How Circulating Melanoma Cells Usurp the Leukocyte Transmigration Mechanism for Successful Metastasis

大多数实体肿瘤的死亡都是由于转移--癌细胞从原发肿瘤中分离出来， 移动到身体的远处，并在那里建立新的生长。癌症患者减少了数百万 每天将肿瘤细胞(CTC)循环到他们的血液中。有很多关于恶性程度的信息 细胞从原发肿瘤中分离出来，通过结缔组织进入淋巴管和血管。 几乎所有的CTC都会在血管系统中死亡，或者被身体的炎症反应消除。然而，那些 设法从血液中渗出并进入组织的肿瘤细胞有可能生长为 转移的菌落将癌症扩散到远远超出原发灶的地方。不幸的是，几乎什么都没有 知道这一步。我们有证据表明很大一部分黑色素瘤细胞可以穿过血管 使用与白血球相同的分子机制。 在不增加血管通透性或诱导的情况下，白细胞在内皮细胞(EC)间迁移 组织损伤。它们通过与内皮细胞上的分子进行一系列分子相互作用来实现这一点。 它们从称为侧缘回收隔室(LBRC)的周边细胞器中招募膜。 来自LBRC的膜增加了连接的表面积，允许白细胞通过而不需要 损害内皮细胞；连接屏障仍然严密，基底膜没有外露。我们的 初步数据显示，很大一部分黑色素瘤细胞(1/3到1/2)可以跨内皮细胞迁移。 此外，我们可以使用一种新的细胞通透性抑制剂选择性地阻止它们在体外和体内的迁移。 LBRC运动的一部分。令人惊讶的是，使用该抑制剂治疗的小鼠体内形成的转移集落数量 比外渗被阻断的程度低得不成比例。这表明那些黑色素瘤 使用LBRC转生的细胞具有生存优势。我们假设阻止招募 黑色素瘤细胞的LBRC将是阻断成功转移的有效途径。我们将测试我们的 机械论研究中的一种假说，确定不同的驱动程序突变对能力的影响程度 来自患者的黑色素瘤细胞系和恶性黑色素瘤细胞招募LBRC进行转移 (目标I)。我们将使用我们的新型LBRC运动选择性抑制剂来阻断小鼠黑色素瘤的转移 同基因野生型小鼠以及NSG人患者来源的黑色素瘤异种移植瘤(PDX)的转移 小鼠(AIM II)。黑色素瘤不表达白细胞用来招募LBRC的分子；AIM III将 确定肿瘤细胞招募LBRC的机制。这将揭示治疗的靶点 选择性地阻断转移而不抑制免疫反应。初步数据显示，抑制剂 活性氧物种和内皮细胞EP4受体在体外阻止黑色素瘤的转移，提示 黑色素瘤分泌的ROS和PGE2招募了LBRC。我们将测试小鼠的转移瘤是否 在可诱导的内皮细胞特异性TRPM2或EP4基因敲除小鼠中，黑色素瘤和PDX减少。

Most deaths from solid tumors are due to metastases—cancer cells breaking off the primary tumor, traveling to distant parts of the body and establishing new growths there. Patients with cancer shed millions of circulating tumor cells (CTCs) into their bloodstream every day. There is a good deal known about how malignant cells break away from the primary tumor and move through connective tissue into lymphatics and blood vessels. Almost all CTCs die in the vasculature or are eliminated by the body’s inflammatory response. However, those tumor cells that manage to extravasate out of the bloodstream and into tissues have the potential to grow as metastatic colonies that spread the cancer far beyond the primary site. Unfortunately, there is almost nothing known about this step. We have evidence that a substantial fraction of melanoma cells can cross blood vessels using the same molecular machinery that white blood cells do. Leukocytes migrate across endothelial cells (EC) without increasing vascular permeability or inducing tissue damage. They do this through a series of molecular interactions with molecules on the endothelial cells that recruit membrane from a perijunctional organelle called the lateral border recycling compartment (LBRC). Membrane from the LBRC increases the surface area of the junction allowing leukocytes to pass across without harming the EC; the junction barriers remain tight and there is no exposure of the basement membrane. Our preliminary data show that a major fraction (1/3 to ½ ) of melanoma cells can migrate across endothelial cells. Moreover, we can selectively block their transmigration in vitro and in vivo using a novel cell-permeable inhibitor of LBRC movement. Strikingly, the number of metastatic colonies that develop in mice treated with the inhibitor is disproportionately lower than the degree to which extravasation is blocked. This suggests that those melanoma cells that transmigrate using the LBRC have a survival advantage. We hypothesize that blocking recruitment of the LBRC by melanoma cells would be an efficient way to block successful metastases. We will test our hypothesis in mechanistic studies that determine the extent to which different driver mutations affect the ability of melanoma cell lines and malignant melanoma cells derived from patients recruit the LBRC to transmigrate (Aim I). We will use our novel selective inhibitor of LBRC movement to block metastases of murine melanomas in syngeneic wild-type mice as well metastases from human patient-derived melanoma xenografts (PDX) in NSG mice (Aim II). Melanomas do not express the molecules used by leukocytes to recruit the LBRC; Aim III will determine the mechanisms used by tumor cells to recruit the LBRC. This will reveal therapeutic targets for selectively blocking metastasis without inhibiting the immune response. Preliminary data show that inhibitors of reactive oxygen species as well as endothelial EP4 receptors block melanoma transmigration in vitro, suggesting that ROS and PGE2 secreted by melanomas recruits the LBRC. We will test whether metastases from murine melanoma and PDX are reduced in inducible endothelial cell-specific TRPM2 or EP4 knockout mice.