Exosomes for the Early Detection of Ovarian Cancer

Abstract

Epithelial ovarian cancer (EOC) is a silent killer that strikes with few, if any, symptoms. By the time a woman is diagnosed with EOC, it is often at an advanced stage where the outlook is grim. However, if caught early, the prognosis is excellent as it can be cured in up to 90% of patients. Therefore, developing a highly specific blood-based test is extremely appealing for pre-symptomatic screening and early detection of EOC. However, all blood biomarkers to date lack the necessary sensitivity/specificity for early detection of this disease. A fundamental challenge in biomarker discovery is the extremely low concentrations released from developing tumors into the circulation at pre-clinical stages, which can be 104-fold lower than the clinically detectable levels. For this reason there is a pressing need to uncover novel biomarkers, and apply new strategies to propel the advancement of EOC diagnostics. We have focused our efforts on extracellular vesicles (EVs), primarily exosomes derived from the endolsosomal pathway that play important roles in intercellular communication, immune responses and cancer pathogenesis via transfer of a selective repertoire of biomolecules. A proteomic analysis was performed on plasma and EV depleted plasma obtained from patients diagnosed with stage III/IV serous ovarian cancer (n=14). Bead-based Luminex flow cytometry assays were performed on the complete or EV/exosome-free plasma samples to examine circulating or EV-associated levels of 23 growth factors, cytokines, and other cancer related molecules. In addition, similar experiments were completed using immune-purified CA125+ (EOC associated marker) subpopulation of cell culture derived exosomes. Immunoprecipitation was also utilized to isolate three subtypes (CA125+, EpCAM+, and FAP+) of extracellular vesicles, primarily exosomes, from plasma. Our proteomic analysis indicated that the levels of several circulating biomarkers decrease upon the removal of EVs from total plasma (TRAIL, leptin, and OPN), likely indicating a direct association between these analytes and EVs. Three of the 23 analytes were specifically detectable in immunoisolated CA125+ cell culture derived exosomes (OPN, HE4, CYFRA-21) and undetectable in the unfractionated exosome population. Of the 23 analytes investigated 21 were detectable at varying levels in the three subtypes of EVs immuno-captured from plasma. When the growth factors, cytokines, and other cancer related molecules were examined in the subpopulations of EVs derived from cases and controls we found that using CA125 or EpCAM as the capture agents, and subsequently measuring CA125 and CYFRA21-1, respectively, may offer improved specificity and sensitivity for ovarian cancer. This study is the first to report free versus microvesicle-associated proteins in EOC plasma samples, and further characterize a limited number of intravesicular proteins in subtypes of EOC EVs. Our work presents the framework for developing an ovarian cancer specific assay to capture and evaluate protein signatures in tumor-derived EVs, primarily exosomes, with the goal of significantly improving the detection of this deadly disease at a stage when curable.