The tumor microenvironment (TME) is a key determinant of metastatic efficiency. We performed a quantitative high-throughput screen (qHTS) of diverse medicinal chemistry tractable scaffolds (44,420 compounds) and pharmacologically active small molecules (386 compounds) using a layered organotypic, robust assay representing the ovarian cancer metastatic TME. This 3D model contains primary human mesothelial cells, fibroblasts, and extracellular matrix, to which fluorescently labeled ovarian cancer cells are added. Initially, 100 compounds inhibiting ovarian cancer adhesion/invasion to the 3D model in a dose-dependent manner were identified. Of those, eight compounds were confirmed active in five high-grade serous ovarian cancer cell lines and were further validated in secondary in vitro and in vivo biological assays. Two tyrosine kinase inhibitors, PP-121 and milciclib, and a previously unreported compound, NCGC00117362, were selected because they had potency at 1 μmol/L in vitro. Specifically, NCGC00117362 and PP-121 inhibited ovarian cancer adhesion, invasion, and proliferation, whereas milciclib inhibited ovarian cancer invasion and proliferation. Using in situ kinase profiling and immunoblotting, we found that milciclib targeted Cdk2 and Cdk6, and PP-121 targeted mTOR. In vivo, all three compounds prevented ovarian cancer adhesion/invasion and metastasis, prolonged survival, and reduced omental tumor growth in an intervention study. To evaluate the clinical potential of NCGC00117362, structure–activity relationship studies were performed. Four close analogues of NCGC00117362 efficiently inhibited cancer aggressiveness in vitro and metastasis in vivo. Collectively, these data show that a complex 3D culture of the TME is effective in qHTS. The three compounds identified have promise as therapeutics for prevention and treatment of ovarian cancer metastasis.

中文翻译：

---

使用器官模型进行的定量高通量筛选鉴定了抑制卵巢癌转移的化合物。

肿瘤微环境 (TME) 是转移效率的关键决定因素。我们使用代表卵巢癌转移性 TME 的分层器官型、稳健分析对多种药物化学易处理支架（44,420 种化合物）和药理活性小分子（386 种化合物）进行了定量高通量筛选 (qHTS)。该 3D 模型包含原代人类间皮细胞、成纤维细胞和细胞外基质，其中添加了荧光标记的卵巢癌细胞。最初，确定了 100 种以剂量依赖性方式抑制卵巢癌粘附/侵袭 3D 模型的化合物。其中，八种化合物在五种高级浆液性卵巢癌细胞系中被证实具有活性，并在二次体外和体内生物测定中得到进一步验证。两种酪氨酸激酶抑制剂，选择 PP-121 和 milciclib 以及以前未报告的化合物 NCGC00117362，因为它们在体外具有 1 μmol/L 的效力。具体而言，NCGC00117362和PP-121抑制卵巢癌的粘附、侵袭和增殖，而milciclib抑制卵巢癌的侵袭和增殖。使用原位激酶分析和免疫印迹，我们发现 milciclib 靶向 Cdk2 和 Cdk6，而 PP-121 靶向 mTOR。在体内，在一项干预研究中，所有三种化合物均能预防卵巢癌的粘附/侵袭和转移，延长生存期，并减少网膜肿瘤的生长。为了评估 NCGC00117362 的临床潜力，进行了构效关系研究。NCGC00117362 的四种紧密类似物在体外有效抑制癌症侵袭性和体内转移。总的来说，这些数据表明 TME 的复杂 3D 培养在 qHTS 中是有效的。确定的三种化合物有望作为预防和治疗卵巢癌转移的治疗剂。