PROJECT SUMMARY/ABSTRACT Extracellular vesicles (EVs) comprise a heterogeneous group of secreted membranous structures that contain a variety of biomolecular cargo such as nucleic acids, proteins and lipids. EVs are ideal for liquid biopsy because EVs are often more highly secreted by cancer cells than by normal cells, can be detected in body fluids of cancer patients, and protect their cargo from degradation. Because EV cargo often reflects the genetic and biological status of the cell of origin, constituents of EV cargo are promising biomarkers for cancer diagnosis, prognosis and recurrence. Of these constituents, miRNAs have attracted substantial attention as potential biomarkers. However, several studies indicate that a substantial proportion of EVs do not contain significant miRNA copy numbers. These studies have implicated the existence of a subpopulation(s) of EVs that is enriched in miRNAs, but this subpopulation has not been identified. The overarching goal of this study is to develop approaches to isolate subpopulations of EVs that are enriched in miRNAs for liquid biopsy purposes. Our preliminary studies have identified several distinct subpopulations of EVs based on their surface protein expression, and implicate that the surface protein repertoire could identify EVs that are enriched in miRNAs. In this study, we will firstly evaluate EVs that are secreted by human cancer cells and present in body fluids of patients with three major types of cancers (ovarian, colorectal, renal) for the prevalence of EV subpopulations defined by their surface protein repertoire. Secondly, we will determine the total miRNA content in each subpopulation of EVs that are secreted by human cancer cells and present in cancer patient body fluids. Thirdly, we will evaluate the detection of miRNAs in EVs that are isolated by immunocapture of different EV surface proteins from body fluids of tumor- bearing mice and cancer patients. If successful, our study will provide answers to critical gaps-in-knowledge in the EV biomarker field and, importantly, develop an approach for isolating miRNA-rich EVs that can be readily used in a clinical laboratory setting for liquid biopsy purposes.