Fig. 3: Mini-colons support intratumour and intertumour heterogeneity.

a, Schematic of the experimental workflow followed for single-cell and lineage-tracing analysis of mini-colons. b, Unsupervised uniform manifold approximation and projection (UMAP) clustering of the main cell types in mini-colons 7 days after tumorigenic induction. c, The expression (Exp.) of representative cell-type-specific markers in the different cell populations comprising mini-colons. d, Unsupervised clustering (UMAP) of healthy (top) and tumour (bottom) clonal populations in mini-colons. The cell type (left; colour coded as in b) and clonal identity (right) are indicated. e, The relative cell type abundance in healthy and tumour mini-colon clonal populations. Data are mean ± s.e.m. n = 16 and 18 for healthy and tumour clones, respectively. f, Healthy and tumour mini-colon clonal population sizes. Statistical analysis was performed using two-tailed Mann–Whitney U-tests; **P = 0.0011. n = 16 and 18 for healthy and tumour clones, respectively. The box plots show the median (centre lines), the first and third quartiles (box limits) and the minimum and maximum values (whiskers). Each point represents one clonal population. g, The correlation between Gpx2 expression and cancer stem cell transcriptional signature enrichment (Cd44, Lgr5, Sox9). Statistical analysis was performed using two-sided Pearson correlation tests; P < 0.0001. n = 540 cells. Each point represents one cell. CSC, cancer stem cell; ES, enrichment score. h, Bright-field and immunofluorescence images showing the abundance of GPX2 (magenta) and nuclei (cyan) in healthy (right) and tumour (left, indicated by arrows) crypts in a mini-colon. Scale bar, 35 μm. i, Expression of the indicated genes in the indicated tumour clones. Statistical analysis was performed using two-sided Wilcoxon rank-sum tests; ***P = 1.77 × 10⁻¹⁷ (Il1a, clone 1), 1.00 × 10⁻⁷⁸ (Cdkn2a, clone 14), 3.67 × 10⁻²² (Cdkn2a, clone 48). n = 540 cells. Each point represents one cell.

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