Intrahepatic cholangiocarcinoma (iCCA) is a deadly disease with rising incidence and few treatment options. An altered expression and/or activation of NOTCH1-3 receptors has been shown to play a role in iCCA development and progression. In this study, we established a new CCA patient-derived xenograft model, which was validated by immunohistochemistry and transcriptomic analysis. The effects of Notch pathway suppression by the Crenigacestat (LY3039478)-specific inhibitor were evaluated in human iCCA cell lines and the PDX model. In vitro, LY3039478 significantly reduced Notch pathway components, including NICD1 and HES1, but not the other Notch receptors, in a panel of five different iCCA cell lines. In the PDX model, LY3039478 significantly inhibited the Notch pathway and tumor growth to the same extent as gemcitabine. Furthermore, gene expression analysis of iCCA mouse tissues treated with LY3039478 revealed a downregulation of VEGFA, HES1, and MMP13 genes. In the same tissues, DLL4 and CD31 co-localized, and their expression was significantly inhibited in the treated mice, as it happened in the case of MMP13. In an in vitro angiogenesis model, LY3039478 inhibited vessel formation, which was restored by the addition of MMP13. Finally, RNA-sequencing expression data of iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database demonstrated that NOTCH1, HES1, MMP13, DLL4, and VEGFA genes were significantly upregulated in tumors compared with adjacent nontumorous tissues. These data were confirmed by our group, using an independent cohort of iCCA specimens. Conclusion: We have developed and validated a new iCCA PDX model to test in vivo the activity of LY3039478, demonstrating its inhibitory role in Notch-dependent angiogenesis. Thus, the present data provide new knowledge on Notch signaling in iCCA, and support the inhibition of the Notch cascade as a promising strategy for the treatment of this disease.

中文翻译：

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Crenigacestat 是一种选择性 NOTCH1 抑制剂，通过阻断 VEGFA/DLL4/MMP13 轴来减少肝内胆管癌的进展。

肝内胆管癌 (iCCA) 是一种致命的疾病，发病率不断上升，治疗选择很少。NOTCH1-3 受体的表达和/或激活的改变已被证明在 iCCA 的发展和进展中起作用。在这项研究中，我们建立了一个新的 CCA 患者来源的异种移植模型，并通过免疫组织化学和转录组学分析进行了验证。在人 iCCA 细胞系和 PDX 模型中评估了 Crenigacestat (LY3039478) 特异性抑制剂对 Notch 通路抑制的影响。在体外，LY3039478 显着减少了五个不同 iCCA 细胞系中的 Notch 通路成分，包括 NICD1 和 HES1，但不减少其他 Notch 受体。在 PDX 模型中，LY3039478 显着抑制 Notch 通路和肿瘤生长，其抑制程度与吉西他滨相同。此外，用 LY3039478 处理的 iCCA 小鼠组织的基因表达分析显示 VEGFA、HES1 和 MMP13 基因下调。在相同的组织中，DLL4 和 CD31 共定位，并且它们的表达在处理过的小鼠中被显着抑制，就像 MMP13 的情况一样。在体外血管生成模型中，LY3039478 抑制血管形成，而添加 MMP13 可恢复血管形成。最后，从 GEO 数据库下载的 iCCA 患者和匹配的周围正常肝组织的 RNA 测序表达数据表明，与邻近的非肿瘤组织相比，NOTCH1、HES1、MMP13、DLL4 和 VEGFA 基因在肿瘤中显着上调。我们小组使用独立的 iCCA 样本队列证实了这些数据。结论：我们开发并验证了一种新的 iCCA PDX 模型，用于在体内测试 LY3039478 的活性，证明其在 Notch 依赖性血管生成中的抑制作用。因此，目前的数据提供了关于 iCCA 中 Notch 信号传导的新知识，并支持抑制 Notch 级联作为治疗这种疾病的有希望的策略。