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

项目总结 肿瘤通过化疗和靶向治疗存活的主要机制被假设为 是肿瘤内异质性的结果。肿瘤的异质性被认为与肿瘤的发生有关 通过不同机制的进化和治疗规避：产生抗性亚克隆，合作 在次要亚克隆群体之间，次要亚克隆产生有利于肿瘤的基质信号 进展等。作为肿瘤异质性基础的遗传和表观遗传多样性是具有系统多样性的 被传统的基因组方法遗漏了。该项目的总体目标是开发方法来 确定不同肿瘤亚群的功能依赖性和特定转录程序的特征， 使用单细胞分子图谱和分析，特别强调导致复发的克隆和利基 或者进步。这个项目的第一个目标(1)是开发和实施捕获肿瘤的新方法。 克隆表达，整合大规模表达和单细胞突变分析。这将是 通过进行深层外显子组和RNA测序以及单细胞图谱来确定点 肿瘤细胞中的突变、插入和基因融合。该项目(2)的第二个目标是确定 表观遗传学上截然不同的细胞生态位，具有相同的突变谱，要么逃脱治疗，要么可以 让肿瘤再生。克隆性扩增的肿瘤细胞在基因突变方面可能是相同的 在(1)中识别，但代表不同的表观遗传状态，以不同的基因表达特征为特征。 在(1)中开发的方法将应用于在PDX模型中治疗后的残留肿瘤细胞 阐明表观遗传差异细胞中耐药的驱动因素。最后，第三个目标(3)是确定 在异种肿瘤中，实现细胞间通讯过程的分子相互作用- 微环境环境。这将通过开发新的信息论方法来实现 对调节单细胞通讯和相互作用的特定配体和受体进行优先排序。这些 方法学将专门应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技术。

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.