PROJECT SUMMARY/ABSTRACT The focus of this renewal remains colorectal cancer (CRC), the 2nd leading cause of cancer mortality worldwide. During the last cycle of funding, seminal advances were made related to early and late events in colorectal neoplasia, utilizing cutting-edge technologies and uncovering paradigm-shifting concepts applicable to other solid tumors. In the next cycle, we will concentrate on microsatellite stable (MSS) CRC, which accounts for 85% of CRC and does not respond to immune checkpoint blockade (ICB) in marked contrast to microsatellite unstable (MSI-H) CRC. As MSS tumors are derived from a stem/progenitor epithelial cell population, we propose studies involving the identification of a novel marker for a quiescent stem cell population, Tob2, as well as determining the nonredundant roles of Lrig1 and Lrig3 in colonic tumor formation as they are critical regulators of intestinal homeostasis. In separate studies using a novel NSC-seq platform, we have identified Tob2 that is expressed early in mouse embryogenesis and marks a population of quiescent stem cells that will be probed for its ability to impact colonic tumor formation as well. Also, we have discovered a 4-gene immune-exclusion (IEX) score in MSS CRC that impacts clinical outcome. Three of the genes encode secreted proteins (DDR1, TGFBi, DPEP1) that may be responsible for IEX and are therapeutically tractable. A clinical trial has already started combining a neutralizing antibody to DDR1 and ICB. A major area of investigation has been small extracellular vesicles (sEVs) and an amembranous nanoparticle, termed supermeres, which we recently discovered. Remarkably, DPEP1 and TGFBi are the two most abundant proteins in CRC sEVs and supermeres, respectively; DDR1 is also enriched in supermeres. Of note, these proteins are markedly increased in the respective plasma-derived fractions of CRC patients versus healthy controls so they may serve as CRC biomarkers. We propose mechanistic studies to determine the function of these proteins, including that of a novel isoform of DPEP1 whose overexpression results in tumor formation in mice. Our discovery of supermeres has opened the possibility for new biomarkers and ways to target tumor progression through cell-cell communication, which we will explore through understanding supermere biogenesis and determining supermere function in immunomodulation of the CRC tumor microenvironment. In this renewal, we intend to continue to make paradigm-shifting discoveries that impact the ways of thinking about cancer, its origins, how it progresses, as well as implementing practice- changing strategies to diagnose and treat MSS CRC.