A Microfluidic Chip Enables Isolation of Exosomes and Establishment of Their Protein Profiles and Associated Signaling Pathways in Ovarian Cancer

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.

Application of exosomes as liquid biopsy in clinical diagnosis

“…Apart from these classic extraction methods, multiple new extraction and detection technologies have been reported in recent years, such as the microfluidic chip 147,148 and a method of integrated extraction and quantitative analysis of exosomal nucleic acids and proteins, 149 with high specificity and intact yield of exosomes, small required sample volume and simple, time-saving operation. Moreover, a novel urine-based EV extraction and enrichment method was established by Woo et al 150 .…”

Exosomes promote pre-metastatic niche formation in ovarian cancer

“…To this effect, various malignancies have been studied including melanoma, nasopharyngeal carcinoma, breast cancer, colorectal cancer, and ovarian cancer [97]. Very recently, a microfluidic chip-based liquid biopsy was able to isolate exosomes with detailed protein and signaling pathway profiles in ovarian cancer [99].…”

The cancer exosomes: Clinical implications, applications and challenges

“…Reategui et al 86 established a microfluidic platform named (EV)HB-Chip which captured glioblastoma multiformederived characteristic exosomal RNA within 3 hr, contributing to identifying specific genes and subtypes of this disease. Dorayappan et al 87 demonstrated a specific microfluidicbased internal separation device for the establishment of exosomal protein profiles of high-grade serous ovarian cancer (HGSOC) within 20 min, overcoming contamination problems and making new findings for early exosome-based detection. Consequently, the microfluidic chip undoubtedly lays a solid foundation for early diagnosis and prognosis of cancer and it is substantially better than many other existing methods.…”