Colorectal cancer (CRC) is the second most deadly cancer in the United States, affecting over 140,000 people each year, killing approximately 50,000 in the US, largely by metastatic progression. The intestine hosts the body’s largest collection of immune cells, maintained in close proximity to foreign antigens from the diet, enriched in the proximal regions, and microbiota, which accumulate in the distal regions. The vast and highly connected gut lymphatic vessels form a major cell- and antigen transport route to the draining lymph nodes, responsible for the initiation of adaptive immunity, which could play four roles in regulating colorectal cancer metastasis: immune cells could (i) prevent CRC progression and early dissemination or metastasis by immune-cell killing (ii) promote CRC progression and metastasis through release of inflammatory cytokines, (iii) prevent metastatic colonization in the liver, or (iv) promote metastatic colonization in the liver. We provide data that compartmentalization of intestinal lymphatic drainage to functionally distinct lymph nodes facilitates the simultaneous induction of immune-suppressive and inflammatory immune responses in the gut; however, the relevance of gut lymphatics to CRC and metastasis remains underexplored. In addition to compartmentalized lymphatic networks, the gut also hosts a large number of enteric neurons functionally tuned to each region they occupy. Using retrograde tracing from distinct intestinal regions and specific cell-sorting independent transcriptomics, we uncovered novel neuronal circuits and a role for enteric neurons in sensing perturbations in the intestinal tissue; whether enteric neurons sense and modulate CRC metastatic progression remains unknown. Overall, Project 2 is focused on understanding how enteric-associated neurons sense and provide signals that regulate CRC progression and liver dissemination and how compartmentalized intestinal lymphatic drainage of primary tumor and metastatic liver sites regulate anti-tumor responses and early dissemination. In Aim 1, we hypothesize that primary CRC, as well as liver metastasis, are specifically sensed by populations of enteric-associated neurons, which in turn, influence tumor and metastatic progression. This question will be addressed using tools to visualize and circuit- map, single cell and active translating transcriptomics, and chemogenetic approaches targeting specific neuronal subsets. In Aim 2, we hypothesize that the intestinal lymphatic system communicates primary CRC and early metastatic seeding to the local and systemic immune system, modulating anti-tumor responses. This will be addressed combining modern clearing and live imaging techniques, single cell transcriptomics and novel immune cell-interaction approaches. By combining these approaches, expertise of Mucida lab, with Sohail Tavazoie lab’s expertise in cancer and metastasis biology (Project 1), the Birsoy lab’s expertise in in vitro screenings and cell metabo...