Fig. 8: NPY1R-expressing stem cells serve as cancer origins in the distal colon following conditional mutation in vivo.

a, The Npy1r-eGFP-IRES-CreERT2 driver mouse line was crossed with the Rosa26-LSL-tdTom, Apc^fl/fl and LSL-Kras^G12D mouse lines. Mice were bred to homozygosity and LSL-Kras^G12D was kept heterozygotic. Mice were injected three times 2 d apart with 4 mg tamoxifen per 30 g bodyweight and harvested 8 weeks after injection (n = 3). b, β-catenin and pMAPK IHC showed localized nucleocytoplasmic accumulation in the rectum and pMAPK hyperactivation throughout the rectal epithelium. Co-IF for β-catenin and pMAPK showed co-localization of nucleocytoplasmic β-catenin and pMAPK hyperactivation. Co-IF for β-catenin and Ki-67 showed hyperproliferation in the crypts accumulating β-catenin following Apc deletion and Kras hyperactivation (n = 3). c, The p53^fl/fl allele was incorporated into our mouse model. Mice were injected with three consecutive tamoxifen doses at 4 mg per 30 g body weight at 2-d intervals and harvested 3 months after induction (n = 1). Tumours presented with adenocarcinoma features, with β-catenin-accumulating E-cadherin⁺ epithelial cells invading the subepithelial layer. These cells were hyperactivated for pMAPK and devoid of p53. Scale bars, 25 μm.