The focus of this multi-PI R01 application is to characterize and target a new metabolic vulnerability of ovarian cancer stem cells (CSCs) discovered by our collaborative team. By using hyperspectral-stimulated Raman scattering (SRS) imaging of single living cells and mass spectrometry analysis of extracted lipids we identified increased levels of unsaturated fatty acids (UFAs) in ovarian CSCs compared to non-CSCs. We demonstrated that UFAs are critical to the survival, proliferation, and tumorigenicity of ovarian CSCs. Here we propose to analyze the mechanisms by which increased lipid unsaturation mediated by Δ9 desaturase (stearoyl-coA desaturase, SCD1) regulates retinoic acid signaling in ovarian CSCs to determine cellular fate and promote tumorigenicity. We will analyze whether the balance between saturated and unsaturated lipids enhance the survival of drug-tolerant cells after chemotherapy. We will use SCD1 knock down and chemical inhibitors to eradicate drug-tolerant cells persisting after treatment with platinum in ovarian xenografts and patient derived xenografts (PDX). Lipid unsaturation will be visualized in CSCs in situ by using a multimodal high-speed SRS microscope. Label-free molecular imaging will quantify CSCs and unsaturated lipids in human tumors and xenografts before and after treatment with platinum or desaturase inhibitors. Ultimately, in depth characterization of fatty acid metabolism in CSCs will reveal key pathways linked to stemness and persistence of chemotherapy-tolerant cells. In the long run, our studies will develop new strategies to attack deadly ovarian cancer.