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

RFA-RM-21-016：新创新者奖-项目摘要。杰勒特·T·高布卢姆 项目总结 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到的信号的影响 和微环境因素。例如，肿瘤通过沉默T细胞来逃避免疫介导的清除， 它们接收来自癌细胞、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不是最常用的体外研究所能捕捉到的 在药物筛选治疗靶点期间。要广泛地绘制出 肿瘤微环境和导致免疫抑制的细胞间信号转导 环境，我将开发和应用一种描述转录和蛋白质组状态的方法 原位细胞的数量。一旦绘制了肿瘤微环境图，识别驱动肿瘤微环境的信号基因至关重要。 免疫抑制。靶向免疫调节信号可以产生治疗突破，这是有证据证明的。 免疫检查点抑制的最新进展。最近开发的体内集合式CRISPR屏幕是一种 用于识别基因调节器的强大的高通量模式。然而，到目前为止，这些检测需要细胞 在分析之前从它们的天然组织环境中分离。我们的建议是开创一种筛查方法 这使得能够在原生的单细胞水平上光学原位识别CRISPR扰动 组织环境，使我们能够研究组织结构和细胞间的相应变化 互动。我们的方法准备阐明机械论的洞察力，并选择最有希望的 治疗靶点。治疗性治疗实质上是对未受干扰的肿瘤的扰动。 然而，在目前的体内筛查中，肿瘤是由对筛选基因感到不安的细胞产生的，因此 危及正常的肿瘤发生和自然肿瘤微环境的发展。另一把钥匙 我们建议的创新是将肿瘤诱导和我们干扰信号的时间脱钩 我们正在筛选的基因。为了实现时间解耦，我们在a处引入CRISPR扰动 肿瘤发展的理想阶段。我们的创造性策略允许不受抑制的肿瘤发生 过程，允许正常的血管系统、细胞外基质和免疫相互作用的发展，类似 与患者肿瘤的发展过程有关。在我们创新框架的第一次部署中，我们的目标是 优先考虑导致致命性癌症--肝细胞癌模型的治疗靶向基因 由于患者的5年存活率很低， 目前只有一小部分患者从免疫中受益 检查点疗法。最近的研究表明，联合疗法在治疗肝癌方面可能比 单一特工。测试所有有希望的目标的成对组合的成本高得令人望而却步 在实验上很难解决。利用我们提出的方法，我们将确定有希望的 通过在体内进行更高级别的遗传CRISPR筛查来组合治疗靶点。 1

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