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      Clinical application of a microfluidic chip for immunocapture and quantification of circulating exosomes to assist breast cancer diagnosis and molecular classification

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          Abstract

          Increasing attention has been attracted by exosomes in blood-based diagnosis because cancer cells release more exosomes in serum than normal cells and these exosomes overexpress a certain number of cancer-related biomarkers. However, capture and biomarker analysis of exosomes for clinical application are technically challenging. In this study, we developed a microfluidic chip for immunocapture and quantification of circulating exosomes from small sample volume and applied this device in clinical study. Circulating EpCAM-positive exosomes were measured in 6 cases breast cancer patients and 3 healthy controls to assist diagnosis. A significant increase in the EpCAM-positive exosome level in these patients was detected, compared to healthy controls. Furthermore, we quantified circulating HER2-positive exosomes in 19 cases of breast cancer patients for molecular classification. We demonstrated that the exosomal HER2 expression levels were almost consistent with that in tumor tissues assessed by immunohistochemical staining. The microfluidic chip might provide a new platform to assist breast cancer diagnosis and molecular classification.

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          Most cited references29

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          Extracellular Vesicle-Mediated Transfer of a Novel Long Noncoding RNA TUC339: A Mechanism of Intercellular Signaling in Human Hepatocellular Cancer.

          Although the expression of long noncoding RNA (lncRNA) is altered in hepatocellular cancer (HCC), their biological effects are poorly defined. We have identified lncRNA with highly conserved sequences, ultraconserved lncRNA (ucRNA) that are transcribed and altered in expression in HCC. Extracellular vesicles, such as exosomes and microvesicles, are released from tumor cells and can transfer biologically active proteins and RNA across cells. We sought to identify the role of vesicle-mediated transfer of ucRNA as a mechanism by which these novel lncRNA could influence intercellular signaling with potential for environmental modulation of tumor cell behavior. HCC-derived extracellular vesicles could be isolated from cells in culture and taken up by adjacent cells. The expression of several ucRNA was dramatically altered within extracellular vesicles compared to that in donor cells. The most highly significantly expressed ucRNA in HCC cell-derived extracellular vesicles was cloned and identified as a 1,198-bp ucRNA, termed TUC339. TUC339 was functionally implicated in modulating tumor cell growth and adhesion. Suppression of TUC339 by siRNA reduced HCC cell proliferation, clonogenic growth, and growth in soft agar. Thus, intercellular transfer of TUC339 represents a unique signaling mechanism by which tumor cells can promote HCC growth and spread. These findings expand the potential roles of ucRNA in HCC, support the existence of selective mechanisms for lncRNA export from cells, and implicate extracellular vesicle-mediated transfer of lncRNA as a mechanism by which tumor cells can modulate their local cellular environment. Intercellular transfer of functionally active RNA molecules by extracellular vesicles provides a mechanism that enables cells to exert genetic influences on other cells within the microenvironment.
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            Microfluidic filtration system to isolate extracellular vesicles from blood.

            Extracellular vesicles are released by various cell types, particularly tumor cells, and may be potential targets for blood-based cancer diagnosis. However, studies performed on blood-borne vesicles to date have been limited by lack of effective, standardized purification strategies. Using in situ prepared nanoporous membranes, we present a simple strategy employing a microfluidic filtration system to isolate vesicles from whole blood samples. This method can be applied to purify nano-sized particles from blood allowing isolation of intact extracellular vesicles, avoiding the need for laborious and potentially damaging centrifugation steps or overly specific antibody-based affinity purification. Porous polymer monoliths were integrated as membranes into poly(methyl methacrylate) microfluidic chips by benchtop UV photopolymerization through a mask, allowing precise positioning of membrane elements while preserving simplicity of device preparation. Pore size could be manipulated by changing the ratio of porogenic solvent to prepolymer solution, and was tuned to a size proper for extraction of vesicles. Using the membrane as a size exclusion filter, we separated vesicles from cells and large debris by injecting whole blood under pressure through the microfluidic device. To enhance isolation purity, DC electrophoresis was employed as an alternative driving force to propel particles across the filter and increase the separation efficiency of vesicles from proteins. From the whole blood of melanoma-grown mice, we isolated extracellular vesicles and performed RT-PCR to verify their contents of RNA. Melan A mRNA derived from melanoma tumor cells were found enriched in filtered samples, confirming the recovery of vesicles via their cargo. This filtration system can be incorporated into other on-chip processes enabling integrated sample preparation for the downstream analysis of blood-based extracellular vesicles.
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              Exosomes serve as tumour markers for personalized diagnostics owing to their important role in cancer metastasis

              Exosomes, membrane vesicles of 40–100 nm in diameter, are derived from endosomes in various cells. The bioactive molecules specifically packed into exosomes can be horizontally transferred into recipient cells changing their biological properties, by which tumour cells continuously modify their surrounding microenvironment and distant target cells favouring cancer metastasis. It has been suspected for a long time that exosomes participate in the whole process of tumour metastasis. Although there is much unknown and many controversies in the role of cancer exosome, the major contribution of tumour-associated exosomes to different steps of cancer metastasis are demonstrated in this review. Mainly because these exosomes are easily accessible and capable of representing their parental cells, exosomes draw much attention as a promising biomarker for tumour screening, diagnosis and prognosis. Currently, researchers have found numerous biomarkers in exosomes with great potential to be utilized in personalized medicine. In this article, we summarize the roles of biomarkers, which are validated by clinical samples. Even though many conundrums remain, such as exosome extraction, large multicentre validation of biomarkers and data interpretation, exosomes are certain to be used in clinical practice in the near future as the field rapidly expands.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                3 April 2017
                2017
                : 12
                : 4
                : e0175050
                Affiliations
                [1 ]College of Stomatology, Dalian Medical University, Dalian, China
                [2 ]Department of Urology, the Second Affiliated Hospital, Dalian Medical University, Dalian, China
                [3 ]Department of Nuclear Medicine, the First Affiliated Hospital, Dalian Medical University, Dalian, China
                [4 ]Department of Biochemistry and Molecular Biology, Liaoning Provincial Core Lab of Glycobiology and Glycoengineering, Dalian Medical University, Dalian, China
                [5 ]Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
                [6 ]State Key Laboratory of Fine Chemicals, Department of Chemical Engineering, Dalian University of Technology, Dalian, China
                University of South Alabama Mitchell Cancer Institute, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: TL WN.

                • Data curation: SF HT Xiancheng Li DJ Xiaojie Li JK CY XY.

                • Formal analysis: HT Xiancheng Li DJ Xiaojie Li JK CY XY.

                • Funding acquisition: TL WN.

                • Investigation: SF.

                • Methodology: Y. Lu Y. Luo BL.

                • Project administration: TL.

                • Resources: SF.

                • Writing – review & editing: TL SF.

                Author information
                http://orcid.org/0000-0001-5850-789X
                Article
                PONE-D-16-46859
                10.1371/journal.pone.0175050
                5378374
                28369094
                a66f1738-a491-4d47-85c0-c756c1bc5f57
                © 2017 Fang et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 26 November 2016
                : 20 March 2017
                Page count
                Figures: 5, Tables: 0, Pages: 13
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 81571767
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 81171538
                Award Recipient :
                This work was supported by National Natural Science Foundation of China (81571767) and National Natural Science Foundation of China (81171538).
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Exosomes
                Engineering and Technology
                Fluidics
                Microfluidics
                Medicine and Health Sciences
                Oncology
                Cancers and Neoplasms
                Breast Tumors
                Breast Cancer
                Medicine and Health Sciences
                Diagnostic Medicine
                Cancer Detection and Diagnosis
                Medicine and Health Sciences
                Oncology
                Cancer Detection and Diagnosis
                Research and Analysis Methods
                Biological Cultures
                Cell Cultures
                Medicine and Health Sciences
                Clinical Medicine
                Clinical Immunology
                Major Histocompatibility Complex
                Biology and Life Sciences
                Immunology
                Clinical Immunology
                Major Histocompatibility Complex
                Medicine and Health Sciences
                Immunology
                Clinical Immunology
                Major Histocompatibility Complex
                Biology and Life Sciences
                Immunology
                Major Histocompatibility Complex
                Medicine and Health Sciences
                Immunology
                Major Histocompatibility Complex
                Biology and Life Sciences
                Biochemistry
                Biomarkers
                Research and Analysis Methods
                Imaging Techniques
                Fluorescence Imaging
                Custom metadata
                All relevant data are within the paper.

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