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

项目摘要/摘要 非整倍体的定义是细胞中存在异常数量的染色体，长期以来，它一直是一种 癌症的定义特征。例如，90%的实体肿瘤显示出染色体的增减。收获 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抵抗力的亚群，提供新的逃避机制 免疫细胞，促进在具有挑战性的微环境中的生长和增殖，以及其他优势。 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、后果和可能的长期生存能力 进化论。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 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 当参与的细胞来自免疫系统时。 在这项拟议的研究中，我们解决了两个方面的问题：(1)提供了一种简单、有效的方法 为了容易地识别活细胞中的非整倍体，以及(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记者， 我们只将荧光蛋白融合到构成表达的基因的单个等位基因上。为了这些 适当的基因，我们发现荧光丢失等同于染色体丢失--从 从基因组聚合酶链式反应到深度单细胞测序。我们进一步寻求扩展此工具包，使其可供 更合适的鼠标模型。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 促进最初的巨噬细胞免疫反应。这表明非整倍体的初始阶段(癌症之前) 可以适应和进化以耐受它)对巨噬细胞清除敏感。虽然看起来是两个截然不同的 想法，我们最终计划结合我们的记者的方法来分析巨噬细胞是如何表型塑造的 通过我们可以很容易识别的非整倍体细胞。巨噬细胞是否吞噬/清除这些报告阴性的非整倍体细胞？ 报告阴性细胞是更容易受到巨噬细胞的影响，还是更容易受到旁分泌信号的影响？ 涉及到的路径吗？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 巨噬细胞在这种情况下的反应，我们最终也会将这些研究与 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及其对 免疫景观。

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.