Fig. 6: WNT inhibitor effectively suppresses 5-FU-induced enrichment of CSCs and tumor regrowth in CRC patients.

a Immunoblots of indicated proteins in PDCs and PDTOs with or without 5-FU treatment (1.5 μg/ml, 48 h). b, c Relative mRNA levels of WNT3 (b) and LGR5 (c) in PDCs and PDTOs with or without 5-FU treatment. (n = 3 biologically independent samples per group). d Immunoblots of indicated proteins in PDCs and PDTOs co-treated with 5-FU and LGK-974 (5 μM, 48 h). e Confocal images of β-catenin (green) in PDTOs co-treated with 5-FU and LGK-974 (left panel) and quantification of the percentage of β-catenin⁺ population among the cells in each tumor organoid (upper right panel) (n = 4 biologically independent samples per group) and the mean intensities (lower right panel). (n = 10 biologically independent samples per group) Scale bars = 50 μm. f, g PDC#1 xenograft mice were treated with 5-FU (25 mg/kg) alone or co-treated with 5-FU (25 mg/kg) and LGK-974 (5 mg/kg) for 16 days and tumors were extracted and analyzed. f Tumor growth in mice during the treatments is shown. (Control n = 8, LGK-974 n = 6, 5-FU n = 8, 5-FU+LGK-974 n = 8 biologically independent animals) g Relative mRNA levels of LGR5, CD44, CD133, and CD166 in isolated tumors after treatment. (n = 3 biologically independent samples per group) h, i Tumor organoids derived from patient#2 were treated with 5-FU (1.5 μg/ml) alone or co-treated with 5-FU and LGK-974 daily for 10 days followed by discontinuation of treatment for 28 days. h Growth of PDTO#2 at indicated days. (n = 10 in D0, n = 3 in D1, n = 7 in D3, D6, D10, n = 4 in D17, n = 3 in D24, D31 and D38 biologically independent samples per group). i Bright field image of PDTO#2 at 28 days after discontinuation of treatment of 5-FU alone or co-treatment of 5-FU and LGK-974 (left panel) and measurement of cell growth in PDTO#2 by cell titer assay (right panel) (n = 3 biologically independent samples per group). Scale bars = 4 mm. j Schema depicting the mechanism by which 5-FU induces activation and enrichment of CSCs via p53. The mean intensities were measured by Zen software 3.1. Data are mean ± s.d., two-sided Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source data file.