Fig. 5: CAFs compression of cancer cells triggers YAP nuclear exit.

a Patterned cancer cells (Cell tracker, Green) cultured on a collagen-coated polyacrylamide gel growing freely, under confinement (by a PDMS stencil), surrounded by control or Myosin IIA KO CAFs (F-Actin, phalloidin, magenta), or treated with blebbistatin (quantified in right dot plot). Cells are stained for YAP. Right upper panel: YAP nuclear correlation ranging from 0.5 (red, high correlation) to −0.5 (blue, anti-correlation). Lower panel: schematic representation of how correlations are performed. White line outlines the boundary of the cancer cell cluster. Scale bar, 100 µm. Right dot plot: mean YAP nuclear correlation of cancer cells in vitro. Data represented as mean ± SD. Free growth (n = 20 clusters, N = 5), confinement (n = 19 clusters, N = 5), CAFs control (n = 42 clusters, N = 10), CAFs αSMA:Myosin IIA^−/− (n = 17 clusters, N = 4), Blebbistatin (n = 12 clusters, N = 3). **p < 0.01, ***p < 0.001, Kruskal–Wallis ANOVA test, Dunns multiple comparisons test. b Tumors from control or αSMA:Myosin IIA KO mice showing cancer cells (nuclear-GFP, green), CAFs expressing myosin IIA (membrane-tdTomato, magenta) and myosin IIA KO CAFs (membrane-GFP, green), stained for YAP (yellow) and DNA (DAPI, blue). Bottom panel, YAP nuclear correlation. Scale bar, 100 µm. Right dot plot: mean YAP nuclear correlation in cancer cells in vivo. Data represented as mean ± SD, each dot is the average of 3–6 image quantifications per tumor of n = 7 control and n = 6 myosin IIA KO mice, from N = 2 independent experiments. **p < 0.01, Mann Whitney two-tailed test. c Cancer cells (Cell tracker, Green) surrounded by CAFs treated with SiRNA Control and SiRNA fibronectin. Scale bar, 100 µm. Right dot plot: mean YAP nuclear correlation in cancer cells. Data represented as mean ± SD, n = 12 (SiRNA Control), n = 13 (SiRNA Fibronectin) clusters per condition, from N = 3 independent experiments. ***p < 0.001, Mann Whitney two-tailed test. d Left graph: Average radial traction peaks plotted against YAP nuclear correlation for all conditions. Right graph: Cancer cell nuclear area plotted against YAP nuclear correlation for all conditions. Correlations were tested by linear regression (p < 0.05). Data represented as mean ± SD. n and N values correspond to the ones in (a, c). e Left graph: CAF normal displacement upon ablation (respect to the ablation) plotted against YAP nuclear correlation in control and αSMA:MyosinIIA^−/− tumors. Right graph: Cancer cell nuclear area plotted against YAP nuclear correlation in control and αSMA:MyosinIIA^−/− tumors. Data represented as mean ± SD. n and N values correspond to the ones in (b).