Project 3: Modeling tumor evolution and drug response in bladder cancer organoids

项目总结/摘要 肿瘤异质性和克隆进化在癌症进展和对化疗的反应中起关键作用。 疗法然而，克隆进化在实体瘤如膀胱癌中一直难以研究，部分原因在于， 因为缺乏可靠的模型系统。在本项目中，我们建议使用患者来源的膀胱肿瘤 类器官作为一种新的模型系统，用于纵向研究肿瘤演变及其在药物治疗中的作用 反应和抵抗。在初步研究中，我们已经开发了用于建立 来自膀胱癌组织的患者来源的类器官系，其范围从乳头状非侵入性肿瘤到 肌肉浸润性癌症，并已表明，这些类器官线概括了组织病理学和 其相应亲本肿瘤的分子特征。特别是，我们发现这些类器官 在培养物中连续传代期间，细胞系显示突变谱的变化，这与克隆的突变谱一致。 进化我们已经进一步证明，这些线可用于测定药物反应，并且这些线可用于测定药物反应。 可以在来自这些类器官的原位异种移植物中在体内验证应答。 我们现在将使用这些患者来源的膀胱癌类器官模型来研究克隆进化 通过追求三个特定目标来研究膀胱癌的异质性和药物反应：1）分析异质性和肿瘤 通过检查连续传代过程中异质性的维持来研究膀胱癌类器官的演变 以及研究基因组不稳定性和表观遗传失调如何驱动克隆进化; 2） 通过分析核苷酸的作用研究膀胱癌类器官的化疗反应 切除修复途径基因ERCC 2对顺铂治疗的差异反应;和3）克隆性ERCC 2基因的分析。 使用慢病毒介导的条形码和单细胞技术研究膀胱癌类器官的耐药性演变 RNA测序用于在药物出现期间对克隆肿瘤群体进行纵向分析 文化中的抵抗这些研究将极大地促进了患者源性类器官系的生物库 以及将由膀胱癌模型核心、组织病理学和靶向外显子组产生的异种移植物 测序由分子病理学核心进行，生物信息学和生物统计学支持来自 行政核心。我们的工作也将通过我们的分析与项目1和2高度整合， 表观遗传调节因子KDM 6A和ARID 1A在驱动肿瘤异质性和进化中的潜在作用， 同时，我们对患者来源的膀胱肿瘤类器官的研究将提供有价值的试剂， 和其他项目的见解。

Project Summary/Abstract Tumor heterogeneity and clonal evolution play key roles in cancer progression and the response to therapy. However, clonal evolution has been difficult to study in solid tumors such as bladder cancer, in part due to the lack of amenable model systems. In this Project, we propose to use patient-derived bladder tumor organoids as a new model system to pursue longitudinal studies of tumor evolution and its role in drug response and resistance. In preliminary studies, we have developed culture conditions for the establishment of patient-derived organoid lines from bladder cancer tissues that range from papillary non-invasive tumors to muscle-invasive cancer, and have shown that these organoid lines recapitulate the histopathological and molecular features of their corresponding parental tumors. In particular, we have found that these organoid lines display changes in mutational profiles during serial passaging in culture that are consistent with clonal evolution. We have further demonstrated that these lines can be used to assay drug response, and that these responses can be validated in orthotopic xenografts from these organoids in vivo. We will now use these patient-derived bladder cancer organoid models to investigate clonal evolution and drug response in bladder cancer by pursuing three specific aims: 1) Analysis of heterogeneity and tumor evolution in bladder cancer organoids by examining the maintenance of heterogeneity during serial passaging and by investigating how genome instability and epigenetic dysregulation drive clonal evolution; 2) Investigation of chemotherapy response in bladder cancer organoids by analyzing the role of the nucleotide excision repair pathway gene ERCC2 in differential response to cisplatin treatment; and 3) Analysis of clonal evolution in drug resistance of bladder cancer organoids using lentiviral-mediated barcoding and single-cell RNA sequencing to perform longitudinal analyses of clonal tumor populations during the emergence of drug resistance in culture. These studies will be greatly facilitated by the biobank of patient-derived organoid lines and xenografts that will be generated by the Bladder Cancer Models Core, histopathology and targeted exome sequencing performed by the Molecular Pathology Core, and bioinformatic and biostatistical support from the Administrative Core. Our work will also be highly integrated with Projects 1 and 2 through our analyses of the potential roles of the epigenetic regulators KDM6A and ARID1A in driving tumor heterogeneity and evolution, while at the same time, our studies of patient-derived bladder tumor organoids will provide valuable reagents and insights for the other Projects.