ABSTRACT Although most melanoma deaths are caused by distant metastasis, the mechanisms that regulate metastasis are poorly understood. During metastasis, cancer cells encounter oxidative stress in the blood, and successful metastasis requires that cancer cells adapt to this hostile environment by undergoing metabolic changes. One of the ways in which oxidative stress can kill cells is through ferroptosis, marked by the accumulation of lipid reactive oxygen species. Recent work from Dr. Ubellacker’s studies discovered that melanoma cells in lymph are protected against ferroptosis. These studies revealed that lymph protects from ferroptosis is by having high levels of the monounsaturated fatty acid (MUFA) oleic acid, which shields cells from lipid oxidation by reducing the abundance of polyunsaturated fatty acids (PUFAs) that undergo lipid oxidation in membranes. These observations suggest that it may be possible to target lipid metabolism in cancer cells to prevent cancer spread from lymph nodes. However, we do not know to what extent the lymph node microenvironment protects melanoma cells from lipid oxidation. Nor do we know what lipidomic adaptations melanoma cells undergo in the lymph node microenvironment enable them to metastasize more efficiently. The objective in this project is to identify and exploit lipidomic vulnerabilities in melanoma cells in the lymphatic microenvironment to prevent early- stage melanoma metastasis. To accomplish this objective, this project will use a clinically relevant model of melanoma metastasis of patient-derived xenografts, as well as a murine melanoma model in immunocompetent mice and human clinical samples The central hypothesis is that melanoma cells in the lymph node microenvironment undergo specific lipid adaptations to withstand oxidative stress and that targeting these adaptations will prevent melanoma metastasis from lymph nodes. The central hypothesis will be tested in three specific aims: 1.) To investigate the extent to which melanoma cells depend on N-acylglycine synthesis for survival in the lymph nodes, and whether this synthesis pathway can be targeted to decrease melanoma cell survival; 2.) To assess the contribution of dietary and intracellular fatty acids to increasing lipid oxidation and increasing death of melanoma cell metastases in lymph nodes; 3.) To determine the extent to which ferroptosis resistance can be targeted to reduce survival of melanoma cells in lymph nodes. By identifying the influence of the lymph node microenvironment on lipidomic changes in melanoma cells, we expect to determine lipids in melanoma cells that serve as prognostic biomarkers and to identify feasible lipidomic targets and dietary interventions to prevent the spread of early-stage melanoma and improve disease outcome in patients.