Mechanics of Cells & Tissues impact Chromosome Instability & Phagocytic Interactions

项目摘要-项目2 细胞和组织的力学影响染色体不稳定性和吞噬相互作用 肿瘤通过从具有高水平遗传变异的多样化细胞群体中进行选择而发生遗传进化。一 常见的原因是由于有丝分裂分离受损导致的染色体不稳定性（CIN）。奇怪的是，有丝分裂错误 通常与有丝分裂核心过程中涉及的基因突变无关。我们的整体 假设是外部机械因素-特别是3D组织/肿瘤结构及其刚度- 有助于CIN和非整倍体的免疫雕刻进化。传统的癌细胞2D培养 在阐明大多数肿瘤抑制基因和癌基因的作用方面受到限制。我们的体外和体内研究 因此，将合作扩展到3D的一些关键的DNA损伤/有丝分裂和免疫信号的研究 以及项目3（Shin/Haldar）对肿瘤的骨髓中心效应。中央 我们研究的组成部分也利用了两个独特的核心（布莱克，切诺维斯）。 组织结构破坏，这在上皮癌中很常见，并且粘附的丧失导致有丝分裂， 错误，但这些细胞外信号如何耦合到内部有丝分裂过程尚不清楚。我们建议 当张力从细胞外基质传播到皮质再到 有丝分裂纺锤体并最终转化为动粒。我们将检验粘附力收紧机械结构的假设， 通过操纵细胞外或运动舞蹈微管张力的偶联，使用哺乳动物人工 染色体（MACs，Black）和化学光遗传学工具（Chenoweth）。我们还将测试 组织结构和整合素功能的丧失产生了脆弱性，因为细胞更依赖于其他细胞， 维持张力的途径，如皮质变圆或纺锤体内的微管交联。 包括细胞质RNA积累在内的基因组不稳定性输出将是主要措施， 信号通路（与格林伯格）通过先天免疫。 实体瘤充满了巨噬细胞，它们对来自附近肿瘤细胞的许多信号做出反应， 实体肿瘤的限制和收缩刚度的耦合效应是未知的。我们将调整， 通过“巨噬细胞检查点”破坏（癌细胞上的CD 47; 巨噬细胞），以测试这种基本的巨噬细胞相互作用（已经在临床试验中）调节 并受癌症基因组变异的调节。单细胞分析将评估免疫亚型和信号传导 我们将干扰（与Shin/Haldar）的相互作用。我们将主要研究这些过程， 免疫活性的、同基因的B16小鼠黑素瘤模型中，而且在卵巢癌模型中（具有 Greenberg）。我们试图确定3D肿瘤组织硬度是否会增加肿瘤的遗传变异， 特别是通过CIN启动的信号传导，例如通过微核（与Greenberg）。我们还将确定 巨噬细胞检查点破坏的影响（包括来自Chenowith的拮抗剂肽），基于 初步数据已经证明了抗肿瘤IgG的持久治疗。

Project Summary – Project 2 Mechanics of Cells & Tissues impact Chromosome Instability & Phagocytic Interactions Tumors evolve genetically via selection from a diverse population of cells with high levels of genetic variation. A common cause is chromosome instability (CIN) due to impaired mitotic segregation. Paradoxically, mitotic errors do not typically associate with mutations in genes involved in the core processes of mitosis. Our overall hypothesis is that extrinsic mechanical factors – particularly 3D tissue/tumor architecture and its rigidity – contribute to CIN and to the immune-sculpted evolution of aneuploidy. Conventional 2D cultures of cancer cells are limited in elucidating roles for most tumor suppressor genes and oncogenes. Our in vitro and in vivo studies will therefore collaboratively extend to 3D some of the key DNA damage/mitotic and immune signaling studies of project 1 (Greenberg) as well as the myeloid-centric effects on tumors of project 3 (Shin/Haldar). Central components of our studies also make use of two unique cores (Black, Chenoweth). Disrupted tissue architecture, which is common in epithelial cancers, and loss of adhesion lead to mitotic errors, but how these extracellular signals couple to internal mitotic processes is unclear. We propose that the external and internal mechanics are linked as tension propagates from extracellular matrices to the cortex to the mitotic spindle and ultimately to kinetochores. We will test the hypothesis that adhesion tightens the mechanical coupling by manipulating either extracellular or kinetochore-microtubule tension, using mammalian artificial chromosomes (MACs, with Black) and chemical optogenetic tools (with Chenoweth). We will also test whether loss of tissue architecture and integrin function creates a vulnerability as cells are more dependent on other pathways for maintaining tension, such as cortical rounding or microtubule crosslinking within the spindle. Genome instability outputs including cytoplasmic RNA accumulation will be primary measures, extending to signaling pathways (with Greenberg) via innate immunity. Solid tumors are filled with macrophages that respond to numerous signals from nearby tumor cells, but coupled effects of the confining and constricting rigidity of solid tumors are unknown. We will modulate and visualize phagocytic interactions via ‘macrophage checkpoint’ disruption (CD47 on the cancer cell; SIRPa on the macrophage) to test the hypothesis that this basic macrophage interaction (already in clinical trials) modulates and is modulated by cancer genome variation. Single cell analyses will assess immune subtypes and signaling interactions, which we will perturb (with Shin/Haldar). We will study the processes primarily in immunocompetent, syngeneic B16 mouse melanoma model but also in an ovarian cancer model (with Greenberg). We seek to determine whether 3D tumor tissue rigidity increases a tumor’s genetic variation, particularly via CIN-initiated signaling, such as by micronuclei (with Greenberg). We will also determine coupled effects of macrophage checkpoint disruption (including antagonist peptides from Chenowith), based on preliminary data that already demonstrates durable cures with production of anti-tumor IgG.