Fig. 4: Invasion into the cancerous CAF stroma and subsequent gene upregulation.

a HT29 distance of invasion and b HCT116 distance of invasion into the stroma within tumouroid models at D21 and surface are of invasion within c HT29 and d HCT116 tumouroids at D21. Tumouroids contained either HDF cells or one of six patient-specific CAF tissue samples within the stromal compartment of the constructs. All mean ± SEM with n = 3 with four technical repeats and showing Kruskal–Wallis with Dunn’s multiple-comparison test P values, with values 0.05 = *, 0.005 = **, 0.0005=*** and 0.00005 = ****. e Representation of average invasive bodies at day 21 in a “normal“ HDF-containing tumouroid in comparison with a (f) CAF-containing tumouroid. Scale bar = 100 µm for top and 500 µm for bottom image, with red = CK20 and blue = DAPI. g ACTA2 (α-smooth muscle actin), h HGF (hepatocyte growth factor) and i TIMP1 (metallopeptidase inhibitor 1) gene expression in tumouroids at day 21 of growth, comparing HDF- and CAF-containing stroma. The value shown is normalised to HPRT1 mRNA levels (mean ± SEM) with n = 3 and three technical repeats. Ordinary one-way ANOVA Dunnett’s multiple-comparison test with P values 0.05 = *, 0.005 = **, 0.0005 = *** and 0.00005 = **** with DOF = 20 for all and F value for ACTA2 for HT29 = 4.213 and HCT116 = 12.31, F value for HGF = 3.836 for the HT29 group and 16.3 for the HCT116 group and finally for TIMP1 F value for HT29 = 37.21 and HCT116 = 11.25.