Mechanical properties of the tissue microenvironment, such as confinement and extracellular matrix (ECM) stiffness, play a key role in regulating cell behavior. During tumor progression, ECM stiffening alters tissue mechanics with poorly understood consequences for chromosome segregation, genome stability, and immune function. Here, we investigated whether mechanical confinement promotes genome instability and alters immune cell dynamics in a 3D engineered tissue culture model. Using cancer cells engineered to express a GFP-tagged Lamin B1 reporter on one allele of chromosome 5 (ChReporter), we cultured spheroids in methylcellulose or agarose hydrogels of tunable stiffness. In stiff hydrogels reminiscent of tumor-like ECM, we observed increased chromosome missegregation, micronuclei formation, and inter-spheroid variance, as visualized by confocal microscopy and flow cytometry. Colonies of reporter-negative cells emerged in a manner consistent with Luria and Delbrück’s theory of heritable genetic change, indicating that mechanical stress promotes genetic diversification. We also found that a known mechanosensor, myosin-II, contributes to chromosome loss without affecting spheroid growth, consistent with its role as a putative tumor suppressor. Pan-cancer genomic analyses revealed that chromosome number variation across patients correlates with elevated collagen-I expression, linking matrix stiffening to genome instability in clinical samples. We also investigated immune behavior by tracking macrophage migration through mechanically-confined tumor spheroids, and found that confinement altered macrophage dynamics and their ability to engulf cancer cells, revealing potential effects on immune surveillance. Altogether, these findings reveal that ECM stiffness and confinement suppress mitosis, promote genome instability, and disrupt immune function, contributing to tumor heterogeneity. Moving forward, this biophysical framework will be applied to tissues that undergo cyclical remodeling and mechanical changes, such as those relevant to women’s health, with the goal of developing predictive models of tissue dysfunction across health and disease.
