Abstract Prostate cancer (PCa) is the most common cancer amongst males and becomes incurable once it advances to the secondary site. The most common site of metastasis from PCa is the adipocyte-rich bone marrow. Adipocytes are metabolically active cells capable of secreting a variety of adipokines and free fatty acids, which can be utilized by tumor cells to enhance proliferation and cause resistance to chemotherapies. Our lab has shown that adipocytes promote PCa progression and therapy evasion through modulation of tumor metabolism and activation of pro-survival signaling; however, the molecular mechanisms behind tumor-promoting effects of fat cells are not understood. It is well known that high levels of iron within the tumor microenvironment help the growth of PCa cells. However, since iron overload can cause an increase in reactive oxygen species (ROS), which are harmful to cell structure, tumor cells have developed defense mechanisms to protect them from this oxidative damage. Intriguingly, our data show that the expression of ferritin, an iron storing protein, is reduced in PC3 and ARCaP(M) cells in co-culture with adipocytes. Since low ferritin levels have been associated with induction of ferroptosis, an iron and oxidative stress-regulated cell death process, we examined protein levels of ferroptosis markers GPX4 and NCOA4 in PCa cells. Surprisingly, our data show that interaction with adipocytes increases GPX4 and reduces NCOA4 expression in PCa cells suggesting an adipocyte-mediated defense mechanism against ferroptosis. The aim of the present study was to establish that adipocytes promote a defense mechanism against ferroptosis in PCa cells by the upregulation of GPX4 activity via the mTOR pathway. GPX4 is known to be an essential antioxidant enzyme that utilizes glutathione (GSH) to reduce lipid-peroxidation-induced ROS. We demonstrate that PCa cells exposed to adipocytes have reduced lipid-peroxidation induced ROS, which can be reversed with ferroptosis inducers. We also show that exposure of PC3 and ARCaP(M) cells to adipocytes leads to depletion of GSH, which may be indicative of higher GPX4 activity. It has been suggested that an increase in GPX4 can result from an interplay between ferroptosis and mTOR pathways, specifically mTORC1, which may mediate cysteine-induced GPX4 protein synthesis. Surprisingly, treatment of PCa cells co-cultured with adipocytes with mTORC1 inhibitor Everolimus (EVO) increases the protein and gene levels of GPX4. This coincides with reduced mRNA and protein expression of SLC7A11, transporter involved in the cysteine/glutamate antiporter system, which typically fuels GPX4 activity by bringing in the GSH precursor cysteine. Understanding the mechanisms of fat cell contribution to dysregulation of the mTOR pathway and escape from ferroptosis may have therapeutic implications for metastatic PCa. Citation Format: Alexis Wilson, Shane Mecca, Mackenzie Herroon, Laimar Garmo, Izabela Podgorski. Defense mechanism against oxidative damage in metastatic prostate cancer: Adipocyte-mediated modulation of mTOR and ferroptosis pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6319.