A SYNTHETIC BIOMARKER TO UNIVERSALLY ASSESS THE RELATIVE CONTRIBUTION OF HEATHY AND CANCEROUS TISSUE TO CIRCULATING EV POOL

Extracellular vesicles (EVs) are cell-derived membrane-bound structures released into extracellular spaces that navigate the bodily fluids and appear to support intercellular communication. Cancer cells release significantly higher numbers of EVs then their normal counterparts. Because the EV contents are derived from the cell of origin, molecular profiling of circulating EVs are being scrutinized as a non-invasive means for early cancer diagnosis, monitoring disease progression, and assessing response to treatment. However, EV-based clinical diagnostics have been limited by inadequate rigor and reproducibility of samples to specifically discriminate, isolate, and characterize normal and disease-associated EVs. Three analytical and conceptual challenges have prevented the identification of specific and reproducible EVassociated cancer biomarkers with clinical relevance: 1) no unbiased strategy has been developed to evaluate the limits of cancer detection using EVs; 2) the relative contribution of healthy tissues to the pool of circulating EVs is not known; and 3) a systematic analysis of the number and composition of circulating cancer-derived EVs during tumor development has not been performed. These complications have prompted us to design a general platform capable of evaluating the contribution of specific tissues to the pool of circulating EVs in otherwise health animals compared to animals undergoing cancer development. The system is based on an engineered EV marker developed from the tetraspanin protein CD63 (enCD63), which facilitates collection, visualization, and quantification of EVs released by specific cells and tissues. By restricting the expression of enCD63 to specific normal or neoplastic cells and tissues of genetically engineered mouse models, we will unambiguously examine the efficacy of EVs as biomarkers. We propose to use our innovative platform to: 1) perform an unbiased calibration correlating the number of EVs with their cells of origin (Aim 1); 2) define the relative contribution of healthy tissues to the pool of circulating EVs (Aim 2); and 3) to assess the specificity and sensitivity of EVs in cancer detection (Aim 3). This application focuses on Pancreatic Ductal Adenocarcinoma (PDAC), a deadly neoplastic disease with low survival rate that lacks specific and sensitive diagnostic tests. Completion of the proposed studies will contribute to the development of standardized procedures for the preparation, selection, and analysis of EV-based biomarkers.