Fig 2: Znhit1 deletion disrupts postnatal generation of Lgr5+ ISC. a Znhit1 in situ was performed in intestinal section of 8-week-old C57BL/6 mouse. Scale bar, 50 μm. b Body weight comparison between Znhit1fl/+; Villin-cre and Znhit1fl/fl; Villin-cre mice at indicated time. The data represent mean ± s.d. (n = 5 mice per group). Wilcoxon’s rank sum test: **P < 0.01. *P < 0.05. c Kaplan–Meier survival curves of Znhit1fl/+; Villin-cre and Znhit1fl/fl; Villin-cre mice (n = 19 mice per genotype) and body size comparison between survived mice at P30. Scale bar, 2 cm. d Paraffin-embedded intestine tissues were stained with hematoxylin and eosin. e Ki67 and Krt20 staining of intestinal sections from Znhit1fl/+; Villin-cre and Znhit1fl/fl; Villin-cre mice at P9. f Intestinal crypts were isolated from Znhit1fl/+; Villin-cre and Znhit1fl/fl; Villin-cre mice at P9, embedded in Matrigel (100 crypts per well) and cultured for 3 days. The statistical analysis of organoid numbers (n = 5 mice per genotype) was shown as mean ± s.d. Student’s t-test: ***P < 0.001. g GFP staining of intestinal sections from Znhit1fl/+; Villin-cre; Lgr5-EGFP-IRES-creERT2 and Znhit1fl/fl; Villin-cre; Lgr5-EGFP-IRES-creERT2 mice at P9. Arrows: Lgr5+ ISCs. h Lgr5 and Olfm4 in situ were performed in intestinal sections at P9. i Intestine was harvested from Znhit1fl/+; Villin-cre (fl/+) and Znhit1fl/fl; Villin-cre (fl/fl) mice at P0 to examine the expression of Znhit1, Lgr5, Ascl2, and Olfm4 using qRT-PCR. j Intestine was harvested from Znhit1fl/+; Villin-cre (fl/+) and Znhit1fl/fl; Villin-cre (fl/fl) mice at indicated time to examine Lgr5 expression using qRT-PCR. For qRT-PCR, histone H3 was used as an internal control. The statistical data represent mean ± s.d. (n = 3 mice per genotype). Student’s t-test: ***P < 0.001. *P < 0.05. All images are representative of n = 3 mice per genotype. Scale bar, 50 μm

Fig 3: Organoid culture possesses a better efficiency to reproduce primary colorectal tumors than sphere-forming assay. (A) Representative hematoxylin and eosin images of xenografts generated from primary CRC cells (PDX), organoid-derived cells (ODX), sphere-derived cells (SDX) and their parental tumor (primary cancer). Scale bars, 100 µm. (B) Immunofluorescence staining of CK20 in a primary CRC tumor and its corresponding PDX, ODX and SDX. Scale bars, 200 µm. The nuclei of CRC cells were counterstained with DAPI, and the 'Merge' in top panel of (B) indicates the merging of DAPI (in blue) for nuclei and CK20 (in red) for differentiated CRC cells. The cells used in (A and B) were derived from CRC6. (C) Representative images of organoids and spheres derived from primary CRCs (CRC5, CRC6, and CRC9). (D) A total of 1,000 single purified primary CRC cells per well were respectively processed for the sphere-forming assay and organoid culture; 7 days later, the organoids or spheres were photographed in (C). Then, the organoid- or sphere-forming efficiency of primary CRCs were quantified. **P<0.01 and ***P<0.001. CRC, colorectal cancer; PDX, xenografts of primary CRC cells; ODX, xenografts of organoid-derived cells; SDX, xenografts of sphere-derived cells; CK20, cytokeratin 20.

Fig 4: Immunofluorescence of biomarkers in patient-derived CRC spheroids. Representative confocal images (maximum intensity projections of z-stacks) of cancer spheroids stained at day 4 post spheroid embedding in collagen for the (A) CRC biomarkers CK20 and CDX2; (B) cancer stem cell biomarkers ESA and CD133 at day 4; (C) active form of ß-catenin, i.e., a molecular marker known to be active in CRC. (D) Representative confocal images of the proliferation marker Ki-67 at day 0 and day 7 (scale bar 100 µm) (- = control; M = MET; A = ASA; C = ASA/MET combination).

Fig 5: Differentiation of colon organoids after IFN-? treatment AQuantification of KRT20 immunofluorescence of human colon organoids cultured either in full or basic medium and treated with IFN-?. Left: relative abundance of KRT20+ cells per organoid; Right: Fluorescence intensity of KRT20+ cells, indicating an increase in KRT20 expression in response to IFN-?. Data are presented as mean ± SD, *: P = 0.05, **: P = 0.001, ***: P = 0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test (more details see Appendix Table S2).BMeasurement of IFN-? responsiveness by qRT–PCR for the IFN-? target gene IFIT3 at concentrations from 5 pg/ml to 100 ng/ml.CImmunofluorescence staining for E-cadherin (green) and actin (red) of organoids grown in full medium (left) or basic medium (right) and additionally treated with IFN-? (lower), indicating increased columnar cell height as a feature of differentiation upon IFN-? treatment. Scale bar: 25 µm. Quantification of cell length on the right. Data are presented as mean ± SD, *: P = 0.05, **: P = 0.001, ***: P = 0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparison test (more details see Appendix Table S2).DAlcian blue–nuclear fast red staining of human colon organoids cultured in full medium (upper panel) or differentiation medium (lower panel), with or without IFN-? treatment to visualize goblet cells. Scale bar: 50 µm.EComparison of the differentiation markers ALPI (enterocytes, left) and ITF (goblet cells, right) in organoids grown in full medium or in basic medium and either untreated or treated with IFN-?. Data are presented as mean ± SD, *: P = 0.05, **: P = 0.001, ***: P = 0.0001, as determined by one-way ANOVA followed by Tukey's multiple comparison test.