Fig. 1

Wnt activity is important for the self-renewal of CCICs. a Colospheres were enriched from 3 primary colorectal cancer tissues, colorectal cancer HT29 cells, and SW620 cells on the 5th day in low-adhesive and serum-free culture medium. Colosphere formation occurred after the serial dilution of cells on the 5th day. Single colosphere cells formed organoids in growth factor-deficient Matrigel medium at the 5-7th day (bottom). b Tumorigenicity of colospheres. Different numbers of colosphere cells from primary colorectal cancer tissues (P1 cells) were injected into NOD/SCID mice and tumor formation was quantified after 8 weeks. The results showed that colospheres exhibited significantly increased tumorigenicity (p < 0.05, Multivariate logistic analysis). The same number of parental cells was used as a control. c Wnt activity in colospheres. Colosphere or parental cells (control) were transfected with Top/Fop flash reporters, and the Wnt pathway activity was determined at 24 h post-transfection. The fold-change was calculated relative to controls. The values are represented as the mean ± SD from triplicate samples. *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA). d The levels of Wnt signaling downstream of c-Myc were detected by western blotting in HT29, P1 and SW620 colospheres (Spheres), with their parental cells as controls. e Quantification of organoid formation after Wnt pathway activation. The number of organoids was counted from 3 primary colorectal cancer cells (100 cells/well) by the treatment of recombinant Wnt3a (200 ng/mL) for 7 days; cells without treatment of Wnt3a were used as controls. The number of organoids significantly increased after Wnt pathway activation. *p < 0.05 (ANOVA). f Quantification of colosphere formation after CTNNB1 knockdown. The colosphere number was counted in CTNNB1-knockdown HT29, P1, and SW620 cells under low-adhesive and serum-free conditions for 7 days. The colosphere number significantly decreased in these cells after knockdown of CTNNB1. **p < 0.01; ***p < 0.001 (ANOVA). g Quantification of colosphere formation after Wnt pathway inactivation. Colospheres were counted in HT29, P1, and SW620 cells treated with Wnt inhibitor (7.2 μM) or β-catenin inhibitor (3.6 μM) for 7 days, with the solvent dimethyl sulphoxide (DMSO) as a control. The colosphere number significantly decreased after the inhibition of Wnt activity. *p < 0.05; **p < 0.01; (ANOVA)