Cancer Biology Research Test-Bed Unit 2: Effects of cell-intrinsic and cell-extrinsic variations in lipid metabolism on metastasis patterns

项目摘要 转移是一个非常低效的过程，在这个过程中几乎没有扩散的癌细胞存活下来。我们发现 黑色素瘤的转移受到氧化应激的限制。活性氧物种(ROS)在 黑色素瘤细胞通过血液转移。存活下来的稀有细胞经历可逆的 赋予氧化应激抵抗能力的代谢变化。与此一致的是，大型临床试验发现 服用抗氧化剂的患者比对照组患者更有可能死于癌症。癌细胞， 包括黑色素瘤，通常在全身转移之前通过淋巴管转移。 通过鲜血。我们最近发现，淋巴中的黑色素瘤细胞经历的氧化应激较少， 在血液中形成比黑色素瘤细胞更多的转移。患者来源的黑色素瘤在 免疫功能低下的小鼠以及在免疫功能正常的小鼠中生长的小鼠黑色素瘤。氧化的 应激通过诱导铁下垂杀死血液中的黑色素瘤细胞，铁下垂是一种细胞死亡形式，其特征是 过氧化脂质的积聚。淋巴预防铁性下垂的方法之一是 单不饱和脂肪酸(MUFA)油酸的水平，它通过以下方式保护细胞免受脂质氧化 减少膜磷脂中多不饱和脂肪酸(PUFAs)的丰度。越多 膜磷脂中含有丰富的多不饱和脂肪酸，细胞对铁性下垂越敏感。黑色素瘤细胞 因此，当它们转移时，暴露在不同位置的不同脂质环境中，这些细胞- 外在的变化会影响它们的生存。黑色素瘤之间也有细胞固有的差异， 影响他们对这些环境的反应：不同患者的黑色素瘤发生在不同的部位 它们在异种移植时转移。我们假设这些转移模式的差异是由于 脂类代谢中的细胞固有差异影响其对脂类反应中铁下垂的敏感性 它们在转移过程中遇到的环境。尽管如此，我们始终无法想象命运 对体内转移的黑色素瘤细胞的研究，限制了我们对氧化应激如何影响这些细胞的理解。 一个关键的障碍是对整个器官的有效成像，以在早期阶段识别罕见的黑色素瘤细胞 转移。整个器官亚细胞分辨率的缺乏也削弱了我们探索 氧化应激会影响细胞的存活和增殖。这两个限制都将通过 该联盟的技术开发单位(TDU)正在开发执行高性能 通过完整、透明的器官成像来评估罕见肿瘤的存活、增殖和定位 黑色素瘤细胞转移后。我们将通过流式细胞仪对这些成像数据进行验证。通过将单个 细胞成像(目标1)通过代谢分析，我们将表征细胞外源性(目标2)和细胞内在(目标1， 3)调控肿瘤转移早期阶段的机制。

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.