Fig. 4
High fibrillar collagen content resulting from Hh pathway activation promotes mechano-signaling and breast cancer stemness. a–d Concomitant expression analysis of collagen content and organization, integrin/focal adhesion activation and CSC-like characteristics at the tumor−stromal interface of M6 tumor models ± SMOi (100 mg/kg/bid; n = 3 biological replicates). a Representative multiphoton SHG imaging (scale bars: 100 μm) and quantitative analysis of collagen abundance. Statistical significance was determined using unpaired two-tailed Student’s t test with equal s.d. Corresponding graphs comparing fiber orientation (top right panel) and quantifying GLCM (bottom right panel). Unpaired two-tailed nonparametric Mann–Whitney U test was used for determining statistical significance across distributions. For GLCM analysis, statistical significance was determined using unpaired two-tailed Student’s t test with equal s.d. b Collagen I and III deposition detected and quantified by picrosirius red staining. Scale bars, 200 μm. Statistical significance was determined using unpaired two-tailed Student’s t test with equal s.d. c Representative immunofluorescence images and quantification of phospho-FAK and d CK6 expression. Scale bars, 100 μm. Statistical significance was determined using Kruskal−Wallis test. e M6-Ctrl and M6-Hh single cells were embedded and grown for 12 days in 3D Alginate IPNs containing increasing concentrations of collagen. Quantification of the number of colonies was normalized to the mean colony count in 0% collagen IPNs. n = 3 biological replicates with at least six technical replicates per condition. Statistical significance was determined using unpaired two-tailed Student’s t test with equal s.d. Representative phase contrast images of M6 colonies on polyacrylamide substrata after 12 days of culture. Scale bars: 100 μm. f Relative mRNA expression of the CSC markers Id3, Igtb3, and Krt6b in M6-Ctrl and M6-Hh cells cultured within 3D alginate-collagen I IPNs. n = 3 biological replicates with six technical replicates per experiment. Statistical significance was determined using unpaired two-tailed Student’s t test with equal s.d. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Bars represent mean ± s.e.m.
