Extended Data Fig. 4: Mini-colon tumours display in vivo-like functional and transcriptional features.

a, Brightfield and fluorescence images of a mini-colon where blue light exposure has been targeted to a specific area (dashed blue line). Red and green signals correspond to healthy and mutated cells, respectively. Images were taken 36 h after induction. Scale bar, 75 μm. b, Multiplicity of tumours emerged in mini-colons cultured in the indicated conditions. *P = 0.0122; **P = 0.0035; ***P = 0.0002 (two-way ANOVA and Sidak’s multiple comparisons test; n = 4 mini-colons for each condition). c, Distribution of tumour morphologies in mini-colons cultured in the indicated conditions. **P = 0.0024 (two-way ANOVA and Sidak’s multiple comparisons test; n = 4 mini-colons for each condition). d, Brightfield images of the indicated colon organoid lines cultured for 3 days in full organoid medium. Scale bar, 200 μm. e, Metabolic activity (measured using resazurin) of the indicated colon organoid lines cultured in BMGF medium for the indicated time. Numeric labelling (1-8) is used to facilitate cell line identification. ***P = 0.0002 (mini-colon AKP #iii), <0.0001 (all other conditions) (two-way ANOVA and Sidak’s multiple comparisons test; n = 3 cultures for each line). f, Volcano plot of the differentially expressed genes between “in vivo” and “organoid AKP” cell lines. g, Top enriched functional clusters in the differentially expressed genes identified in panel f. h, Enrichment of the “in vivo AKP” transcriptional signature identified in panel f across the different “mini-colon” and “organoid AKP” lines. *P = 0.0137; **P = 0.0032 (#iii), 0.0075 (#iv); ***P = 0.0001 (Brown-Forsythe ANOVA and Dunnett’s T3 multiple comparisons test (two-sided), n = 6 and 3 cultures for “organoid AKP” and the rest of cell lines, respectively). Each dot represents one culture. i, Main enriched functional terms in the differentially expressed genes between “mini-colon AKP” lines # i and # v. Significant terms are highlighted in red (one-sided Fisher’s exact test, adjusted P values). In b, c, e, and h, data represent mean ± SEM. GPL, glycerophospholipid; EL, ether lipid; PG, proteoglycans; SC, stem cell.

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