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细胞的相互作用 肿瘤通过从遗传变异水平较高的不同细胞群体中进行选择来进行遗传进化。一个 常见的原因是染色体不稳定(CIN)，这是由于有丝分裂分离受损所致。矛盾的是，有丝分裂错误 通常不会与涉及有丝分裂核心过程的基因突变联系在一起。我们的整体 假设是外在机械因素--特别是3D组织/肿瘤结构及其刚性-- 有助于CIN和非整倍体的免疫雕刻进化。癌细胞的常规2D培养 在阐明大多数抑癌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研究 项目1(Greenberg)以及项目3(Shin/Haldar)的以髓系为中心的肿瘤效应。中环 我们研究的组件还利用了两个独特的核心(Black，Chenoweth)。 组织结构紊乱，这在上皮性癌症中很常见，粘连丧失会导致有丝分裂 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血管时，外部和内部机制相互联系 有丝分裂纺锤体，最终形成动粒。我们将检验粘合使机械密封的假设 通过操纵细胞外或动粒-微管张力进行偶联，使用哺乳动物人工 染色体(Mac，黑色)和化学光遗传工具(Chenoweth)。我们还将测试 组织结构和整合素功能的丧失造成了脆弱性，因为细胞更依赖于其他 维持张力的途径，例如纺锤体内的皮质圆化或微管交联。 包括细胞质RNA积累在内的基因组不稳定输出将是主要衡量标准，扩展到 信号通路(与格林伯格一起)通过先天免疫。 实体瘤充满了巨噬细胞，这些巨噬细胞对附近肿瘤细胞发出的大量信号做出反应，但 实体瘤的约束和收缩刚性的耦合效应尚不清楚。我们将调整和调整 通过破坏巨噬细胞检查点可视化吞噬细胞的相互作用(癌细胞上的CD47；癌细胞上的SIRPA 巨噬细胞)来测试这种基本的巨噬细胞相互作用(已经在临床试验中)调节的假设 并受癌症基因组变异的影响。单细胞分析将评估免疫亚型和信号 互动，我们将扰乱(与Shin/Haldar)。我们将主要在以下方面研究这些过程 免疫活性的同基因B16小鼠黑色素瘤模型，以及卵巢癌模型(与 格林伯格)。我们试图确定3D肿瘤组织刚性是否会增加肿瘤的遗传变异， 特别是通过CIN启动的信号，例如微核(与Greenberg一起)。我们还将确定耦合 巨噬细胞检查点破坏的影响(包括来自Chenowith的拮抗肽)，基于 初步数据已经证明，通过生产抗肿瘤免疫球蛋白可以持久治愈。

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.