Abstract Pancreatic ductal adenocarcinoma (PDAC) is non-resectable in 85% of cases and highly resistant to chemotherapy, resulting in a poor 5-year survival (5-7%). Understanding the metabolic vulnerabilities of PDAC in the harsh tumor microenvironment (TME) may lead to novel therapeutic approaches with improved clinical efficacy. The pancreatic TME is characterized by widespread desmoplasia represented by alpha-smooth muscle actin-positive fibroblasts, pancreatic stellate cells and extracellular matrix components, among others. Up to 90% of the pancreatic tumor mass consists of non-neoplastic cells, and high interstitial pressures and poor perfusion both result in severe hypoxia, leading to a more malignant PDAC phenotype. We hypothesized that these conditions lead to specific metabolic constrains in oncogene-driven, rapidly proliferating PDAC cells that experience high levels of stress, in contrast to the surrounding quiescent stromal cells. We used co-culturing of PDAC (MIAPaCa2) and stromal (NIH/3T3) cells in transwell systems as a robust and reproducible model of cell contact-independent interactions in the pancreatic TME. A commercial metabolic profiling platform (Metabolon) and 13C-based flux assays were used to study changes in metabolite levels in both cell types in normoxia or hypoxia (1% O2). We found that hypoxia induced similar metabolic changes in the PDAC and stromal cells, suggesting that hypoxia-regulated metabolic rewiring is independent of cell-type. Interestingly, the metabolic effects of co-culturing were predominantly observed in the stromal compartment, e.g. enhanced glycogenolysis, metabolite changes indicative of gluconeogenesis, and increased dipeptide levels, all reminiscent of a ‘starvation’ phenotype. In contrast, the tumor cells maintained a mixed anabolic and catabolic phenotype, as shown by elevated intracellular levels of essential amino acids and ribonucleotide triphosphates, representative of a ‘feeding’ phenotype. Importantly, a unique dependence on complex lipid species was observed in cancer cells with reciprocal changes in stromal cells. These data suggest that pancreatic cancer cells reprogram stromal cells to ‘feed off’ the metabolic capacity of non-neoplastic cells in the tumor microenvironment, thereby inducing the release of diffusable metabolites to satisfy their specific catabolic needs. Citation Format: Petrus R. de Jong, Alejandro D. Campos, Sean-Luc Shanahan, Adam Richardson, Garth Powis.{Authors}. Pancreatic cancer cells scavenge complex lipids from stroma in the hypoxic tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A25.