Extended Data Fig. 2: Atrx deletion leads to tumour invasion.
From: Loss of colonic fidelity enables multilineage plasticity and metastasis
(a) Subcutaneous tumour volume growth of mice injected with AKP Control or AKP AtrxKO organoid cells. (b) Volumes of tumours generated via subcutaneous injection of AKP Control or AKP AtrxKO, n = 5 vs 5 tumours. (c) Representative images of H&E-stained subcutaneous tumours in mice subcutaneously injected with AKP Control or AKP AtrxKO organoid cells. Scale bars, 100 μm overview and 50 μm zoom. (d) Representative images of β-catenin-stained subcutaneous tumours in mice subcutaneously injected with AKP Control or AKP AtrxKO organoid cells. β-catenin staining is used to identify tumour cells. Black arrows indicate β-catenin positive tumour cells invading into surrounding stroma. Scale bars, 100 μm overview and 50 μm zoom. (e) Quantification of invasive area of AKP Control vs AKP AtrxKO tumours, n = 5 vs 5 tumours. (f) Representative images of CDH1 stained subcutaneous tumours in mice subcutaneously injected with AKP Control or AKP AtrxKO organoid cells. Tumour core and invasive region of AKP AtrxKO tumour shown. Scale bars, 100 μm overview and 50 μm zoom. (g) Quantification of CDH1 staining intensity (H-score analysed with QuPath) in AKP Control vs AKP AtrxKO tumours, n = 5 vs 5 tumours. (h) Representative images of TWIST1 stained subcutaneous tumours in mice subcutaneously injected with AKP Control or AKP AtrxKO organoid cells. Scale bars, 100 μm overview and 50 μm zoom. (i) Quantification of TWIST1 staining in AKP Control vs AKP AtrxKO tumours, n = 5 vs 5 tumours. (j) RT-qPCR analysis of EMT marker expression in AKP Control vs AKP AtrxKO tumours, n = 5 vs 5 tumours. For (b), (e) and (i) data are mean ± SD. P values were calculated using two-tailed Mann-Whitney test. For (g) data are mean ± SD. P values were calculated using ordinary one-way ANOVA with multiple comparisons. For (j) gene expression was normalised to Actb and levels relative to AKP Control tumours calculated using the ΔΔCt method. Data are mean ± SD. P values were calculated using two-tailed student’s ttest. (k) Fluorescence microscopy of phalloidin stained AKP Control and AKP AtrxKO2 organoids following treatment with 5 ng/ml TGF-beta. Scale bars, 200 μm. (l) Quantification of the percentage of AKP Control vs AKP AtrxKO2 organoids adopting spindle-like morphology following TGF-beta treatment, n = 3 vs 3 independent experiments. Data are mean ± SD. P value was calculated using two-tailed student’s ttest.
