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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要肝内胆管癌 (ICC) 是第二常见的肝癌形式，通常是诊断时已处于晚期或转移期。对于不可切除的晚期 ICC，唯一可用的治疗方法是全身化疗通常可将患者的生存期延长不到两年。 ICC的一个标志是致密肿瘤反应性基质 (TRS)，由招募的内皮细胞、巨噬细胞和肌成纤维细胞组成（MF）来自周围的肝脏。 TRS 与 ICC 肿瘤发生有关，特别是 TRS MF 激活越多，预后越差。 ICC的另一个特点是它的异质性，没有大多数患者常见的致癌突变。然而，是相关蛋白 (YAP) Hippo 信号通路的主要下游效应器已被证明在大多数细胞中被激活人类 ICC 组织样本。我们的实验室开发了一种新型同基因小鼠移植 ICC 模型，其中分离小鼠胆管细胞，培养为肝类器官，并在人类 ICC 患者中发现突变介绍了使用 CRISPR 技术生成 ICC 类器官。这些 ICC 类器官可以移植皮下或肝内产生 ICC 肿瘤，再现了人类的主要特征疾病，包括招募密集的 TRS。有趣的是，我们观察到最高的 YAP 活动在我们的模型中，TRS 的 MF 与 ICC 肿瘤细胞相反。因此，我们假设 YAP 激活 TRS 对于 ICC 肿瘤发生至关重要。我们建议通过删除 YAP 来检验这一假设 TRS，以及特别是在 TRS MF 中删除 YAP，我们假设这将减少 ICC 肿瘤负担在我们的体内小鼠模型中。为了阐明 YAP 驱动的 TRS MF 和 ICC 细胞串扰的机制，我们将开发一种新型 ICC 类器官和 TRS MF 体外共培养系统，我们假设 TRS MF 将以 YAP 依赖性方式促进 ICC 细胞生长和侵袭。最后，我们将进行单细胞RNA 对对照和 YAP 删除的 TRS MF 进行测序，以鉴定这些细胞中 YAP 驱动的分泌配体。我们然后将在我们的 ICC 类器官中进行 CRISPR-Cas9 筛选，针对与我们顶部对应的受体 TRS MF 中分泌的配体命中。该 CRISPR-Cas9 功能丧失筛选将在我们的实验室中进行体外共培养系统，因此我们将能够识别介导串扰的信号通路 TRS MF 和 ICC 单元之间。我们的屏幕中确定的途径可以在我们的体内 ICC 中进行验证小鼠模型，增加了我们研究结果的有效性。这项工作不仅将确定 ICC 肿瘤细胞与其相关肝基质细胞之间的串扰，但将确定新的治疗靶点可以研究新的 ICC 治疗策略。