A microfluidic chip enables isolation of exosomes and establishment of their protein profiles and associated signaling pathways in ovarian cancer

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

doi:10.1158/0008-5472.CAN-18-3538

A microfluidic chip enables isolation of exosomes and establishment of their protein profiles and associated signaling pathways in ovarian cancer

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^*

^*Corresponding author for this work

Biomedical Engineering

Research output: Contribution to journal › Article › peer-review

86 Scopus citations

Abstract

Because of limits on specificity and purity to allow for indepth 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. Significance: A unique platform utilizing a microfluidic device enables the discovery of new exosome-based biomarkers in ovarian cancer.

Original language	English (US)
Pages (from-to)	3503-3513
Number of pages	11
Journal	Cancer Research
Volume	79
Issue number	13
DOIs	https://doi.org/10.1158/0008-5472.CAN-18-3538
State	Published - Jul 1 2019

Funding

The authors are thankful to the undergraduate students Puneet Modgil, Roshan Sivakumar, Marissa Werner, and Audrey Whitaker for the cell culture and basic assay. We thank Dr. Zhang Liwen for proteomic support and Alex Cornwell for Image Stream Analysis. This work was funded by NCI RO1-CA176078 grant (to K. Selvendiran. and D.E. Cohn) and KOH Ovarian Cancer Foundation grant (to K.D.P. Dorayappan), the Beucler Family Fund (D.E. Cohn), NCI R01GM122436 (M.A. Freitas), and Pelotonia fellowship (M.L. Gardner).

ASJC Scopus subject areas

Oncology
Cancer Research

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1158/0008-5472.CAN-18-3538

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Dorayappan, K. D. P., Gardner, M. L., Hisey, C. L., Zingarelli, R. A., Smith, B. Q., Lightfoot, M. D. S., Gogna, R., Flannery, M. M., Hays, J., Hansford, D. J., Freitas, M. A., Yu, L., Cohn, D. E., & Selvendiran, K. (2019). A microfluidic chip enables isolation of exosomes and establishment of their protein profiles and associated signaling pathways in ovarian cancer. Cancer Research, 79(13), 3503-3513. https://doi.org/10.1158/0008-5472.CAN-18-3538

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abstract = "Because of limits on specificity and purity to allow for indepth 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. Significance: A unique platform utilizing a microfluidic device enables the discovery of new exosome-based biomarkers in ovarian cancer.",

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

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Dorayappan, KDP, Gardner, ML, Hisey, CL, Zingarelli, RA, Smith, BQ, Lightfoot, MDS, Gogna, R, Flannery, MM, Hays, J, Hansford, DJ, Freitas, MA, Yu, L, Cohn, DE & Selvendiran, K 2019, 'A microfluidic chip enables isolation of exosomes and establishment of their protein profiles and associated signaling pathways in ovarian cancer', Cancer Research, vol. 79, no. 13, pp. 3503-3513. https://doi.org/10.1158/0008-5472.CAN-18-3538

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AU - Dorayappan, Kalpana Deepa Priya

AU - Gardner, Miranda L.

AU - Hisey, Colin L.

AU - Zingarelli, Roman A.

AU - Smith, Brentley Q.

AU - Lightfoot, Michelle D.S.

AU - Gogna, Rajan

AU - Flannery, Meghan M.

AU - Hays, John

AU - Hansford, Derek J.

AU - Freitas, Michael A.

AU - Yu, Lianbo

AU - Cohn, David E.

AU - Selvendiran, Karuppaiyah

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A microfluidic chip enables isolation of exosomes and establishment of their protein profiles and associated signaling pathways in ovarian cancer

Abstract

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UN SDGs

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