The focus of this R01 application is to characterize and target a new metabolic vulnerability of ovarian cancer stem cells (CSCs) described by our collaborative team. By using hyperspectral stimulated Raman scattering imaging of single living cells and mass spectrometry analysis of extracted lipids we identified increased levels of unsaturated lipids in ovarian CSCs compared to non-CSCs. Based on these results, we hypothesized that altered lipid metabolism is critical to the survival and the unique functions of ovarian CSCs and can be targeted to eradicate recalcitrant cells responsible for tumor recurrence. In this project we propose to analyze the mechanism by which increased lipid unsaturation mediated by Δ9 and Δ6 fatty acid desaturases regulates NF-Kand retinoic acid signaling to promote ovarian CSC survival. Effects of Δ9 and Δ6 fatty acid desaturases inhibitors will be tested in ovarian xenografts and in patient derived xenografts (PDX) enriched in CSCs by treatment with platinum. Label free chemical imaging will use a multimodal high-speed hyperspectral stimulated Raman spectroscopy (SRS) microscope to visualize lipid metabolism in CSCs in situ. SRS microscopy will study for the first time changes induced by platinum treatment in human tumors or by treatment with desaturase inhibitors in ovarian xenografts or PDXs. We envision that the in depth characterization of the fatty acid metabolism in CSCs will reveal key survival mechanisms linked to stemness and persistence of resistant cells after chemotherapy. In the long run, our studies will be instrumental in developing new strategies to attack deadly ovarian cancer.