Project Summary Pancreatic cancer, most commonly diagnosed as pancreatic ductal adenocarcinoma (PDAC), is amongst the top three leading causes of cancer death in the United States. These tumors are notoriously resistant to apoptosis and richly populated by stromal and immune cells that create a therapy-resistant microenvironment. Immunotherapy shows no benefit to patient survival, and despite advances in combinational chemotherapy the 5-year survival rate sits around 11%. Therefore, investigating alternative forms of cell death represents a unique strategy for targeting PDAC. Ferroptosis is a recently described form of cell death distinct from apoptosis. It is dependent on an accumulation of lipid peroxides, generated by free labile iron, resulting in osmotic cell lysis. Targeting ferroptosis can potently induce cell death in cultured PDAC cells. Yet, in PDAC tumors in mice, triggering ferroptosis has a modest response with distinct cases of intrinsic and acquired resistance currently limiting its therapeutic utility. It is therefore urgent that we address the gap between the in vitro and in vivo responses for future clinical studies. In pursuit of this, I am taking a reductionist approach to distill the mechanisms that regulate ferroptosis by osmotic and reductive-oxidative (redox) forces. Mechanistically, it is known that ferroptosis requires osmotic pressure to induce cell lysis, but this fact is not well studied within the current growing literature on ferroptosis in cancer. We hypothesize that cell death via ferroptosis requires an imbalance of redox metabolism and sufficient osmotic pressure, to which PDAC cells are particularly vulnerable. AIM I studies the biochemical dependencies between osmolarity and redox metabolism. AIM II distinguishes changes in the external osmotic pressure from the cellular biochemical adaptations, scaling up from monolayer to 3D culture techniques. Together, these aims investigate whether ferroptotic cell death can be simplified to a balance of redox and osmotic pressure. The overall goals of this study are to outline the boundary of these parameters and explore their application for targeting ferroptosis in PDAC.