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Genomic heterogeneity drives mechanical adaptation in human tumor cells [RNA-seq]

ABSTRACT: The progression of many solid tumors is accompanied by temporal and spatial changes in the stiffness of the extracellular matrix (ECM). Cancer cells adapt to soft and stiff ECM through mechanisms that are not fully understood. In particular, it is well known that there is significant genetic heterogeneity from cell to cell in tumors, but how ECM stiffness as a parameter might interact with that genetic variation is not known. Here, we used the method of experimental evolution to study response of genetically variable and clonal tumor cell populations to ECM stiffness. Cell fitness increased on soft ECM over a period of several weeks in genetically variable but not clonal populations. DNA barcode-enabled clonal tracking revealed that sustained culture on soft ECM selects for a few genetic variants. These data provide the first evidence that ECM stiffness exerts natural selection on genetically variable tumor populations. Genome-wide analysis including RNA-seq, ATAC-seq and DNA methylation profiling reveal substantial differences in gene expression between selected populations and ancestral cells which are partially explained by epigenetic modifications. Soft-selected cells are highly migratory with enriched oncogenic signatures and exhibit highly unusual behaviors like spreading and traction force generation on ECMs as soft as 1 kPa. Cell spreading is the directly selected trait but not levels of integrins or adhesion proteins like talin. Overall, these data show that ECM stiffness in solid tumors may drive malignant behaviors through evolution by natural selection.

ORGANISM(S): Homo sapiens

PROVIDER: GSE255829 | GEO | 2024/06/17

REPOSITORIES: GEO

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Project description:Background: The differentiation of pericytes into myofibroblasts causes microvascular degeneration, extracellular matrix (ECM) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel two-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions. Methods: Primary microvessels were cultured in the TGM2D medium. Stiff ECM was prepared by incubating ECM solution in regular culture dishes for one hour followed by PBS wash. Soft ECM with Young’s modulus of approximately 6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, qRT-PCR, western blotting, and knockdown experiments were performed on the cells. Results: Primary microvascular pericytes differentiated into myofibroblasts (NG2+αSMA+) on stiff ECM, even with the TGFβ signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined two pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As Integrins are the major mechanosensor, we performed qRT-PCR screening of Integrin family members selected from RNA sequencing data. We found that Integrin β1 (Itgb1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF, to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation. Conclusions: Microvascular ECs mediate ECM stiffness-induced pericyte-myofibroblast differentiation through paracrine signaling.

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Genomic heterogeneity drives mechanical adaptation in human tumor cells [RNA-seq]

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