The treatment of pancreatic ductal adenocarcinoma (PDAC) remains a major hurdle, with a 5-year survival rate of only 9%. Resistance of PDAC to chemotherapy and immunotherapy is thought to arise primarily due the immunosuppressive tumor microenvironment (TME), characterized by a dense fibrotic stroma and high infiltrates of immunosuppressive cells, including tumor associated macrophages (TAM). TAMs block effector T cell function and trigger exhaustion. Immune responses in the TME are likely controlled by multiple mechanisms, including host intrinsic factors and, more recently appreciated, changes in the gut microbiome. However, the molecular mechanisms and pathways by which the gut microbiome impact the immune responses in the PDAC TME remain poorly understood. Our new preliminary work indicates that the gut microbiota can influence the immune response through microbial metabolites that enter the blood stream to modulate TAM activity. In an untargeted metabolomic screen on circulating metabolites, we discovered the most significant change was a dramatic reduction (>80-fold) in trimethylamine N-oxide (TMAO) levels in PDAC-bearing mice treated with antibiotic metronidazole. TMAO is formed in two steps: the gut microbial TMA lyase degrades choline to trimethylamine (TMA), which then undergoes oxidation in the liver to form TMAO. Intraperitoneal administration of physiologically relevant amounts of TMAO to PDAC-bearing mice significantly suppressed tumor growth. This was accompanied by a significant decrease in tumor-infiltrating, immunosuppressive myeloid cells, an increase in a pro-inflammatory TAM phenotype, and a striking increase in infiltrates of activated CD8+ T cells in the PDAC TME. These and other data support our hypothesis that dietary choline and the gut microbial TMA lyase are key factors contributing to circulating TMAO levels and the pro-inflammatory TAM phenotype. We propose that supplementing TMAO or enacting strategies that increase it will improve response to immunotherapy in PDAC. Aim 1 tests the hypothesis that dietary choline and the gut microbial TMA pathway contribute to the acquisition of a pro-inflammatory TAM phenotype and induction of anti-tumor immune response. We will employ supplementation of choline diet, depletion of TMAproducing bacteria, and inhibition of TMA lyase to evaluate the mechanistic links between dietary precursors of TMAO, the gut microbial TMA pathway, and immune responses in the PDAC TME. Aim 2 tests the hypothesis that increasing TMAO sensitizes PDAC tumors to immunotherapy. We will evaluate direct administration of TMAO or enrichment of TMA-producing gut bacteria such as Clostridium sporogenes in combination with immune checkpoint blockade. This aim will also evaluate the clinical significance of the TMAO biology by determining the association between plasma TMAO levels and clinical outcome of survival and disease progression in PDAC. In the longer term, this work may form the basis for developing new, microbiome-based therapies for the aggressive and hard to treat PDAC.