Investigating the role of stromal YAP activation in intrahepatic cholangiocarcinoma

研究基质 YAP 激活在肝内胆管癌中的作用

• 批准号: 10200632
• 负责人: Jacquelyn Olivia Russell
• 金额: $ 6.6万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200632
• 关键词: Automobile Driving; Biliary; CRISPR screen; CRISPR/Cas technology; Cell Survival; Cells; Coculture Techniques; Combination Drug Therapy; Complex; Diagnosis; Doxycycline; Endothelial Cells; Excision; Fibroblasts; Gene Expression; Generations; Genes; Growth; Heterogeneity; Histologic; Human; Immunocompromised Host; In Vitro; Incidence; Inflammatory; Intrahepatic Cholangiocarcinoma; KRASG12D; Libraries; Ligands; Liver; Malignant neoplasm of liver; Mediating; Minority; Modeling; Molecular; Morphology; Mus; Mutation; Myofibroblast; Nuclear; Oncogenic; Operative Surgical Procedures; Organ Size; Organoids; Pathway interactions; Patients; Pattern; Prognosis; Proteins; Recurrence; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Smooth Muscle Actin Staining Method; Stromal Cells; Survival Rate; System; Tamoxifen; Testing; Tissue Sample; Tissues; Transcription Coactivator; Transplantation; Tumor Burden; Tumor-Derived; Unresectable; Work; carcinogenesis; cell growth; cell stroma; chemotherapy; density; design; effective therapy; experimental study; human disease; in vivo; intrahepatic; loss of function; macrophage; mortality; mouse model; mutant; neoplastic cell; new therapeutic target; novel; organ regeneration; organoid transplantation; protein activation; receptor; recruit; single-cell RNA sequencing; subcutaneous; therapeutic target; transplant model; treatment strategy; tumor; tumor growth; tumorigenesis; tumorigenic

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.

项目总结/摘要 肝内胆管细胞癌（ICC）是肝癌的第二种最常见形式，并且通常是肝癌的第二种最常见形式。 诊断为晚期或转移性阶段。对于不能切除的晚期ICC，唯一可用的治疗方法是 全身化疗通常将患者生存期延长不到两年。国际刑事法院的一个特点是， 致密的肿瘤反应性基质（TRS），由募集的内皮细胞、巨噬细胞和肌成纤维细胞组成 (MFs)从周围的肝脏。TRS与ICC肿瘤发生有关，特别是TRS 更多的激活的MF与更差的预后相关。ICC的另一个特点是其异质性， 没有大多数患者常见的致癌突变。然而，是相关蛋白（雅普）， Hippo信号通路的主要下游效应物，已显示在大多数 人ICC组织样品。我们的实验室已经开发了一种新的移植ICC的同基因小鼠模型，其中 分离小鼠胆管细胞，将其培养为肝类器官， 引入了使用CRISPR技术来产生ICC类器官。这些ICC类器官可以移植 皮下或肝内注射以产生ICC肿瘤，其概括了人类肿瘤的关键特征。 疾病，包括募集密集的TRS。有趣的是，我们已经观察到最高的雅普活性， 与我们模型中的ICC肿瘤细胞相反，TRS的MF。因此，我们假设，在细胞内雅普的激活可能与细胞内的细胞凋亡有关。 TRS对ICC肿瘤发生至关重要。我们建议通过在细胞中缺失雅普来检验这一假设。 TRS，以及在TRS微纤维中特异性缺失雅普，我们假设这将减少ICC肿瘤负荷 在我们的体内小鼠模型中。为了阐明YAP驱动的TRS MF和ICC单元串扰的机制，我们将 开发一种新的ICC类器官和TRS MF体外共培养系统，我们假设TRS MF将 以YAP依赖的方式促进ICC细胞生长和侵袭。最后，我们将进行单细胞RNA 对对照和YAP缺失的TRS MF进行测序，以鉴定这些细胞中YAP驱动的分泌配体。我们 然后，我们将在我们的ICC类器官中进行CRISPR-Cas9筛选，靶向与我们的顶部相对应的受体， TRS MF中的分泌配体命中。这项CRISPR-Cas9功能丧失筛查将在我们的研究中进行。 体外共培养系统，我们将因此能够确定介导串扰的信号通路 TRS MF和ICC细胞之间的关系。在我们的筛选中确定的途径可以在我们的体内ICC中得到验证 小鼠模型，增加我们的研究结果的有效性。这项工作不仅将确定分子机制， ICC肿瘤细胞和其相关的肝基质细胞之间的串扰，但将确定新的治疗靶点 这可以被研究用于新的ICC治疗策略。