SPATIALLY MAPPING OF POOLED IN VIVO CRISPR SCREENS IN THE TUMOR MICROENVIRONMENT

肿瘤微环境中体内 CRISPR 筛选池的空间映射

• 批准号: 10473150
• 负责人: Jellert Gaublomme
• 金额: $ 148.05万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-26 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10473150
• 关键词: Architecture; Automobile Driving; Award; Biological Assay; CRISPR screen; Cell Communication; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Combined Modality Therapy; Development; Environment; Extracellular Matrix; Genes; Genetic; Health; Immune; Immune system; Immunosuppression; In Situ; In Vitro; Knowledge; Longevity; Malignant Neoplasms; Maps; Mediating; Methodology; Methods; Modality; Names; Optics; Pathogenesis; Pathogenicity; Patients; Pharmacologic Substance; Primary carcinoma of the liver cells; Process; Proteomics; Research Proposals; Signal Transduction; Stromal Cells; T-Lymphocyte; Testing; Therapeutic; Time; Tissues; Tumor stage; cancer cell; cell behavior; checkpoint inhibition; checkpoint therapy; cost; immunoregulation; in vivo; innovation; insight; invention; liver cancer model; macrophage; neoplastic cell; patient subsets; screening; therapeutic target; transcriptomics; tumor; tumor microenvironment; tumorigenesis

RFA-RM-21-016: New Innovator Award– Project Summary. Jellert T. Gaublomme PROJECT SUMMARY To understand how cellular behavior informs pathogenesis we need to study cells in their native tissue environment. Only by doing so can we elucidate the influence of signals received through cell-to-cell interactions and microenvironmental factors. For example, tumors evade immune mediated clearing by silencing T-cells, which receive signals from cancer cells, stromal cells, macrophages, and the extracellular matrix, to name a few. These numerous tissue microenvironmental influences are not captured by in vitro studies most often used during pharmaceutical screens for therapeutic targets. To extensively chart the cellular architecture of the tumor microenvironment, and the intercellular signaling that leads to an immunosuppressive environment, I will develop and apply an approach that profiles the transcriptomic and proteomic state of cells in situ. Once the tumor microenvironment is mapped, it is crucial to identify signaling genes that drive immunosuppression. Targeting immunomodulatory signaling can yield therapeutic breakthroughs, as evidenced by recent advances in immune checkpoint inhibition. Recently developed in vivo pooled CRISPR screens are a powerful high-throughput modality to identify genetic regulators. However, to date, these assays require cells to be isolated from their native tissue context prior to analysis. Our proposal is to pioneer a screening method that enables optical in situ identification of the CRISPR perturbation at the single-cell level in the native tissue context, enabling us to study the corresponding changes in tissue architecture and cell-to-cell interactions. Our approach is poised to elucidate mechanistic insights, and selection of the most promising therapeutic targets. A therapeutic treatment is in essence a perturbation on a tumor that developed unperturbed. In current in vivo screens however, tumors arise from cells that are perturbed for the screening genes, thus compromising normal tumorigenesis and development of a native tumor microenvironment. Another key innovation of our proposal is to decouple tumor induction and the time at which we perturb the signaling genes we are screening for. To achieve temporal decoupling, we induce the CRISPR perturbation at a desired stage of tumor development. Our inventive strategy allows for an uninhibited tumorigenesis process, allowing normal vasculature, extracellular matrix and immune interaction development, similar to the tumor development process in patients. In a first deployment of our innovative framework we aim to prioritize therapeutically targetable genes driving a model of hepatocellular carcinoma (HCC), a deadly cancer with poor 5-year survival in patients, where currently only a small subset of patients benefits from immune checkpoint therapy. Recent studies demonstrate that combination therapies in HCC can be more effective than single agents. Testing all pairwise combinations of promising targets is prohibitively costly and experimentally intractable. Leveraging our proposed methodology, we will identify promising combinations of therapeutic targets by performing higher order genetic CRISPR screening in vivo. 1

RFA-RM-21 - 016：新创新者奖-项目摘要。Jellert T.戈布隆 项目摘要 为了了解细胞行为如何影响发病机制，我们需要研究天然组织中的细胞 环境只有这样，我们才能阐明通过细胞间相互作用接收的信号的影响 微环境因素。例如，肿瘤通过沉默T细胞来逃避免疫介导的清除， 其接收来自癌细胞、基质细胞、巨噬细胞和细胞外基质的信号，仅举几例。 这些众多的组织微环境影响并没有被最常用的体外研究所捕获 在药物筛选治疗靶点时。为了广泛地绘制出 肿瘤微环境，以及导致免疫抑制的细胞间信号传导 环境，我将开发和应用一种方法，配置文件的转录组和蛋白质组状态 细胞在原位。一旦肿瘤微环境被绘制出来，关键是要识别出驱动肿瘤微环境的信号基因。 免疫抑制靶向免疫调节信号传导可以产生治疗突破， 免疫检查点抑制的最新进展。最近开发的体内合并CRISPR筛选是一种新的方法。 强大的高通量模式来识别遗传调节因子。然而，迄今为止，这些测定需要细胞 在分析之前与其天然组织背景分离。我们的建议是开创一种筛选方法 这使得能够在天然细胞中在单细胞水平上光学原位鉴定CRISPR扰动， 组织背景，使我们能够研究组织结构和细胞间的相应变化 交互.我们的方法是准备阐明机制的见解，并选择最有前途的 治疗目标治疗性治疗实质上是对未受干扰地发展的肿瘤的干扰。 然而，在目前的体内筛选中，肿瘤产生于被筛选基因干扰的细胞， 损害正常肿瘤发生和天然肿瘤微环境的发展。另一个关键 我们方案的创新之处在于将肿瘤诱导与我们干扰信号传导的时间解耦 我们正在筛选的基因为了实现时间解耦，我们在一个时间点处诱导CRISPR扰动。 肿瘤发展的理想阶段。我们的发明策略允许不受抑制的肿瘤发生 过程，允许正常的脉管系统，细胞外基质和免疫相互作用的发展，类似 肿瘤在患者体内的发展过程。在我们创新框架的首次部署中，我们的目标是 优先考虑驱动肝细胞癌（HCC）模型的治疗靶向基因， 患者的5年生存率很低， 目前只有一小部分患者受益于免疫治疗， 检查点疗法最近的研究表明，在HCC中，联合治疗可能比 单一代理测试有希望的靶的所有成对组合是极其昂贵的， 实验上难以处理利用我们提出的方法，我们将确定有前途的 通过在体内进行高阶遗传CRISPR筛选，来确定治疗靶点的组合。 1