PROJECT SUMMARY Ovarian cancer is a silent killer that strikes with few, if any, symptoms. By the time a woman knows she has it, the cancer is often advanced and the outlook grim. However, if epithelial ovarian cancer is caught early the prognosis for the patient is excellent. Developing non-invasive and highly specific blood-based tests for pre- symptomatic screening and early detection of ovarian cancer is therefore crucial. This is especially essential since obtaining a biopsy is difficult, costly, and sometimes not even an option. In addition, most blood biomarkers to date lack the necessary sensitivity and specificity for early detection of this silent killer. A fundamental challenge is the extremely low concentrations of circulating biomolecules released from the developing tumors at pre-clinical stages which can be 10,000-fold lower than their clinically detectable levels. Therefore, there is a pressing need to uncover novel biomarkers, apply new strategies, and develop robust technologies to propel the advancement of cancer diagnostics, especially in a disease such as ovarian cancer. We have focused our efforts on small extracellular vesicles (sEVs), primarily small and large exosomes derived from the endolysosomal pathway, which play important roles in cellular communication, immune response, and cancer progression via transfer of a selective repertoire of biomolecules. sEVs/exosome release is significantly increased in most neoplastic cells, including ovarian cancer and occurs continuously at all stages of tumor development. Tumor- derived sEVs accumulate in human blood and malignant effusions. These vesicles carry enriched subsets of biomolecules mirroring the tumor cells of origin, such as signaling proteins, tumor antigens, and functional RNAs (mRNA and miRNAs), which offer a new strategy to surmount the challenge in reliable detection of intrinsically low-level serum markers during early malignant transformation. Thus, the constitutive release and enrichment of certain tumor markers within sEVs present distinctive opportunities for early cancer diagnosis. We hypothesize that circulating sEVs, much akin to circulating tumor cells but more robust due to their active release and incredible stability in bodily fluids, represent a greater source for the discovery of exo-biomarkers for early detection, potentially while still confined to the fallopian tube. In addition, sEVs can serve as a 'liquid biopsy’ to assess benefits and treatment responses in real time in cancer patients. Our innovative application merges EV biology with nano-material/microfluidic technology to develop an advanced microfluidic platform to capture and detect ovarian cancer-derived sEVs with high specificity and sensitivity from the circulation. This approach will integrate validated fallopian tube and ovarian cancer associated exo-protein biomarkers (SA1), which will be applied to a second-generation nano-engineered EV analysis chip (SA2). These studies will be fol...