Project Summary/Abstract Colorectal cancer (CRC) is the second leading cause of cancer mortality in the United States, and obesity, which affects 40% of the population, not only increases the risk of developing CRC, but also increases CRC mortality through unknown mechanisms. Our preliminary studies demonstrate that CRC grows faster in mice rendered obese through a high fat diet (HFD), and that the tumor associated macrophages (TAMs) in these mice exhibit higher expression of the acid sensing receptor, GPR65, which is known to dampen the immune response. Moreover, in HFD-induced obese mice lacking GRP65, TAMs secrete more TNF-α and tumor growth is retarded. Given that TAMs but not normal tissue macrophages of obese mice exhibit increased GPR65, we examined the pH of the tumors in these mice and found them to be more acidic. On the basis of these findings, we hypothesize that excess lipids in the HFD alter tumor cell metabolism resulting in increased acid production, which potentiates GPR65 expression and signaling in TAMs, causing them to become immunosuppressive and promote tumor growth. To test this hypothesis we will pursue the following specific aims: 1) Determine the contribution of GPR65 signaling to TAM function and CRC growth under conditions of obesity by determining if the cAMP- PKA signaling axis, which functions downstream of GPR65, is activated in TAMs of obese mice and controls TNF-α production. We will also analyze GPR65 expression and the cytokine secretion capacity of macrophages from healthy blood donors and TAMs in CRC samples from non-obese and obese patients; 2) Identify the mechanism by which HFD promotes GPR65 signaling in CRC TAMs by testing the ability of HFD and oleic acid, a dietary triglyceride that is highly enriched in HFD tumors, to alter the oxidative potential, fatty acid oxidation capacity and acid production of human tumor cells via flow cytometry, CyTOF and Seahorse assays. We will also determine if a high-oleic-acid diet is sufficient to modify GPR65 expression and cytokine production by TAMs, and examine if tumor acidity is required for the blunted inflammatory response of TAMs; and verify the role of GPR65 in human macrophages and 3) Assess the effects of targeting GPR65 for tumor immunotherapy in obese and nonobese mice with CRC and other tumor types, namely hepatocellular carcinoma and melanoma, and assess the effects of checkpoint blocking antibodies on tumor growth and anti- tumor immunity in GPR65+ and GPR-/- mice bearing these tumors. The results of these studies are expected to not only reveal a critical mechanism responsible for accelerated tumor growth in the setting of obesity, but also identify a novel target for the treatment of these cancers.