Fig 2: Combination of DMBA and Ad-Cre induces dysplasia and oncogenic transformation in Ppp2r1aflox/flox intestinal organoid culture. In vitro culture of Ppp2r1aflox/flox intestinal organoids without (control) or with DMBA or/and Ad-Cre-GFP (Ad-Cre) infection in the presence of EGF, Noggin and R-spondin 1 (500 single cells/well). (A) Colony (organoid)-forming efficiency was calculated at day 7. At least 100 organoids were counted in each group. (B) Bright-field and fluorescence images of organoid culture at day 50. Scale bar, 100 µm. (C) H&E staining and histologic characterization of cystic stratified epithelium with nuclear pleomorphism (arrow indicated). Scale bar, 50 µm. (D) Dysplasia index at day 50 (experiments were repeated twice with n = 3 microscopic fields containing viable organoids). (E) Dissociated cells in Matrigel (500,000 cells/100 µL) were injected s.c. into NOD-SCID mice. In vivo tumor formation 45 days later (for those without tumor formation, observation was extended for up to 3 months, experiments were repeated twice with n = 3). (F) Tumor size 45 days after s.c. implantation (n = 3). (G) H&E staining and immunofluorescence of CK20 and SMA for tumor sections. Scale bar, 100 µm. *, p < 0.05; ***, p < 0.0001 as determined with one-way ANOVA.

Fig 3: Combination of DMBA and okadaic acid (OA) induces dysplasia and oncogenic transformation in wild-type intestinal organoid culture. In vitro culture of wild type intestinal organoids without (control) or with DMBA or/and tamoxifen (TAM) in the presence of EGF, Noggin, and R-spondin 1 (500 single cells/well). (A) Colony (organoid)-forming efficiency was calculated at day 7. Experiment has been carried out in triplicate and each time 100 organoids were counted in each group. (B) Bright-field of organoid culture at day 50. Scale bar, 100 μm. (C) H&E staining and histologic characterization of cystic stratified epithelium with nuclear pleomorphism (arrow). Scale bar, 50 μm. (D) Dysplasia index at day 50 (experiment were repeated twice with n = 3 microscopic fields containing viable organoids). (E) Dissociated cells in Matrigel (500,000 cells/100 μL) were injected s.c. into NOD-SCID mice. In vivo tumor formation 45 days later (For those without tumor formation, observation was extended for up to 3 months, experiment were repeated twice with n = 3). (F) Tumor size 45 days after s.c. implantation (n = 8). (G) H&E staining and immunofluorescence of CK20 and SMA for tumor sections. Scale bar, 100 μm. *, p < 0.05; ***, p < 0.0001 as determined with one-way ANOVA.