Fig. 7: NOX1-expressing stem cells serve as colon cancer origins following conditional mutation in vivo.
From: NOX1 and NPY1R mark regional colon stem cell populations that serve as cancer origins in vivo
a, The Nox1-2A-CreERT2 driver mouse line was crossed with the Rosa26-LSL-tdTom and Apcfl/fl mouse lines. Mice were bred to homozygosity. Mice were injected twice 2 d apart with 4 mg tamoxifen per 30 g bodyweight and harvested 1 month after injection (n = 4). β-catenin IHC showed nucleocytoplasmic accumulation in the caecum and middle and distal colon 1 month after induction. b, Co-IF for β-catenin and the proliferation marker Ki-67 showed hyperproliferation in the crypts of the caecum and middle and distal colon following Apc deletion (n = 4). c, Nox1-2A-CreERT2, Apcfl/fl, Rosa26-LSL-tdTom mice were bred with the inducible LSL-KrasG12D strain. LSL-KrasG12D was kept heterozygotic. Mice were injected with 1 mg tamoxifen per 30 g bodyweight and harvested 1 month later (n = 3). β-catenin IHC showed nucleocytoplasmic accumulation throughout the caecum epithelium, with hyperactivation of Kras shown by an increase of pMapk. Only one polyp over four different colons was detected with nucleocytoplasmic β-catenin accumulation and pMapk hyperactivation. The rest of the colon presented with normal membranous β-catenin with no nucleocytoplasmic accumulation. d, A p53fl/fl allele was incorporated into the Nox1-2A-CreERT2, Apcfl/+, LSL-KrasG12D mouse model. Mice were injected with 2 mg tamoxifen per 30 g bodyweight and harvested 3 months after induction (n = 4). Tumours in the caecum presented with adenocarcinoma features with high levels of β-catenin accumulation, validating the loss of Apc heterozygosity. E-cadherin+ cells had invaded the submucosa layer until the muscularis for the most advanced stage. Invading tumours were hyperactivated for KRAS (pMAPK+) and devoid of p53 expression, highlighting efficient allelic recombination. Scale bars, 25 μm. D, day.
