Robert D Bent Professor University of Pennsylvania Philadelphia, Pennsylvania, United States
Introduction: : The mechanical microenvironment undergoes dynamic changes during development and disease, shaping how cells perceive and respond to physical cues. In cancer, progressive stiffening of the extracellular matrix (ECM) is associated with chromosomal instability, as solid tumors often display aberrant chromosome numbers. Although chromosome loss is a hallmark of solid tumors, it remains unclear whether ECM stiffness drives mitotic errors that lead to heritable chromosome loss. Here, we used a 3D engineered tumor spheroid model to investigate if ECM stiffness regulates chromosome missegregation and loss, thereby contributing to tumor heterogeneity.
Materials and
Methods: : Spheroids of lung adenocarcinoma (H23) and mouse melanoma (B16) cells, each engineered to express a GFP-tagged Lamin B1 allele (ChReporter), were embedded in methylcellulose and agarose hydrogels with tunable stiffness to mimic that of tumors (Figure 1A-C). Chromosome missegregation, micronuclei formation, and reporter loss were quantified using confocal imaging and flow cytometry. Chromosome reporter loss was validated using single nucleotide polymorphism arrays (SNPa). To investigate mechanosensitive pathways, myosin-IIA was disrupted by pharmacological inhibition with blebbistatin or by shRNA knockdown (shMYH9). In parallel, statistical analyses based on Luria-Delbrück models were used to assess inter-spheroid variation in chromosome loss. Additionally, pan-cancer patient data from the Cancer Genome Atlas (TCGA) were analyzed to explore potential links between ECM composition, particularly collagen-I expression, and chromosomal instability across tumor types.
Results, Conclusions, and Discussions:: To determine whether ECM stiffness drives heritable chromosome loss, we cultured ChReporter-expressing lung adenocarcinoma (H23) or mouse melanoma (B16) spheroids in hydrogels of tunable stiffness. Confocal microscopy revealed that spheroids in stiffer matrices exhibited increased chromosome missegregation as evidenced by micronucleus formation. Similarly, confocal microscopy and flow cytometry revealed that a loss of the ChReporter signal (Figure 1D), which is an indicator of chromosome loss, increased in stiffer hydrogels (Figure 1E). Our data suggest that chromosome losses accumulated over time and were maintained across generations (Figure 1D), indicating stable genomic alterations. Given the role of MYHIIa as a putative mechanosensitive tumor suppressor, we perturbed myosin-IIa using blebbistatin or shMYH9 knockdown. This increased chromosome loss in a stiffness-dependent manner, implicating actomyosin contractility as a key mediator of stiffness-induced mitotic error. To further assess how ECM stiffness contributes to population-level genetic diversity, we applied Luria-Delbruck fluctuation analysis (Figure 1F) and observed increased inter-spheroid variation in chromosome loss in stiff hydrogels (Figure 1G), which was consistent with elevated rates of heritable genomic alterations. Finally, pan-cancer analysis of TCGA data revealed that high collagen-I expression, that is correlated with increased ECM stiffness, is associated with chromosomal instability across solid tumors. Together, these findings support a model in which ECM stiffness drives mitotic errors and promotes chromosome loss. By linking mechanical regulation of genome integrity to tumor heterogeneity, this work uncovers a new dimension of cancer progression shaped by the physical microenvironment.
