Genomic heterogeneity drives mechanical adaptation in human tumor cells [RRBS]
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. Overall design: Because selection on soft ECM may be mediated at least in part by epigenetic modifications in cells, we determined the methylation levels at CpG sites in 4 sample groups  (ancestral cultured on stiff, ancestral cultured on soft, stiff-selected cultured on stiff, soft-selected cultured on soft). Methylation status was assessed using enhanced reduced representation bisulfite sequencing  (ERRBS), a technique that combines enzymatic digestion with bisulfite conversion to achieve quantitative, base-pair resolution of cytosine methylation at CpG sites throughout the genome.