Exosomes are bi-layered lipid microvesicles containing DNA, RNA and proteins, which have emerged as an important avenue for cell-cell communication in cancer. Our team has performed pioneering studies on the utility of circulating exosomes for genomic and transcriptomic profiling of visceral cancers that are challenging to sample longitudinally. Specifically, we have demonstrated the remarkable integrity of the nucleic acid cargo (exoDNA and exoRNA) contained within exosomes, which makes them readily amenable to next generation sequencing (NGS). The objective of this proposal is to establish the utility of liquid biopsies that enrich for high quality exosomes as a platform for therapeutic stratification and disease monitoring in advanced colorectal cancer (CRC). In Aim 1 will meticulously evaluate the most appropriate methodology for reproducible and sensitive isolation of circulating exosomes from metastatic CRC patients using gradient ultracentrifugation versus two of the most commonly used commercial kits. We will develop perform rigorous biological and technical reproducibility assays on the isolated exosomes and nucleic acid cargo that will develop standards necessary for translation of exosomal-based “liquid biopsy” to a clinical molecular diagnostics laboratory (MDL). We will also perform mass spectrometry-based proteomic profiling of exosomes isolated from a panel of CRC organoid models versus control organoids and cell lines, in order to identify the surface proteins (“surfaceome”) present on CRC-derived exosomes. The surfaceome data will serve as an invaluable tool for enrichment of cancerspecific exosomes in low-volume tumor settings, such as minimal residual disease monitoring. In Aim 2, we will compare targeted mutation panel and copy number profiles of tissue samples in patients undergoing surgical debulking for metastatic (m)CRC, with that of corresponding plasma exoDNA-derived profiles. The remarkable preservation of exoRNA within exosomes will allow us to perform RNA-Seq and compare the consensus molecular subtype (CMS) classification obtained by shed exosomes versus metastatic tissues. Finally, in Aim 3, we will longitudinally monitor cohorts of surgically resected CRC patients, or mCRC patients on two prospective clinical trials, using exosomes as a molecular tool for identifying disease recurrence and emergence of treatment resistance, respectively. The first trial will evaluate for emergence of low frequency KRAS mutations and other secondary drivers of resistance to EGFR-based therapies. The second trial is an immunotherapy trial of multivalent peptide vaccine, where exoRNA data will also be used to predict, and then monitor, expressed neoantigens in mCRC samples, using bioinformatics tools we have developed for mapping transcript data to HLA-presented peptides on the cancer cell surface. The long-term goal of these studies is to generate the compendium of standards and assays needed for successful translation of exosomes as a liquid biopsy platform to the clinical realm.