Figure 5.

NOTCH3 signaling regulates tumor cell invasion and survival. (a) Schematic depiction of the strategy used for the generation of Trp53^−/−Akt^E17K organoids with doxycycline-inducible Notch3 knockdown. RNA was isolated and used for RNA sequencing after culture in doxycycline-containing medium for 10 d. (b) Representative morphology of Trp53^−/−Akt^E17KshCo and Trp53^−/−Akt^E17KshNotch3 organoids without doxycycline and after culture in doxycycline-containing medium for 10 d. Scale bars = 1,000 µm. (c) Relative Notch3 mRNA expression in Trp53^−/−Akt^E17KshCo and Trp53^−/−Akt^E17KshNotch3 organoids after culture in doxycycline-containing medium for 10 d by qRT-PCR. Data are mean ± SD; ****, P ≤ 0.0001 by one-way ANOVA followed by Dunnett’s multiple comparison test. Data are shown for a dataset of n = 1 experiment performed in triplicate. (d) Differentially regulated pathways in response to Notch3 silencing. Predictions were made based on RNA-sequencing results of Trp53^−/−Akt^E17KshCo and Trp53^−/−Akt^E17KshNotch3 organoids after culture in doxycycline-containing medium for 10 d using IPA. P < 0.05 with right-tailed Fisher’s exact test; activation: z-score > 0, inhibition: z-score < 0, no pattern: pathway is differentially regulated, but z-score is not available. (e) Schematic depiction of the strategy used for subcutaneous injection of Trp53^−/−Akt^E17KshCo or Trp53^−/−Akt^E17KshNotch3 organoids and doxycycline administration in the drinking water. (f) Representative growth curves of subcutaneous tumors in NBSGW mice transplanted with Trp53^−/−Akt^E17KshCo or Trp53^−/−Akt^E17KshNotch3 organoids. Tumor size is shown starting from the first day of doxycycline administration (day 0). Doxycycline was administered for 12 d. Experiment was performed in three independent sets for 10 or 12 d, data are shown for one set of 12 d. Data are mean ± SEM. Not significant (ns) by one-way ANOVA followed by Dunnett’s multiple comparison test. n ≥ 4 per group. (g) Representative images of H&E-stained sections of subcutaneous tumors from NBSGW mice transplanted with Trp53^−/−Akt^E17KshCo or Trp53^−/−Akt^E17KshNotch3 organoids at day 10 after the start of doxycycline administration. Stars mark large cysts filled with liquid. Scale bars = 5 mm. (h) Representative images of tGFP immunohistochemistry (IHC) on sections from subcutaneous tumors of NBSGW mice transplanted with Trp53^−/−Akt^E17KshCo or Trp53^−/−Akt^E17KshNotch3 organoids at day 10 after the start of doxycycline administration. Scale bars = 200 µm.