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

大多数死于实体瘤的人都是因为癌细胞从原发肿瘤上脱落， 移动到身体的远处并在那里建立新的生长。癌症患者会流出数百万 循环肿瘤细胞（CTC）进入他们的血液每天。有很多人知道 细胞从原发性肿瘤脱离并穿过结缔组织进入血管和血管。 几乎所有的CTC都在脉管系统中死亡或被身体的炎症反应消除。但这些 设法从血流中渗出并进入组织的肿瘤细胞有可能生长， 转移性集落将癌症扩散到原发部位以外。不幸的是，几乎没有什么 了解这一步。我们有证据表明相当一部分黑色素瘤细胞可以穿过血管 使用与白色血细胞相同的分子机制。 白细胞迁移穿过内皮细胞（EC），而不增加血管通透性或诱导 组织损伤它们通过与内皮细胞上的分子发生一系列分子相互作用来实现这一点 这些细胞从一个被称为侧缘回收室（LBRC）的连接周细胞器中募集膜。 来自LBRC的膜增加了交界处的表面积，允许白细胞穿过而不 损害EC;结屏障保持紧密并且没有基底膜暴露。我们 初步数据显示大部分（1/3至1/2）的黑素瘤细胞可以迁移穿过内皮细胞。 此外，我们可以选择性地阻止他们的迁移在体外和体内使用一种新的细胞渗透性抑制剂 LBRC运动引人注目的是，在用抑制剂治疗的小鼠中形成的转移性集落的数量 不成比例地低于外渗被阻断的程度。这表明那些黑色素瘤 使用LBRC迁移的细胞具有存活优势。我们假设，阻止 通过黑素瘤细胞的LBRC将是阻断成功转移的有效方式。我们将测试我们的 机制研究中的假设，确定不同驱动突变影响能力的程度 的黑色素瘤细胞系和来自患者的恶性黑色素瘤细胞募集LBRC以进行易位 (Aim（一）。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 在同系野生型小鼠中以及在NSG中来自人患者来源的黑素瘤异种移植物（PDX）的转移中 小鼠（Aim II）。黑色素瘤不表达白细胞用来募集LBRC的分子; Aim III将 确定肿瘤细胞招募LBRC的机制。这将揭示治疗靶点， 选择性阻断转移而不抑制免疫应答。初步数据显示， 活性氧自由基和内皮EP 4受体在体外阻断黑色素瘤的迁移，这表明 黑色素瘤分泌的ROS和PGE 2募集LBRC。我们将测试是否从小鼠转移 黑色素瘤和PDX在可诱导内皮细胞特异性TRPM 2或EP 4敲除小鼠中减少。

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.