Fig. 7: Loss of Hsf1 in colon fibroblasts impairs ECM secretion.

a–f Colons from day 15 AOM-DSS-treated WT or Hsf1 null mice were excised, and fibroblasts were isolated and allowed to recover in culture for 5 days (see the “Methods” section). a, b Colon fibroblasts were induced to secrete ECM by 3–5 days incubation with MC38-conditioned media supplemented by growth factors and insulin. Representative SHG images are shown in a. The average area of collagen covered is quantified in b for n = 6 WT and n = 5 Hsf1 null mice combined from three different experiments. Scale bar—50 µm. c Colon fibroblasts were cultured for 5 days in the presence or absence of MC38-conditioned media, and the amount of collagen secreted by the cells was quantified by Sirius red staining (see the “Methods” section). n = 5 WT and 4 Hsf1 null mice combined from two different experiments. d–f Representative images of fibronectin d, MMP7 e, and MMP9 f and DAPI (nuclear) staining of colon fibroblasts cultured for 5 days in the presence of MC38-conditioned media. For d–f n = 6 WT and n = 5 d, e or 3 f Hsf1 null mice, examined over two independent experiments. Scale bar—100 µm. g The molecular structure of (–)-aglaroxin C (CMLD011866). h, i WT colon fibroblasts were incubated with MC38-conditioned media supplemented by growth factors and insulin in the presence or absence of 3 nM CMLD011866 for 3–5 days, after which SHG imaging was performed. Representative images are shown in h. The average area of collagen covered is quantified in i for n = 6 WT mice and n = 5 Hsf1 null mice, combined from three different experiments. Scale bar—50 µm. j–l Colons from day 15 j–k or day 20 l AOM-DSS-treated WT or Hsf1 null mice were excised and decellularized. MC38 cancer cells were added and allowed to re-cellularize the matrices (see the “Methods” section) after which SHG imaging was performed. Scale bar—50 µm. j, l Representative SHG + IF images taken from the mucosal side for j (upper panels) and the muscularis externa side for l. SHG is shown in red, DRAQ5 nuclear staining is shown in blue. Heatmaps depicting the depth of invasion of cancer cells into the matrix are shown in j (bottom panels). k The average number of MC38 cells was calculated per mouse from 3 to 7 images averaged per area, each experiment was normalized to WT and the average of three biological replicates is presented. White arrows in l point to “holes” in the matrix where cancer cells have invaded. Results of c are presented as mean ± SEM, analyzed by two-way ANOVA and Bonferroni correction for multiple comparisons. For b, i, and k, Results presented as mean ± SEM, analyzed by an unpaired Student’s t-test.