This proposal leverages trans-institutional collaboration and multidisciplinary expertise in extracellular vesicle (EV) biology, together with established clinical expertise in oncology, to identify EV-associated biomarkers for the detection, diagnosis and prognosis of early prostate cancer (PC). EVs shed by the tumor and its microenvironment are a very promising source of biomarkers, with the potential to assist with the detection, diagnosis and prognosis of cancer in the clinical setting. This is particularly true for PC where there is an immediate need for improved diagnostics to reduce unnecessary biopsies and over-diagnosis of indolent disease, and for a methodology that will allow thousands of under-diagnosed men who have clinically significant PC to receive appropriate early diagnosis. Unfortunately, the rigor and reproducibility of detecting EV-associated biomarkers with appropriate specificity and sensitivity, as well as a detection approach that provides reproducible results across all clinical settings in North America, have not been achieved. Our recent studies on large oncosomes (LO), which are atypically large EVs (1-10 m), have identified a previously unexplored pool of EV-associated biomarkers that we find predictive of clinically significant PC. As a team with cross-cutting expertise in EV biology, PC, and biomarker discovery, we are collaborating to pursue EV-associated biomarkers that can detect PC with sufficient sensitivity and specificity to diagnose PC early and to distinguish between indolent disease and lethal disease. Our preliminary studies reveal that tumorderived EVs are readily detected in PC patients. Moreover, an assessment of known markers demonstrates enormous potential for EV-associated biomarkers in the differential diagnosis of early PC. We hypothesize that incorporating robust EV-associated markers into the existing standard of care for PC can reduce cancerrelated deaths caused by lethal PC and avoid the overtreatment of indolent disease by improving early detection and differential diagnosis of these patient categories. This multidisciplinary team will address this hypothesis through three Specific Aims: 1) To refine methods leading to reproducible analysis of LO from plasma and serum. 2) Discovery of candidate (palmitoyl)protein biomarkers from purified LO for early identification of clinically significant PC and 3) Clinical testing to assess the diagnostic and prognostic value of LO in early prostate cancer. The project incorporates several next generation technologies and resources to identify cancer-specific biomarkers in circulating LO and assess their performance in independent clinical cohorts of PC patients from the collaborating institutions. Our objective is to implement and validate a rigorous and reproducible methodology for EV analysis with high clinical value.