PROJECT SUMMARY Novel diagnostic and prognostic biomarkers are urgently needed to improve clinical outcomes of cancer. Circulating exosomes are emerging as a new paradigm of “liquid biopsy” for non-invasive cancer diagnosis and monitoring. Exosomes are small membrane vesicles of 30-150 nm in size that are secreted by most cells, and growing evidence has shown important biological roles and clinical relevance of exosomes. In various cancers, tumor-derived exosomes have been found to be accumulated in human biofluids, such as blood, and enriched with a set of biomolecules from the cells of origin, such as specific proteins and RNAs, which may constitute a “tumor signature”. However, the evaluation of clinical value of exosomes has been limited in part due to challenges in isolation and analysis of such small, dynamic and molecularly diverse vesicles. To address this obvious gap in both analytical technologies and precision medicine, here we propose to an Exosome Fingerprinting Nano-Device (ExoFIND) system that offers unprecedented analytical capabilities for measuring intravesicular protein markers carried by tumor-derived exosomes present in circulation. This new technology will be adapted to examining exosomes derived from cell lines, animal models and human biofluids to assess exosomal proteins, including Hu antigen R (HuR), as potential liquid biopsy-based markers for early diagnosis, prognosis, and targeted therapy of cancer. The proposed studies will be carried out to accomplish four Specific Aims: 1) Develop a general, high-resolution inkjet colloidal printing technology for standardized and scalable fabrication of 3D nanoengineered bioassay devices; 2) Establish a 3D nano-engineered ExoFIND system for integrative analysis of exosomal fingerprints of tumors; 3) Adapt and validate the ExoFIND system as a blood-based detection platform to assess colorectal cancer (CRC)-derived exosomes as biomarkers for tumor initiation and progression; 4) Adapt and validate the ExoFIND system as a companion assay to monitor tumor response, drug resistance and prognosis of HuR-targeted therapy. Successfully carried out, our study will provide important impetus to: (1) develop next-generation technologies with transformative and reproducible analytical capabilities and minimal sample requirement, and (2) catalyze the exploration of exosomes as a novel paradigm of liquid biopsy for precision oncology. Our overarching goal is to ultimately provide a transformative platform technology for basic and translation investigation of a wide range of malignancies and diseases.