of size exclusion
chromatography (SEC) and density gradient the L1CAM appears. We also immunocapture
L1CAM
from CSF and plasma and perform Western blots for the internal and external domains
of
L1CAM.
Results: Simoa and Western blot analysis of SEC and density gradient fractions
demonstrated that while the EV markers peaked all together, L1CAM eluted in the free
protein
fractions along with Albumin in both CSF and plasma. When immunoprecipitation was
performed,
Western blotting revealed different isoforms of L1CAM in CSF and plasma.
Summary/Conclusion: Our data utilize a multitude of distinct techniques that
converge to demonstrate that L1CAM is not associated with EVs in CSF or plasma. Furthermore,
our data suggest that the isoforms present in CSF and plasma are distinct, which indicates
that the L1CAM in plasma is likely not coming from the brain. This data call into
question
the utility of L1CAM as a NDEV marker and point to the need to find novel candidates
for
immunoprecipitation of NDEVs.
Funding: Chan Zuckerberg Initiative
PS15.06 = OP3.06
An in vitro and in vivo perspective on the role of
erythrocyte-derived extracellular vesicles in Parkinson’s disease pathology
Helena L. Denisa
, Melanie
Alpaughb, Manon Leclercc, Vicky Caronc, Michel
Panissetd, Alexandre Prate, Frédéric Calonc, Éric
Boilardf and Francesca Cicchettib
aCentre de Recherche du CHU de Québec and Faculté de
médecine, Département de psychiatrie & neurosciences, Université Laval, Québec, QC,
Canada, Québec, Canada; bCentre de recherche du CHU de Québec; Faculté de
médecine, Département de psychiatrie & neurosciences, Université Laval, Québec, QC,
Canada, Québec, Canada; cCentre de recherche du CHU de Québec; Faculté de
Pharmacie, Université Laval, Québec, QC, Canada, Québec, Canada; dCentre de
recherche du CHU de Montréal; Département de médecine de Université de Montréal, Montréal,
Québec, Canada, Montréal, Canada; eCentre de recherche du CHU de Montréal;
Département de médecine de Université de Montréal, Montréal, Québec, Canada, Montréal,
Canada; fCentre de recherche du CHU de Québec; Faculté de médecine, Département
de microbiologie-infectiologie et d’immunologie, Université Laval, Québec, QC, Canada,
Québec, Canada
Introduction: In Parkinson’s disease (PD), α-synuclein (α-Syn) aggregates
known as Lewy bodies (LB) are present in both the central and peripheral nervous system.
Furthermore, data showing that α-Syn can spread from PD patients to transplanted tissue
has
led to a new theory postulating that pathological forms of α-Syn can drive disease
by
“infecting” healthy cells and corrupting normal proteins. The exact routes and mechanisms
involved in such spreading are yet to be fully understood but it is known that α-Syn
can be
secreted from cells and transported via extracellular vesicles (EV). EV derived from
erythrocytes (EEV) are of particular interest in this regard as they have been shown
to
contain α-Syn.
Methods: We first optimized a protocol for the isolation of fluorescently
labelled human EEV. The capacity of these EEV to cross the blood-brain barrier (BBB)
was
then evaluated in vitro using a Boyden chamber composed of primary human brain endothelial
cells. Next, EEV were added to a more complex and physiologically relevant 3D human
BBB
model including iPSC-derived brain microvascular endothelial cells. In both in vitro
protocols, flow cytometry was performed on media collect from each compartment to
determine
the number of EEV. Immunofluorescence was performed to assess the localization of
fluorophore tagged EEV. We are also using an in vivo paradigm for the extraction and
testing
of EEV spread and an in situ cerebral perfusion (ISBP) model in WT mice to investigate
if
and how EEV cross the BBB using confocal microscopy.
Results: In both in vitro models, flow cytometry analyses showed that
fluorescently tagged EEV added to the luminal side traversed the endothelial cell
barrier.
Confocal analysis revealed that some EEV could also be found within endothelial cells
themselves. Ongoing experiments are being conducted in our newly developed 3D BBB
to further
confirm these results. Our preliminary in vivo experiments showed that fluorescently
labelled beads, similar in size to EEV, used in the ISBP experiments are detectable
in the
brain parenchyma of injected WT mice using confocal microscopy. Preliminary work also
includes ISBP injections of EEV in 6-month-old WT mice, (n = 6/groups) derived from
PD
patients (at different stage of the disease) and a healthy individual as a control.
Summary/Conclusion: Our preliminary data suggests that EEV can indeed move
across the BBB in both in vitro and in vivo experimental setups. Ongoing experiments
will
determine the dynamics and processes involved in this transport and whether EEV can
precipitate and/or exacerbate disease-related features.
Funding: FRQS
PS15.07 = OP3.07
Exosomes from N-Myc amplified neuroblastoma cells induce
migration and confer chemoresistance to non-N-Myc amplified cells: implications of
intra-tumour heterogeneity
Pamali Fonsekaa
, Michael
Liemb and Suresh Mathivananb
aLa Trobe Institute for Molecular Sciences, La Trobe
University, Melbourne, Australia; bLa Trobe University, Melbourne, Australia
Introduction: Neuroblastoma accounts for 15% of childhood cancer mortality.
Amplification of the oncogene N-Myc is a well-established poor prognostic marker for
neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour
aggression and resistance to treatment, the role of N-Myc in the aggressiveness of
the
disease is poorly understood. Exosomes are released by many cell types including cancer
cells and are implicated as key mediators in cell-cell communication via the transfer
of
molecular cargo. Hence, characterising the exosomal protein components from N-Myc
amplified
and non-amplified neuroblastoma cells will improve our understanding on their role
in the
progression of neuroblastoma.
Methods: In this study, comparative proteomic analysis, nanoparticle tracking
analysis, transmission electron microscopy, RNAi-based knockdown, migration and cellular
survivability assays were performed to understand the role of exosomes isolated from
cells
with varying N-Myc amplification status.
Results: Label-free quantitative proteomic profiling revealed 968 proteins
that are differentially abundant in exosomes released by the N-Myc amplified and
non-amplified neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment
of proteins involved in cell communication and signal transduction in N-Myc amplified
exosomes. Treatment of less aggressive SH-SY5Y cells with N-Myc amplified SK-N-BE2
cell-derived exosomes increased the migratory potential, colony forming abilities
and
conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from
N-Myc
knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced
apoptosis.
Summary/Conclusion: These findings suggest that exosomes could play a pivotal
role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between
cells.
PS15.08 = OP3.08
Dissecting the heterogeneity of extracellular vesicle
sub-populations at single vesicle level
Anudeep Yekulaa
, Koushik
Muralidharana, Bob Carterb and Leonora Balajc
aMassachusetts General Hospital, Harvard Medical School,
Boston, USA; bMassachusetts General Hospital, Cambridge, USA;
cMassachusetts General Hospital and Harvard Medical School, Cambridge, USA
Introduction: Quantification and characterization of single extracellular
vesicles (sEVs) based on surface markers can aid in dissecting the heterogeneous landscape
of EV subpopulations. We and others have demonstrated the potential of imaging flow
cytometry (IFC) to perform sEV characterization. We recently showed release of
protoporphyrin (PpIX) positive sEVs by 5-aminolevulinic acid (5-ALA) dosed glioma
cells, in
vitro and in vivo. Rickfels et al. also used IFC to demonstrate the enrichment of
CD63+/CD81+ EVs in the plasma of glioma patients. Herein, we performed in vitro studies
to
characterize EV subfractions using 5-ALA as well as EV and CNS specific surface markers.
Methods: We use IFC to characterize EVs released by glioma using 5-ALA,
fluorescently labelled EV (CFDA-SE, CD81) and glioma specific (tenascin C and epidermal
growth factor receptor vIII, EGFRvIII) markers. Furthermore, we characterized EVs
released
by EGFRvIII positive glioma cells treated with dexamethasone, a steroid commonly used
in
glioma patients, to determine the effect of steroids on EV release. EVs were quantified
by
IFC and results were confirmed by qPCR for the levels of EGFRvIII mRNA.
Results: Firstly, we optimized protocols to label glioma sEVs using
fluorescently labelled EV markers (CFDA-SE, CD81) and tumour specific markers (tenascin
C
and EGFRvIII). Of the total EVs (CFDA-SE), we demonstrate that 58% are tenascin C
positive,
2.7% are EGFRvIII positive and 1.6% are 5-ALA positive. There was only a minor overlap
(<16%) between the sub-populations. Finally, we show that dexamethasone treated glioma
cells release lower total EVs (2.5-fold), tumour specific EVs (2.8-fold; EGFRvIII),
EGFRvIII
mRNA compared to mock treated cells.
Summary/Conclusion: We demonstrate the potential of IFC to monitor sEVs
released by glioma cells exposed to different stimuli. This allows the characterization
of
EV sub-populations providing a working model to understand the dynamics of tumour
EVs at a
single vesicle level.
Funding: This work is supported by grants U01 CA230697 (BSC, LB), UH3 TR000931
(BSC), P01 CA069246 (BSC).
PS15.09 = OP3.09
Proteomic analysis of EVs from the filamentous fungal plant
pathogens Fusarium graminearum and Fusarium oxysporum f. sp. vasinfectum
Donovan Garcia-Cerona
, Charlotte
Dawsonb, Mark Bleackleyc and Marilyn Andersona
aLa Trobe University, Melbourne, Australia;
bCambridge University, Cambridge, UK; cIncannex, Melbourne,
Australia
Introduction: F. graminearum (Fgr) and F. oxysporum f. sp. vasinfectum (Fov)
are severe fungal pathogens of cereals and cotton, respectively. Fgr and Fov cause
economic
losses and threaten food and fibre supplies worldwide. Understanding host-pathogen
interactions is crucial for developing new strategies for disease control. We are
determining whether extracellular vesicles (EVs) have a role in the interaction between
fungal pathogens and their host plant.
Methods: We isolated EVs from Fgr and Fov by size-exclusion chromatography and
characterized them by NTA and TEM. EVs from Fgr and Fov are between 100–300 nm and
have
morphology similar to EVs reported for other fungi. We performed label-free quantitative
proteomics to describe the protein cargo of EVs from Fgr and Fov, including a comparative
study of EVs from Fov grown on different media: Czapek Dox (CD) and Saboraud’s Dextrose
Broth (SDB).
Results: A total of 658 proteins were detected in Fgr EVs and, according to
prediction software EffectorP, 12.5% of these were potential effectors. Similarly,
70% of EV
proteins do not contain signal peptide indicating that packaging into EVs is a novel
mechanism of secretion for these proteins. Notable Fgr EV proteins include lipases,
proteases and synthases for toxins and chitin. Fov produced EVs in similar quantities
in
both growth media tested, but EV protein cargo differed between them. There was a
39%
overlap in proteins identified in the 465 CD and the 658 SBD EV proteins. In general,
EV
proteins were involved in metabolism, cell wall architecture and oxidoreduction, with
15.4%
and 12.9% of potential effectors, respectively. Polyketide and toxin synthases, proteases
and effectors were present in both types of Fov EVs.
Summary/Conclusion: This new fungal EV isolation method was rapid, yielded
high-quality EVs, and did not submit particles to high centrifugal forces. Our data
revealed
that both Fgr and Fov produce EVs enriched with proteins that could alter host immune
responses or facilitate fungal infection. Furthermore, the protein composition of
Fov EVs
was dependant on culture conditions. This supports a potential role for fungal EVs
in
disease progression in plants and provides the foundations to pursue the role of EVs
in
plant-fungal interactions with the potential to identify new targets for disease
control.
Funding: Australian Research Council DP160100309
PS15.10 = OP3.10
Trypanosoma cruzi releases different types of extracellular
vesicles that distinctly modulate host cells
Yifat Ofir- Birina, Rafael Pedro Madeirab,
Sergio Schenkmanc, Ori-Yam Revachd, Ori Avinoamd, Yael
fridmann-sirkisd, Ziv Poratd, Mattia Morandid, Neta
Regev-rudzia, Ana Claudia Torrecilhasc and Ana Claudia
Trocoli Torrecilhase
aWeizmann Institute of Science, Rehovot, Israel;
bUniversidade Federal de Sao Paulo, Sao Paulo, Brazil; cUNIFESP, Sao
Paulo, Brazil; dWIS, Rehovot, Israel; eDepartment of Biological
Sciences, Universidade Federal de Sao Paulo, Sao Paulo, Brazil
Introduction: Extracellular Vesicles (EV) released by infective forms of
Trypanosoma cruzi, the agent of Chagas’ disease, modulate inflammatory response of
macrophages through the activation of Toll 2 receptor (TLR2) via mitogen-activated
protein
kinase pathway. This induces the production of nitric oxide (NO) and expression of
the
cytokines TNF-α, IL-12 and IL-6, which could explain the inflammation observed in
experimental Chagas’ disease, and eventually in the progression of human disease.
EVs
released by the parasite are heterogeneous and it is unknown which factor, or factors
present in the different vesicle populations act during the interaction with host
cells.
Objectives. The goal of the present work was to characterize and isolate the different
populations of EVs released by T. cruzi and test their effects on macrophages.
Methods: EV released by trypomastigotes forms of T. cruzi (Y strain) were
purified by Asymmetric flow field-flow fractionation (AF4) and characterized by
Nanoparticles tracking analysis (NTA). The different populations of EVs were incubated
with
host human monocytes cells (THP-1) and cytokines production determined by ELISA and
qPCR.
The different EV populations were also incubated with LLCMK-2 epithelial cells and
the
infection by T. cruzi determined.
Results: We found two distinct populations of EVs. A population with 50 to
50 nm (EV1) and another with 100 to 120 nm (EV2). EV1 induced more TNF-alpha, IL-6,
IP-10
and CCL20 than EV2. It was also more effective in promoting T. cruzi infection in
epithelial
cells.
Summary/Conclusion: T. cruzi released two EV populations that affects
differently host cells. Identification of these EVs composition might help to better
understand the role of EVs in the modulation of T. cruzi infection.
Funding: FAPESP, CNPq and CAPES.
PS15.11 = OP3.11
Commensal bacterial extracellular vesicles act as carriers for
norovirus
Sutonuka Bhar, Melissa Jones, Annalise
Galbraith and Mariola Edelmann
University of Florida, Gainesville, USA
Introduction: Human norovirus (HuNoV) are one of the most common causes of
gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have
a
massive economic impact resulting in approximately 60 USD billion each year in healthcare
costs and missed worker productivity. Development of anti-viral therapies for HuNoV
has been
hampered by the lack of robust in vitro cultivation systems. Several cell types support
viral replication but only produce modest amounts of virus due to unknown reasons,
making
these systems insufficient for use in drug development and infectivity assays.
Noroviruses are known to attach to gram-negative enteric bacteria and this facilitates
infection in vitro. However, the microbiome- norovirus-host communication link is
missing.
Noroviruses infect immune cells present in lamina propria during acute infection,
but
bacteria themselves are large enough to cross the mucosal and the tight epithelial
barrier
which separates gut lumen from lamina propria. We hypothesized that binding of noroviruses
to bacteria enhances extracellular vesicles (EV) production. Because commensal bacterial
EVs
by themselves do not have any detrimental effects on host cells, we believe using
EVs in in
vitro culture will enhance norovirus infection, thus producing higher titre of viruses
for
vaccine and anti-viral drug development.
Methods: Attachment assay: Purified norovirus was incubated with Enterobacter
cloacae, Lactobacillus acidophilus and Bacteroides thethiotaomicron, and grown to
produce
EVs. The attachment was confirmed via qPCR.
Isolation of EVs: Clarified media supernatants were subjected to ultracentrifugation
at
varying speeds and 0.2um filtration. Co-purification of norovirus with the EVs was
checked.
EV quantification and characterization: EV total protein content was measured by microBCA.
The number of vesicles were quantified by Nanoparticle tracking analysis. Scanning
and
Transmission electron microscopy was performed to check quality of EV preparation
and
determine if virus was attached to the vesicles. Internal EV protein content was evaluated
using MS-HPLC. The EVs were also check for infectivity via TCID50 assay.
Results: Incubation of noroviruses with commensal bacteria resulted in
significant increases in production of EVs compared to uninfected controls. Murine
norovirus
(MNV), used as a surrogate, was found to be associated with EVs. EM analysis determine
association of viruses with the bacteria as well as the MVs, while also showing certain
surface structural changes in virus attached bacteria compared to mock bacteria. The
EVs
were found to cause infection in naive macrophages.
Summary/Conclusion: Changes in EV production and content by bacteria exposed
to noroviruses will provide insight into its pathogenesis and possible solutions to
the low
viral output from HuNoV culture systems.
PS15.12 = OP3.12
Detection of bacterial extracellular vesicles in blood from
healthy volunteers
Kylie Krohmalya
, Claire
Hoptayb, Andrea Hahna and Robert Freishtata
aChildren’s National Hospital, Washington, USA;
bChildrens National Hospital, Washington, USA
Introduction: Bacteria constitutively produce biologically active
extracellular vesicles (EVs), which contain RNA, DNA, and/or proteins. Bacteria use
these
EVs for communication with other bacteria and recent research suggests bacterial EVs
can
also affect host cells. Given these findings, it is necessary to examine the role
of
bacterial EVs in human disease. Current methods of bacterial EV isolation from human
specimens cannot distinguish between bacterial species. However, there is utility
in
examining EVs from specific species, as bacterial species and their EVs may have unique
contributions to human disease. Our objective was to isolate circulating EVs specifically
from Escherichia coli (EEVs) and Haemophilus influenzae (HEVs), two known colonizers
and
pathogens in the gut and airway, respectively.
Methods: Total EVs were isolated from the blood of six healthy volunteers via
precipitation and size exclusion chromatography. EVs were then selected via a novel
latex
bead-based fluorescent antibody construct targeting species-specific outer membrane
proteins. We used flow cytometry to evaluate the isolated EVs.
Results: The constructs were saturated with EEVs at an antibody concentration
of 11.5 µg/mL of plasma, as geometric means ≥11.5 µg/mL were nearly equal. HEVs were
detected at 48 µg/mL of plasma, but saturation is yet to be determined. EEVs were
imaged by
a FEI Talos F200X electron microscope and measured between 40–90 nm, and HEVs were
between
60–160 nm. Both types of EVs were spherical.
Summary/Conclusion: Using this novel technique, we were able to isolate,
detect, and visualize EEVs and HEVs. This technique enables the study of specific
bacterial
EVs. In the future, EV contents will be assayed. Furthermore, this technique will
be
modified so that specific bacterial EVs from body fluids can be used for downstream
functional applications. This is the first time that bacterial EVs from targeted bacterial
species have been detected in blood from healthy humans.
LBS01: Late Breaking: Cancer Biology and Biomarkers Chair: Josep
Domingo-Domenech – Thomas Jefferson University Chair: Al Charest – Harvard
University
LBS01.01
Nasopharyngeal carcinoma exosomes modify the metabolism status
of human dendritic cells and favour their recruitment through the CCL20
chemokine
Anthony Lefebvre
a, Sarah Renaud,
William Laineb, Benjamin Hennart, Delphine Allorge, Jérôme Kluzab,
Nadira Delhem and Olivier Moralesc
aCNRS UMR8161 IRCV team, Lille, France; bINSERM
UMR-S 1172 centre de recherche Jean-Pierre Aubert, Lille, France; cCNRS, Lille,
France
Introduction: Nasopharyngeal Carcinoma (NPC) is characterized by a large
presence of regulatory T cells (Tregs) and the production of tumour-derived exosomes
with
immunosuppressive properties. Our team showed that NPC-derived exosomes favour the
suppressive activity and recruitment of human Tregs via CCL20 chemokine, thus contributing
to NPC immune escape (Mrizak et al., JNCI, 2015). More recently, our team has shown
that
NPC-exosomes could induce Tregs by altering the maturation of dendritic cells (DCs)
and
promoting tolerogenic dendritic cells (tDCs) (Renaud et al., HerPas congress 2017).
Our main
objectives in this study are (i) to define and compare the metabolic status of mature
dendritic cells (mDCs), control tDCs and tDCs generated in the presence of NPC-exosomes
(ExoCNPtDC) and (ii) to evaluate the chemoattractive potential of NPC-exosomes on
ExoCNPtDCs, and notably to investigate the involvement of CCL20 in this recruitment.
Methods: DCs are generated from human monocytes in the presence or absence of
NPC-exosomes. The maturation status of DCs was evaluated at a phenotypic level by
studying
the expression of maturation markers using flow cytometry and at a functional level
by
analysing cytokines secretion using ELISA. This cytokine analyse has been performed
in both
conditions, on treated DCs and during co-culture assays of autologous CD3 T lymphocytes
with
treated DCs. In a second step, a mitochondrial metabolic and glycolytic study was
performed
using the Seahorse technology (OCR and ECAR measurement). Finally, the chemoattractive
potential of NPC-derived exosomes on the different induced DCs was analysed (i) using
Boyden
chamber chemoattraction assays or real-time videomicroscopy (Chemotaxis µSlide IBIDI)
and
(ii) using RT-qPCR analysis of the receptor expression of CCL20 (CCR6).
Results: NPC-exosomes alter DC maturation, which gives rise to tolerogenic DCs
that favour the induction of Tregs. In addition, the metabolic analysis of DCs seems
to put
foward a specific metabolic signature of the tDCs induced by NPC-exosomes. And finally,
chemoattraction assay suggests that NPC-exosomes preferentially attract tDCs and ExoCNPtDCs
in a CCL20-dependant manner.
Summary/Conclusion: Taken together our results should allow us to characterize
the major role of NPC tumour exosomes on the maturation and the recruitment of DC
and so
identify them as anti-tumoural therapeutic targets.
LBS01.02
Cytotoxic T lymphocyte EV that prevents tumour metastasis by
collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle
or apoptotic body.
Naohiro Seo
a, Junko
Nakamuraa, Tsuguhiro Kanedaa, Takanori Ichikib, Asako
Shimodac, Kazunari Akiyoshic and Hiroshi Shikua
aMie University Graduate School of Medicine, Mie, Japan;
bThe University of Tokyo, Bunkyo, Japan; cKyoto University, Kyoto,
Japan
Introduction: Recently, instead of ultracentrifugation, development of new
preparation protocol is demanded for research of reliable bioactivity and drug discovery
of
extracellular vesicles (EVs). In this study, we propose a novel method for large scale
preparation of high-performance extracellular vesicles focusing on membrane negative
charge.
Methods: Murine cytotoxic T lymphocyte (CTL) EVs in supernatant were
concentrated more than 20 times at over 97% purity without leaking by 750 kDa MWCO
ultrafiltration, and subjected to ion exchange DEAE column chromatography after replacing
with PBS. After ion exchange, EVs were characterized by BCA assay, NTA assay, cryoTEM
observation, proteome analysis, DNA content measurement, miRNA microarray analysis,
zeta
potential measurement, lectin array analysis, and target cell analysis.
Results: Murine CTL EVs were broadly divided into two populations that were
eluted at low salt (L-s: 0.15 M-0.3 M NaCl) and high salt (H-s: 0.3 M-0.5 M NaCl)
concentrations. L-s CTL EVs were abundant in late endosome-related proteins, integrins,
Rabs, and effective miRNAs, indicating exosome characteristics, and had biological
activity
for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations
by
intratumoral administration (See Seo et al., Nat. Commun. 9: 435, 2018). Contrary,
H-s CTL
EVs were rich in DNA, core histones, ribosomal proteins, cytoskeleton proteins, and
housekeeping proteins, considering microvesicles and apoptotic bodies, and easily
phagocytosed by a Kupffer cell line (KUP5: Kitani et al., Results Immunol. 4: 68–74.
2014).
In addition, there were noticeable differences between L-s and H-s CTL EVs in the
negative
zeta potential width and membrane glycan structure.
Summary/Conclusion: Thus, ion exchange can be an optimal mass fractionation
method for discriminating bioactive exosomes from cargos for nucleic acids in EVs.
Funding: CryoTEM was conducted in Nara Institute of Science and Technology
(NAIST), supported by Nanotechnology Platform Program (Synthesis of Molecules and
Materials:
2019 #04) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT).
This
work was supported by grants from the Japan Agency for Medical Research and Development
(Translational Research Network Program (Nagoya Univ. Seeds A64)) and the Japan Science
and
Technology Agency (CREST [JPMJCR17H2]).
LBS01.03
CLIC4 is essential for breast cancer metastatic competence and
predicts disease outcome
Vanesa C. Sanchez
a, Alayna
Craig-Lucasa, Wendy Duboisb, Justin Lackc, Howard
Yangb, Maxwell Leeb, Ji Luoa, Kent Huntera and
Stuart Yuspaa
aNational Institutes of Health (NIH), Bethesda, USA;
bLaboratory of Cancer Biology and Genetics, Center for Cancer Research,
National Cancer Center, National Institutes of Health. Bethesda, MD USA 20812, Bethesda,
USA; cNIAID Collaborative Bioinformatics Resource, Frederick National Laboratory
for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702, Bethesda,
USA
Introduction: Metastatic breast cancer is a consequence of complex
interactions between cancer cells and the host. CLIC4, a member of a conserved gene
family
in the glutathione-S-transferase superfamily, mediates crosstalk between tumour and
host in
breast cancer. TCGA and METABRIC data indicated that elevated CLIC4 expression was
associated with breast cancers from young women, those with poor prognosis, and those
with
early stage metastatic disease.
Methods: Since bulk tumour analysis does not distinguish between cancer and
host stromal cells, we used genetic modifications of established syngeneic breast
cancer
mouse models to evaluate the contributions of CLIC4 in the host or tumour cells to
develop
metastases.
Results: Experimentally, the essential Clic4 host contributions for metastatic
competence were related to circulating levels of pro-metastatic soluble factors,
neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation,
and
leukocyte migration. CLIC4 was detected as cargo in circulating extracellular vesicles
(EVs)
from breast cancer patients. Similarly, circulating EVs from tumour-bearing mice have
abundant CLIC4 in comparison to those from mice bearing tumours that lack CLIC4. Tumour
cells released EVs that induced myofibroblast conversion of wildtype but not Clic4
ablated
lung fibroblasts.
Summary/Conclusion: These results illuminate CLIC4 expression as a prognostic
marker for breast cancer patients, and experimentally, CLIC4 is a critical host factor
for
metastatic competence and potential target within host tissues for anti-metastatic
therapy.
Funding: This work was supported by the intramural program of the National
Cancer Institute under Project ZIA BC 005445.
LBS01.04
The application of flow cytometry in an EV-based liquid biopsy
for the detection of Cancer Multidrug Resistance in Myeloma
Gabriele De Rubis
a, Krishna
Sunkaraa, Sabna Rajeev Krishnan and Mary Bebawyb
aLaboratory of Cancer Cell Biology and Therapeutics,
Discipline of Pharmacy, Graduate School of Health, The University of Technology Sydney,
Australia, Sydney, Australia; bThe University of Technology Sydney, Sydney,
Australia
Introduction: Multiple Myeloma (MM) is an incurable cancer of bone-marrow
plasma cells. It is characterized by unpredictable and highly variable therapeutic
response
and poor survival, attributed to the development of multidrug resistance (MDR) to
chemotherapy. Presently, no clinical procedures allow for a continuous, minimally
invasive
monitoring of MDR. We identified unique extracellular vesicle (EV) populations in
the blood
of myeloma patients, which serve as biomarkers of disease evolution and MDR to combination
chemotherapy. We describe approaches used to optimise the use of flow cytometry (FCM)
for EV
biomarker detection and analysis and detail strategies for cross-platform analytical
validation.
Methods: We conducted a cross-platform analysis using two commercially
available flow cytometers designed for EV detection. Scatter resolution, enumeration
accuracy and precision were determined across both platforms by analysing submicron
silica
beads (ApogeeMix, 180–1300 nm) of known concentration. We detected large EVs, as established
by reference size beads, electron microscopy, expression of phosphatidylserine and
the
presence of integral membrane proteins of cell of origin. We analysed EVs isolated
from
plasma by high-speed centrifugation (18,900 g) as well performing analysis by direct
plasma
labelling followed by validation by detergent lysis of vesicular constituents. A clinical
operating range was defined which ensures linearity and avoids swarm detection.
Results: We observed comparable scatter resolution, enumeration accuracy
(error ≤15%) and precision (CV ≤5%) across both platforms used. We defined two EV
size
gates: a “Latex” gate (300 to 1100 nm polystyrene latex beads), and a “Silica” gate
(180 to
1300 nm silica beads) for EVs at the lower end of our size range of interest. To improve
detection sensitivity, we identified common contributors to signal noise and applied
workflow strategies to minimize these. Finally, we identified linear ranges which
avoid
swarm detection, and which ensures reproducible EV counts (CV <20%) across both
instruments.
Summary/Conclusion: We present an optimised, standardised and cross-platform
reproducible working protocol which supports the use of FCM in an EV-based liquid
biopsy
application.
Funding: The project is funded by SPARK OCEANIA and UTS Innovation
Commercialisation Seed Fund Scheme to MB.
LBS01.05
Metabolomic profiling of serum and exosomes isolated from head
and neck cancer patients after radiotherapy
Anna Wojakowska
a, Lukasz
Marczakb, Aneta Zebrowskac, Agata Skowronekc, Tomasz
Rutkowskid, Piotr Widlakd and Monika Pietrowskad
aInstitute of Bioorganic Chemistry Polish Academy of
Sciences, Poznan, Poland; bInstitute of Bioorganic Chemistry, Polish Academy of
Sciences, Poznan, Poland; cMaria Sklodowska-Curie National Research Institute of
Oncology, Gliwice Branch, Gliwice, Poland; dMaria Sklodowska-Curie Institute –
Oncology Centre, Gliwice Branch, Gliwice, Poland
Introduction: Cancer radiotherapy (RT) induces the response of the whole body
that could be detected at the blood level. Searching for new molecular signatures
which
could correlate with treatment response in cancer patients is of particular importance.
Radiation-induced changes in proteome and transcriptome of serum have been widely
described.
However, metabolomic changes in serum, exosomes and other classes of small extracellular
vesicles (EV) of cancer patients after RT have not been given as much attention.
Metabolomics of serum and EV of cancer patients could provide a valuable insight into
the
response of both tumour and whole organism to the treatment. The aim of the study
was to
compare serum and EV metabolomic profiles in head and neck cancer (HNC) patients before
and
after RT.
Methods: Serum samples from 10 HNC patients were taken before (A) and after
(B) RT. 10 healthy volunteers were used as a control group (C). EV were isolated from
1 ml
of serum using size-exclusion chromatography (SEC). Selected SEC fractions were subjected
to
extraction of metabolites. A mixture of MeOH/H2O was used for extraction of metabolites
from
serum and EV samples. Samples were analysed by gas chromatography-mass spectrometry
(GC-MS).
The study protocol adhered to the tenets of the Declaration of Helsinki and was approved
by
the Bioethical Committee of the Maria Skłodowska-Curie National Research Institute
of
Oncology, Branch Gliwice, Poland (permit nr. DO/DGP/493/4/06/1/2016/G).
Results: An untargeted GC-MS-based approach allowed the detection of 189
metabolites in serum samples and 50 exosomal small molecules, of which 32 joint. The
identified compounds included amino acids, fatty acids, carboxylic acids, sugars,
and
others. There were 49 metabolites which levels discriminated compared groups (A,B,C)
of
serum samples and 12 compounds that discriminate the EV isolated from HNC serum before
and
after RT from HC.
Summary/Conclusion: RT caused significant changes in levels of serum and EV
metabolites witch are involved in amino acid metabolism, lipids metabolism, energy
metabolism and oxidative stress response.
Funding: This study was supported by the National Science Centre, Poland,
Grant 2017/26/D/NZ2/00964 (for AW, LM) and 2015/17/B/NZ5/01387 (for TR and PW).
LBS01.06
Proteomic profiling of small extracellular vesicles secreted by
human pancreatic cancer cells implicated in cellular transformation
Karoliina Stefanius, Kelly Servage and Kim
Orth
UT Southwestern Medical Center, Dallas, USA
Introduction: Small extracellular vesicles (sEV) secreted from tumour cells
are functional vehicles capable of contributing to intercellular communication and
metastasis. Numerous studies have focused on elucidating their role in cancer progression.
We recently showed that sEVs isolated from pancreatic cancer cells can function as
an
initiator in malignant cell transformation. Here, using a mass spectrometry (MS)-based
proteomics approach, we analysed the differences in the protein cargo of sEVs secreted
from
normal pancreatic and cancer cells to better understand their biological
characteristics.
Methods: sEVs were isolated from 3 human pancreatic cancer cell lines
(Capan-2, MIA PaCa-2, and Panc-1) and normal pancreatic epithelial cells (HPDE) using
a
combined ultrafiltration-ultracentrifugation method coupled with a sucrose density
gradient
purification. Proteomic profiling of sEVs was carried out using an LC-MS/MS method.
Protein
identification from resulting MS/MS spectra was conducted using proteome database
search
software followed by Gene Ontology (GO) enrichment and Reactome pathway analysis.
Results: A total of 4,907 unique proteins were identified confidently across
the combined samples. The proteins present in all four sEV types (1,135 proteins)
consist of
general housekeeping proteins. 348 proteins were uniquely found in all cancer sEVs
but not
in the normal HPDE sEVs. This group contains an enrichment of proteins that function
in the
endosomal compartment of cells responsible for vesicle formation and secretion and
suggest
their important role in driving the increased production of sEVs from cancer cells
relative
to normal cells. Moreover, this group includes a set of proteins that have been implicated
in malignant cell transformation, consistent with our previous work showing that each
of the
cancer sEVs analysed here could initiate malignant transformation of NIH/3 T3 cells.
Conversely, there were 313 proteins uniquely found in normal HPDE sEVs. This group
includes
a number of immune response proteins that are not found in any of the pancreatic cancer
cell
sEVs.
Summary/Conclusion: The differences in the proteomes of cancer and normal sEVs
may be indicative of their varying roles in cell transformation and helpful in delineating
the types of EVs that are being produced. In addition, these differences point towards
their
potential value as cancer biomarkers.
LBS01.07
Proteomic profile of tumour-derived exosomes in plasma of
melanoma patients
Aneta Zebrowskaa, Marta Gawinb, Lukasz
Marczakc, Priyanka Sharmad, Justyna Mikae, Joanna
Polanskae, Piotr Widlakb, Theresa L. Whitesidef and
Monika Pietrowska
b
aMaria Sklodowska-Curie National Research Institute of
Oncology, Gliwice, Poland; bMaria Sklodowska-Curie Institute – Oncology Centre,
Gliwice Branch, Gliwice, Poland; cInstitute of Bioorganic Chemistry, Polish
Academy of Sciences, Poznan, Poland; dUPMC Hillman Cancer Center, Pittsburgh PA,
USA, Pittsburgh, USA; eDepartment of Data Science and Engineering, Silesian
University of Technology, Gliwice, Poland; fDepartment of Pathology, University
of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh,
USA
Introduction: Exosomes released by cancer cells referred to as “tumor-derived
exosomes” (TEX) play a key role in tumour-induced suppression of immune effector cells
and
in the promotion of tumour growth by autocrine or paracrine mechanisms. Molecular
profiling
of extracellular vesicles circulating in human body fluids, including plasma exosomes,
is a
promising non-invasive strategy to identify cancer biomarkers. However, specific targeting
of TEX remains a real challenge because there is no antigens specific for exosomes
produced
by cancer cells in general. Here we took advantage of CSPG4 antigen frequently overexpressed
in melanoma cells for the immunocapturing of melanoma-derived exosomes (MTEX) present
in the
plasma of melanoma patients and their separation from non-malignant cell-derived exosomes
(non-MTEX) present in the specimen.
Methods: Blood samples were obtained from 15 patients with melanoma treated at
the UPMC Hillman Cancer Center Melanoma Program Outpatient Clinic. Total exosomes
were
isolated by the mini-SEC method. Fraction #4 containing the majority of exosomes was
separated into MTEX and non-MTEX fractions using the immunoaffinity capture method
with
biotin-labelled anti-CSPG4 mAb. The full MS/data-dependent acquisition was performed
with
the use of the Dionex UltiMate 3000 RSLC nanoLC system connected to the Q Exactive
Plus
Orbitrap mass spectrometer.
Results: An untargeted MS/data-dependent approach allowed the detection of
about 650 proteins. Proteins detected in the material from at least 8 patients were
considered, which resulted in 488 proteins included in the analysis. To identify proteins
enriched in MTEX, the individual patient ratio between MTEX and non-MTEX fraction
was
analysed for each protein. We found 49 proteins specific/characteristic for MTEX.
These
included several enzymes exemplified by ribosomal protein S6 kinase alpha-3,
D-3-phosphoglycerate dehydrogenase, phosphomannomutase 2, hypermethylated in cancer
2
protein, acidic mammalian chitinase. Moreover, among components enriched in the MTEX
fraction there were several proteins associated with immune-related functions exemplified
by
podocalyxin-like protein 2, transforming protein RhoA, tyrosine-protein kinase Yes.
Summary/Conclusion: Using full MS/dd-MS/MS mode we found specific MTEX
proteomic profile of tumour-derived exosomes, which needs further evaluation by targeted
methods.
Funding: This study was supported by the National Science Centre, Poland,
Grant 2016/22/M/NZ5/00667.
LBS01.08
Isolation and characterization of prostate-derived extracellular
vesicles as a liquid biopsy strategy in cancer diagnosis
Susann Allelein, Paula Medina-perez, Ana
Leonor Heitor Lopes, Andreas Kölsch, Sabrina Rau and Dirk Kuhlmeier
Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig,
Germany
Introduction: In the past years, extracellular vesicles (EVs) have attracted
considerable interest due to their ability to provide valuable diagnostic information
from
liquid biopsies. The high abundance in all bodily fluids and their cargo stability
confers
EVs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible
tissues, therapy response and monitoring, but also to reduce infection risks of conventional
highly invasive biopsies.
Virtually all cells continuously release vesicles into the extracellular environment,
diverse in size, content and features depending on the biogenesis, origin and function.
This
heterogeneity adds a layer of complexity when attempting to isolate and characterize
tissue-specific vesicles.
Methods: Hence, we aimed to use a immunomagnetic capture approach for
prostate-derived EVs from cell culture supernatants, with further investigation into
human
plasma and urine samples. Analysis was performed by nanoparticle tracking analysis,
western
blotting and electron microscopy. Additionally, an in-house spotted antibody microarray
is
in development. Here, we intend to detect different EV sub-populations based on their
surface markers.
Results: Isolated immunocaptured EV populations based on the classical EV
marker CD9 show an increased signal for the luminal protein TSG101. EV populations
targeting
the tissue-specific marker prostate specific membrane antigen (PSMA), were found positive
for TSG101 in a lower extent indicating a sub-population of EVs. The microarray uses
less
than 100 µL of sample (concentrated cell culture supernatant, human plasma, urine)
and leads
to a faster characterization within 3 h for EV surface marker as compared to western
blot.
Summary/Conclusion: Immunomagnetic isolation might be a promising approach for
liquid biopsy and thereby the microarray could be valuable to identify potential capture
targets. The current design for 6 different surface marker from 16 samples simultaneously
could be easily extended for sample size and surface profiling allowing for a more
economical way to multiplex samples.
LBS01.09
Paving the way for implementing a feasible and reliable
technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer
in
clinical practice
Andrés Vega-Avalosa, Alejandro
Mercado-Camperob, Eliana Andahurb, Catherine
Sánchez
b and Catherine A. Sánchezc
aUniversity of Chile, Santiago, Chile; bLas Condes
Clinic, Santiago, Chile; cClinica las Condes, Santiago, Chile
Introduction: Extracellular vesicles (EV) in urine have been proposed as
biomarkers for bladder cancer (BC). However, at present there are no standardized
methods
for EV isolation or urine sampling. Our goal was to evaluate the EV isolation performance
between different methods, the effect of the sampling time and the importance of urinary
creatinine (UCr) normalization.
Methods: Two urine samples of 120 mL were collected from 5 patients with non
muscle-invasive BC: one from the first micturition and another from any time of the
day.
Twenty mL were used for UCr measurement and 100 mL were used for EV isolation by either
precipitation with polyethylene glycol (PEG), concentration by filtration (UF, Centricon
Plus-70, 10 k, Millipore), sepharose size exclusion column (SEC), or combinations
of these
methods. Additionally, the effect of protease inhibitors (PI) and DTT treatment after
collection or during processing was analysed. Size and number of particles were evaluated
by
Nanosight and the presence of exosomal markers was evaluated by Western Blot.
Results: Among the methods evaluated, UF + SEC showed the best performance
retrieving the highest number of particles in the range of 50–200 nm, and the highest
protein expression of exosomal proteins. UF alone showed the highest concentration
of EV,
but with a tendency to isolate larger particles. Particle concentration was positively
correlated with UCr, reflecting the importance of UCr normalization before comparing
between
patients. Finally, no differences in the performance according to the time of collection,
nor in the use of PI or DTT were observed.
Summary/Conclusion: UF + SEC gave the highest EV yield and was not affected by
the time of urine collection. The use of PI and DTT can be avoided, and normalization
to UCr
should be considered when implementing this technique for assessing EVs as biomarkers
for BC
in clinical practice.
Funding: PIDA 2019.
LBS01.10
The role of small extracellular vesicles secreted by cells with
extra centrosomes in PDAC microenvironment remodelling
Judit B. Csere
a, Susana A.
Godinhoa, Sophie Adamsa, Teresa Arnandisb, Judith
Simona and John F. Marshalla
aBarts Cancer Institute, London, UK; bBarts Cancer
Institute, Valencia, Spain
Introduction: Human tumours, including pancreatic ductal adenocarcinoma
(PDAC), often harbour a subpopulation of cancer cells with extra centrosomes. We found,
that
these cells secrete an increased number of small extracellular vesicles (sEVs), within
the
20–120 nm size range. SEVs play a role in cancer signalling and progression and are
widely
studied for their diagnostic potential. We aim to understand the role of sEVs secreted
by
cells with extra centrosomes in shaping PDAC-associated stroma, particularly fibrosis.
Methods: To study the sEV mediated changes in the PDAC microenvironment, we
purified sEVs through serial ultracentrifugation and Size Exclusion Chromatography,
characterised the content through SILAC-based proteomics, and assessed phenotypic
changes in
pancreatic stellate cells (PSCs) and extracellular matrix (ECM) production through
immunofluorescence staining.
Results: Our data indicates, that the sEVs secreted by cells with extra
centrosomes are exosomes due to their endocytic origin, and we found, that they can
activate
PSCs, key mediators of fibrosis in PDAC. Indeed, we observed an increased level of
collagen
I produced by PSCs activated by sEVs from cells with extra centrosomes as compared
to cells
without extra centrosomes. Interestingly, we found, that PSC activation through sEVs
is not
mediated by TGF-β, assessed by the level of nuclear SMAD2 accumulation downstream
of TGF-β
activation, suggesting a novel mechanism of PSCs activation.
Summary/Conclusion: PDAC cells with extra centrosomes contribute to a novel
type of PSC reprogramming, which could alter their ECM deposition and contribute to
the
extensive fibrosis observed in PDAC. We are currently characterising the signalling
pathways
associated with sEV mediated PSC activation and how it impacts PADC progression to
better
understand the role of centrosome amplification in the cancer-stromal crosstalk.
Funding: Barry Reed Cancer Research Fund, The Lister Institute, Medical
Research Council, Cancer Research UK.
LBS01.11
Exosomal Carboxypeptidase E confers and CPE-shRNA loaded
exosomes inhibit growth and invasion of hepatocellular carcinoma cells.
Y. Peng Loh
a, Sangeetha
Hareendrana, Bassam Albraidya, Xuyu Yanga and Jennifer
Jonesb
aNICHD, NIH, Bethesda, USA; bLaboratory of
Pathology, National Cancer Institute, National Institutes of Health, Bethesda, USA
Introduction: Carboxypeptidase E (CPE) is associated with growth and
metastasis of liver, pancreatic and colorectal cancers. Here, we examined if exosome-based
CPE plays a role in promoting malignant properties of liver hepatocellular carcinoma
(HCC)
cells, and if CPE-shRNA loaded exosomes can be used to target growth and invasion
of HCC
cells.
Methods: Exosomes were isolated from the culture media of high metastic
HCC97 H cells and incubated with low metastatic HCC97 L cells. In other experiments,
CPE-shNA loaded exosomes from HEK293cells were incubated with HCC97 H cells. The recipient
cells were analysed for proliferation using MTT assay, colony formation, and Matrigel
invasion.
Results: Analysis of exosomes derived from HCC97 H cells revealed CPE-WT mRNA
and protein. Exosomes released from HCC97 H cells were able to enhance proliferation
and
invasion of HCC97 L cells. When CPE expression was suppressed in the HCC97 H cells
before
exosome isolation, the exosomes had no effect on proliferation and invasion. These
data
demonstrate the ability of exosomes to confer growth and invasion in HCC cells and
the role
of exosomal CPE in driving the process. Previously it was shown that down-regulation
of CPE
expression by shRNA can reverse tumour growth and metastasis in an HCC mouse model.
We
therefore loaded CPE-shRNA into exosomes by infecting HEK293 (Human Embryonic Kidney)
cells
with adenovirus carrying CPE-shRNA-GFP. These modified exosomes were used to transfer
CPE-shRNA to HCC97 H cells, resulting in significant reduction in proliferation and
colony-forming ability of these cells. CPE-shRNA loaded exosomes were found to down-regulate
the expression of Cyclin D1 and c-MYC, two genes with high relavance to tumour growth
and
metastasis.
Summary/Conclusion: Our results demonstrate the ability of exosomal CPE to
enhance proliferation and invasion in low metastatic HCC cells and the potential to
use
shRNA loaded exosomes to target CPE as a therapeutic strategy to treat liver cancer.
Funding: Intramural Program of the Eunice Kennedy Shriver National Institute
of Child Health and Human Development, and National Cancer Institute, National Institutes
of
Health, Bethesda, Md. 20892.
LBS01.12
Stress hormones promote prostate cancer aggressiveness through
modulation of mir-628-5p expression and exosome release
Leslimar Rios-Colon, Elena Arthur and Deepak
Kumar
North Carolina Central University, Durham, USA
Introduction: Despite proactive screening and steady declines in mortality,
prostate cancer (PCa) remains one of the most prevalent cancers among men. Evidence
suggests
that chronic activation of stress signalling pathways can result in an altered miRNAs
transcriptome and affect exosomal content and release. Here, we study the interaction
between leptin and miR-628-5p expression, previously shown to be downregulated in
PCa
patients. In addition, explored the effect of stress hormones cortisol and leptin
on
exosomal release and content from PCa cells.
Methods: We utilized normal prostate cell line RWPE-1, and PCa cells PC3,
LNCaP and MDA-PCa-2b. Proliferation of cells treated with leptin in the presence or
absence
of miR-628-5p mimic or negative control was assessed by MTT, colony formation, wound
healing, and expression of targets affected by miR-628-5p was assessed by western
blotting.
Moreover, exosomes were isolated via differential centrifugation from PCa cells treated
with
leptin or cortisol and exosome number was determined by Nanotracking Analysis. Exosome
content was determined by western blotting and proteomic analysis by mass spectrometry.
Results: We observed that leptin significantly decreased expression of
miR-628-5p in RWPE-1 cells. Co-treatment with miR-628-5p mimic and leptin abrogated
these
effects in a cell dependent manner. We also observed that co-treatment with leptin
affected
miR-628-5p target JAG1 and other molecules involved in epithelial to mesenchymal transition.
In parallel, we demonstrated that cortisol increases exosome secretion particularly
in PC3
cell exosomes with a 2.6-fold increase at 5 nM Cortisol compared to untreated. Western
blotting revealed the presence of GR in exosomes particularly at 5 nM Cortisol.
Summary/Conclusion: Understanding epigenetic regulation through miRNAs and
exosomes may be the key to understand stress hormone influence in PCa progression.
These
findings suggest that stress hormones effectively affect miR-628-5p expression and
exosomal
release and signalling.
LBS02: Late Breaking: New Technologies and Methods Chair: Alicia
Llorente – Department of Molecular Cell Biology, Institute of Cancer Research, Oslo
University Hospital, The Norwegian Radium Hospital Chair: Wyatt Vreeland – National
Institute of Standards and Technology
LBS02.01
Exogenous microRNA loading into extracellular vesicles via
producer cell transfection
Alex Eli Pottash
a, Daniel
Levyb and Steven M. Jayc
aUniversity of Maryland, College Park, USA;
bUniversity of Maryland – College Park, Washington, USA; cUniversity
of Maryland, College Park, College Park, USA
Introduction: Extracellular vesicles (EVs) are promising drug delivery
vehicles for therapeutic microRNA (miRNA). For the loading of exogenous cargo, researchers
broadly seek to either manipulate the EVs directly or the cell that produce them.
Electroporation, sonication, and direct EV transfection are common methods that work
by
physical disruption or irreversible chemical addition, which may irreparably damage
the
molecules intended for therapy. On the other hand, transfection into the producer
cells is a
simple option that does not imperil EV integrity.
Methods: There are multiple factors that contribute to EV loading efficiency,
including transfection reagent used, timing, and dosage. Thus, we sought to establish
a
basic protocol and improve understanding of the underlying dynamics involved in a
basic
system consisting of HEK293 T cells and miR-146a-5p mimic.
Results: In this work, we examined how different reagents lead to variable EV
loading. Then we looked at variable dosages, specifically the relationship between
RNA
amount added to reagent, amount present in cell, and amount exported to EVs.
Summary/Conclusion: These results will help future studies produce EVs with
exogenously loaded small RNA, and suggest future optimizations.
Funding: National Institutes of Health. R01 and T32 (Host Pathogen
Interactions at University of Maryland).
LBS02.02
Single extracellular vesicle trapping by Aptamer-Au Nanoparticle
Mediated Au Superlattices
Seung Hee Baek
a, Hye Jin
Leeb and Sung-Wook Namc
aSchool of Medicine, Kyungpook National Univiersity, Daegu,
Republic of Korea; bDepartment of Chemistry and Green Nano Materials Research
Centre, Kyungpook National University, Daegu, Republic of Korea; cDepartment of
Molecular Medicine, School of Medicine, Kyungpook National University, Repulic of
Korea,
Daegu, Republic of Korea
Introduction: xtracellular vesicles (EVs), secretory vesicles of 30–100 nm
size from cells, are carriers of proteins and miRNAs. Despite a variety of EV isolation
tools, it is still challenging to separate individual EVs. We report a single EV trapping
method via aptamer-mediated assembly between Au nanoparticle (AuNP) and Au superlattice
template. We propose a chip-based EV trapping technique based on semiconductor
processes.
Methods: We introduce aptamer coated Au nanoparticle (AuNP) and Au
superlattices as a template to capture EVs. First, we fabricated poly(methyl methacrylate)
(PMMA) hole pattern on Au-coated Si substrates by using electron beam lithography
(EBL). We
designed 200 nm-diameter hole patterns to capture one EV in each hole. To connect
the AuNP
and the Au superlattice template, we used an aptamer molecule as a linker strand.
Also, to
capture individual EVs, the aptamer molecule is designed to have a hairpin structure
to
specifically bind to CD63, a protein marker of EV. We modified 5ʹ-terminal and 3ʹ-terminal
of the CD63 aptamer with thiol group for the formation of self-assembly monolayer
(SAM) on
both AuNP and Au superlattice surface.
Results: First, we coat the CD63 aptamer on the surface of AuNP. Afterwards,
we load the aptamer-coated AuNP into Au superlattice template. EV solution is specifically
bound to CD63 aptamer. After washing step, each EV is expected to locate within a
single
hole due to the size confinement of the hole. To separate the EVs from the aptamer,
we use
restriction enzyme, BamHI, to recognize specific DNA sequence and cleave them.
Summary/Conclusion: In this report, we propose a AuNP -linked Au superlattice
chip by aptamer molecules for trapping EVs. We selected CD63 aptamer for specifically
binding with CD63 in EVs. In addition, we designed CD63 aptamer as a linker strand
to
connect AuNP to Au superlattice chip. Using this chip, we differentiate the captured
single
EV which includes various biological information such as proteins and miRNAs.
Funding: This research was supported by the Bio & Medical Technology
Development Program of the National Research Foundation (NRF) funded by the Ministry
of
Science & ICT (2017M3A9G8083382).
LBS02.03
Edit the use of a DLS-based clinical platelet instrument beyond
the blood transfusion laboratory
Sam Law
a, Melissa Lima,
Chantelle Blythb, Michael Whitmorea, Jancy Johnsonc and
Gregor Lichtfussb
aExopharm Ltd, Melbourne, Australia; bExopharm,
Melbourne, Australia; cExopharm Ltd/University of Melbourne, Parkville,
Australia
Introduction: A hallmark of platelet activation is the release of internal
granules as extracellular vesicles/microparticles. ThromboLUX is a
dynamic-light-scattering-based (DLS) instrument that was developed for use in clinical
setting to check for platelet activation before transfusion. Compared to traditional
DLS,
the ThromboLUX requires no cleaning (single-use capillary) and requires very little
sample
(70 µL). Hence the ThromboLUX may be a useful instrument beyond platelet pack test
in blood
transfusion laboratory. We have evaluated its use as an in-process monitoring tool
for
industrial EV manufacturing, for both quantifying cells (input) and EVs (output).
Methods: The ThromboLUX was used to test the activation status of expired
platelet packs (donated by ARCBS for research purpose). The readout was compared with
platelet swirling test and flow cytometry data (surface marker). Furthermore, the
ThromboLUX
was also tested for process development and EV manufacturing monitoring purposes at
different stages of the process for its ability to rapidly obtain particle presence
and size
information on EVs. Time to result was also compared between different particle analysis
methods.
Results: The ThromboLUX was a better predictor of platelet packs variability
compared to the traditional platelet swirling method. However, we did not observe
a strong
correlation between the activation status and the flow cytometry-based activation
marker
data. The ThromboLUX was able to provide a useful estimation of particle presence
and sizing
of EVs in-process. Results are obtained rapidly, within minutes, with minimal sample
prep.
Summary/Conclusion: Although we did not observe a significant direct
correlation between flow cytometry activation data and the % microparticles (within
a small
sample size), the ThromboLUX has shown potential to become a useful tool for in-process
monitoring for EV manufacturing and other EV research, in particular through its speed
and
ease of use.
Funding: All funding was through Exopharm Ltd (ASX:EX1).
LBS02.04
Secreted protein of MSC: Adipose vs Umbilical cord
tissue
Yanni Dirgantaraa, Ajeng Diantinib, Cynthia
Retna Sartikac, Endah Dianty Pratiwic, Emilia Rahmadania
Utamic, Sheila Fawziyya Jundanc, Sheila Mutiac, Zahira
Halifac, Angliana Chouwd, Geofanny Faciciliaa and Annisa
Nur Arofahc
aPT Prodia StemCell Indonesia, Jakarta, Indonesia;
bFaculty of Pharmacy, Padjadjaran University, Jl. Eijkman No. 38, Bandung,
Indonesia, Bandung, Indonesia; cPT. Prodia Stemcell Indonesia, DKI Jakarta,
Indonesia; dPT. Prodia StemCell Indonesia, Jakarta, Indonesia
Introduction: Mesenchymal stem cells (MSC) has been widely used in both
clinical and pre-clinical trials as an alternative teraphy for degenerative diseases1.
The
therapeutic effect of MSC derived through paracrine effects that secreted protein
as
signalling molecules that support the process of cell and tissue regeneration3. Secreted
proteins from MSC can be different depends on source of the cells, such as umbilical
cord,
adipose, dental pulp, and other potential sources. Therefore, it is considered quite
important to know the better source of MSC to produce the protein that can be used
for cell
and tissue regeneration.
Methods: This study compared the total protein produced by MSC from umbilical
cord and adipose tissue from various passages. MSC were cultured using growth medium
until
reach 70–80% of confluency. Afterwards, growth medium were replace with serum free
media for
24–48 haours to harvest the secreted protein.
Results: Total protein produced by adipose-derived MSC is ranged from
55.7–188.2 μg/ml, it is higher compared to the total protein from umbilical cord-derived
MSC
which ranged from 55.1–141.2 μg/ml. Total protein produced by MSC is increased along
with
the passage in both sources of MSC. MSC release signalling molecules for cells
communication, where in vitro condition it can be found in their culture medium. Protein
expression from the culture medium is increased along with the passage of the cells.
This is
related to the process of cell maturation, where the more mature cells become more
active to
metabolize and secrete proteins. Adipose-derived MSC secreted a higher concentration
of
protein compared to umbilical cord-derived MSC. The more mature tissue, the more protein
secreted by the cells.
Summary/Conclusion: Adipose-derived MSC secreted a higher concentration of
protein compared to umbilical cord-derived MSC.
LBS02.05
Development of scalable monolith chromatography processes for
the purification of exosomes from a clinically relevant stem cell product
Ivano L. Colao
a, Daniel
Bracewellb, Randolph Cortelingc and Ivan Walld
aUniversity College London, Reading, UK;
bUniversity College London, LONDON, UK; cReNeuron Limited, Pencoed
Business Park, Pencoed, Bridgend, Wales, CF35 5HY, UK, Bridgend, UK; dUniveristy
College London, London, UK
Introduction: A major manufacturing challenge related to exosome bioprocessing
is that of robust and scalable purification. As efforts to translate exosomes into
clinics
grows, the more important the design of quality systems which can reproducibly purify
the
product becomes. The current gold-standard, ultracentrifugation, was adopted from
the viral
vaccine industry, but remains imperfect in terms of scale up and manufacturing due
to labour
and time intensive process requirements. In order to follow the preferential adoption
of
more standard bioprocesses, as previously achieved by the viral vaccine industry,
we show
the development of two monolith chromatography steps which can be used to purify exosomes
from a clinically relevant, allogeneic stem cell product (CTX0E03).
Methods: T-flask expansion of CTX0E03 cells was performed to yield batches of
5–15 L of conditioned medium. The medium was subsequently clarified by bench-top
centrifugation, and concentrated into a crude concentrate by tangential flow filtration
[TFF], using a combination of 0.22 µm dead-end filtration prior to concentration in
a 300kDa
hollow-fibre TFF system. TFF retentate was loaded onto 1 mL HIC or AEx monoliths,
for
further purification. Potency was assessed by a fibroblast wound healing assay in
vitro.
Results: Exosome presence was verified in the TFF material by detection of CD
81 and CD 63. Exosomes recovered in this manner could achieve full wound closure in
vitro
over 72 hours, when dosed at 20 µg. Further purification by monolith chromatography
showed
high levels of reduction of albumin, detected by western blot, as well as heightened
ratios
of particles to both total protein, and total DNA. The results indicate that neither
AEx nor
HIC steps cause detrimental loss to product function, either alone or in combination
with
one another.
Summary/Conclusion: Monolith chromatography can achieve scalable and
reproducible purification of stem cell derived exosomes, whilst maintaining their
functional
capacity.
Funding: Engineering and Physical Science Research Council (EPSRC) Industrial
Doctoral Training Centre in Bioprocess Engineering Leadership (EP/G034656/1).
LBS02.06
Using degron-tagged reporters to specifically label and track
extracellular vesicles in vivo
Katharina Beera, Gholamreza Fazelib and
Ann M. Wehman
c
aRudolf Virchow Centre at the University of Würzburg,
Würzburg, Germany; bRudolf Virchow Centre for Experimental Biomedicine,
University of Wuerzburg, Germany, Wuerzburg, Germany; cUniversity of Denver,
Denver, USA
Introduction: To clarify the roles of extracellular vesicles (EV) in vivo, it
is important to visualize and track EVs from the source cells to their destination.
However,
since most EV reporters are also present in the releasing cell, it is challenging
to
visualize EVs in vivo.
Methods: To tackle these problems, we developed a degradation-based technique
to remove background fluorescence. We use degradation motifs called degrons to target
proteins for ubiquitination and degradation in the cytosol, while leaving EVs labelled.
We
re-purposed an endogenous zinc finger (ZF1) degron in the nematode model organism
Caenorhabditis elegans, but show that the technique can also be applied to mammalian
cells
with the auxin-inducible degron (AID). To specifically label MVs in C. elegans, we
ZF1-tagged the PI4,5P2-binding PH domain of the cytosolic phospholipase PLC1∂1, which
is
primarily found at the plasma membrane.
Results: The ZF1-tagged plasma membrane reporter is released in MVs outside
the cell and remains fluorescent because intervening membranes hinder the proteasomal
degradation of the ZF1 reporter in MVs. In the end, MVs maintain fluorescence, while
the
reporter is removed from inside the source cell. This increased the visibility of
MVs from
the neighbouring cells, enabling the visualization of MVs with a normal light microscope.
Using this ubiquitous plasma membrane reporter, we could visualize released MVs during
C.
elegans embryogenesis, which helped us to identify new MV release inhibitors. Additionally,
we show that this technique can be used to determine MV cargo in vivo.
Summary/Conclusion: Ultimately, our approach boosts the visualization and
tracking of EVs in vivo. Since degron-mediated degradation is a widely used tool to
analyse
loss of function effects in cell culture and model organisms, we propose that this
technique
can be readily adapted to EV experiments and will help to visualize and track MVs
in a broad
range of experimental systems.
Funding: KBB and AMW are funded by DFG grant WE5719/2-1. GF is funded by DFG
grant FA1046/3-1.
LBS02.07
Heterogeneity and batch variation of HEK293 extracellular
vesicles
Richard Kelwick
a, Amelie
Heliota and Paul Freemontb
aImperial College London, London, UK; bThe London
DNA Foundry, Imperial College London, London, UK
Introduction: Extracellular vesicles (EVs), are emerging as a potentially
powerful new class of multi-modal therapeutics and drug delivery vehicles. However,
challenges remain – EVs are highly heterogeneous and differ in terms of their biogenesis,
size (~30-1000 nm) and complex molecular compositions (lipid bilayers, ncRNAs, proteins
and
small molecules). Furthermore, EVs exist within complex cell secretomes and body fluids.
Thus, navigating EV heterogeneity through the use of robust isolation and characterisation
methods (metrology) is beneficial to several research applications – including, therapeutic
EV manufacturing. To this end, we isolated and characterised batches of HEK293 EVs
that were
harvested from a hollow fibre bioreactor.
Methods: HEK293 cells are an industrially important mammalian cell line that
have been previously used to manufacture therapeutic antibodies and EVs. To prepare
EV
batches, HEK293 cells were cultured for 30 days within a hollow fibre bioreactor that
was
configured with a 20 kDa cartridge to concentrate HEK293 cells (up to ~10e9 cells)
and their
secretomes (>20 kDa proteins and EVs) within ~20 ml harvest volumes. EVs were isolated,
for comparative purposes, using ultracentrifugation (UC), tangential flow filtration
(TFF)
and immunocapture (IC)-based methods. Isolated EVs were characterised using DLS,
high-throughput NTA, dot blot array and partially using nanoflow cytometry, ExoView
and
TRPS.
Results: EV batches harvested at different intervals were characterised.
EV/particle sizes (NTA): UC 140 ± 10.7 nm, TFF 134 ± 13.3 nm and IC 126 ± 3.9 nm.
EV/particle concentrations (NTA): UC 17 ± 25x10e11/ml, TFF 0.2 ± 0.1x10e11/ml and
IC
49 ± 35x10e11/ml). Total protein (Qubit): UC 0.8 ± 0.6 mg/ml, TFF 0.2 ± 0.2 mg/ml
and IC
4 ± 0.6 mg/ml. EV batch diversity is likely due to a number of interacting factors
including
changes in cell density, cell growth rate and EV isolation or characterisation
methodology.
Summary/Conclusion: EV heterogeneity varies across different EV batches and
must be carefully monitored and assessed in terms of any potential impacts on scalable
EV
manufacturing.
Funding: RK is supported by the Cancer Research UK Imperial Centre Development
Fund and until recently a BBSRC-funded RSE Enterprise Fellowship. We also acknowledge
the
support of Imperial Confidence in Concept (MRC/EPSRC), Imperial College London EPSRC
Impact
Acceleration Account (EP/R511547/1), EPSRC grant (EP/L011573/1), and BBSRC grants
(BB/L027852/1), Follow-on-Fund [BB/T017147/1].
LBS03: Late Breaking: Specific Cell Derived EVs Chair: Dimitrios
Kapogiannis – Laboratory of Clinical Investigation, National Institutes of
Ageing
LBS03.01
Blunted postprandial suppression of phosphoenolpyruvate
carboxykinase in kidney-derived urinary exosomes in early insulin resistance in
humans
Rajni Sharmaa, Manju Kumaria, Pawan
Kumara, Ashish Awasthib and Swasti
Tiwari
a
aSanjay Gandhi Post Graduate Institute of Medical Sciences,
Lucknow, India; bPublic Health Foundation of India, Delhi, India
Introduction: Renal resistance to insulin’s action could be an independent
risk for chronic kidney disease (CKD) and diabetes. The existing methods to estimate
kidney
– specific insulin resistance in humans are not feasible for clinical and epidemiological
studies or use in routine clinical practice. Blunted action of insulin on insulin-responsive
genes in the kidneys, such as Phosphoenolpyruvate carboxykinase (PEPCK), could be
an
important indicator for impaired kidney-specific insulin sensitivity.
Methods: PEPCK, Glucose 6-phosphatase (G6Pase) and Fructose 1,6-bisphosphatase
(FBPase) were estimated in kidney-derived exosomes in human urine (UE). Fold mRNA
expression
were estimated using qRT-PCR and related to fasting serum insulin levels. Proteins
were
estimated by ELISA and normalized to urine creatinine. Urinary exosomal protein after
overnight fast and at 2 hours of oral glucose tolerance test (2 h-OGTT, postprandial)
was
compared. Subjects were categorized based on HOMA-IR values for the analysis. In vivo
and in
vitro models with PEPCK induction was used to study PEPCK regulation in kidney and
in
kidney-derived exosomes.
Results: Human UE had detectable protein and mRNA levels of PEPCK, FBPase and
G6Pase, with FBPase having the highest abundance. Fasting insulin levels strongly
predicted
PEPCK mRNA levels in human UE. Also, Pepck protein in UE showed a significant suppression
in
the fed state, relative to the fasted state. HOMA-IR values significantly predicted
the
expression of the three Gng enzymes in uE from the fed state. Subgroup analysis showed
blunted Pepck suppression in subjects with lower insulin sensitivity relative to subjects
with better insulin sensitivity respectively. Immuno-blotting showed significantly
higher
PEPCK protein band-density for PEPCK protein in UE from pre-diabetic and diabetic
subjects
relative to non-diabetic controls. Renal PEPCK induction in rat using short-term acidosis,
or in human proximal tubule (hPT) by glucocorticoid stimulation, resulted in higher
PEPCK
levels in rat UE and hPT-derived exosomes, respectively.
Summary/Conclusion: The urine-based approach would ease regular screening of
kidney-specific insulin sensitivity in humans.
Funding: Funded by ICMR, HRD & SGPGI intramural.
LBS03.02
Differential characterization of extracellular vesicles in
neuroprotective human platelet lysate preparations
Balasubramaniam Namasivayam
a,
Liling Delilab, Yu-Wen Wub, Alain Rouleauc, Annie
Frelet-Barranda, Celine Elie-Caillea, Thierry Burnoufd
and Wilfrid Boireau
aFEMTO-ST Institute, Besançon, France; bCollege of
Biomedical Engineering, Taipei Medical University, Taipei, Taiwan (Republic of China);
cFEMTO-ST Institute, UBFC, CNRS, Besancon, France, Besancon, France;
dCollege of Biomedical Engineering, Taipei Medical University, Taipei City,
Taiwan (Republic of China)
Introduction: Custom-made Platelet Pellet Lysate (PPL) and Heat-treated PPL
(HPPL) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic LUHMES
neuronal cell culture. This effect is significantly enhanced using HPPL, which was
also
highly protective of TH-expressing neurons in mice Parkinson’s disease (PD) model.
The role
of their EVs in neuroprotection is unclear. Our NanoBioAnalytical (NBA) platform can
help to
characterize platelet EVs phenotype, size, and morphology, and unveil the influence
to
neuroprotective functions of platelet lysates.
Methods: PPL and HPPL have been prepared by freeze-thaw lysis of purified
platelets. HPPL underwent an additional treatment at 56°C for 30 min. Proteins were
analysed
by Western blot (WB). HPPL neuroprotective function was studied by cell viability
assays of
LUHMES cells exposed to erastin. The concentration and size of EVs were evaluated
in
solution by several complementary approaches. In the multiscale NBA system, EVs were
captured onto specific antibodies grafted on the biochip, quantified by Surface Plasmon
Resonance imaging (SPRi) and then studied in situ by Atomic Force Microscopy (AFM)
to unveil
EVs subsets.
Results: WB revealed higher expression of CD9, CD41 and CD61 markers in PPL
versus HPPL. HPPL exerted increased viability of erastin-exposed LUHMES cells compared
to
PPL. EVs concentration was in the order of 10e11/mL. SPRi results were consistent
with WB,
with higher capture PPL EVs on aCD9, aCD41, and aCD61 immunoarrays compared to HPPL.
Evidence of EVs was confirmed by AFM that revealed spherical EVs and dimensions of
the
objects captured on spots. The density of EVs was consistent with the differential
capture
in SPRi.
Summary/Conclusion: HPPL exerts superior neuroprotection in in vitro and in
vivo PD models. Our work confirms the presence of EVs in HPPL and PPL, suggesting
the
applicability of NBA platform for complex sample analysis. CD41 and CD61 markers seemed
affected by PPL heat treatment. NBA platform can be valuable in identification of
the role
played by EVs in the beneficial neuroprotective effects of HPPL that can be exerted
by the
EVs cargo (growth factors, mRNA, miRNA). The proteomic and genomic contents of EVs
present
in both platelet lysates remains to be studied.
Funding: MicroMPs, 2017 from Franche-Comte region (France); 107–2314-B-038-084
from MOST, Taiwan; DP2-107-21121-01 N-09 from MoE and TMU, Taiwan
LBS03.03
GMP compatible angiogenic exosome processing towards therapeutic
for treating stroke
Jieun Lee
a, Wei Guob,
Mike Westc and Dana Laroccac
aStem Cell Team, Seoul, Republic of Korea;
bUniversity of Pennsylvania, Philadelphia, USA; cAgeX Therapeutics
Inc., Alameda, USA
Introduction: There is a critical unmet medical need for new therapies to
treat age-related diseases including cardiovascular diseases such as stroke. Exosome
derived
from stem cells have shown intrinsic therapeutic potential in a variety of animal
models of
ischaemic diseases. We have identified scalable exosome production cell lines (PureStem)
as
a source of angiogenic exosomes and are aiming to generate good manufacturing practice
(GMP)
grade therapeutic exosomes that can effectively mediate angiogenesis and tissue
regeneration.
Methods: We are developing exosome production and purification protocols that
combine methods of Tangential filtration flow (TFF) and size exclusion chromatography
(SEC).
The particle number and size were measured by both tunable resistive pulse sensing
(TRPS) as
well as nanoparticle tracking analysis (NTA) for comparison. Exosomes were characterized
by
detection of exosome surface markers and absence of cellular markers. Purity was assessed
by
measuring particles per ug of total protein content. The angiogenic activity of
PureStem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing
and tube formation assays. We further investigated the molecular cargo of PureStem-exosomes
by screening miRNAs targets, RNA-seq analysis, and mass spectrometry analysis.
Results: The isolated PureStem-exosomes using our developed protocols were
highly purified, resulting purity in the range of 1E10-5E10 particles/ug. We selected
angiogenic exosome-producing cell lines from our PureStem library by screening for
functional activity and characterizing their molecular cargo. We found that PureStem
progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal
stem
cell (MSC)-derived exosomes. Furthermore, angiogenic microRNAs such as miR-126 were
enriched
in PureStem-exosomes from certain producer cell lines.
Summary/Conclusion: These data demonstrate the potential for using PureStem
lines as a highly scalable source of therapeutic exosomes. We were able to obtain
highly
pure exosomes that retain their angiogenic activity. We anticipate that PureStem-exosomes
will be a valuable resource for developing EV therapies for stroke and other ischaemic
diseases. We have developed purification methodologies aimed at achieving a robust
and
scalable exosome production compatible with GMP for clinical grade PureStem-exosomes.
These
developments have great potential as therapeutic agents for future preclinical in
animal
model of stroke and clinical trials.
LBS03.04
Neuronal-origin plasma EVs provide biomarkers for Parkinson’s
Disease
Joseph M. Blommer
a, Toni
Pitcherb, Maja Mustapicc, Wassilios Meissnerb, Tim
Andersonb and Dimitrios Kapogiannisa
aNational Institute on Aging, Intramural Research Program,
Laboratory of Clinical Investigations, Baltimore, USA; bNew Zealand Brain
Research Institute, Christchurch, New Zealand; cNIH/National Institute on Aging,
Baltimore, USA
Introduction: The hallmark of Parkinson’s Disease (PD) is a-synuclein
accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. PD
is also
associated with insulin resistance, a condition characterized by phosphorylated insulin
receptor substrate-1 (IRS-1). Besides motor symptoms, some PD patients develop Mild
Cognitive Impairment (PD-MCI) or dementia (PD-D). Given the importance for prognosis,
there
is an urgent need to develop biomarkers for distinguishing PD with normal cognition
(PD-N)
from PD-MCI/D. Neuronal-origin Extracellular vesicles (NEVs) contain cell signalling
and
pathogenic proteins (including a-synuclein), which may serve as biomarkers for Alzheimer’s
disease, PD and other dementias.
Methods: From 0.5 ml of plasma from 104 PD-N, 83 PD-MCI, and 39 PD-D patients,
we immunocaptured NEVs using anti-L1CAM antibody. Then, IRS-1pSer312 and IRS-1pTyr20
and
a-synuclein were measured in NEVs using electrochemiluminescence immunoassays.
Results: A-synuclein was lower in PD-MCI and PD-D compared to PD-N
(p < 0.005) and significantly decreased with increasing motor symptom severity measured
by MDS-UPDRS III score (p = 0.005). IRS-1pSer312 was lower in PD-D than in PD-N. IRS-1pTyr20
significantly decreased with increasing MDS-UPDRS III score (p < 0.005). No biomarker
was
associated with disease duration.
Summary/Conclusion: PD patients with cognitive impairment exhibited lower NEV
levels of a-synuclein than cognitively intact PD patients, whereas a-synuclein and
IRS-1pTyr20 were inversely associated with PD motor symptom severity. Additional biomarkers
and measurements will be available by the time of ISEV. Plasma NEVs is a valuable
tool for
discovering biomarkers in PD and investigating aspects of disease progression.
Funding: This research was funded in part by the Intramural Research Program
of the NIH, National Institute on Aging.
LBS03.05
Urinary extracellular vesicles as biomarkers of kidney allograft
injury: optimization of isolation protocol and characterization
Ivana Sedej
a, Magda Tušek
Žnidaričb, Vita Dolžanc, Miha Arnold and Metka
Lenassie
aUniversity Medical Centre Ljubljana, Ljubljana, Slovenia;
bNational Institute of Biology, Ljubljana, Slovenia, Ljubljana, Slovenia;
cInstitute of Biochemistry, Faculty of Medicine, University of Ljubljana,
Slovenia, Ljubljana, Slovenia; dUniversity Medical Centre Ljubljana (UMCL),
Slovenia, Ljubljana, Slovenia; eUniversity of Ljubljana, Faculty of Medicine,
Institute of Biochemistry, Ljubljana, Slovenia
Introduction: Despite decades-long advancement in transplant medicine, there
is a necessity for personalized approach regarding early kidney allograft injury recognition
and immunosuppression therapy towards improved transplant outcomes. Biopsy, a gold
standard
for assessment of kidney allograft injury, cannot be serially used for the diagnosis
of
subclinical injury due to it’s invasiveness and possible sampling errors. Instead,
urine is
easily obtainable and bearing extracellular vesicles (EVs), potential carriers of
pathological signals related to kidney injury. Our aim was to set up a urinary EV
(uEV)
isolation protocol that would allow consistent and reliable identification of their
characteristics and cargo.
Methods: Second morning urine sample (25 mL) was collected from 7 patients and
processed within 4 hours. Oxalate precipitation, pH and dilution variability, uromodulin
polymerization and high protein content were taken into account. Isolated EVs were
defined
by Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA).
uEV
specific proteins and miRNAs were analysed by Western blot and qPCR, respectively.
Results: The optimal protocol relied on low speed urine centrifugation (2.000
x g, RT) for cell removal and storage at −80°C prior to further analyses. After urine
thawing at RT, added EDTA averted cryoprecipitate and uromodulin polymer formation,
while
concentrated PBS neutralized the pH. Filtration through 0.22 µm pores was used for
large
particle removal, while centrifugal 100 kDa membrane units (Amicon®, Milipore) served
for
sample concentration followed by particle separation on size-exclusion chromatography
(SEC;
qEVoriginal, Izon Q). Protein vacant SEC fractions (as rated at A280) were pooled
and
concentrated to a volume of 70 µl. TEM micrographs revealed high sample purity and
cup-shaped morphology of uEVs. As per NTA results, the average mean size of EVs was
129,9 nm
with concentration range of 1 × 109 particles/mL of starting urine. uEVs were positive
for
the tested marker proteins Hsc70, flotillin, tubulin, GADPH and CD63. qPCR verified
miRNA
presence in uEVs, with CT for miR let-7i at 20.
Summary/Conclusion: We successfully isolated pure uEVs. The set up protocol
will be used to assess uEVs as non-invasive biomarkers of allograft injury in kidney
transplant recipients.
LBS03.06
Astrocyte-derived extracellular vesicles regulate dendritic
spine formation and neuronal network connectivity
Hyejin Lee
a, Amanda
Troutb, Raha Dastgheybc, Saja Khuderd and Norman
Haugheye
aDepartment of Neurology, Johns Hopkins University School of
Medicine, Baltimore, Maryland, Baltimore, USA; bDepartment of Neurology, Johns
Hopkins University School of Medicine, Baltimore, USA; cJohns Hopkins University,
Baltimore, USA; dJohns Hopkins University School of Medicine, Baltimore, USA;
eDepartment of Neurology, Johns Hopkins University School of Medicine,
Baltimore, USA
Introduction: Recent advancements in the biology of extracellular vesicles
have begun to implicate glial released microvesicles as mediators of glia to neuron
communication, suggesting that alterations in the release and/or composition of astrocyte
microvesicles could impact neuronal function.
Methods: Astrocytes were allowed to constitutively release extracellular
vesicles (ADEV-CR), or stimulated with ATP (ADEV-ATP). ADEVs were isolated by
ultracentrifugation followed by proteomic analysis. We developed a normative whole
transcriptome database using primary neurons exposed to ADEV-CR, and identified changes
in
neuronal gene expression produced by exposure of neurons to ADEV-ATP. We identified
a number
of pathways associated with the biological response of synapse, spine and neurite
outgrowth
that were regulated by ADEV-ATP.
The molecular cargo of ADEV-ATP responsible for regulating synaptic functions in neurons
were characterized by biochemical, molecular, and functional assays.
Results: ADEV-ATP enhanced the maturation of dendritic spines and produced
functional enhancements in neuronal activity and network connectivity. The mechanism
for
this effect involved the delivery of Integrin-☑ 1 and EphA2 that were enriched in
ADEV-ATP.
Integrin-☑ 1 facilitated binding of ADEVs to the neuronal surface, and EphA2-receptor
signalled through Ephrin to the tyrosine kinase ERBB2/4 that regulated the phosphorylation
and activation of TrkB without increasing expression of the natural ligands BDNF or
NTF3.
This direct activation of TrkB increased the expression of the synaptic scaffolding
proteins
Disc1, Arc, and Cplx3 to promote the maturation of dendritic spines. This increase
in mature
dendritic spines was associated with increased neuronal activity and network connectivity
demonstrating a functional strengthening of synapses.
Summary/Conclusion: These data identify a molecular mechanism whereby
modifications in ADEV protein cargo produced by the stimulation of astrocytes with
ATP
regulates synaptic maturation through activation of TrkB in a manner independent of
growth
factors.
LBS03.07
Enhancement of immunomodulatory functions of MSC-derived
extracellular vesicles through modification of up-stream parameters
Stephanie Kronstadt and Steven M. Jay
University of Maryland, College Park, College Park, USA
Introduction: Mesenchymal stem cell extracellular vesicles (MSC-EVs) have been
shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders.
Despite this, clinical translation of EV therapies is hindered by potentially low
potency in
vivo and the lack of a scalable biomanufacturing process. Cell culture parameters
are
critical in modulating both yield and bioactivity of EVs. Thus, we hypothesized that
the
combination of chemical priming and 3D dynamic culture would enhance the yield and
potency
of immunosuppressive MSC-EVs.
Methods: Bone marrow-derived MSCs cultured in flasks were chemically primed
using ethanol or curcumin. MSCs were also cultured using a 3D-printed scaffold-perfusion
bioreactor using a flow rate of 5 ml/min. Anti-inflammatory effects were assessed
following
application of MSC-EVs to lipopolysaccharide (LPS)-stimulated murine macrophages.
Subsequent
inhibition of the production of the pro-inflammatory cytokine IL-6, quantified using
an
ELISA, was used to characterize EVs as anti-inflammatory. In addition, both chemical
priming
and the bioreactor will be simultaneously utilized to potentially uncover any synergistic
effects on EV immunomodulation abilities. Nanoparticle tracking analysis (NTA) was
used to
assess EV size and concentration while protein mass was measured via a BCA assay.
Results: Preliminary data suggests that priming MSCs with 100 µM ethanol for
24 hours prior to EV collection results in a strong inhibition of IL-6 production
in
stimulated murine macrophages. NTA revealed that MSC-EV yield increased by about two
orders
of magnitude in the bioreactor (1.40E12 ± 7.92E10) when compared with flasks
(2.28E10 ± 2.81E9). Protein measurements also indicated that EV production in the
bioreactor
(~7600 µg) was much greater compared with production in the flasks (~2400 µg). Additionally,
average protein content per EV was reduced in the bioreactor when compared with flask
EVs.
Summary/Conclusion: Although further investigation is required, our results
potentially promise an effective and inexpensive priming agent (i.e., ethanol) for
the
production of anti-inflammatory MSC-EVs. This, combined with the significant increase
in
yield via 3D dynamic culture, presents practical solutions to both EV manufacturing
scalability and potency issues.
LBS03.08
Donor source affects potency of mesenchymal stem cell-derived
extracellular vesicles
Daniel Levy
a and Steven M.
Jayb
aUniversity of Maryland – College Park, Washington, USA;
bUniversity of Maryland, College Park, College Park, USA
Introduction: Mesenchymal stem cell (MSC) therapies have been heavily
investigated for their utility in applications such as wound healing and regenerative
medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. Recently,
MSC-derived extracellular vesicles (EVs) have been implicated as primary effectors
in
MSC-based therapies via protein and nucleic acid cargo transfer to patient cells.
MSC EVs
represent a superior alternative to MSC-based therapies, as they lack the ability
to
replicate and are much smaller in size, circumventing related safety concerns such
as
immunogenicity, teratoma formation and blood vessel occlusion. However, a key drawback
with
MSC therapies in general is their variable therapeutic potency, which is dependent
on donor
source. As a cell derived therapeutic, this crucial limitation is hypothesized to
exist in
MSC EVs as well. Here, we demonstrate the varying bioactivities of isolated MSC EVs
from
differing donors and tissue sources.
Methods: Six separate MSC lines were obtained from different donors, with
three MSC lines derived from donor adipose tissue, and the other three from the bone
marrow
of separate individuals. EVs were isolated from each MSC line at passage 3 via differential
centrifugation and ultrafiltration. These isolated MSC EVs were then characterized
for
size/concentration via nanoparticle tracking analysis, and EV markers (TSG101, ALIX,
CD63)
via western blot. Pro-vascularization capacities of MSC EVs were determined by a gap
closure
assay using human umbilical cord vein endothelial cells (HUVECs).
Results: Characterization of MSC EVs revealed similar sizes and EV marker
expression across donor groups, regardless of tissue source. Furthermore, comparison
of
adipose tissue-derived (AD) MSC EVs from three donors indicates varying pro-vascularization
bioactivity between those donors evaluated in vitro via gap closure assay. Similar
results
were observed for the bone marrow-derived (BM) MSC EV donor groups.
Summary/Conclusion: This work highlights the need for screening of donor
derived-MSCs before use for therapeutic EV production. Additionally, standardized
criteria
for MSC donor selection are needed before isolated MSC EVs can be used as a large-scale,
repeatable therapeutic treatment.
LBS03.09
Analysis of extracellular vesicle populations from
malaria-infected erythrocytes by field-flow fractionation reveal distinct
sub-sets
Alicia Rojas
a, Paula
Abou-Karama, Anna Rivkina, Yael fridmann-sirkisb, Yifat
Ofir- Birinc and Neta Regev-rudzic
aDepartment of Biochemical Sciences, Weizmann Institute of
Sciences, Rehovot, Israel, Rehovot, Israel; bWIS, Rehovot, Israel;
cWeizmann Institute of Science, Rehovot, Israel
Introduction: Malaria is one the most devastating infectious disease in the
world and Plasmodium falciparum (Pf) represents the deadliest species. This parasite
invades
human red blood cells (RBCs) and releases extracellular vesicles (EVs) carrying DNA,
RNA and
protein cargo components which are involved in the pathogenesis of the disease. Recently,
it
has been shown in mammalian systems that EVs are subdivided into different subpopulations,
each with a distinct biological function. However, it is still unknown whether Pf-infected
RBCs (Pf-EVs) release different EV subpopulations with distinct cargo.
Methods: We isolated EVs from Pf- infected and uninfected RBCs, Pf-EVs or
ui-EVs, respectively, using differential centrifugation. The EV pellet was subjected
to
field flow fractionation (FFF). The different subpopulations were collected, concentrated
with size-exclusion filters and evaluated by Nanoparticle Tracking Analysis. Additionally,
the presence of EV markers (SR1 and HSP90) were examined by Western blot analysis.
Results: The FFF analysis showed four particle subpopulations derived from the
Pf-EVs and five in the ui-EVs. The first three subpopulations were similar in their
detection signals in both samples, but the fourth subpopulation was consistently higher
in
ui-EVs than in Pf-EVs. Moreover, HSP90 was detected in subpopulations 3 and 4 of both
Pf-EVs
and ui-EVs, whereas SR1 only in subpopulation 3.
Summary/Conclusion: Pf-EV and ui-EV have similar separation profiles and
proteins markers in their subpopulations, consistent with the fact that both samples
are
derived from host RBCs. Additional data regarding the DNA and RNA cargo, as well as
microscopic observations of the Pf-EV and ui-EV subpopulations is necessary. This
will
clarify how malaria parasites sort their components into EVs and which fractions are
associated to immune evasion and pathogenesis.
LBS03.10
Evidences on microalgal extracellular vesicles: a morphological
assessment
Marko Jeran
a, Darja
Božiča, Zala Janb, Urška Štiblerc, Apolonija Bedina
Zavecd, Matej Hočevare, Barbara Šetina Batiče, Nicolas
Touzetf, Mauro Mannog, Gabriella Pocsfalvih, Antonella
Bongiovannii, Ales Igličj and Veronika Kralj-Igličj
aLaboratory of Clinical Biophysics, Faculty of Health
Sciences, University of Ljubljana, Ljubljana, Slovenia; Laboratory of Physics, Faculty
of
Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia;
bLaboratory of Clinical Biophysics, Faculty of Health Sciences, University of
Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia; cLaboratory of Physics,
Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia, Ljubljana,
Slovenia; dNational Institute of Chemistry, Ljubljana, Slovenia;
eDepartment of Physics and Chemistry of Materials, Institute of Metals and
Technology, Ljubljana, Slovenia, Ljubljana, Slovenia; fInstitute of Technology
Sligo (ITSligo), Sligo, Ireland; gInstitute of Biophysics (IBF) – National
Research Council (CNR), Palermo, Italy; hInstitute of Biosciences and
BioResources (IBBR) – National Research Council (CNR), Naples, Italy; iInstitute
for Research and Biomedical Innovation (IRIB) – National Research Council (CNR), Palermo,
Italy; jUniversity of Ljubljana, Ljubljana, Slovenia
Introduction: We have established a small size laboratory production of the
microalgae culture in order to harvest the extracellular vesicles (EVs) for pharmaceutical
and medical uses. In this work we report on globular particles in the isolates from
media of
microalgae of two types, that we recognize as EVs. We observed changes in their production
at different temperatures and conditions.
Methods: Samples were fixed by various combinations of aldehyde fixatives
and/or osmium tetroxide. They were dehydrated in a graded series of ethanol,
hexamethyldisilazane, and air dried. They were Au/Pd coated for inspection with Scanning
Electron Microscopes (SEM) Crossbeam 550 FIB-SEM Gemini II (ZEISS, Germany) and JSM-6500 F
Field Emission Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan).
Results: Microalgae were incubated overnight at 22°C and 37°C in growth medium
and in growth medium supplemented with detergent. The samples obtained from the microalgae
culture contained particles that we recognized as extracellular vesicles, however,
these
particles do not correspond to characteristic shapes of membrane enclosed entities
without
internal structure. Increased temperature and/or presence of surfactant (Triton X-100
and
sodium dodecyl sulphate) stimulated formation of EVs of different shapes and sizes.
The
isolates of these samples were rich with EVs. In the presence of surfactant, the cell-walls
detached from the cell and collapsed upon dehydration. This was documented by SEM.
Summary/Conclusion: Focused Ion Beam technique revealed complex internal
structure of the algae. It seems from the shapes of the observed structures that the
particles deposited on the surface of the microalgae do not derive from budding of
the
membrane surface, but are instead shed by the cells from the cell interior upon the
rupture
of the cell wall.
Funding: European Union’s Horizon 2020 research and innovation program under
grant agreement No. 801338 (VES4US project) & Slovenian Research Agency, ARRS grands:
P2-0232, P3-0388, P2-0132, J1-9162, Z2-9215.