胆管癌（CCA）是原发性硬化性胆管炎（PSC）的可怕并发症，难以诊断且死亡率高。缺乏概括硬化性胆管炎肝脏微环境的 CCA 动物模型阻碍了新疗法的开发。在这里，我们试图在小鼠中开发这种与 PSC 相关的 CCA 模型。对患有先天性 PSC 样疾病的 10 周龄 Mdr2-/- 小鼠和健康的野生型同窝小鼠进行改良的逆行胆汁滴注或水动力尾静脉注射具有激活的 AKT (myr-AKT) 和 Yap (YapS127A) 原癌基因 (SB AKT/YAP1) 的睡美人转座子-转座酶质粒系统。通过 ALK5 抑制剂 (SB-525334) 给药来探究 TGFβ 的作用。通过组织学和 RNA-seq 分析肿瘤表型、负荷和促纤维增生反应。虽然通过逆行胆道注射 SB AKT/YAP1 质粒在 Mdr2-/- 中引起肿瘤，但这些肿瘤中只有 26.67% (4/15) 是 CCA。或者，与健康小鼠相比，水动力尾静脉注射 SB AKT/YAP1 导致所有具有高 CCA 负荷的纤维化 Mdr2-/- 小鼠发生强烈的肿瘤发生。肿瘤在表型上与人类 CCA 相似，表达多种 CCA（但不包括肝细胞癌）标记物，并表现出严重的促纤维增生反应。 RNA-seq 分析揭示了在类似 PSC 的环境中进化的 CCA 发生了深刻的转录变化，并在多个免疫途径中发生了特定的改变。与安慰剂相比，药理学 TGFβ 抑制导致免疫细胞肿瘤浸润增强、肿瘤负荷减轻并抑制促纤维增生性胶原积累结论：我们在小鼠中建立了一种新的高保真胆管癌模型，称为 SB CCA.Mdr2-/-，它概括了敏感性增加的情况在 PSC 中观察到的胆道损伤和纤维化的情况下对 CCA 的影响。通过转录组学和药理学研究，我们发现多种免疫途径和 TGFβ 信号传导失调是 PSC 样微环境中 CCA 的潜在驱动因素。目前缺乏原发性硬化性胆管炎 (PSC) 相关胆管癌 (PSC-CCA) 的动物模型。我们开发并表征了一种新的 PSC-CCA 小鼠模型，称为 SB CCA.Mdr2-/-，其特点是在类似 PSC 的胆道损伤和纤维化背景中可靠地诱导肿瘤。确定了全局基因表达改变，并建立了标准化工具，包括用于肿瘤负荷和特征分析的自动化全幻灯片图像分析方法，以实现对 PSC-CCA 生物学和正式临床前药物测试的系统研究。版权所有 © 2024。由 Elsevier 出版B.V.

Cholangiocarcinoma (CCA) is a dreaded complication of primary sclerosing cholangitis (PSC), difficult to diagnose and associated with high mortality. Lack of animal models of CCA recapitulating the hepatic microenvironment of sclerosing cholangitis hinders development of novel treatments. Here we sought to develop such PSC-associated CCA model in mice.Ten-week-old Mdr2-/- mice with congenital PSC-like disease, and healthy wild-type littermates were subjected to either modified retrograde biliary instillation or hydrodynamic tail vein injection of sleeping beauty transposon-transposase plasmid system with activated AKT (myr-AKT) and Yap (YapS127A) protooncogenes (SB AKT/YAP1). The role of TGFβ was interrogated via ALK5 inhibitor (SB-525334) administration. Tumor phenotype, burden and desmoplastic reaction were analyzed histologically and via RNA-seq.While SB AKT/YAP1 plasmids via retrograde biliary injection caused tumors in Mdr2-/-, only 26.67% (4/15) of these tumors were CCA. Alternatively, hydrodynamic tail vein injection of SB AKT/YAP1 resulted in robust tumorigenesis in all fibrotic Mdr2-/- mice with high CCA burden compared to healthy mice. Tumors phenotypically resembled human CCA, expressed multiple CCA (but not hepatocellular carcinoma) markers, and exhibited a profound desmoplastic reaction. RNA-seq analysis revealed profound transcriptional changes in CCA evolving in PSC-like context, with specific alterations in multiple immune pathways. Pharmacological TGFβ inhibition led to enhanced immune cell tumor infiltration, reduced tumor burden and suppressed desmoplastic collagen accumulation compared to placebo CONCLUSION: We established a new high-fidelity cholangiocarcinoma model in mice, termed SB CCA.Mdr2-/-, which recapitulates the increased susceptibility to CCA in the setting of biliary injury and fibrosis observed in PSC. Through transcriptomics and pharmacological studies, we show dysregulation of multiple immune pathways and TGFβ signaling as potential drivers of CCA in PSC-like microenvironment.There is a lack of animal models for primary sclerosing cholangitis (PSC) related cholangiocarcinoma (PSC-CCA). We have developed and characterized a new mouse model of PSC-CCA, termed SB CCA.Mdr2-/-, which features reliable tumor induction in PSC-like background of biliary injury and fibrosis. Global gene expression alterations were identified and standardized tools, including automated whole slide image analysis methodology for tumor burden and feature analysis, were established to enable systematic research into PSC-CCA biology and formal pre-clinical drug testing.Copyright © 2024. Published by Elsevier B.V.