Poster T5 - Extracellular matrix stiffness increases chromosome loss and genetic variation in cancer spheroids

Introduction: Chromosome losses often appear early in cancer, with solid tumors displaying more chromosome number variations compared to liquid and soft tumors. Stiff microenvironments physically restrain cell proliferation; however it is unclear if these lead to mistakes in division and chromosome losses. We hypothesized that increased extracellular matrix (ECM) stiffness induces chromosome loss and tested this hypothesis using live cell chromosome reporters (ChReporters) in cancer spheroids that are grown in 3D hydrogels of tunable stiffness. We find that matrix stiffness suppresses growth and size variation of cancer spheroids, but greatly increases mitotic aberrations and heritable chromosome loss with high variation – in agreement with seminal studies of Luria-Delbruck.

Materials and

Methods: H23 lung adenocarcinoma cells were engineered using the CRISPR/Cas9 system to express a GFP-tagged version of the constitutively expressed Lamin B1 on one allele of chromosome 5. H23 cells carrying the Lamin B1-GFP reporter were cultured in viscous methylcellulose or in agarose hydrogels of varying stiffnesses to form cancer spheroids. Confocal microscopy was employed to evaluate chromosome loss, mitotic events, and micronuclei formation within each spheroid. Flow analysis was used to verify chromosome loss for each population. Myosin-IIA was suppressed using blebbistatin or a knockdown approach, where non-muscle myosin-II isoform myosin-IIA (MYH9) was knocked down using shRNA (shMyh9) to assess its role in chromosome loss. Luria-Delbruck’s theory of heritable genetic changes was used to compare genetic variation rates of spheroids cultured in different stiffnesses. Additionally, pan-cancer data from the Cancer Genome Atlas were analyzed to determine how collagen expression correlates with chromosome number variations.

Results, Conclusions, and Discussions: We find that ECM stiffness suppresses mitosis but increases micronucleus formation (Figure 1B), which is a signature of chromosome missegregation. Consistently, stiffness increases chromosome loss, as evidenced by the loss of ChReporters, while colonies of ChReporter-negative cells reveal heritable genetic change (Figure 1B-C). Inter-spheroid variance in chromosome loss exceeds Poisson statistics and align with Luria-Delbruck’s theory for genetic changes. Our data suggest that the knockdown of a known mechanosensor, myosin-II, increases chromosome loss without affecting spheroid growth (Figure 1D). This is consistent with the role of myosin-II as a putative tumor suppressor, however our finding that myosin-II protects genomic integrity is novel. Futhermore, our pan-cancer analyses demonstrate that the inter-patient mean for chromosomal changes increases with median collagen-I expression, which correlates with tissue stiffness. Altogether, ECM stiffening in cancer initiation or progression suppresses growth but also induces heritable genetic changes and diversification. Our findings highlight mechano-evolution that drives tumor heterogeneity -a deeper understanding of which could contribute to synergistic cancer therapies.

Acknowledgements (Optional):