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.

项目摘要/摘要 非整倍性被定义为细胞中异常数量的染色体的存在，长期以来一直是一个 定义癌症的特征。例如，所有实体瘤中的90％均显示出染色体的收益/损失。获得 和/或失去染色体会在肿瘤内产生表型异质性，这可能有益于生存 通过创建可能对治疗有抵抗力的亚群，提供了新的机制来逃避 免疫细胞，支持挑战微环境的生长和增殖，以及其他优势。 但是，我们目前很难轻松地识别非整倍性细胞 - 从通常的角度来看，我们需要杀死它们进行测序， 这意味着我们只能最多可以推断出可能的原因，后果和可能的长期生存能力以及 进化。这也限制了我们确定非整倍体细胞与其他细胞相互作用的能力，并研究了如何以及如何 这些相互作用与其他“较少”的非整倍体细胞不同。这些互动变得越来越重要 当引人入胜的细胞来自免疫曲目时。 在这项拟议的研究中，我们解决了非整倍性的两个方面：（1）提供一种简单，有效的方法 为了轻松识别活细胞中的非整倍性，（2）了解各个方倍性如何调节巨噬细胞 - 介导的癌细胞吞噬作用。我们已经成功产生了几个染色体记者， 其中，我们将荧光蛋白融合到仅表达基因的单个等位基因。为此 适当的基因，我们发现荧光损失等于染色体损失，从而从染色体损失。 基因组PCR至深单细胞测序。我们进一步寻求扩展此工具包，以使其可访问 更合适的鼠标模型。在免疫生物学结束时，我们已经发现高水平的非整倍体帮助 促进初始巨噬细胞免疫反应。这表明非整倍性的初始阶段（癌症之前 可以适应和发展以耐受它）容易受到巨噬细胞清除的影响。虽然看似两个不同 想法，我们终于计划合并我们的记者方法，以分析巨噬细胞如何表型模制 通过非整倍体细胞，我们可以轻松识别。巨噬细胞会吃/清除这些记者阴性的非整倍体细胞吗？ 是报告基因阴性细胞更容易受到巨噬细胞的影响或是深层的旁分泌信号传导 涉及的途径？我们将把研究扩展到体外和体内模型，以更好地表征 在这种情况下，巨噬细胞反应，我们还将最终将这些研究与 强大的单细胞测序，以查看各向酶雕刻巨噬细胞行为及其对整体影响 免疫景观。