Extended Data Fig. 4: Atrx deletion in BPN model results in tumour invasion.

(a) Targeted DNA sequencing of targeted Atrx locus in BPN Atrx^KO organoid lines. (b) Representative pictures of BPN Control and BPN Atrx^KO organoid culture. Scale bars, 1000 µm. (c) RT-qPCR analysis of the EMT marker Twist1 in BPN Control vs BPN Atrx^KO organoid culture, n = 3 vs 3 independent experiments. (d) Fluorescence microscopy of phalloidin stained BPN Control and BPN Atrx^KO organoids following treatment with 5 ng/ml TGF-beta. Zoomed area outlined in white box. Scale bars, 400 μm overview and 200 μm zoom. (e) Average number of clusters of BPN Control and BPN Atrx^KO organoids per well exhibiting a spindle-like morphology following TGF-beta treatment, n = 5 vs 4 independent experiments. (f) RT-qPCR analysis of EMT and epithelial marker expression in BPN Atrx^KO organoids either untreated or treated with 5 ng/ml TGF-beta, n = 3 vs 3 independent experiments. (g) Representative H&E and β-catenin-stained images of primary tumours in mice orthotopically transplanted with BPN Control or BPN Atrx^KO organoid cells. β-catenin is used to identify tumour cells. Magnification highlights the distinct morphological features. Scale bars, 1 mm overview and 100 µm zoom, n = 6 vs 8 mice. (h) Quantification of tumour areas with glandular morphology in BPN Control and BPN Atrx^KO primary tumours, n = 6 vs 8 mice. (i) Representative H&E-stained image of BPN Atrx^KO metastasis. Scale bars, 1 mm. (j) Summary data indicating presence or absence of metastases. Number of mice with metastases or no metastases indicated on graph, n = 6 vs 8 mice. For (c) and (f) gene expression was normalised to Actb and relative levels were calculated using the ΔΔCt method. Data are mean ± SD. For (c), (e) and (f) p values were calculated using two-tailed student’s ttest. For (e) p = 0.000023. For (h) p value was calculated using two-tailed Mann-Whitney test. (k) Normalised enrichment scores of significantly enriched gene sets in TGF-beta treated AKP Control vs AKP Atrx^KO organoids. (l) RT-qPCR analysis of representative EMT markers induced in AKP Atrx^KO organoids following treatment with TGF-beta, n = 3 vs 3 independent experiments. (m) Normalised enrichment scores of significantly enriched gene sets in untreated AKP Control vs AKP Atrx^KO organoids. (n) RT-qPCR analysis of representative genes important for colonic lineage specification and function, n = 3 vs 3 independent experiments. (o) RT-qPCR analysis of representative genes important for colonic lineage specification and function in BPN Control vs BPN Atrx^KO organoids, n = 3 vs 3 independent experiments. For (l) gene expression was normalised to 18S rRNA and levels relative to untreated AKP control organoids calculated using the ΔΔCt method. For Twist1, Irx2 and Tfap2c gene expression was normalised to Actb and levels relative to untreated AKP control organoids calculated using the ΔΔCt method. For (n) gene expression was normalised to Actb and levels relative to untreated AKP Control organoids calculated using the ΔΔCt method. For (o) gene expression was normalised to Actb and levels relative to BPN Control organoids calculated using the ΔΔCt method. For (l) and (n) data are mean ± SD. P values were calculated using ordinary one-way ANOVA with multiple comparisons. For (l) Fgf2 p = 0.00000006, Serpine2 p = 0.000006, Col1a1 p = 0.000005, Col6a1 p = 0.000045, Col6a2 p = 0.000004, Pcolce p = 0.000015, Twist1 control p = 0.00000026, Twist1 + TGFβ1 p = 0.00000004. For (n) Hnf4a control p = 0.00000001, Cdx1 control p = 0.000000000000051, Cftr control p = 0.00000008. For (o) data are mean ± SD. P values were calculated using two-tailed student’s ttest. (p) Representative images of LY6D, KRT5 and EPCAM stained oesophagus and colon from Human Protein Atlas (Human Protein Atlas proteinatlas.org).

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