Extracellular vesicles (EVs) are nanosized (~30-1000 nm) lipid-enclosed particles released by all cell types. The cargo contained within the EV is representative of the cell of origin containing lipids, proteins, glycans, and nucleic acids. Evidence of EV presence in biological fluids has led to considerable efforts focused on identifying their cargo and determining their utility as a non-invasive diagnostic platform for cancer. Direct enumeration of tumor-derived EVs and/or profiling of their molecular cargo in patient body fluids have been shown to provide valuable information about the biology of the tumor. Given their nanosized properties, EVs are difficult to isolate and study. In complex biological samples, this difficulty is amplified by other small particles and contaminating proteins making the discovery and validation of EV-based biomarkers challenging. Developing new strategies to analyze and isolate EVs from complex biological samples is of significant interest. This thesis focuses on developing nanoscale flow cytometry for EV analysis and isolation in the context of prostate cancer. Prostate cancer screening strategies have not advanced since the introduction of the Prostate Specific Antigen (PSA) blood test, despite prostate cancer being the most commonly diagnosed cancer in Canadian males. Relying on imprecise detection methods, such as the PSA blood test, has deeply hampered our ability to detect and treat clinically relevant prostate cancer early, which is the most important factor for decreasing mortality rates. Therefore, alternative, non-invasive biomarkers capable of identifying high-risk prostate cancer are urgently needed to improve the detection and prognostication of prostate cancer patients. iv The work described in this thesis utilizes nanoscale flow cytometry to enumerate circulating STEAP1 (six-transmembrane epithelial antigen of the prostate 1)-positive EVs in the plasma of prostate cancer patients and healthy males and demonstrates a diagnostic capability far superior to the PSA blood test. To improve the prognostication capabilities of our test and detect high-risk prostate cancer, we then developed a method to isolate subpopulations of EVs directly from human plasma using nanoscale flow cytometry without additional EV isolation strategies. The isolation and enrichment of unique EV populations have significant implications in the discovery and validation of biomarkers with clinical utility.
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