Project Summary Metastasis is a highly inefficient process in which few disseminating cancer cells survive. We discovered that melanoma metastasis is limited by oxidative stress. Reactive Oxygen Species (ROS) increase dramatically in melanoma cells as they metastasize through the blood. The rare cells that survive undergo reversible metabolic changes that confer oxidative stress resistance. Consistent with this, large clinical trials found that patients administered anti-oxidants were more likely to die of cancer than control patients. Cancer cells, including melanoma, often metastasize regionally through lymphatic vessels before metastasizing systemically through the blood. We recently discovered that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood. This was true of patient-derived melanomas growing in immunocompromised mice as well as mouse melanomas growing in immunocompetent mice. The oxidative stress kills melanoma cells in the blood by inducing ferroptosis, a form of cell death marked by the accumulation of lipid peroxides. One of the ways in which lymph protects from ferroptosis is by having high levels of the monounsaturated fatty acid (MUFA), oleic acid, which protects cells from lipid oxidation by reducing the abundance of polyunsaturated fatty acids (PUFAs) in membrane phospholipids. The more abundant PUFAs are in membrane phospholipids, the more sensitive cells are to ferroptosis. Melanoma cells are thus exposed to different lipid environments in different locations as they metastasize and these cell- extrinsic changes influence their survival. There are also cell-intrinsic differences among melanomas that influence their response to these environments: melanomas from different patients differ in the sites to which they metastasize upon xenografting. We hypothesize that these differences in metastasis patterns result from cell-intrinsic differences in lipid metabolism that influence their sensitivity to ferroptosis in response to the lipid environments they encounter as they metastasize. Nonetheless, we have never been able to image the fates of metastasizing melanoma cells in vivo, limiting our understanding of how oxidative stress affects these cells. A critical barrier is the efficient imaging of entire organs to identify rare melanoma cells at the earliest stages of metastasis. The lack of subcellular resolution in whole organs also impairs our ability to explore the ways in which oxidative stress influences cell survival and proliferation. Both of these limitations will be addressed by the Technology Development Unit (TDU) of this consortium, which is developing the ability to perform high throughput imaging of whole, cleared organs to assess the survival, proliferation, and localization of rare melanoma cells after metastasis. We will validate these imaging data by flow cytometry. By combining single cell imaging (Aim 1) with metabolic assays, we will characterize cell-extrinsic (Aim 2) and cell-intrinsic (Aims 1, 3) mechanisms that regulate the earliest stages of metastasis.