Project II. Immune regulatory circuits in primary colon cancer and lymph node and liver metastases Experimental Lead: Rudensky Computational Lead: Leslie Experimental Co-Lead: Ganesh PROJECT SUMMARY Colorectal cancer (CRC) represents the third most common cancer in the US and worldwide. Despite major advances in treatment of some cancer afforded by immunotherapy, mismatch repair-proficient (pMMR) CRC, which accounts for the vast majority (85%) of cases, fails to respond with ~15% 5-year survival rate. We hypothesize that this failure is due to the activity of CRC-promoting local circuits of immune and stromal accessory cells, in which suppressive regulatory T (Treg) cells and non-inflammatory tissue-supporting Th17 cells serve as essential components. We also propose that the architecture and cellular composition of these T cell circuits are distinct in primary CRC and in proximal lymph node and distal liver metastases. In this project, we will dissect the dynamics of the main immunomodulatory cells states including Treg and Th17 cell and their interactions with cellular components (“immunomodulatory neighborhoods”) in primary tumors and lymph node and liver metastases in the same pMMR CRC patients alongside a novel mouse genetic model of spontaneously metastasizing pMMR CRC (Aim 1). Our perturbation studies will elucidate roles of these key immunomodulatory cells and their partners in metastatic disease progression and resistance to immunotherapy (Aim 2) and examine the impact of key immune cell-derived factors on cancer cells in human primary and metastatic CRC organoids (Aim 3). We will employ cutting edge “omics” analyses at a single cell resolution combined with spatial transcriptomics and highly multiplexed optical imaging and microscopy and flow cytometry coupled to experimental perturbations iteratively with novel computational modeling and analyses to deconstruct archetypal T cell-based cell circuits in CRC and conserved chromatin states and gene regulatory programs of their cellular constituents. These studies will enable better mechanistic understanding of emergence of pro-metastatic cell states in pMMR CRC and inform development of orthogonal rational combination immunotherapies for this disease and elucidate the therapeutic actionability of targeting immune-epithelial circuits in preventing and treating metastasis in patients with advanced CRC. .