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，其中很少有扩散的癌细胞存活。我们发现 黑色素瘤转移受氧化应激的限制。活性氧（ROS）在体内急剧增加， 黑色素瘤细胞通过血液转移。存活下来的稀有细胞经历了可逆的 代谢变化，赋予氧化应激抗性。与此相一致的是，大型临床试验发现， 服用抗氧化剂的患者比对照组患者更可能死于癌症。癌细胞， 包括黑色素瘤，在全身转移之前， 通过血液。我们最近发现淋巴中的黑色素瘤细胞经历较少的氧化应激， 比血液中的黑色素瘤细胞形成更多的转移。这是真实的病人来源的黑色素瘤生长在 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素瘤。氧化 压力通过诱导铁凋亡来杀死血液中的黑色素瘤细胞，铁凋亡是一种细胞死亡形式， 脂质过氧化物的积累。其中一种方法，淋巴保护从ferroptosis是通过具有高 单不饱和脂肪酸（MUFA），油酸，保护细胞免受脂质氧化， 减少膜磷脂中多不饱和脂肪酸（PUFA）的丰度。越 膜磷脂中多不饱和脂肪酸含量越高，细胞对铁凋亡越敏感。黑素瘤细胞 因此，当它们转移时，在不同的位置暴露于不同的脂质环境，这些细胞- 外在变化影响其生存。黑色素瘤之间也存在细胞内在差异， 影响他们对这些环境的反应：来自不同患者的黑色素瘤的部位不同， 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 脂质代谢的细胞内在差异，影响其对脂质引起的铁凋亡的敏感性 他们在转移时遇到的环境。尽管如此，我们还是无法想象 这限制了我们对氧化应激如何影响这些细胞的理解。 一个关键的障碍是整个器官的有效成像，以确定罕见的黑色素瘤细胞在早期阶段， 转移在整个器官中缺乏亚细胞分辨率也削弱了我们探索细胞内的途径的能力。 该氧化应激影响细胞存活和增殖。这两个限制将通过以下方式解决： 该联盟的技术开发单位（TDU）正在开发高性能的能力， 对整个透明器官进行通量成像，以评估罕见肿瘤的存活、增殖和定位。 转移后的黑色素瘤细胞。我们将通过流式细胞术验证这些成像数据。通过组合单个 细胞成像（Aim 1）与代谢测定，我们将表征细胞-外在（Aim 2）和细胞-内在（Aim 1， 3)调节转移的最早阶段的机制。

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.