Microfluidics chip based exosome proteomic profiling from A Microfluidic Chip Enables Isolation of Exosomes and Establishment of Their Protein Profiles and Associated Signaling Pathways in Ovarian Cancer

Dorayappan, Kalpana Deepa Priya; Gardner, Miranda L.; Hisey, Colin L.; Zingarelli, Roman A.; Smith, Brentley Q.; Lightfoot, Michelle D.S.; Gogna, Rajan; Flannery, Meghan M.; Hays, John; Hansford, Derek J.; Freitas, Michael A.; Yu, Lianbo; Cohn, David E.; Selvendiran, Karuppaiyah

doi:10.1158/0008-5472.22421355.v1

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Microfluidics chip based exosome proteomic profiling from A Microfluidic Chip Enables Isolation of Exosomes and Establishment of Their Protein Profiles and Associated Signaling Pathways in Ovarian Cancer

journal contribution

posted on 2023-03-31, 02:25 authored by Kalpana Deepa Priya Dorayappan, Miranda L. Gardner, Colin L. Hisey, Roman A. Zingarelli, Brentley Q. Smith, Michelle D.S. Lightfoot, Rajan Gogna, Meghan M. Flannery, John Hays, Derek J. Hansford, Michael A. Freitas, Lianbo Yu, David E. Cohn, Karuppaiyah Selvendiran

Supplementary Figures: S1 is a work flow for exosome isolation in cancer cells. S2-S5 is protomic profiling in cancer cells Vs normal cells using different techniques. S6-S8 is The top Canonical pathways identified in the exosome protein

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ARTICLE ABSTRACT

Because of limits on specificity and purity to allow for in-depth protein profiling, a standardized method for exosome isolation has yet to be established. In this study, we describe a novel, in-house microfluidic-based device to isolate exosomes from culture media and patient samples. This technology overcomes contamination issues because sample separation is based on the expression of highly specific surface markers CD63 and EpCAM. Mass spectrometry revealed over 25 exosome proteins that are differentially expressed in high-grade serous ovarian cancer (HGSOC) cell lines compared with normal cells—ovarian surface epithelia cells and fallopian tube secretory epithelial cells (FTSEC). Top exosome proteins were identified on the basis of their fold change and statistical significance between groups. Ingenuity pathway analysis identified STAT3 and HGF as top regulator proteins. We further validated exosome proteins of interest (pSTAT3, HGF, and IL6) in HGSOC samples of origin-based cell lines (OVCAR-8, FTSEC) and in early-stage HGSOC patient serum exosome samples using LC/MS-MS and proximity extension assay. Our microfluidic device will allow us to make new discoveries for exosome-based biomarkers for the early detection of HGSOC and will contribute to the development of new targeted therapies based on signaling pathways that are unique to HGSOC, both of which could improve the outcome for women with HGSOC. A unique platform utilizing a microfluidic device enables the discovery of new exosome-based biomarkers in ovarian cancer.