Fig. 5
Deletion of Ap4 decreases stemness in adenomas and tumor organoids. a Left panel: in situ hybridization of Lgr5 mRNA. Scale bars represent 100 µm. Right panel: quantification of Lgr5-positive area in % in the adenomas from two male and one female mice in at least six adenomas per genotype. b Left panel: representative pictures of small intestinal tumor organoids 6 days after isolation (passage 0), upper panel, or 4 days after passaging (passage 1), lower panel. Organoids were isolated from three tumors per mouse from two female and two male mice per genotype. Scale bars represent 500 µm. Right panels: number of tumor organoids per drop of 50 µl Matrigel. A total of 24 drops (passage 0) or 15 drops (passage 1) was analyzed per genotype. c, d qPCR analysis of the indicated mRNA derived from tumor organoids. e Western blot analysis of the indicated proteins. f Representative pictures of tumor organoids derived from small intestinal epithelial cells obtained from Lgr5-CreERT2+/-/Apcfl/fl and Lgr5-CreERT2+/-/Apcfl/fl/Ap4fl/fl mice after treatment with 4-OHT. After isolation, organoids were kept in ENR media (contains EGF, Noggin and RSPO1). Forty-eight hours after isolation, organoids were treated with 4-OHT in a concentration of 100 nM for 48 h to delete Apc or Apc in addition to Ap4 in Lgr5-positive intestinal stem cells (ISC). Additional 48 h later, organoids were passaged and EN media (containing EGF and Noggin, but without RSPO1) was used, which selectively allowed Apc-deficient tumoroids to expand (passage 1). Pictures were taken 7 days after passaging in case of passage 1, and 6 days after passaging in case of passage 5. g Mean tumor organoid number per drop of 25 µl Matrigel calculated as a mean of a total of 20 drops of 25 µl Matrigel each for passage 1, 3–5 or a total of 11 drops (Ap4 wt) and 6 drops (Ap4 ko) for passage 2. h Mean tumor organoid size was measured and calculated from Matrigel drops as depicted exemplarily in d. a, b, c, d, g, h Results represent the mean ± SD. Results were subjected to an unpaired, two-tailed Student’s t-test with p-values * < 0.05, ** < 0.01, *** < 0.001, n.s.: not significant. See also Supplementary Fig. 4
