Mechanics of Cells & Tissues impact Chromosome Instability & Phagocytic Interactions

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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.