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Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.

转移主要依赖于肿瘤细胞通过血管系统的血源性扩散，这极大地限制了实体瘤患者的预后。为了在远处定植，循环肿瘤细胞必须破坏内皮屏障并穿过血管壁迁移。在这里，我们进行了高通量筛选，以鉴定通过在跨内皮侵袭共培养测定中测试 3520 种化合物来阻止肿瘤细胞外渗的药物。通过一系列测定、允许三维（3D）可视化肿瘤细胞外渗的斑马鱼模型以及肺转移的小鼠模型，对命中化合物进行了进一步的表征和验证。初始筛选推进了 38 种化合物作为潜在的命中化合物，其中 4 种化合物增强了内皮屏障稳定性，同时抑制了肿瘤细胞的迁移能力。两种化合物尼克罗酰胺和福司可林显著减少了斑马鱼中的肿瘤细胞外渗，尼克罗酰胺大大降低了小鼠中的转移。由于尼克罗酰胺以前与对屏障功能的影响无关，单细胞 RNA 测序揭示了药物对肿瘤细胞和内皮细胞的机制作用。重要的是，尼克罗酰胺影响了同质和异质信号，这些信号对细胞间连接、细胞-基质相互作用和细胞骨架调节至关重要。蛋白质组学分析表明，尼克罗酰胺处理的小鼠也显示出激肽原水平降低，激肽原是通透性调节剂缓激肽的前体。我们的研究结果将尼克罗酰胺指定为一种有效的药物，它通过调节信号通路、趋化因子和肿瘤-内皮细胞相互作用来限制肿瘤细胞的外渗。