Live cell reporters of genetic changes in stiff vs soft surroundings - Causes & Consequences

僵硬与柔软环境中遗传变化的活细胞报告 - 原因

• 批准号: 10594852
• 负责人: Dennis E. Discher
• 金额: $ 6.66万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594852
• 关键词: Affect; Alleles; Aneuploid Cells; Aneuploidy; Behavior; Cells; Characteristics; Chromosomal Gain; Chromosomal Instability; Chromosomal Loss; Chromosome abnormality; Chromosomes; Eating; Engineering; Evolution; Fluorescence; Genes; Genomic Instability; Genomics; Growth; Heterogeneity; Immune; Immune response; Immune system; Immunobiology; Immunotherapy; In Vitro; Malignant Neoplasms; Mediating; Methods; Molds; Mutation; Paracrine Communication; Phagocytes; Phagocytosis; Phenotype; Proteins; Reporter; Research; Resistance; Signal Pathway; Solid Neoplasm; Therapeutic; cancer cell; chromosome number abnormality; in vivo Model; macrophage; mouse model; response; single cell sequencing; tumor

Project Summary/Abstract Aneuploidy is defined as the existence of an abnormal number of chromosomes in a cell, and it has long been a defining characteristic of cancers. 90% of all solid tumors, for example, show chromosomal gains/losses. Gaining and/or losing chromosomes generates phenotypic heterogeneity within tumors that can be beneficial to survival by creating subpopulations that may become resistant to therapeutics, providing new mechanisms to evade immune cells, facilitating growth and proliferation in challenging microenvironments, among other advantages. However, we currently struggle to easily identify aneuploid cells—typically we need to kill them for sequencing, meaning that we can only at best infer possible causes, consequences, and possible long-term viability and evolution. This also limits our ability to identify how aneuploid cells interact with other cells and study if and how these interactions differ from other “less” aneuploid cells. These interactions become increasingly important when the engaging cell is from the immune repertoire. In this proposed research, we tackle two aspects of aneuploidy: (1) providing a simple, effective method to easily identify aneuploidy in live cells and (2) understanding how aneuploidy can modulate macrophage- mediated phagocytosis of cancer cells. We already have successfully generated several chromosome reporters, in which we fuse fluorescent proteins to only single alleles of constitutively expressed genes. For these appropriate genes, we find that fluorescence loss equates to chromosomal loss—thoroughly characterized from genomic PCR to deep single-cell sequencing. We further seek to expand this toolkit to make it accessible to more appropriate mouse models. On the immunobiology end, we already find that high levels of aneuploid help promote an initial macrophage-immune response. This suggests that initial stages of aneuploidy (before a cancer can adapt and evolve to tolerate it) are susceptible to macrophage clearance. Although seemingly two distinct ideas, we finally plan to merge our reporter approach in analyzing how macrophages are phenotypically molded by aneuploid cells that we can easily identify. Do macrophages eat/clear these reporter-negative aneuploid cells? Are reporter-negative cells simply more susceptible to macrophages or are profound paracrine signaling pathways involved? We will extend our studies to both in vitro and in vivo models to better characterize macrophage response under these circumstances, and we will also ultimately combine these studies with powerful single-cell sequencing to see how aneuploidy sculpts macrophage behavior and their overall effect on the immune landscape.

项目概要/摘要 非整倍体被定义为细胞中存在异常数量的染色体，长期以来一直被认为是一种非整倍体。 定义癌症的特征。例如，90% 的实体瘤都显示染色体增加/丢失。获得 和/或丢失染色体会在肿瘤内产生表型异质性，这可能有利于生存 通过创造可能对治疗产生耐药性的亚群，提供新的逃避机制 免疫细胞，在具有挑战性的微环境中促进生长和增殖等优点。 然而，我们目前很难轻松识别非整倍体细胞——通常我们需要杀死它们进行测序， 这意味着我们最多只能推断可能的原因、后果以及可能的长期生存能力和 进化。这也限制了我们识别非整倍体细胞如何与其他细胞相互作用以及研究是否以及如何相互作用的能力 这些相互作用不同于其他“较少”的非整倍体细胞。这些互动变得越来越重要 当参与细胞来自免疫库时。 在这项拟议的研究中，我们解决了非整倍性的两个方面：（1）提供了一种简单、有效的方法 轻松识别活细胞中的非整倍性，以及（2）了解非整倍性如何调节巨噬细胞 介导癌细胞的吞噬作用。我们已经成功生成了几个染色体报告基因， 其中我们将荧光蛋白仅与组成型表达基因的单个等位基因融合。对于这些 适当的基因，我们发现荧光损失等同于染色体损失——彻底表征为 基因组 PCR 到深度单细胞测序。我们进一步寻求扩展该工具包，使其易于使用 更合适的鼠标型号。在免疫生物学方面，我们已经发现高水平的非整倍体有助于 促进初始巨噬细胞免疫反应。这表明非整倍体的初始阶段（在癌症之前） 可以适应并进化以耐受它）容易受到巨噬细胞清除的影响。虽然看似截然不同的两种 想法，我们最终计划合并我们的报告方法来分析巨噬细胞的表型塑造方式 通过我们可以轻松识别的非整倍体细胞。巨噬细胞会吞噬/清除这些报告基因阴性的非整倍体细胞吗？ 报告阴性细胞只是更容易受到巨噬细胞的影响还是具有深刻的旁分泌信号传导 涉及途径？我们将把我们的研究扩展到体外和体内模型，以更好地表征 在这种情况下巨噬细胞的反应，我们最终也会将这些研究与 强大的单细胞测序来了解非整倍体如何塑造巨噬细胞的行为及其对 免疫景观。