(G-242) Matrix stiffness suppresses growth but induces genomic instability and variation in cancer spheroids

Introduction:: Genomic instability, the inability of a cell to pass on its genetic information accurately, is a hallmark of cancer. Aneuploidy, or an abnormal number of chromosomes, is observed in approximately 85% of solid tumors. During cancer progression, changes in the mechanical microenvironment can physically confine cancer cells and induce errors in DNA replication or mitosis. Using chromosome reporter cell lines, we investigated whether a three-dimensional (3D) tumor microenvironment contributes to aneuploidy and found that increased matrix stiffness increases mitotic aberrations that may lead to chromosome missegregation. Our findings indicate that stiff microenvironments suppress mitosis, increase micronuclei, and enhance chromosome loss, thus highlighting a potential role for mechanical confinement in chromosome segregation.

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 media or in agarose hydrogels of varying stiffnesses to generate cancer spheroids. Confocal microscopy was employed to assess mitotic events, micronuclei formation, and chromosome loss in each spheroid.

Results, Conclusions, and Discussions:: Using chromosome reporter cell lines, we had previously demonstrated that physical confinement prevents mitotic rounding and results in chromosome missegregation in lung adenocarcinoma cells. Here we demonstrate that increased matrix stiffness leads to decreased spheroid growth while enhancing micronuclei and chromosome loss. We also show that stiff microenvironments induce genomic variation and heterogeneity, a factor that is critical in tumor treatment response. In addition, our data demonstrate that chromosome loss is heritable and can be propagated through generations. Overall, our findings indicate that the mechanical properties of the tumor microenvironment regulate genomic instability, a deeper understanding of which could contribute to synergistic treatments for cancer. Future work will uncover the mechanotransduction pathways that regulate chromosome loss in stiff microenvironments.


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