Deconvoluting myeloid gene regulation through functional genomics

项目摘要 巨噬细胞（MF）是肿瘤病变中最大的免疫细胞成分之一，它们的数量 甚至可以超过肿瘤细胞。尽管MF对于组织稳态和感染控制很重要，但在 肿瘤MF采取赋予它们促肿瘤发生和免疫抑制功能的表型状态。 因此，MF是癌症治疗的目标。然而，调节肿瘤相关MF已经证明， 难这在很大程度上是因为我们仍然没有一个完整的了解基因，控制 MF生物学的许多关键方面，因此我们仍然缺乏用于调节相关MF功能的最佳靶标。 本项目的目的是鉴定驱动人类效应免疫活性和抑制免疫活性的基因。 MF。在人非小细胞肺癌（NSCLC）的单细胞RNA-seq分析中，我的申办方实验室确定了 与邻近健康肺组织中的MF相比，肿瘤相关MF中上调的基因的大模块。 这包括许多具有免疫抑制功能的基因，但对于大多数已鉴定的基因， 这些职能，特别是可能与豁免有关的职能，不得而知。几乎一半的已识别基因 编码在MF生物学中具有潜在上游调节作用的因子，如转录因子（TF）， 信号成分和细胞表面受体。 在这个项目中，我将测试的假设，即免疫抑制活性的肿瘤相关的MF驱动 通过调节因子和信号成分上调肿瘤内MF。为了验证我的假设，我 将利用我的赞助实验室开发的一种新型CRISPR基因组学平台， 多个CRISPR敲除同时和单细胞分辨率的表型分析（Wroblewska，Dhainaut 等人。Cell 2018）。我将系统性地敲除肿瘤相关的MF标记基因，并确定如何 干扰影响细胞的关键表型和功能，包括检查点配体的表达， 调节T细胞活化。通过鉴定控制MF身份以及效应子和抑制子功能的基因， 该项目的结果将为主要细胞之一的分子力学提供新的见解 控制肿瘤微环境中免疫应答的方向，并提供分子靶点 用于旨在增强抗肿瘤免疫力的治疗干预。

PROJECT SUMMARY Macrophages (MF) are one of the largest immune cell components of tumor lesions, where their numbers can even exceed tumor cells. Though MF are important for tissue homeostasis and control of infections, in tumors MF adopt a phenotypic state which confers them with pro-tumorigenic and immunosuppressive functions. Because of this, MF are a target for cancer treatment. However, modulating tumor-associated MF has proved difficult. This is in large part because we still do not have a complete understanding of the genes that control many key aspects of MF biology, and thus we still lack an optimal target for modulating relevant MF functions. The objective of this project is to identify genes that drive effector and suppressor immune activity of human MF. In single cell RNA-seq analysis of human non-small cell lung carcinoma (NSCLC), my sponsor labs identified a large module of genes upregulated in tumor-associated MF compared to MF in adjacent healthy lung tissue. This included a number of genes with immunosuppressive functions, but for a majority of the identified genes, the functions, particularly as they may relate to immunity, are not known. Almost half of the identified genes encode for factors with potential upstream regulatory roles in MF biology, such as transcription factors (TF), signaling components, and cell surface receptors. In this project, I will test the hypothesis that the immunosuppressive activity of tumor-associated MF is driven by the regulatory factors and signaling components upregulated on intratumoral MF. To test my hypothesis, I will utilize a novel CRISPR genomics platform my sponsor lab developed, which enables high-dimensional phenotyping of multiple CRISPR knockouts simultaneously and with single cell resolution (Wroblewska, Dhainaut et al. Cell 2018). I will systematically knockout the tumor-associated MF signature genes and determine how perturbation impacts key phenotypes and functions of the cells, including expression of checkpoint ligands and modulation of T cell activation. By identifying genes that control MF identity and effector and suppressor function, the outcome of this project will provide new insights in to the molecular mechanics of one of the major cells controlling the direction of the immune response in the tumor microenvironment and provide molecular targets for therapeutic interventions aimed at enhancing anti-tumor immunity.