Investigating the role of stromal YAP activation in intrahepatic cholangiocarcinoma

项目摘要/摘要 肝内胆管细胞癌(ICC)是第二种最常见的肝癌，通常 确诊处于晚期或转移阶段。对于无法切除的晚期ICC，唯一可用的治疗方法是 全身化疗，通常将患者的生存期延长不到两年。国际刑事法院的一个标志是它的 致密的肿瘤反应性间质(TRS)，由重新招募的内皮细胞、巨噬细胞和肌成纤维细胞组成 (MFS)从周围的肝脏。TRS与ICC肿瘤的发生有关，尤其是TRS。 MFS激活程度越高，预后越差。ICC的另一个特点是它的异构性， 大多数患者没有常见的致癌基因突变。然而，是相关蛋白(YAP)， 河马信号通路的主要下游效应器，已被证明在大多数 人ICC组织标本。我们实验室开发了一种新的移植ICC的同源小鼠模型，其中 分离小鼠胆汁细胞，作为肝脏器官培养，并在人类ICC患者中发现突变 介绍了利用CRISPR技术生成ICC有机化合物。这些ICC有机化合物可以移植到 皮下或肝内引起ICC肿瘤，这些肿瘤概括了人类的主要特征 疾病，包括招募密集的TRS。有趣的是，我们观察到了YAP在 在我们的模型中，TRS的MFS与ICC肿瘤细胞相反。因此，我们假设YAP在 TRS在ICC的发生发展中起着至关重要的作用。我们建议通过删除YAP中的 TRS，以及在TRS MFS中特定的YAP的缺失，我们假设这将减少ICC肿瘤负担 在我们体内的小鼠模型中。为了阐明YAP驱动的TRS中频和ICC细胞串扰的机制，我们将 建立了一种新型的ICC类有机物与TRS-MFS体外共培养体系，我们假设TRS-MFS将 以YAP依赖的方式促进ICC细胞的生长和侵袭。最后，我们将进行单细胞RNA 对对照和YAP缺失的TRS MFS进行测序，以确定这些细胞中YAP驱动的分泌配体。我们 然后在我们的ICC有机体中进行CRISPR-Cas9筛选，目标是与我们的TOP对应的受体 TRS MFS中的分泌配体命中。此CRISPR-CAS9功能丧失屏幕将在我们的输入中执行 体外共培养系统，因此我们将能够确定介导串扰的信号通路 TRS MFS和ICC细胞之间的相互作用。我们屏幕中识别的通路可以在我们的活体ICC中得到验证 小鼠模型，增加了我们研究结果的有效性。这项工作不仅将确定 ICC肿瘤细胞与其相关的肝基质细胞之间的串扰，但将确定新的治疗靶点 这可能为新的ICC治疗策略提供研究。

Project Summary/Abstract Intrahepatic cholangiocarcinoma (ICC) is the second most common form of liver cancer and is often diagnosed at an advanced or metastatic stage. The only available treatment for unresectable, late-stage ICC is systemic chemotherapy which typically extends patient survival by less than two years. A hallmark of ICC is its dense tumor reactive stroma (TRS), consisting of recruited endothelial cells, macrophages, and myofibroblasts (MFs) from the surrounding liver. The TRS has been implicated in ICC tumorigenesis, and in particular a TRS with more activated MFs is associated with a worse prognosis. Another hallmark of ICC is its heterogeneity, with no oncogenic mutations common to the majority of patients. However, Yes Associated Protein (YAP), the major downstream effector of the Hippo signaling pathway, has been shown to be activated in the majority of human ICC tissue samples. Our lab has developed a novel syngenic mouse model of transplanted ICC, where mouse biliary cells were isolated, cultured as liver organoids, and mutations found in human ICC patients were introduced using CRISPR technology to generate ICC organoids. These ICC organoids can be transplanted subcutaneously or intrahepatically to give rise to ICC tumors which recapitulate key features of the human disease, including the recruitment of a dense TRS. Interestingly, we have observed the highest YAP activity in the MFs of the TRS as opposed to ICC tumor cells in our model. Thus, we hypothesize that YAP activation in the TRS is crucial for ICC tumorigenesis. We propose to test this hypothesis through deletion of YAP in the TRS, as well as deletion of YAP specifically in TRS MFs, which we hypothesize will reduce ICC tumor burden in our in vivo mouse model. To elucidate the mechanism of YAP-driven TRS MF and ICC cell crosstalk, we will develop a novel ICC organoid and TRS MF in vitro co-culture system, where we hypothesize that TRS MFs will promote ICC cell growth and invasion in a YAP-dependent manner. Finally, we will perform single-cell RNA sequencing on control and YAP-deleted TRS MFs to identify YAP-driven secreted ligands in these cells. We will then perform in our ICC organoids a CRISPR-Cas9 screen targeting the receptors corresponding to our top secreted ligand hits in the TRS MFs. This CRISPR-Cas9 loss-of-function screen will be performed in our in vitro co-culture system, and we will thus be able to identify the signaling pathways that mediate crosstalk between TRS MFs and ICC cells. The pathways identified in our screen can be validated in our in vivo ICC mouse model, increasing the validity of our findings. This work will not only identify molecular mechanisms of crosstalk between ICC tumor cells and its associated liver stromal cells, but will identify new therapeutic targets which could be investigated for novel ICC treatment strategies.