The Metabolic Regulation of Melanoma Metastasis

摘要 大多数癌症死亡是由远处转移引起的。然而，调节远处转移的机制是 不太了解。转移是一个非常低效的过程，其中很少有播散的癌细胞存活， 甚至更少的扩散，但不知道为什么。我们开发了一种患者来源的异种移植物（PDX）测定法， 黑色素瘤非常有效地移植并自发转移。通过这种分析，我们发现 不同患者黑色素瘤转移潜能的内在差异。一些第三阶段 黑色素瘤是“有效的转移因子”，在患者和NSG中自发形成远处转移 而其他小鼠是“无效的转移者”，其不会在患者或患者中形成远处大转移。 NSG小鼠在相同的实验条件下。通过这种检测，我们发现黑色素瘤细胞 在转移期间经历活性氧（ROS）的峰值，并且远端转移是有限的 氧化应激。成功转移的细胞在转移过程中经历可逆的代谢变化 这增加了他们抵抗氧化应激的能力，包括增加叶酸途径依赖性。 然而，不同来源的黑色素瘤转移潜能差异的机制不同， 患者尚未确定。我们假设有效和无效转移的黑色素瘤 具有内在的代谢差异，可降低有效转移者的氧化应激。一个障碍， 对这一假设的检验是来自患者的黑素瘤在已知培养物中以克隆密度生长不良 条件，阻止某些研究癌症代谢的方法和使用CRISPR基因编辑 （因为不能筛选或扩增单细胞衍生的克隆）。我们花了几年时间培养一种文化 培养基，其中来自患者的单个黑素瘤细胞形成肿瘤类器官（PDO）。这种能力提高了 关于代谢和氧化应激抗性在PDX和PDX中是否受到类似调节的一般性问题， PDO。为了解决这个问题，我们将比较，并排，生物学特性和代谢 PDX和PDO中有效和无效转移剂的调节。我们将测试是否有效转移 黑色素瘤具有较低的ROS水平或氧化应激标志物，或叶酸或戊糖的使用增加 磷酸途径，与低效转移剂相比，以及这是否促进PDX的转移 或迁移/入侵的问题。我们还将测试MCT 1，一种乳酸转运蛋白，是否促进转移， 有效的转移剂是否部分地通过乳酸盐交换减少氧化应激。最后，我们将测试 在有效和无效的转移剂之间存在线粒体功能的内在差异， 减少ROS的产生。通过比较黑色素瘤的生物学特性和代谢调节， PDX和PDO检测试剂盒，我们将评估PDX和PDO检测试剂盒的优势和劣势， 癌症代谢和患者之间的生物学差异。这些结果有可能发现新的 调节转移的机制，癌症代谢的新方面，以及阻断进展的策略。

ABSTRACT Most cancer deaths are caused by distant metastasis. Yet the mechanisms that regulate distant metastasis are poorly understood. Metastasis is a very inefficient process in which few disseminated cancer cells survive, and even fewer proliferate, but it is not known why. We developed a patient-derived xenograft (PDX) assay in which melanomas engraft very efficiently and spontaneously metastasize. Using this assay, we discovered intrinsic differences in metastatic potential among melanomas from different patients. Some stage III melanomas are “efficient metastasizers” that spontaneously form distant metastases in patients and in NSG mice while others are “inefficient metastasizers” that do not form distant macrometastases in patients or in NSG mice under the same experimental conditions. Using this assay, we discovered that melanoma cells experience a spike in reactive oxygen species (ROS) during metastasis and that distant metastasis is limited by oxidative stress. Successfully metastasizing cells undergo reversible metabolic changes during metastasis that increase their capacity to withstand oxidative stress, including increased folate pathway dependence. However, the mechanisms that confer differences in metastatic potential upon melanomas from different patients have not yet been identified. We hypothesize that efficiently and inefficiently metastasizing melanomas have intrinsic metabolic differences that reduce oxidative stress in efficient metastasizers. One impediment to testing this hypothesis is that melanomas from patients grow poorly at clonal density in known culture conditions, preventing certain approaches for studying cancer metabolism and the use of CRISPR gene editing (because single cell-derived clones could not be screened or expanded). We spent years developing a culture medium in which single melanoma cells from patients form tumor organoids (PDOs). This capability raises the general question of whether metabolism and oxidative stress resistance are regulated similarly in PDXs and in PDOs. To address this question, we will compare, side-by-side, the biological properties and metabolic regulation of efficient and inefficient metastasizers in PDXs and PDOs. We will test if efficiently metastasizing melanomas have lower ROS levels or markers of oxidative stress, or increased use of the folate or pentose phosphate pathways, as compared to inefficient metastasizers, and whether this promotes metastasis in PDXs or migration/invasion in PDOs. We will also test if MCT1, a lactate transporter, promotes metastasis and whether efficient metastasizers reduce oxidative stress partly through lactate exchange. Finally, we will test if there are intrinsic differences in mitochondrial function between efficient and inefficient metastasizers that reduce ROS generation. By comparing the biological properties and metabolic regulation of each melanoma in PDX and PDO assays, we will assess the strengths and weaknesses of PDX and PDO assays for studying cancer metabolism and biological differences among patients. These results have the potential to identify new mechanisms that regulate metastasis, new aspects of cancer metabolism, and strategies to block progression.