PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is a second cause of cancer-related mortality because of metastatic disease represented by liver and peritoneal metastasis. This disease is largely insensitive to modern immunotherapies and is with limited curative options. Immune mechanisms regulating CRC tumor microenvironment (TME) are not well understood and are likely to hold the key for better immune-based therapies. We surprisingly found that the levels of Interleukin (IL)27 cytokine are increased in human CRC and relevant mouse models of CRC and that its increased levels correlate with poor prognosis. As a part of preliminary data we found that genetic inactivation of IL27R (receptor) or neutralization of IL27 cytokine in sporadic CRC models reduces tumor growth, progression and metastasis. This proposal aims to understand how IL27 regulates CRC TME during metastasis development and how its inactivation drives anti-tumor immunity exerted by innate and adaptive arms of immune responses. Our hypothesis is that IL27 suppressed anti-tumor immune responses mediated by adaptive immune T cells and possible cells of innate immunity. We will use genetic models of IL27/IL27R deficiency as well as neutralization of IL27 cytokine combined with cutting edge preclinical models of CRC spread and metastasis and histological, cutting-edge imaging and molecular biological/advanced transcriptomics methods in mouse and human CRC. Proposal will be based on three interconnected Specific Aims: 1) Examine effects of IL27 on immunosuppressive TME of CRC liver and peritoneal metastasis; 2) Uncover cell type specificity of IL27 signals within TME of CRC metastasis; 3) Test whether IL27 neutralization can aid other immunotherapeutic modalities, particularly focuses on reactivation of adaptive (anti-PD1/anti-LAG3) and innate myeloid cell anti—cancer immune responses (anti-CD47). Our experiments will therefore establish an unique role for IL27 signaling in liver and rarely studied peritoneal CRC metastasis, uncover underlying molecular and cellular mechanisms and establish and test rationale and feasibility of IL27 pathway blockade to facilitate anti-cancer immunity in difficult-to-treat advanced cancer.