Genetic variation drives cancer cell adaptation to ECM stiffness

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. 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 employed experimental evolution to study the response of genetically variable and clonal populations of tumor cells to variable ECM stiffness. Proliferation rates of genetically variable populations cultured on soft ECM increased over a period of several weeks, whereas clonal populations did not evolve. Tracking of DNA barcoded cell lineages revealed that soft ECM consistently selected for the same few variants. These data provide evidence that ECM stiffness exerts natural selection on genetically variable tumor populations. Soft-selected cells were highly migratory, with enriched oncogenic signatures and unusual behaviors such as spreading and traction force generation on ECMs with stiffness as low as 1 kPa. Rho-regulated cell spreading was found to be the directly selected trait, with yes-associated protein 1 translocation to the nucleus mediating fitness on soft ECM. Overall, these data show that genetic variation can drive cancer cell adaptation to ECM stiffness.

Keywords: ECM stiffness; cancer; mechanoadaptation; mechanobiology.

Fig. 1.

Evidence of cancer cell evolution on soft model ECMs. (A) Schematic of the evolution experiment. (B) MDA-MB-231 human breast carcinoma cells were cultured on collagen type I conjugated soft (red, E = 1 kPa), intermediate stiff (Int. stiff; hollow, E = 22 kPa), or stiff (blue, E = 308 kPa) hydrogels for up to 75 to 90 d. Mean growth rate (GR) is shown as a measure of fitness of selected lines measured at different times during the sustained culture in genetically variable ancestral populations (Left) or clonal lines (Right). Error bars, SEM (10 replicate lines on all stiffness). Statistically significant differences between growth rates at each time-point were determined by the Mann–Whitney U test, *P < 0.05; nonsignificant (NS): P > 0.05. (C) Nonlinear regression of equation from deterministic selection theory (dashed line) to the generation-dependent growth rate of populations (circles) measured on soft ECM in B. Error bars, SEM (10 replicates). (D) MDA-MB-231 cells labeled with heritable DNA barcodes underwent the same selection as in A. Mean growth rate is shown of barcoded MDA-MB-231 lines cultured on the stiff (blue) and soft (red) ECM, measured at multiple time points during sustained 7-wk culture. Error bars, SEM (five replicates on each stiffness). *P < 0.05; NS: P > 0.05 by the Mann–Whitney U test. (E) Abundance of barcoded MDA-MB-231 clones was identified with targeted amplification and sequencing of DNA barcode tags, shown in dot plot of sequences ordered by total abundance within the ancestral population. Dots are sized according to percent abundance. The bar plot to the right is the total number of stiffness-selected samples each lineage is found in. The four labeled barcodes have a higher average change in abundance from the ancestral population in the soft-selected cells compared to the stiff-selected cells. (F) Unique clonal barcode sequences (lineages) in the ancestral population (black), soft-selected group (red; five replicates), and stiff-selected group (blue; five replicates). (G) Percent total abundance of four clones with a positive average log₂(fold change) on soft ECM. Dots represent percent abundances of individual replicates in the respective populations.

Fig. 2.

Soft-selected cells spread, assemble F-actin fibers, and exhibit YAP in the nucleus on soft ECM. (A) Representative images of DNA (blue), F-actin (green), lamin B1 (magenta), and YAP (cyan) in ancestral, soft- or stiff-selected, and clonal cells cultured on stiff or soft ECM (here, soft refers to 1 kPa, and stiff refers to 308 kPa gels). (Scale bar: 50 μm.) Corresponding quantification of cell spreading area (B) and nuclear to cytoplasmic YAP intensity ratio (C) is shown of ancestral cells and soft- or stiff-selected MDA-MB-231 cells cultured on stiff and soft ECM. Mean values are calculated from >100 cells from three replicate lines. Error bars, SEM. *P < 0.05; NS: P > 0.05 by ordinary one-way ANOVA. (D) Representative images of DNA (blue), F-actin (green), and β1-integrin (ITGB1; magenta) in ancestral and soft-selected cells both cultured on soft ECM. Enlarged views show F-actin fibers terminating in integrin-marked adhesions. (Scale bar: 50 μm.) (E) Mean growth rate is shown of selected populations after 8-wk selection on soft or stiff ECM, followed by culture on soft or stiff ECM. Error bars, SEM (six replicates on both stiffness). *P < 0.05; NS: P > 0.05 by ordinary one-way ANOVA. Quantification of cell spreading area (F) and nuclear to cytoplasmic YAP intensity ratio (G) in clonal cells cultured on soft ECM at day 0 and day 75. Mean values are calculated from >80 cells from three replicate lines. Error bars, SEM. *P < 0.05; NS: P > 0.05 by the Mann–Whitney U test.

Fig. 3.

Rho-regulated cell spreading is the directly selected trait while YAP mediates fitness on soft ECM. (A) Representative differential interference contrast images and bead displacement heatmaps of ancestral and soft-selected cells cultured on soft ECM. (Scale bar: 20 μm.) (B) Normalized surface tension calculated from displacement field. Error bars, SEM (13 replicates). *P < 0.05 by the Mann–Whitney U test. (C) Normalized RhoA activation quantified using the G-LISA^® assay of ancestral and soft-selected cells cultured on tissue culture plastic. Error bars, SEM (data were collected from three replicate lines with two technical replicates per line). *P < 0.05 by the Mann–Whitney U test. (D) Representative images of DNA (blue), F-actin (green), and YAP (cyan) in soft-selected cells cultured on soft ECM under treatment with C3 transferase (C3), AIIB2 antibody, or verteporfin (VP). (Scale bar: 50 μm.) (E) Mean cell spreading area and mean growth rate in soft-selected cells cultured on soft ECM (control) under treatment with C3 transferase (C3), AIIB2 antibody, VP, or Rho activator II (RhoActII). Error bars, SEM. Mean spreading area is calculated from >80 cells in three replicate lines; growth rates were measured in at least four replicate lines. (F) Migration and (G) invasion of MDA-MB-231 cells were quantified using the Boyden chamber assay with a serum gradient. Error bars, SEM (10 replicate lines pooled into five groups for selected lines, and five biological replicates were analyzed for ancestral cells). *P < 0.05; NS: P > 0.05 by ordinary one-way ANOVA. (H) Fold change of RhoA GTPase-activating protein and Guanine nucleotide exchange factor gene expression in soft-selected cells cultured on soft ECM compared to ancestral cells cultured on soft ECM. Error bars, SEM (four replicates). *P < 0.05 by the Mann–Whitney U test. (I) The schematic diagram shows the selection mechanism for certain variants to spread and proliferate more on soft ECM.