PROJECT SUMMARY The treatment of pancreatic ductal adenocarcinoma (PDAC) remains a major hurdle with 5-year survival of 12%. PDAC resistance to chemotherapy and immunotherapy is thought to arise from the immunosuppressive tumor microenvironment (TME), characterized by a fibrotic stroma and high infiltrates of immunosuppressive cells, including tumor associated macrophages (TAMs). TAMs block anti-tumor effector T cell function and trigger exhaustion. It has been postulated that reprogramming TAMs to an immunostimulatory phenotype could improve therapy response. Recent studies suggest that metabolites originating from the gut microbiome influence phenotype of immune cells, including TAMs. However, little is understood about such metabolites, including their identity, the signaling pathways by which they alter TAM phenotype, and whether these metabolites influence cancer development. We are identifying microbial metabolites that modulate TAMs in the PDAC TME. Using unbiased, global, LC-MS/MS metabolomic screens, we found a gut microbe-derived metabolite, trimethylamine N-oxide (TMAO) that induced significant anti-tumor effects in the PDAC TME. Specifically, delivery of TMAO intraperitoneally, or by supplementing diet with the TMAO precursor choline, to PDAC-bearing mice reduced tumor growth and was associated with an immunostimulatory TAM phenotype and activated effector T cell response in the TME. The immunostimulatory macrophage phenotype was due to a direct effect of TMAO and the TMAO-conditioned macrophages could activate CD8+ T cells. Combining TMAO with immune checkpoint blockade reduced tumor burden and improved anti-tumor immune responses. These data support our hypothesis that the diet and microbiome-derived metabolites shape anti-tumor immunity and treatment response in PDAC. Aim 1 will characterize dietary and microbial sources of TMAO for anti-tumor responses in PDAC. We will characterize the (i) dietary (l-carnitine or betaine), and (ii) the microbiome (e.g., Enterococcus asini, engineered E. coliCutC/D) sources of TMAO for their anti-tumor effects. Aim 2 will test the hypothesis that TMAO induces its anti-tumor effects by potentiating the type I IFN and/or STING signaling in TAMs. We will determine (i) the requirement of type-I IFN and/or STING specifically in macrophages for the anti-tumor effects, and (ii) the impact of TMAO on the transcriptional activity of type-I IFN responsive STAT1 and/or STAT3. Aim 3 will test the efficacy of TMAO to improve treatment response in pre-clinical models of PDAC. We will evaluate the translational relevance of TMAO using (i) a genetically engineered mouse model of PDAC and patient derived PDAC organoid cultures, and (ii) a treatment strategy combining TMAO with STING agonists. Our studies will characterize the sources, mechanism of action, and translational relevance of a novel, minimally understood, high impact microbial metabolite, TMAO, in boosting anti-tumor immune responses in the PDAC TME and rendering PDAC responsive to chemo-immunotherapy. In the longer term, this work may lay the groundwork for new diet/microbiome-based treatments for PDAC.

PROJECT NARRATIVE Microbial metabolites have suggested to influence anti-tumor immunity and cancer treatment. This proposal will characterize the sources, mechanisms, and translational relevance of a minimally understood, high impact microbial metabolite, TMAO (trimethylamine N-oxide) in a lethal pancreatic ductal adenocarcinoma (PDAC).