GESTALT Barcoding and Single-cell Transcriptomics of Tumor Cell Evolution in Personalized Tumor Models

项目摘要/摘要 随着肿瘤的发展，不同肿瘤类型的体细胞突变的鉴定呈指数级增长 下一代测序技术。然而，迫切需要确认推定的癌症驱动因素 基因，并从巧合的“乘客”突变中分离它们。此外，越来越明显的是，许多人 癌症在体细胞基因型的多克隆方面具有高度的异质性--通常表现为 同时或在不同的亚群中发生多个驱动突变。此外，护理治疗的标准 通常会引起选择压力，导致经常出现的人口发生重大变化。我们只是在 在许多肿瘤类型中验证驱动基因的开始阶段，研究甚至更落后 这些系统中的进化和重现机制。这项拨款申请的中心主题是 通过对基因组进行编辑，生成与患者派生的“个性化”体细胞突变签名相结合的工具集 用于谱系追踪的合成靶点阵列(格式塔)用于阐明转录机制 导致肿瘤的多样性。具体地说，我们将生成一条分离单细胞转录本和 格式塔条形码可随时间推移对大量肿瘤群体进行分类和谱系映射，包括在 护理标准治疗的选择性压力。在过去的几年里，我们开创了一种 基于电穿孔的体细胞突变方法用于快速、无创、高密度的体细胞转基因 用双重组酶介导盒进行镶嵌分析验证肿瘤驱动基因的吞吐量 交易所(MADR)。我们将使用新的胶质瘤体内MADR模型作为测试案例，以利用 该系统供以后用于不同类型的肿瘤。该提案的总体目标是执行高级 这种MADR-格式塔联合方法的发展允许在不同的肿瘤中广泛使用 并因此展示了这项技术改变癌症研究的潜力。 我们建议分三部分进行这项工作。具体目标1的重点是优化组合 通过转录组和谱系图生成肿瘤细胞分类的MADR-格式塔系统。这个 具体目标2的主要目标是在以下背景下严格验证MADR-格式塔诱导元素 临床标准护理治疗。最后，为广泛传播这些工具做好准备，具体而言 目的3我们将产生并验证带有格式塔元素的敲入小鼠的组织不服从于 电穿孔。这些实验的成功完成将为长期的、 用于了解该病的基本和病理机制以及 提供对进展和转录的细胞和转录机制的明确的、遗传学的见解 在各种癌症中复发。

PROJECT SUMMARY / ABSTRACT Identification of somatic mutations in diverse tumor types has grown exponentially with the development of next-generation sequencing technologies. However, there is a pressing need to validate putative cancer driver genes and separate them from coincidental “passenger” mutations. Further, it is becoming clear that many cancers are highly heterogeneous in terms of the polyclonality of somatic genotypes—often expressing multiple driver mutations simultaneously or in different subpopulations. Moreover, standard of care treatment often induces selective pressures resulting in significant alterations in recurrent populations. We are only in the beginning stages of validating driver genes in many tumor types and are even further behind in studying mechanisms of evolution and recurrence in these systems. The central theme of this grant application is to generate a toolset marrying patient-derived, “personalized” somatic mutation signatures with genome editing of synthetic target arrays for lineage tracing (GESTALT) for the elucidation of the transcriptomic mechanisms leading to tumor diversity. Specifically, we will generate a pipeline for isolating single-cell transcriptomes and GESTALT barcodes to classify and lineage map large numbers of tumor populations over time, including after the selective pressures of standard of care treatment. Over the past several years, we have pioneered an electroporation-based somatic mutation method for rapid, non-invasive, somatic transgenesis for high throughput validation of tumor driver genes using mosaic analysis with dual recombinase-mediated cassette exchange (MADR). We will employ novel in vivo MADR models of glioma as a test case for the utilization of this system for later use with diverse tumor types. The overall objective of the proposal is to perform advanced development of this combined MADR-GESTALT approach to allow for generalized use in diverse tumor contexts and, therefore, demonstrate the potential of this technology to transform cancer research. We propose to carry out this work in three parts. The focus of Specific Aim 1 is to optimize the combined MADR-GESTALT system for generating tumor cell classification by transcriptome and lineage maps. The main goal of Specific Aim 2 is to rigorously validate MADR-GESTALT inducible elements in the context of clinical standard of care treatment. Finally, to prepare for widespread dissemination of these tools, in Specific Aim 3 we will generate and validate knock-in mice with GESTALT elements for tissues not amenable to electroporation. Successful completion of these experiments will create the foundation for a long-lived, cornerstone toolset for understanding both basic and pathologic mechanisms of the disease as well as providing definitive, genetic insights into the cellular and transcriptomic mechanisms of progression and recurrence in a diverse array of cancers.