Diagnosis of pancreatic ductal adenocarcinoma (PDAC) is associated with dismal prognosis
despite current therapies; therefore new treatment strategies are urgently required.
Numerous studies have suggested that epithelial to mesenchymal transition (EMT) contributes
to early-stage dissemination of cancer cells and is pivotal for invasion and metastasis
of PDAC
1-4
. EMT program is associated with phenotypic conversion of epithelial cells into mesenchymal-like
cells in cell culture conditions, albeit such defined mesenchymal conversion (with
spindle shaped morphology) of epithelial cells is rare with quasi-mesenchymal phenotypes
occasionally observed in the tumor (partial EMT)
5,6
. Most studies exploring the functional role of EMT in tumors have depended on cell
culture induced loss-of-function and gain-of-function experiments involving EMT inducing
transcription factors such as Twist, Snail and Zeb1
2,3,7-10
. Therefore, the functional contribution of EMT program for invasion and metastasis
remains unclear
4,6
and genetically engineered mouse models (GEMMs) to specifically address a causal connection
are lacking. Here we functionally probed the role of EMT program in PDAC by generating
PDAC GEMMs with deletion of Snail or Twist, two key transcription factors responsible
for EMT. EMT suppression in the primary tumor did not alter the emergence of invasive
PDAC, systemic dissemination and metastasis. Suppression of EMT led to an increase
in cancer cell proliferation with enhanced expression of nucleoside transporters in
tumors, contributing to enhanced sensitivity to gemcitabine treatment and increased
overall survival of mice. Collectively, our study suggests that Snail or Twist induced
EMT program is not rate-limiting for invasion and metastasis but highlights the importance
of combining EMT inhibition with chemotherapy for the treatment of pancreatic cancer.
We crossed Twist1L/L
or Snai1L/L
mice with Pdx1-Cre; LSL-KrasG12D; P53R172H/+
(KPC) to generate the Pdx1-Cre; LSL-KrasG12D; P53R172H/+; Twist1L/L
(KPC; TwistcKO) and the Pdx1-Cre; LSL-KrasG12D; P53R172H/+; Snai1L/L
(KPC; SnailcKO) mice, respectively. The resultant progeny were born in an expected
Mendelian ratio, without overt phenotypic findings other than the anticipated emergence
of spontaneous pancreatic cancer (
Extended Figure 1A
). Genetic deletion of Snai1 or Twist1 did not significantly delay pancreatic tumorigenesis,
alter tumor histopathology features or local invasion (
Figure 1A-C
and
Extended Table 1
). KPC; TwistcKO and KPC; SnailcKO mice displayed similar tumor burden compared to
KPC control mice (
Extended Figure 1B
), and insignificant difference in overall survival (
Figure 1D
). Loss of Twist1 or Snai1 expression in the pancreas epithelium was confirmed by
in situ hybridization coupled with CK8 epithelial immunolabeling (
Figure 1E
and
Extended Figure 1C
) as well as immunolabeling for Twist and Snail (
Extended Figure 1D
). Suppression of EMT program was significantly noted (
Figure 1F-G, Extended Figure 1E
). Lineage tracing (
Figure 1F
) and immunolabeling of the primary tumor (
Figure 1G
) showed a significant decrease in the frequency of epithelial cells with expression
of the mesenchymal marker αSMA (EMT+ cells) and a decrease in expression of EMT inducing
transcription factor, Zeb1 (
Figure 1H
). Global gene expression profiling of tumors revealed a decrease in expression of
EMT associated genes (including Snai1 and Twist1) in KPC; SnailcKO and KPC; TwistcKO
mice compared to KPC control (
Extended Figure 1F
). Loss of Snail and Twist enhanced E-cadherin expression and suppressed Zeb2 and
Sox4 expression in cancer cells (
Extended Figure 2A-C
). Snai2 (Slug) expression was restricted to early PanIN lesion in all the experimental
groups with no observed expression in advanced tumors and was significantly reduced
in KPC; SnailcKO and KPC;TwistcKO mice compared to KPC control mice (
Extended Figure 2D
).
While desmoplasia, including extracellular matrix (ECM) and myofibroblasts content
(
Figure 1I
and
Extended Figure 2E-F
), tumor vessel density (
Extended Figure 2G
), intratumoral hypoxia (
Extended Figure 2H
), CD3+ T-cell infiltration (
Extended Figure 2I
), and cancer cell apoptosis was unaffected with Twist/Snail deletion in KPC tumors
(
Figure 2A
), the proliferation of cancer cells in mice with suppressed EMT program was significantly
increased (
Figure 2B
), as shown previously in mouse models of breast cancers
11-13
. Immunostaining experiments further revealed that EMT+ cancer cells are largely Ki67−
(
Extended Figure 3A
). Altogether, the data suggests that EMT program driven by Twist/Snail transcription
factors is dispensable for initiation and progression of primary pancreatic cancer.
Next, we investigated whether suppression of EMT program impacts invasion and metastasis.
The number of YFP+ CTCs from lineage traced KPC and KPC; TwistcKO was found unchanged
(
Figure 2C
and
Extended Figure 3B
), and expression of cancer cell specific KrasG12D mRNA in the blood from KPC, KPC;
TwistcKO and KPC; SnailcKO was unaffected (
Figure 2D
), suggesting that suppression of EMT program in pancreatic tumors does not impact
the rate of systemic dissemination of cancer cells. Extensive histopathological analyses,
coupled with CK19 or YFP immunostaining of distant metastatic target organs, namely
the liver, lung and spleen, indicated a similar frequency of metastasis in EMT suppressed
tumors when compared to control tumors (
Figure 2E, Extended Figure 3C, Extended Table 1
, and
Extended Table 2
). The metastases were negative for Twist and Snail, and only a few KPC metastatic
cells expressed αSMA or Zeb1 (
Extended Figure 3D-F
), while being positive for E-cadherin and Ki-67 (
Extended Figure 3G-H
). The proliferation rate of cancer cells in the metastases was similar in KPC, KPC;
SnailcKO and KPC; TwistcKO mice (
Extended Figure 3H
). Collectively, the results indicated that the genetic deletion of Twist1 or Snai1
in PDAC GEMMs did not reduce metastatic disease.
To evaluate whether cancer cells from the pancreas with and without EMT program differentially
benefited from impaired proliferation to form secondary tumors, we isolated cancer
cells from KPC, KPC; TwistcKO and KPC; SnailcKO mice to assay their organ colonization
potential. Twist1 was significantly reduced and Snai1 expression was undetectable
in cancer cells isolated from Twist and Snail deleted tumors, respectively (
Figure 2F
). Short-term potential to form tumor spheres (associated with putative cancer stem
phenotype) appeared similar in TwistcKO and SnailcKO KPC cells when compared to control
KPC cells (
Figure 2G
)
3,8,14-16
. Lung colonization frequency following the i.v. injection of KPC cancer cells (Twist
or Snail deleted) were similar to the control KPC cancer cells (
Figure 2H
). These results suggest that a favored epithelial phenotype of cancer cells (via
suppression of EMT program) did not impact the capacity to form tumor spheres or their
ability for organ colonization
17
.
Cancer cell EMT program is associated with gemcitabine drug resistance in PDAC patients
and in the orthotopic mouse models of PDAC
1,2,8,9,18-23
. Moreover, enhanced frequency of EMT+ cancer cells in pancreatic tumors is associated
with poor survival
24,25
. To determine whether EMT program suppression enhances PDAC sensitivity to gemcitabine
chemotherapy, we tested the gemcitabine sensitivity of cancer cells with suppressed
EMT program in KPC mice. Equilibrative nucleoside transporter ENT1 and concentrating
nucleoside transporter CNT3 were significantly upregulated in cancer cells lacking
Snail and Twist, while ENT2 expression was unchanged (
Figure 3A-C
). KPC, KPC; SnailcKO and KPC; TwistcKO mice were treated with gemcitabine and tumor
burden was monitored by MRI (
Extended Table 3
). Tumor progression was suppressed in KPC; SnailcKO and KPC; TwistcKO mice when compared
to treated KPC control mice (
Figure 3D
). KPC; SnailcKO and KPC; TwistcKO mice treated with gemcitabine showed improved histopathology
and increased survival (
Figure 3E-G
).
Cancer cells isolated from the tumors of KPC; SnailcKO and KPC; TwistcKO mice showed
epithelial morphology (
Extended Figure 4A
) and reduced expression of mesenchymal genes compared to KPC cancer cell lines (
Extended Figure 4B
), however, in tissue culture conditions (2D culture on plastic), equilibrative nucleoside
transporters (ENT1/ENT2/ENT3) showed similar expression pattern and expression of
concentrating nucleoside transporters (CNT1/CNT3) was not detected (
Extended Figure 4B
). Increased proliferation of KPC; SnailcKO and KPC; TwistcKO cancer cells compared
to KPC control cells (
Extended Figure 4C
) likely accounted for the increased sensitivity to gemcitabine and erlotinib in this
setting (
Extended Figure 4D
).
Next, we crossed the Snai1L/L
to the PDAC GEMM, Ptf1a (P48)-Cre; LSL-KrasG12D; Tgfbr2L/L
(KTC) to generate Ptf1a (P48)-Cre; LSL-KrasG12D; Tgfbr2L/L; Snai1L/L
(KTC; SnailcKO). The KTC model offers a reliable and penetrant disease progression
rate with a consistent timeline of death due to PDAC. Similar to the KPC; SnailcKO
mice, KTC; SnailcKO deletion exhibited suppression of EMT program but did not impact
primary tumor histopathology, lifespan, local invasion, desmoplasia and frequency
of apoptosis (
Figure 4F, Extended Figure 5A-E
, and
Extended Figure 6A
). KTC; SnailcKO mice presented with significantly reduced Zeb1 expression in cancer
cells but enhanced proliferation and concentrating nucleoside transporter 3 (CNT3)
expression (
Extended Figure 5E
). ENT2 and ENT1 expression were unchanged in KTC; SnailcKO mice compared to KTC mice
(
Extended Figure 5E
and
Extended Figure 6A
). KTC; SnailcKO mice demonstrated enhanced response to gemcitabine therapy, with
significant normal parenchymal area and reduced tumor tissue (
Figure 4A-C
). Gemcitabine therapy in KTC; SnailcKO reduced tumor burden (
Figure 4D
) and significantly improved overall survival (
Figure 4E
) of mice when compared to gemcitabine treated control KTC mice. Gemcitabine therapy
specifically increased cancer cell apoptosis and removed enhanced proliferation observed
in EMT program suppressed tumors (
Figure 4G
and
Extended Figure 5E
), without impacting the desmoplastic reaction (
Extended Figure 6B
). Overall, these results suggested an enhanced sensitivity of EMT− cancer cells to
gemcitabine. Both the equilibrative nucleoside transporter 2 (ENT2) and the concentrating
nucleoside transporter 3 (CNT3) were upregulated in EMT suppressed tumors (
Figure 4G
). These data support a possible mechanistic connection between EMT program and resistance
to chemotherapy in PDAC.
Collectively, our studies provide a comprehensive functional analysis of EMT program
in PDAC progression and metastasis. Absence of either Twist1 or Snai1 did not alter
cancer progression or the capacity for local invasion or metastasis to lung and liver
in PDAC GEMMs. Metastasis occurs despite a significant loss of EMT program with either
the deletion of Snail or Twist, and in both settings, Zeb1, Sox4, Slug and Zeb2 are
also significantly suppressed. Nevertheless, it is likely that other EMT inducing
factors may compensate for the loss of Snail or Twist to induce invasion and metastasis.
While PDX-1 is expressed during the development of the pancreas (in early pancreatic
buds: all three major lineages of the pancreas-ductal, acinar and beta-islets), its
expression is largely repressed in the adult exocrine pancreas
26,27
. Therefore, deletion of Snail or Twist occurs at the embryonic stage and mice are
born normal and exhibit normal pancreas histology prior to the onset of cancer. The
GEMMs with Snail or Twist deletion develop PanIN lesions at the same frequency as
the control mice. One could argue that suppression of EMT program starting from the
inception of cancer could have launched compensatory mechanisms to overcome EMT program-dependent
invasion and metastasis. However, such compensation is not observed with respect to
chemo-resistance and previous studies have demonstrated that EMT program and cancer
cell dissemination are observed even before PDAC lesions are detected in KPC mice
4
.
Our study demonstrates that EMT program results in suppression of cancer cell proliferation,
and suppression of drug transporter and concentrating proteins, therefore, inadvertently
protecting EMT+ cells from anti-proliferative drugs such as gemcitabine. The correlation
of decreased survival of pancreatic cancer patients with an increased EMT program
is likely due to their impaired capacity to respond to gemcitabine, which is a standard
of care for most patients
28,29
. Such diminished response to Gemcitabine will likely reflect on such patients also
exhibiting higher metastatic disease. Collectively, our study offers the opportunity
to evaluate the potential of targeting EMT program to enhance efficacy of Gemcitabine
and targeted therapies
30
.
Methods
Mice
Characterization of disease progression and genotyping for the Pdx1-Cre; LSL-KrasG12D;
P53R172H/+
(herein referred to as KPC) and Ptf1a (P48)-Cre; LSL-KrasG12D; Tgfbr2L/L
(herein referred to as KTC) mice were previously described
31-33
. These mice were bred to Snai1L/L
(herein referred to as SnailcKO), Twist1L/L
(herein referred to as TwistcKO), and R26-LSL-EYFP
33
. SnailcKO mice were kindly provided by S.J. Weiss, University of Michigan, Ann Arbor.
TwistcKO mice were kindly provided by R. R. Behringer (UT MDACC, Houston, TX) via
the Mutant Mouse Regional Resource Center (MMRRC) repository. The resulting progeny
were referred to as KPC, KPC; SnailcKO, KPC; TwistcKO, KTC, and KTC; SnailcKO mice
and were maintained on a mixed genetic background. Both males and females were used
indiscriminately. Mice were given Gemcitabine (G-4177, LC Laboratories) via intraperitoneal
injection (i.p.) every other day at 50 mg/kg of body weight. Hypoxyprobe was injected
in a subset of mice i.p. at 60 mg/kg of body weight 30 minutes prior to euthanasia.
For in vivo colonization assay, one million KPC, KPC; TwistcKO and KPC; SnailcKO tumor
cells in 100 μL of PBS were injected intravenously via the retro-orbital venous sinus.
Four to eleven mice were injected per cell line. All mice were euthanized at 15 days
post-injection. All mice were housed under standard housing conditions at MD Anderson
Cancer Center (MDACC) animal facilities, and all animal procedures were reviewed and
approved by the MDACC Institutional Animal Care and Use Committee. Tumor growth met
the standard of a diameter less than or equal to 1.5 cm. Investigators were not blinded
for group allocation but were blinded for the assessment of the phenotypic outcome
assessed by histological analyses. No randomization method or statistical sample size
estimation was used.
Histology and histopathology
Histology, histopathological scoring, Masson's Trichrome staining (MTS), and Picrosirius
Red were previously described
19,33
. Formalin-fixed tissues were embedded in paraffin and sectioned at 5 μm thickness.
MTS was performed using Gomori's Trichome Stain Kit (38016SS2, Leica Biosystems).
Picrosirius red staining for collagen was performed using 0.1% picrosirius red (Direct
Red80; Sigma) and counterstained with Weigert's hematoxylin. Sections were also stained
with hematoxylin and eosin (H&E). Histopathological measurements were assessed by
scoring H&E stained tumors for relative percentages of each histopathological phenotype:
normal (non-neoplastic), PanIN, well-differentiated PDAC, moderately-differentiated
PDAC, poorly-differentiated PDAC, sarcomatoid carcinoma, or necrosis. When tumor histology
was missing or of poor quality, the mice were excluded from all analyses and this
was determined blinded from genotype information. A histological invasion score of
the tumor cells into the surrounding stroma was scored on a scale of 0 to 2, with
0 indicating no invasion and 2 indicating high invasion, where invasion is defined
as tumor cell dissemination throughout the stroma away from clearly defined epithelial
“nests”. Microscopic metastases were observed in H&E stained tissue sections of the
liver, lung and spleen. Positivity (one or more lesions in a tissue) was confirmed
using CK19 and YFP immunohistochemistry. This data has been presented as a contingency
table (
Figure 2E
) and represented as the number of positive tissues out of the number of tissues scored.
The “Any” metastasis score is the number of mice positive for a secondary lesion found
anywhere throughout the body out of the total number of mice scored.
Immunohistochemistry and Immunofluorescence
Tissues were fixed in 10% formalin overnight, dehydrated, and embedded in paraffin
and 5 μm thick sections were then processed for analyses. Immunohistochemical analysis
was performed as described
33
. Heat mediated antigen retrieval in 1 mM EDTA + 0.05% Tween20 (pH 8.0) for one hour
(pressure cooker) was performed for Snail and Twist, 10 mM citrate buffer, pH 6.0
was performed for one hour (microwave) for Ki67 or 10 minutes for all other antibodies.
Primary antibodies are as follows: αSMA (M0851, DAKO, 1:400 or ab5694, Abcam, 1:400),
cleaved caspase-3 (9661, Cell Signaling, 1:200), CD3 (A0452, DAKO, 1:200), CD31 (Dia310M,
DiaNova, 1:10), CK8 (TROMA-1, Developmental Studies Hybridoma Bank, 1:50), CK19 (ab52625,
Abcam, 1:100), CNT3 (HPA023311, Sigma-Aldrich, 1:400), ENT1 (LS-B3385, LifeSpan Bio.,
1:100), E-cadherin (3195S, Cell Signaling, 1:400), ENT2 (ab48595, Abcam, 1:200), Ki67
(RM-9106, Thermo Scientific, 1:400), SLUG (9585, Cell Signaling, 1:200), SNAIL (ab180714,
Abcam, 1:100), SOX4 (ab86809, Abcam, 1:200), TWIST (ab50581, Abcam, 1:100), YFP (ab13970,
Abcam, 1:1000), ZEB1 (NBP1-05987, Novus, 1:500), and ZEB2 (NBP1-82991, Novus, 1:100).
Sections for pimonidazole adduct (HPI Inc., 1:50) or αSMA immunohistochemistry staining
were blocked with M.O.M kit (Vector Laboratories, West Grove, PA) and developed by
DAB according to the manufacturer's recommendations. Alternatively, for immunofluorescence,
sections were dual-labeled using secondary antibodies conjugated to Alexa fluor-488
or -594 or tyramide signal amplification (TSA, PerkinElmer) conjugated to FITC. Lineage
traced (YFP positive) EMT analysis was performed on 8 μm thick O.C.T. medium (TissueTek)
embedded frozen sections. Sections were stained for αSMA (ab5694, Abcam, 1:400) followed
by Alexa fluor-680 conjugated secondary antibody. Bright field imagery was obtained
on a Leica DM1000 light microscope or the Perkin Elmer 3DHistotech Slide Scanner.
Fluorescence imagery was obtained on a Zeiss Axio Imager.M2 or the Perkin Elmer Vectra
Multispectral imaging platform. The images were quantified for percent positive area
using NIH ImageJ analysis software (αSMA, Pimonidazole, SLUG, and CD31), percent positive
cells using InForm analysis software (Ki-67 and CD3), or scored for intensity either
positive or negative (CK19, YFP, ZEB1, ZEB2, SOX4, and Cleaved Caspase-3) or on a
scale of 1-3 (E-cadherin) or 1-4 (ENT1, ENT2 and CNT3).
In situ hybridization
In situ hybridization (ISH) was performed on frozen tumor sections as previously described
34
. In brief, 10 μm-thick sections were hybridized with antisense probes to Twist1 and
Snai1 overnight at 65°C. After hybridization, sections were washed and incubated with
AP-conjugated sheep anti-DIG antibody (1:2000; Roche) for 90 min at room temperature.
After three washes, sections were incubated in BM Purple (Roche) until positive staining
was seen. Digoxigenin labeled in situ riboprobes were generated by in vitro transcription
method (Promega and Roche) using a PCR template. The following primers were used to
generate the template PCR product. Twist1; forward (5’-CGGCCAGGTACATCGACTTC-3’) and
reverse (5’-TAATACGACTCACTATAGGGAGATTTAAAAGTGTGCCCCACGC-3’) Snai1: forward (5’-CAACCGTGCTTTTGCTGAC-3’)
and reverse (5’-TAATACGACTCACTATAGGGAGACCTTTAAAATGTAAACATCTTTCTCC-3’)
Gene Expression Profiling
Total RNA was isolated from tumors of KPC control, KPC; TwistcKO and KPC; SnailcKO
mice (n = 3 in each group) by TRIzol (15596026, Life Technologies) and submitted to
the Microarray Core Facility at MD Anderson Cancer Center. Gene expression analysis
was performed using Mouse Ref6 Gene Expression Bead Chip (Illumina). The Limma package
from R Bioconductor
35
was used for quantile normalization of expression arrays and to analyze differentially
expressed genes between cKO and control sample groups (p ≤ 0.05 and fold change ≥
1.2). Gene expression microarray data was deposited in GEO (Accession number GSE66981).
Genes up-regulated in cells acquiring an EMT program were expected to be down-regulated
in the TwistcKO and SnailcKO tumors compared to control tumors.
CTC assays
Blood (200 μL) was collected from KPC;LSL-YFP and KPC; TwistcKO;LSL-YFP (ROSA-LSL-YFP
lineage tracing of cancer cells) mice and incubated with 10 ml of ACK lysis buffer
(A1049201, Gibco) at room temperature to lyse red blood cells. Cell pellets were resuspended
in 2% FBS containing PBS and analyzed for the number of YFP+ cells by flow cytometry
(BD LSRFortessa X-20 Cell Analyzer). The data was expressed as the percent YFP+ cells
from gated cells, with 100,000 cells analyzed at the time of acquisition. Whole blood
cell pellets were also assayed for the expression of KrasG12D
transcripts, using quantitative real-time PCR analyses (described below).
Primary pancreatic adenocarcinoma cell culture and analyses
Derivation of primary PDAC cell lines were performed as previously described
36
. Fresh tumors were minced with sterile razor blades, digested with dispase II (17105041,
Gibco, 4 mg/ml)/collagenase IV (17104019, Gibco, 4 mg/ml)/RPMI for 1 h at 37°C, filtered
by a 70 μm cell strainer, resuspended in RPMI/20%FBS and then seeded on collagen I
coated plates (087747, Fisher Scientific). Cells were maintained in RPMI medium with
20% FBS and 1% penicillin, streptomycin and amphotericin B (PSA) antibiotic mixture.
Cancer cells were further purified by FACS based on YFP or E-Cadherin expression (anti-E-cadherin
antibody, 50-3249-82, eBioscience, 1:100). The sorted cells, using BD FACSAria™ II
sorter (South Campus Flow Cytometry Core Lab of MD Anderson Cancer Center) were subsequently
expanded in vitro. All studies were performed on cells cultivated less than 30 passages.
As these are primary cell lines no further authentication methods were applicable
and no mycoplasma tests were performed.
MTT and drug sensitivity assays
MTT assay was performed to detect cell proliferation and viability by using Thiazolyl
Blue Tetrazolium Bromide (MTT, M2128, Sigma) following the manufacturer's recommendations
with an incubation of two hours at 37°C. For the drug treatment studies, a cell line
derived from each of the KPC, KPC; SnailcKO and KPC; TwistcKO mice was treated with
20 μM Gemcitabine (G-4177, LC Laboratories) or 100 μM erlotinib (5083S, NEB) for 48
hours. The relative cell viability was detected using MTT assay with a cell line derived
from each of the KPC, KPC; SnailcKO and KPC; TwistcKO mice. N value is defined as
biological replicates of a single cell line. Control conditions included 1% DMSO vehicle
for erlotinib. The relative absorbance was normalized and control (time 0 hour or
vehicle treated) arbitrarily set to 1 or 100% for absorbance or drug survival, respectively.
Quantitative real-time PCR analyses (qPCR)
RNA was extracted from whole blood cell pellets following ACK lysis using the PicoPure
Extraction kit as directed (KIT0214, Arcturus), or from cultured primary pancreatic
adenocarcinoma cells using TRIzol (15596026, Life Technologies). cDNA was synthetized
using TaqMan Reverse Transcription Reagents (N8080234, Applied Biosystems) or High
Capacity cDNA Reverse Transcription Kit (4368814, Applied Biosystems). Primers for
KrasG12D
recombination are: KrasG12D
forward (5’ ACTTGTGGTGGTTGGAGCAGC 3’), KrasG12D
reverse (5’ TAGGGTCATACTCATCCACAA 3’). 1/ΔCt values are presented to show KrasG12D
expression in indicated experimental groups, statistical analyses were assayed on
ΔCt. Primer sequences for EMT related genes are listed in Supplemental Table 1, GAPDH
was used as an internal control. The data is presented as the relative fold change
and statistical analyses were assayed on ΔCt.
Tumor sphere assay
Tumor sphere assays were performed as previously described
33
. Two million cultured primary tumor cells were plated in a low-adherence 100mm dish
(FB0875713, Fisherbrand) with 1% fetal bovine serum, Dulbecco's modified Eagle's medium,
and penicillin/streptomycin/amphotericin. Cells were incubated for seven days and
formed spheres were counted at 100x magnification. Three, two and three cell lines
were analyzed for KPC control, KPC; TwistcKO and KPC; SnailcKO group, respectively,
five field of views per cell line were quantified.
MRI Analyses
MRI imaging was performed using a 7T small animal MR system as previously described
37
. To measure tumor volume, suspected regions were drawn blinded on each slice based
on normalized intensities. The volume was calculated by the addition of delineated
regions of interest in mm2 × 1 mm slice distance. None of the mice had a tumor burden
that exceeded 1.5 cm in diameter, in accordance with institutional regulations. All
mice with measurable tumors were enrolled in the study (see
Extended Table 3
). Mice were imaged twice, once at the beginning of the enrollment (Day 0), and a
second time 20 days (Day 19) afterwards. Surviving animals were euthanized at end
point (Day 21) for histological characterization.
Statistical analyses
Statistical analyses were performed on the mean values of biological replicates in
each group using unpaired two-tailed or one-tailed t-tests (qPCR only), one-way ANOVA
with Tukey's multiple comparisons test using GraphPad Prism, as stipulated in the
figure legends. χ2 analyses, using SPSS statistical software, were performed comparing
control to cKO groups for metastatic or colonization frequency across multiple histological
parameters in all mice and mice ≥ 120 days of age. Fisher's Exact P value was used
to determine significance. Results are outlined in
Extended Table 2
. Kaplan-Meier plots were drawn for survival analysis and the log rank Mantel-Cox
test was used to evaluate statistical differences, using GraphPad Prism. Data met
the assumptions of each statistical test, where variance was not equal (determined
by an F-test) Welch's correction for unequal variances was applied. Error bars represent
s.e.m. when multiple visual fields were averaged to produce a single value for each
animal which was then averaged again to represent the mean bar for the group in each
graph. P < 0.05 was considered statistically significant.
Extended Data
Extended Figure 1
A Representative H&E images of small intestine (SmInt), kidney, and heart (scale,
100μm). B Pancreatic mass of (n = 29, 13, and n = 28 mice; s.d.; one-way ANOVA). C
Merge of Twist1 or Snai1 in situ hybridization (black) followed by CK8 (red) immunolabeling
in tumors from KPC and KPC; TwistcKO or KPC; SnailcKO mice, respectively. White arrows
highlight positive cells in the stroma while yellow arrows highlight negative epithelium
(scale, 50 μm). D Twist or Snail immunostaining in KPC and KPC; TwistcKO or KPC; SnailcKO
tumors, respectively. Black arrows highlight positive cells in the stroma while red
arrows highlight negative epithelium (scale, 20 μm). E Channel separations of the
representative images of αSMA immunolabeling in YFP lineage traced tumors found in
Figure 1F
(scale, 50 μm). F EMT gene expression signature analysis in KPC, KPC; TwistcKO and
KPC; SnailcKO cohorts (n = 3 mice). Red arrows indicate reduced Twist1 and Snai1 expression
in KPC; TwistcKO and KPC; SnailcKO cohorts, respectively.
Extended Figure 2
A E-Cadherin immunolabeling and quantification of primary KPC (n = 5 mice), KPC; TwistcKO
(n = 5 mice) and KPC; SnailcKO (n = 4 mice) (scale, 100 μm). B Zeb2 immunolabeling
and quantification of primary KPC (n = 6 mice), KPC; TwistcKO (n = 5 mice) and KPC;
SnailcKO (n = 7 mice) (scale, 50 μm; inset scale, 20 μm). C Sox4 immunolabeling and
quantification of primary KPC (n = 7 mice), KPC; TwistcKO (n = 6 mice) and KPC; SnailcKO
(n = 8 mice) (scale, 50 μm; inset scale, 20 μm). D Slug immunolabeling and quantification
of primary KPC (n = 4 mice), KPC; TwistcKO (n = 4 mice) and KPC; SnailcKO (n = 4 mice)
tumors (scale, 50 μm; inset scale, 20 μm). E Sirius Red staining and quantification
of primary KPC (n = 21 mice), KPC;TwistcKO (n = 8 mice) and KPC;SnailcKO (n = 11 mice)
(scale, 200 μm; s.d.) F αSMA immunolabeling and quantification of primary KPC (n =
5 mice), KPC;TwistcKO (n = 5 mice) and KPC;SnailcKO (n = 5 mice) (scale, 100 μm).
G CD31 immunolabeling and quantification of primary KPC (n = 4 mice), KPC;TwistcKO
(n = 4 mice) and KPC;SnailcKO (n = 3 mice) (scale, 200 μm, inset scale, 100 μm). H
Pimonidazole staining and quantification of primary KPC (n = 4 mice), KPC; TwistcKO
(n = 4 mice) and KPC; SnailcKO (n = 4 mice) (scale, 100 μm). I CD3 immunolabeling
and quantification of primary KPC (n = 5 mice), KPC;TwistcKO (n = 5 mice) and KPC;SnailcKO
(n = 5 mice) (scale, 100 μm; inset scale, 25 μm). Unless otherwise indicated error
bars represent s.e.m, and significance determined by One-way ANOVA. *P < 0.05, **
P <0.01, *** P <0.001. ns, not significant.
Extended Figure 3
A Immunolabeling of primary tumors (n = 3 mice) for αSMA (red), CK8 (green), Ki-67
(white) and DAPI (blue); yellow arrows point to EMT+ cells (scale, 20 μm). B Representative
dot plots of circulating YFP+ cells. C Images of serial sections of KPC; LSL-YFP lung
and liver metastasis stained for H&E or immunolabeled for CK19 or YFP. Yellow dashed
box represents magnified areas in panel below (scale, 200 μm; magnification scale,
100 μm). D KPC metastatic tumors stained for Twist and Snail (n = 3 mice; scale, 50
μm; inset scale, 20 μm). E Zeb1 immunolabeling and quantification of metastatic KPC
(n = 4 mice), KPC; TwistcKO (n = 3 mice) and KPC; SnailcKO (n = 4 mice) (scale, 50
μm; inset scale, 20 μm). F αSMA immunolabeling and quantification of metastatic KPC
(n = 3 mice), KPC; TwistcKO (n = 3 mice) and KPC; SnailcKO (n = 3 mice) (scale, 50
μm; inset scale, 20 μm). G E-Cadherin staining on serial sections of αSMA immunolabeling
and quantification of metastatic KPC (n = 4 mice), KPC; TwistcKO (n = 3 mice) and
KPC; SnailcKO (n = 4 mice) (scale, 50 μm; inset scale, 20 μm).H Ki-67 immunolabeling
and quantification of metastatic KPC (n = 7 mice), KPC; TwistcKO (n = 3 mice) and
KPC; SnailcKO (n = 3 mice) (scale, 50 μm). Unless otherwise indicated error bars represent
s.e.m, percentages indicated represent percent decrease from control, and significance
determined by One-way ANOVA. * P <0.05, ** P <0.01, *** P <0.001. ns, not significant.
Extended Figure 4
A Brightfield micrograph of cultured primary KPC, KPC; TwistcKO and KPC; SnailcKO
cells (scale, 50 μm). B EMT and gemcitabine transport related gene expression shown
by qPCR analysis in KPC (n = 3-4 cell lines), KPC; TwistcKO (n = 5 cell lines) and
KPC; SnailcKO (n = 5-6 cell lines) (s.d., one-tailed t-test, * P < 0.05, numbers list
non-significant P values. nd: not detected, ns: not significant). C MTT assay showing
cell proliferation in KPC, KPC; TwistcKO and KPC; SnailcKO cells (n = 8, 8, and 8
biological replicates of a cell line for each genotype). D Relative cell viability
(MTT assay) in cultured KPC, KPC; TwistcKO and KPC; SnailcKO cells treated with gemcitabine
or erlotinib (n = 8, 8, and 8 biological replicates of a cell line for each genotype).
Unless otherwise indicated error bars represent s.e.m, significance was determined
by one-way ANOVA. ** P <0.01, *** P <0.001, **** P <0.0001.
Extended Figure 5
A Representative H&E images (scale, 100 μm). B Relative percentage of each histological
tissue phenotype of KTC (n = 8 mice) and KTC; SnailcKO (n = 6 mice) primary tumors
(s.d.). C Primary tumor invasiveness in KTC (n = 8 mice) and KTC; SnailcKO (n = 6
mice) (s.d.). D Pancreatic mass in KTC (n = 5 mice) and KTC; SnailcKO (n = 6 mice)
(s.d.). E Immunolabeling and quantification of primary KTC (n = 5 mice), KTC; SnailcKO
(n = 4 mice) for αSMA (red), CK8 (green) and DAPI (blue); white arrows indicate double
positive cells (scale, 20 μm), Zeb1 (scale, 50 μm; inset scale. 20μm), cleaved caspase-3
(scale, 50 μm; n = 4 and 4 mice), Ki-67 (scale, 100 μm), ENT2 (scale, 100 μm) and
CNT3 (scale, 100 μm). Unless otherwise indicated error bars represent s.e.m, and significance
determined by two-tailed t-test. * P <0.05, *** P <0.001. ns, not significant.
Extended Figure 6
A-B Staining and quantification of (A) KTC (n = 5 or 6 mice), KTC; SnailcKO (n = 4
or 5 mice) (B) KTC + GEM (n = 4 or 5 mice), KTC; SnailcKO + GEM (n = 5 mice) for Masson's
Trichrome Stain (MTS) (scale, 200 μm), Sirius Red staining (scale, 200 μm), and ENT1
(scale, 100 μm). Error bars represent s.d. (MTS and Sirius Red) or s.e.m. (ENT1),
and significance determined by two-tailed t-test. ns, not significant.
Extended Table 1
Pathological spectrum of disease and metastasis in KPC, KPC; TwistcKO and KPC; SnailcKO
cohorts.
Pathological Spectrum within cohorts
ID
AGE
PDA
Differentiation
Histology 1
Histology 2
Liver
Lung
Spleen
Any
Moribund
KPC
(104)
1
158
Y
W
S
G
Y
Y
N
Y
Y
2
165
Y
W
G
N
N
N
N
Y
3
148
Y
P
S
G
N
N
-
N
Y
4
135
Y
M
S
G
Y
N
Y
Y
Y
5
95
Y
M
G
N
Y
N
Y
N
6
42
Y
M
G
N
N
N
N
Y
7
55
Y
P
G
S
Y
N
N
Y
Y
8
91
Y
M
G
N
N
N
N
N
9
87
Y
W
G
N
N
N
N
N
10
63
Y
P
G
Y
Y
Y
Y
N
11
108
Y
P
S
G
Y
N
N
Y
FD
12
110
Y
W
G
N
N
N
N
N
13
104
Y
W
G
Y
N
N
Y
Y
14
54
Y
W
S
G
N
N
N
N
Y
15
108
Y
P
S
G
N
Y
N
Y
Y
16
42
Y
P
S
G
N
N
N
N
Y
17
68
Y
W
G
N
N
N
N
N
18
107
Y
P
G
N
N
N
N
N
19
87
Y
P
G
N
N
N
N
N
20
48
Y
P
G
S
N
N
N
N
Y
21
109
Y
P
G
S
Y
Y
N
Y
FD
22
81
Y
P
G
Y
Y
N
Y
Y
23
151
Y
W
G
N
Y
N
Y
Y
24
47
Y
M
G
S
N
Y
N
Y
Y
25
143
Y
P
G
S
N
Y
N
Y
Y
26
122
Y
W
G
Y
N
N
Y
N
27
115
Y
P
G
Y
Y
N
Y
N
28
76
Y
W
G
N
Y
N
Y
N
29
122
Y
M
S
G
Y
N
N
Y
Y
30
97
Y
P
G
N
N
N
N
N
31
107
Y
W
G
N
N
N
N
N
Totals
(Median)
31/31
11/31
11/31
2/30
17/31
%
100.0%
35.5%
35.5%
6.7%
54.8%
TwistcKO
(111)
1
148
Y
W
G
S
Y
N
N
Y
N
2
151
Y
P
S
G
Y
Y
Y
Y
N
3
140
Y
P
G
Y
Y
N
Y
Y
4
53
Y
P
G
S
N
N
N
N
Y
5
43
Y
P
G
N
N
N
N
Y
6
117
Y
P
G
S
N
N
N
N
N
7
90
Y
P
S
G
Y
N
N
Y
Y
8
52
Y
P
G
S
N
N
N
N
Y
9
104
Y
P
G
N
N
N
N
N
10
218
Y
P
G
S
N
N
Y
Y
Y
11
153
Y
P
G
N
Y
N
Y
Y
12
45
Y
P
G
S
N
N
N
N
Y
13
77
Y
P
G
S
Y
N
N
Y
Y
14
126
Y
P
G
S
Y
Y
N
Y
Y
Totals
(Median)
14/14
6/14
4/14
2/14
8/14
%
100.0%
42.9%
28.6%
14.3%
57.1%
SnailcKO
(103)
1
144
Y
W
G
N
Y
N
Y
N
2
51
Y
P
G
S
N
N
N
N
Y
3
105
Y
P
G
S
N
Y
N
Y
Y
4
111
Y
P
G
N
N
N
N
N
5
106
Y
P
G
S
Y
N
Y
Y
Y
6
129
Y
P
G
N
N
N
N
N
7
102
Y
P
G
S
N
Y
-
Y
N
8
98
Y
P
G
S
Y
N
Y
Y
N
9
47
Y
P
G
S
N
N
N
N
Y
10
54
Y
W
G
Y
Y
N
Y
FD
11
59
Y
M
G
Y
N
N
Y
N
12
103
Y
P
G
Y
N
N
Y
N
13
60
Y
P
S
G
Y
N
Y
Y
Y
14
77
Y
P
G
Y
N
N
Y
Y
15
57
Y
M
S
G
Y
N
N
Y
FD
16
130
Y
P
G
Y
Y
N
Y
FD
17
76
Y
P
G
S
N
N
N
N
FD
18
111
Y
P
G
N
Y
N
Y
Y
19
100
Y
P
G
S
Y
N
Y
Y
FD
20
104
Y
P
G
S
Y
N
N
Y
Y
21
124
Y
M
G
N
N
N
N
FD
22
88
Y
P
G
S
N
N
N
N
Y
23
192
Y
W
G
Y
Y
N
Y
Y
24
122
Y
P
G
N
N
N
N
Y
25
60
Y
W
G
S
N
N
N
N
Y
26
112
Y
W
G
N
Y
N
Y
N
27
48
Y
P
G
S
N
N
N
N
Y
28
48
Y
P
G
S
N
N
N
N
Y
29
124
Y
P
G
S
Y
Y
Y
Y
N
30
215
Y
W
G
N
N
N
N
N
Totals
(Median)
30/30
13/30
9/30
5/29
18/30
%
100.0%
43.3%
30.0%
17.2%
60.0%
Key: (Y) yes. (N) no, (W) well, (M) moderate, (P) poor, (G) glandular, (S) sarcomatoid,
(FD) found dead, (-) no tissue
Extended Table 2
Results of χ2 analysis reporting Fisher's Exact P value.
χ2 Analysis
Group
Perameter
Fisher's Exact P value
Differentiation
All Ages
Control vs. TwistcKO
Early Tumor progression
0.458
Control vs. SnailcKO
0.106
Control vs. TwistcKO
Late Tumor progression
0.458
Control vs. SnailcKO
0.106
Control vs. TwistcKO
Sarcomatoid
0.108
Control vs. SnailcKO
0.446
Differentiation
≥120 days
Control vs. TwistcKO
Early Tumor progression
0.580
Control vs. SnailcKO
0.569
Control vs. TwistcKO
Late Tumor progression
0.580
Control vs. SnailcKO
0.569
Control vs. TwistcKO
Sarcomatoid
1.000
Control vs. SnailcKO
0.119
Metastasis
All Ages
Control vs. TwistcKO
Liver Metastasis
0.744
Control vs. SnailcKO
0.358
Control vs. TwistcKO
Lung Metastasis
0.743
Control vs. SnailcKO
0.786
Control vs. TwistcKO
Spleen Invasion
0.581
Control vs. SnailcKO
0.254
Control vs. TwistcKO
Any Metastasis
1.000
Control vs. SnailcKO
0.797
Metastasis
≥120 days
Control vs. TwistcKO
Liver Metastasis
0.627
Control vs. SnailcKO
1.000
Control vs. TwistcKO
Lung Metastasis
0.592
Control vs. SnailcKO
1.000
Control vs. TwistcKO
Spleen Invasion
0.559
Control vs. SnailcKO
1.000
Control vs. TwistcKO
Any Metastasis
0.473
Control vs. SnailcKO
0.608
Extended Table 3
Pathological spectrum of disease and metastasis in KPC, KPC; TwistcKO and KPC; SnailcKO
cohorts treated with Gemcitabine
KPC Gemcitabine cohorts
ID
Start Age (Days)
Start Volume (mm3)
End Volume (mm3)
Survival (Days)
KPC + GEM
(89)
(13)
1
148
1610.351
D
7
2
72
29.736
D
13
3
72
439.795
902.759
21
4
80
44.14
D
14
5
100
536.304
592.31
21
6
89
166.968
D
2
7
94
52.734
D
7
6
122
90.211
D
14
9
164
217.919
D
8
10
143
212.817
D
18
11
84
323.829
897.217
21
12
58
76.734
D
4
13
58
116.186
D
8
Mean
(Median)
301.4
797.4
Stdev
406.9
145.1
TwistcKO + GEM
(79)
(21)
1
117
243.0
644.2
21
2
75
47.2
180.0
21
3
75
45.4
460.9
21
4
78
54.6
47.5
21
5
46
53.7
66.5
21
6
96
63.1
D
13
7
90
23.9
D
13
8
79
101.0
D
14
9
52
28.5
D
14
10
52
49.4
98.706
21
11
104
43.4
127.0
21
12
104
53.5
12.1
21
13
68
56.7
D
15
14
122
650.1
164.1
21
15
104
181.8
78.6
21
Mean
(Median)
113.0
187.9
Stdev
154.8
193.0
SnailcKO + GEM
(96)
(21)
1
188
255.2
D
12
2
181
854.7
D
4
3
127
32.0
59.6
21
4
127
58.7
107.4
21
5
142
109.8
D
14
6
54
33.6
57.2
21
7
89
17.0
D
13
8
78
54.9
39.6
21
9
78
3.1
D
15
10
104
209.7
134.3
21
11
96
220.0
280.2
21
12
96
24.1
46.2
21
13
119
711.0
D
18
14
126
655.6
805.4
21
15
119
168.6
D
18
16
82
453.8
517.4
21
17
82
56.7
74.1
21
18
90
40.0
D
16
19
67
80.5
D
10
20
66
49.5
226.2
21
Mean
(Median)
204.4
213.4
Stdev
250.7
231.7
Key: (D) died
Supplementary Material
Supplemental Table 1