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.

项目总结/摘要 非整倍性是指细胞中存在异常数量的染色体，长期以来， 癌症的定义特征。例如，90%的实体瘤显示染色体获得/丢失。获得 和/或染色体丢失在肿瘤内产生表型异质性， 通过创造可能对治疗产生耐药性的亚群， 免疫细胞，促进在具有挑战性的微环境中的生长和增殖，以及其他优点。 然而，我们目前很难轻易地识别非整倍体细胞--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出可能的原因、后果和可能的长期可行性， 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它“较少的”非整倍体细胞。这些互动变得越来越重要 当接合细胞来自免疫库时。 在这项研究中，我们解决了两个方面的非整倍体：（1）提供一种简单，有效的方法 容易地识别活细胞中的非整倍体和（2）了解非整倍体如何调节巨噬细胞- 介导的癌细胞吞噬作用。我们已经成功地产生了几个染色体报告基因， 其中我们将荧光蛋白融合到组成型表达基因的单个等位基因上。为这些 适当的基因，我们发现，荧光损失等同于染色体损失-彻底的特点， 从基因组PCR到深度单细胞测序。我们进一步寻求扩大这一工具包，使其能够为 更合适的小鼠模型。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应答。这表明非整倍体的初始阶段（癌症发生前） 可以适应和进化以耐受它）对巨噬细胞清除敏感。虽然看似两个截然不同的 想法，我们最终计划合并我们的报告方法，分析巨噬细胞是如何表型塑造 我们可以很容易地识别的非整倍体细胞。巨噬细胞会吃掉/清除这些非整倍体细胞吗？ 嗜酸性粒细胞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 参与的路径？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 巨噬细胞反应在这些情况下，我们也将最终结合联合收割机这些研究， 强大的单细胞测序，以了解非整倍体如何塑造巨噬细胞的行为和它们对巨噬细胞的整体影响。 免疫系统