Ovarian cancer (OvCa) remains the most lethal gynecologic cancer in the United States. Early detection through screening, particularly of high-risk populations, is an appealing strategy for timely intervention yet conventional efforts have not improved survival. Extracellular vesicles (EVs; 30-200 nm in diameter) are actively released by cells into circulation, carrying diverse molecular cargo. Capturing this information could provide a real-time window to monitor tumor burden and dynamic molecular changes and potentially OvCa precursor lesions. We aim to advance our translational EV assay for early OvCa diagnosis with a focus on its most lethal subtype, high-grade serous ovarian cancer (HGSOC). We formed a powerful strategic academic-industry alliance between Massachusetts General Hospital, Brigham and Women's Hospital, and Exosome Diagnostics and have laid out two primary objectives. Technology refinement. We will implement an automated and high-throughput platform, OCEANA (Ovarian Cancer Exosomal Analysis with Nanoplasmonic Array). We will also construct an EV marker panel to enhance the diagnostic power of EV assays. Expanded clinical validation. We will rigorously evaluate OCEANA and EV biomarkers using patient-derived organoids and samples across the clinical spectrum (e.g., benign, early, advanced stages) from a world-class and gynecologic-focused biorepository. We have four Specific Aims to achieve these objectives. Aim 1. We will develop a disk-based cartridge for fast, automated total EV isolation. The underlying technology will be our dual-mode chromatography (DMC) which has demonstrated superior EV extraction from plasma. Aim 2. We will establish an expanded EV-marker panel for HGSOC detection informed by patient-derived organoids and EVs. Specifically, EV protein and mRNA results will be combined to make a multi-analyte marker panel. Aim 3. We will analyze EVs from mice hosting HGSOC patient-derived xenografts to study the relationship between tumor progression and circulating EVs. Aim 4. We will screen circulating EVs in women with benign or malignant ovarian conditions, rigorously evaluating OCEANA's clinical performance. Impact. This project will establish OCEANA as a transformative tool for comprehensive EV analyses. The clinical study will also validate EVs as a potent circulating biomarker for early detection of HGSOC. Ultimately, we envision translating the technology as a monitoring tool for cancer care across research and clinical settings.

NARRATIVE We propose to translate a novel liquid biopsy platform for early cancer detection. Specifically, we will apply a forward-thinking nano-plasmonic platform for comprehensive, high-throughput molecular analyses of extracellular vesicles (EVs). We will rigorously address EVs' clinical utility to detect high- grade serous ovarian cancer in high-risk patient cohorts.