Elucidating time-dependent mechanisms of genetic and epigenetic reprogramming in single cancer cells

项目摘要 肿瘤从化学毒性和靶向治疗中存活的主要机制被假设为 是肿瘤内异质性的结果。肿瘤异质性已被假定为有助于肿瘤 通过不同机制的进化和治疗逃避：耐药亚克隆的产生，合作 在次要亚克隆群体之间，次要亚克隆产生有利于肿瘤生长的基质信号传导， 肿瘤异质性的遗传和表观遗传多样性是系统性的， 被传统的基因组学方法忽略了。该项目的总体目标是制定方法， 表征不同肿瘤亚群中的功能依赖性和特异性转录程序， 使用单细胞分子谱和分析，特别强调诱导复发的克隆和小生境 或进步。本项目的第一个目标（1）是开发和实施捕获肿瘤的新方法 克隆表示，并在单细胞中整合大规模表达和突变分析。这将 通过进行深度外显子组和RNA测序以及单细胞分析来确定点 肿瘤细胞中的突变、插入缺失和基因融合。本项目的第二个目标（2）是确定 表观遗传学上不同的细胞小生境，具有相同的突变谱，要么逃避治疗，要么可以 使肿瘤再生。克隆扩增的肿瘤细胞可能在基因突变方面是相同的 在（1）中鉴定，但代表不同的表观遗传状态，其特征在于不同的基因表达标记。 在（1）中开发的方法将应用于PDX模型中治疗后的残留肿瘤细胞， 阐明表观遗传学不同细胞中耐药性的驱动因素。最后，第三个目标（3）是确定 在异质性肿瘤中实现细胞间通讯过程的分子相互作用， 微环境这将通过开发新的信息理论方法来实现， 介导单细胞通讯和相互作用的特异性配体和受体的优先化。这些 方法学将专门用于阐明新的肿瘤检查点抑制剂和其他机制 与免疫治疗应用相关的研究。

PROJECT SUMMARY A major mechanism by which tumors survive from both chemotoxic and targeted therapies is hypothesized to be the result intra-tumoral heterogeneity. Tumor heterogeneity have been postulated to contribute to tumor evolution and therapy evasion through different mechanisms: generation of resistant subclones, cooperation between minor subclonal populations, minor subclones generating stromal signaling that favors tumor progression, etc. The genetic and epigenetic diversity that underlies tumor heterogeneity is siystematially missed by traditional genomic approaches. The overall goal of this project is to develop approaches to characterize functional dependencies and specific transcriptional programs in distinct tumor subpopulations, using single cell molecular profiling and analysis, with specific emphasis on clones and niches inducing relapse or progression. The first aim (1) of this project is to develop and implement new methods to capture tumor clonal representation and to integrate large-scale expression and mutational analysis in single cells. This will be done by conducting deep exome and RNA sequencing together with single-cell profiling to identify point mutations, indels, and gene fusions in tumor cells. The second aim of this project (2) is to identify epigenetically distinct cell niches with identical mutational spectra that either escape treatment or can regenerate the tumor. Clonally expanded tumor cells may be genetically identical with respect to the mutations identified in (1) but represent distinct epigenetic states, characterized by distinct gene expression signatures. The methods developed in (1) will be applied to residual tumor cells following therapy in PDX models to elucidate the drivers of drug resistance in epigenetically distinct cells. Finally the third aim (3) is to identify the molecular interactions that implement cell-cell communication processes, within a heterogenous tumor- microenviroment milieu. This will be achieved by developing novel information theoretic methodologies for the prioritization of specific ligands and receptors that mediate single cell communication and interaction. These methodologies will be specifically applied to elucidate novel tumor checkpoint inhibitors and other mechanisms that are relevant to immunotherapy applications.