PROJECT SUMMARY/ABSTRACT A major hurdle in the clinical management of pancreatic adenocarcinomas (PDAC) is the profound resistance to chemotherapeutics exhibited by these tumors. PDAC chemoresistance is mediated by the tumor microenvironment (TME) as isolated PDAC cells cultured ex vivo readily respond to therapies while PDAC cells in tumors do not. Thus, identifying the TME factors and mechanisms by which the TME regulates drug response in PDAC is critical to being able to effectively treat this disease. Towards the goal of identifying TME factors regulating PDAC biology, we have taken the strategy of measuring physiological parameters in the PDAC TME and recreating these conditions ex vivo to study in mechanistic detail how PDAC cells are impacted by TME physiology. As part of this approach, we recently developed techniques to isolate interstitial fluid (the local perfusate) from PDAC tumors and quantitative metabolite profiling techniques to measure availability of ~150 major nutrients in the PDAC TME. This provided us with the first quantitative atlas of nutrient availability in PDAC and we found that local nutrient abundance in these tumors was strikingly different than in the bulk circulation or healthy tissues. To study how abnormal access to nutrients in the TME could impact PDAC cells, we developed a novel cell culture model in which PDAC cells are cultured with the precise levels of ~120 major vitamins and nutrients they encounter in the native TME. Using this model, we found that PDAC cells exposed to TME nutrition exhibit resistance to a wide array of clinically used chemotherapies, identifying abnormal tumor nutrient availability as a critical TME factor mediating chemoresistance in this disease. Further analysis of TME nutrient- induced chemoresistant phenotype indicated that: (1) increased availability of the amino acid glycine in the TME causes PDAC cells to become highly chemoresistance and (2) TME glycine does not endow PDAC cells with the ability to evade action of chemotherapies, but rather the ability to tolerate damage induced by therapeutic challenges. Based on these preliminary studies, we developed the hypothesis that TME glycine impairs the ability of PDAC cells to undergo cell death in response to chemotherapeutic insult, thus enabling PDAC cells to tolerate chemotherapeutic treatment. In this proposal, we will determine: (1) the metabolic basis for how glycine accumulates in the PDAC TME and if targeting TME glycine availability can sensitize PDAC tumors to chemotherapy and (2) the mechanism by which PDAC cells gain tolerance to chemotherapeutic stress and if targeting these tolerance mechanisms can synergize with chemotherapy. Therapy resistance is a major contributor to the poor prognosis of PDAC patients. Our work addresses this key therapeutic challenge in a disease with much unmet clinical need and could identify novel therapeutic options for PDAC patients.