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Tracing oncogene-driven remodelling of the intestinal stem cell niche

Abstract

Interactions between tumour cells and the surrounding microenvironment contribute to tumour progression, metastasis and recurrence1,2,3. Although mosaic analyses in Drosophila have advanced our understanding of such interactions4,5, it has been difficult to engineer parallel approaches in vertebrates. Here we present an oncogene-associated, multicolour reporter mouse model—the Red2Onco system—that allows differential tracing of mutant and wild-type cells in the same tissue. By applying this system to the small intestine, we show that oncogene-expressing mutant crypts alter the cellular organization of neighbouring wild-type crypts, thereby driving accelerated clonal drift. Crypts that express oncogenic KRAS or PI3K secrete BMP ligands that suppress local stem cell activity, while changes in PDGFRloCD81+ stromal cells induced by crypts with oncogenic PI3K alter the WNT signalling environment. Together, these results show how oncogene-driven paracrine remodelling creates a niche environment that is detrimental to the maintenance of wild-type tissue, promoting field transformation dominated by oncogenic clones.

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Fig. 1: Red2Onco system: an oncogene-associated multicolour reporter.
Fig. 2: Reduced effective stem cell number leads to accelerated drift of WT clones in crypts that neighbour mutant crypts.
Fig. 3: Comparative single-cell analysis identifies oncogene-driven niche changes.
Fig. 4: Functional validation of oncogene-driven niche remodelling.

Data availability

The scRNA-seq data generated for this study have been deposited in ArrayExpress under E-MTAB-8656. The reference genome sequence was downloaded from Ensembl (http://www.ensembl.org/Mus_musculus) and used for alignment of the scRNA-seq data. To evaluate stem cell priming, scRNA-seq data were obtained from the Single Cell Portal (https://portals.broadinstitute.org/single_cell/study/small-intestinal-epithelium) and used to define gene sets for differentiated sub-lineages of epithelial cells. The lists of marker genes used to annotate types of epithelial, mesenchymal and immune cells in Fig. 3b, f and Extended Data Figs. 6d, 8a–c are given in Supplementary Tables 1, 3. Gene sets used in Fig. 3d, e and Extended Data Fig. 7a, b, k–m are provided in Supplementary Table 2. Source data are provided with this paper.

Code availability

The statistical analysis of the clone fate data, based on a fit to the established modelling scheme, was performed using a FORTRAN (G95 compiler) code developed for this study. The scRNA-seq data were analysed using publicly available R packages. The codes and data used for clonal analysis and scRNA-seq data analysis have been deposited in GitHub (available at https://github.com/BenSimonsLab/Yum_Nature_2021).

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Acknowledgements

We thank S. Alexandrova and members of the Simons laboratory for discussions, C. J. Hindley for reading the manuscript, S. Bae for assistance with graphic illustrations, the Gurdon Institute Imaging Facility for microscopy and image analysis support, and the Cancer Research UK Cambridge Institute Genomics Core Facility for sequencing and library generation. This work was supported by a Cancer Research UK Multidisciplinary Project Award to B.-K.K. and B.D.S. (C52767/A23363), and grants to A.P. (Cancer Research UK, A25636), J.-H.L. (Wellcome Trust, 107633/Z/15/Z; ERC Starting Grant, 679411), B.-K.K. (ERC Starting Grant, 639050; Human Frontier Science Program, RGY0071/2018; Interpark Bio-Convergence Center Grant Program, IBCC-IMBA-2020-01) and B.D.S. (Wellcome Trust, 098357/Z/12/Z and 219478/Z/19/Z), as well as by studentships and fellowships to J.F. (Wellcome Trust), S.-H.S.W. (DOC Fellowship of the Austrian Academy of Sciences), L.C. (Herchel Smith Fund), S.H. (Human Frontier Science Program, LT000092/2016-L; Basic Science Research Program NRF-2014R1A6A3A01005675) and B.D.S. (Royal Society EP Abraham Research Professorship, RP\R1\180165). The research team also acknowledges core grant support from the Austrian Academy of Sciences to the Institute of Molecular Biotechnology, the Wellcome Trust and MRC to the Wellcome–MRC Cambridge Stem Cell Institute, and the Wellcome Trust (092096) and CRUK (C6946/A14492) to the Gurdon Institute.

Author information

Affiliations

Authors

Contributions

M.K.Y., S.H., J.F., B.-K.K. and B.D.S. planned and designed the experiments. M.K.Y. performed lineage tracing, tissue imaging and quantification, flow cytometry and cell sorting, which were supervised by A.P., J.-H.L., B.-K.K. and B.D.S. B.-K.K. conceived the Red2Onco design and the Red2Onco mouse models were established by M.K.Y., S.H., J.F., C.D., T.T., R.M., J.-H.L. and B.-K.K. M.K.Y. and B.D.S. performed the quantitative statistical analysis of the clone size data. M.K.Y. and S.-H.S.W. performed organoid experiments. M.K.Y., L.C. and I.P. performed in situ hybridization experiments. C.D., L.C., R.A. and F.E. performed lineage tracing with oesophagus, stomach corpus, pancreas and lung tissue, respectively. E.L. and J.K.K. aligned raw sequencing data from scRNA-seq experiments. M.K.Y. and S.H. analysed scRNA-seq data, supervised by J.K.K., B.-K.K. and B.D.S. S.H. devised the algorithm for statistical analysis of transcriptomic changes with scRNA-seq data. S.-H.S.W. performed lineage tracing and tissue preparation of LSL-KrasG12D, PIK3CALat-H1047R and Apcfl/fl mice, which were provided by D.E.S. M.K.Y., S.H., B.-K.K. and B.D.S. wrote the manuscript with input from all authors. These authors contributed equally: J.F., S.-H.S.W., C.D., T.T.

Corresponding authors

Correspondence to Bon-Kyoung Koo or Benjamin D. Simons.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Hans Clevers, James DeGregori, Dominic Grün and Toshiro Sato for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Red2Onco system: an oncogene-associated multicolour reporter.

a, Representative confocal images of mutant clones from sections (Red2-Notch1ICD) or whole mounts (Red2-KrasG12D and Red2-PIK3CAH1047R) of small intestine from Villin-CreERT2;Red2Onco mice 2 w after tamoxifen administration. Crypt borders are marked with a grey dashed outline. b, Average clone numbers collected from a single field of image (0.15 mm2) of whole-mount small intestine from Villin-CreERT2;R26R-Confetti or Red2Onco mice 2 d after tamoxifen administration. ce, Representative confocal images (left) and quantification (right) of EdU+ proliferating crypt cells (c), LYZ+ Paneth cells (d) and MUC2+ goblet cells (e) from sections of small intestine from Villin-CreERT2;R26R-Confetti or Red2Onco mice 4 w after tamoxifen administration. f, Representative confocal images of 100-μm-thick sections or whole mounts of tissues from adult R26R-CreERT2; Red2Onco mice (skin and stomach corpus), Sftpc-CreERT2; Red2Onco mice (lung) or Krt5-CreERT2; Red2Onco mice (oesophagus) at 1, 2 and 4 w after tamoxifen administration. White dashed line, epithelial lining. β-catenin stained as a cell membrane marker. SPC marks alveolar type II cells in lung. g, Representative confocal images of sectioned mouse embryonic pancreas tissue from the R26R-CreERT2; Red2-KrasG12D mouse on embryonic day (E)18.5, 6 d after tamoxifen administration. Magnified panel to the right shows an example of acinar cell expansion in developing pancreas. CPA1 marks acinar cells. *P < 0.05, **P < 0.01, ***P < 0.0001 by one-way ANOVA with Games-Howell’s multiple comparisons test (b, c) or unpaired two-tailed t-test (d, e) from biological replicates. Data are mean ± s.d. (b) or mean ± s.e.m. (ce). Exact P values are presented in Source Data. Scale bars: 50 μm (a, cf) or 200 μm (g).

Source data

Extended Data Fig. 2 Oncogenes drive non-neutral clone expansion in the mouse intestinal crypt.

a, Schematic illustration of clonal events within the Red2Onco system (left) and representative tile scan images (right). Images are representative of tissues quantified in hk. Arrow, WT crypt; arrowheads, fixed mutant crypts. b, Representative confocal images of the base and neck of crypts. Images are representative of tissues quantified in c. ce, Strong correlation between clone size at the base and neck of a crypt from Villin-CreERT2;R26R-Confetti (2 w after tamoxifen administration) or Red2Onco mice (1 w after tamoxifen administration). f, Average number of clones per 100 crypts. g, Schematic illustration of mutant (RFP+) and WT (YFP+) clones in crypts, remote from each other. h, Representative confocal images at 4 d, 1 w, 2 w and 3 w after tamoxifen administration. Images are representative of tissues quantified in ik. Red2-Wild-type: remote YFP+ clones; Red2-Mutant: RFP+ clones. i, Heat maps indicate the relative clone fractions of the indicated sizes (columns) at various time points after induction (rows). Data are mean ± s.e.m. j, k, Average clone size (j) and percentage of monoclonal crypts (k) at different time points after tamoxifen administration. Asterisks for statistical significance omitted in graphs in j, k for better visualization. Confocal images of small intestine are from Villin-CreERT2;R26R-Confetti (a, b, h) or Red2Onco mice (a, h). Crypt borders are marked with a white dashed outline (a, b). Significance (*P < 0.05; **P < 0.01; ***P < 0.0001) was determined by unpaired two-tailed Pearson’s correlation test (ce), one-way ANOVA with Games-Howell’s multiple comparisons test (j) or unpaired two-tailed t-test (k) from biological replicates. Data are mean ± s.d. (f) or mean ± s.e.m. (j, k). For exact P values, see Source Data. Scale bars: 100 μm (a, b) or 50 μm (h).

Source data

Extended Data Fig. 3 Biophysical modelling of mutant clone expansion.

a, d, Contour plots showing mean-square differences of clone size distribution between neutral drift model and YFP clone data from Confetti (left), WT crypts remote from mutant crypts in R2KR (middle) and R2P3 (right) in a; and between biased drift model and RFP mutant (MT) clone data from R2KR (left), R2P3 (middle) and R2N1 (right) in d. Plots: scan of loss/replacement rate λ against time delay between injection and induction in a; and drift bias δ with time delay of 0.29 w (R2KR), 0 w (R2P3), and 0.43 w (R2N1) in d. Blue lines in d, constraint λ(1 – δ) = λWT, where λWT is the loss/replacement rate inferred from Confetti (Supplementary Theory). Analysis in a, d based on data in c, f, respectively. b, e, Average clone size (effective stem cell number) from a, d, respectively. Points show data, lines show model prediction at optimal parameter values. In each case, total effective stem cell number N = 5, so that an average clone size of, for example, 2 corresponds to circumferential angle of 360° × 2/5. c, f, Distribution of clone sizes for models from a, d, respectively. Points show data; lines show model prediction at optimal parameter values. g, Representative confocal images of cleaved caspase-3+ apoptotic cells. A single cleaved caspase-3+ apoptotic cell in the villus tip is indicated by the white arrow as a positive control. h, i, Representative confocal images (h) and quantification (i) of EdU+ proliferating crypt base columnar cells. Whole mount of small intestine from Villin-CreERT2;R26R-Confetti or Red2Onco 1 w (g) or 2 w (h, i) after tamoxifen administration. *P < 0.05, **P < 0.01, ***P < 0.0001; unpaired two-tailed t-test (i) from biological replicates. Data are mean ± s.e.m. (b, c, e, f, i). Exact P values in Source Data. Scale bars: 50 μm (g) or 25 μm (h).

Source data

Extended Data Fig. 4 Mutant crypts perturb clonal dynamics of WT cells in neighbouring crypts.

a, Representative confocal images of tissues quantified in b. Fixed (monoclonal) WT crypts are indicated by white arrows. b, Percentage of monoclonal WT small intestinal crypts. c, d, Average clone size (c) and percentage of monoclonal crypts (d) of remote and proximate WT (YFP+) clones at different time points after tamoxifen administration. e, Schematic illustration of proximate WT clones in relation to fixed mutant crypts. f, Heat maps indicate the relative clone fractions of the indicated sizes (columns) at various time points after induction (rows). Data are mean ± s.e.m. g, Average clone size of proximate (inner and outer) WT (YFP+) clones at different time points after tamoxifen administration. h, Average clone size θ/360° of WT (YFP+) clones in crypts neighbouring fixed mutant crypts as a function of time t after induction. Data are mean ± s.e.m. Blue line shows a fit to the square root dependence predicted by the neutral drift model (Supplementary Theory). Orange line shows the 95% confidence interval. i, Schematic illustration of factors that affect rate of clonal drift (Supplementary Theory). j, Representative images (left) and quantification (right) of OLFM4+ ISCs. Arrows, proximate WT crypts; arrowheads, fixed mutant crypts; grey dashed outlines, crypt borders. Confocal images of whole-mount small intestine are from Villin-CreERT2;R26R-Confetti or Red2Onco mice (a) and Lgr5-EGFP-IRES-CreERT2;Red2Onco mice (j) 2 w after tamoxifen administration. *P < 0.05, **P < 0.01, ***P < 0.0001; one-way ANOVA with Games-Howell’s multiple comparisons test (c, g) and unpaired two-tailed t-test (b, d, j) from biological replicates. Data are mean ± s.e.m. (bd, fh) or mean ± s.d. (j). For exact P values, see Source Data. Asterisks for statistical significance omitted in graphs in c, d, g for better visualization. Scale bars, 50 μm (a, j).

Source data

Extended Data Fig. 5 Reduced effective stem cell number leads to accelerated drift dynamics.

a, Original image (left) was thresholded (top right) and outlined (bottom right) to measure crypt size and circularity. Image representative of tissues quantified in be. be, Scatter and violin plots display size (b, c) and circularity (d, e) of WT crypts against distance from nearest fixed mutant (RFP+) crypt. f, g, Illustration (f) and confocal images (g) of clones representative of tissues quantified in h, i. h, Heat maps indicate relative clone fractions of given sizes. Data are mean ± s.e.m. i, Percentage of monoclonal crypts of proximate WT (YFP+) clones. j, k, Confocal images (j) and quantification (k) of EGFP+ (LGR5+) ISCs. Images representative of tissues quantified in k, l. White dashed lines, EGFP+ cells in WT crypts. l, Violin plots display size of WT crypts in relation to multiplicity of neighbouring mutant crypts. n for each group is shown. m, n, Representative confocal images of Red2Onco intestine (m) and fractions of WT crypts from single field (0.15 mm2; n). o, Illustration (left) and representative images (right) of crypt fission and fusion event in ‘8-shaped crypts’30. Images representative of tissues quantified in p. p, Percentage of crypts undergoing crypt fission (upper) or fusion (lower). Whole mount of small intestine from Villin-CreERT2;R26R-Confetti or Red2Onco mice (ai, m, o, p), and Lgr5-EGFP-IRES-CreERT2;Red2Onco mice (j, k) at indicated time points. Proximate WT crypts and fixed mutant crypts indicated by white arrows and arrowheads, respectively (g, j). Crypt borders marked by dashed grey outlines (g, j, o). In b, d, blue shaded area and red dashed line indicate 95% confidence interval of R26R-Confetti controls and average distance between the centre of fixed mutant crypt and proximate WT crypts, respectively. *P < 0.05, **P < 0.01, ***P < 0.0001; unpaired two-tailed t-test (c, e, i, k, l, p). Data are mean ± s.d. (i, k, n) or mean ± s.e.m. (h, p). For exact P values, see Source Data. Scale bars, 50 μm (a, g, j, m, o).

Source data

Extended Data Fig. 6 Oncogene-driven signalling changes.

a, FACS sorting strategy to isolate cells from Confetti and Red2Onco tissue. R1, live; R2, singlet; R3, mesenchymal/immune (EPCAM); R4, epithelial (EPCAM+); R5, mutant-epithelial (RFP+); R6, WT epithelial (YFP+); R7, immune (CD45+); R8, mesenchymal (CD45). b, Box plots showing distributions of Pearson correlation coefficients in averaged log2-transformed normalized UMIs for cell types across all pairs of mice from the same (white) and between different (grey) conditions. c, UMAP of epithelial cells detected by Louvain. k, k nearest-neighbour value. d, UMAPs showing distribution of averaged expression of marker genes. Colour bars, average log2-transformed normalized UMIs. Top left from Fig. 3b. e, Heat map representing marker expression for epithelial cells. Coloured panel (left) groups marker genes (right) for cell types. Colour bar, auto-scaled log2-transformed normalized UMIs. f, Heat maps representing differential gene expression for epithelial cells in Red2Onco compared to Confetti. Parentheses, number of differentially expressed genes. Colour bar, log2(fold change) (Supplementary Table 1). g, UMAPs showing distributions of mutant (RFP+) and WT (YFP+) epithelial cells for Confetti and Red2Onco mice. h, i, Fractions of mutant (h) and WT (i) epithelial cells in Red2Onco and Confetti mice. See Fig. 3c for other WT data. jl, Confocal images (j) of EGFP+ cells, representative of tissues quantified for stem cell number (k) and fraction (l). In j, white arrows indicate WT crypts proximate to mutant (MT) crypts. White dashed lines mark crypts. Scale bars, 25 μm. m, n, FACS plots (m) and quantification (n) of EGFPhi stem cell fractions from R5 or R6 (a). Small intestine from Lgr5-EGFP-IRES-CreERT2;Red2Onco 2 w after induction (clonal dosage (0.2 mg per 20 g body weight) for jl, mosaic dosage (4 mg per 20 g body weight) for m, n). *P < 0.05, **P < 0.01, ***P < 0.0001; n.s., statistically not significant, P > 0.05; two-sided Kolmogorov–Smirnov test (b), two-sided likelihood ratio test (h, i) and one-way ANOVA with Games-Howell’s multiple comparisons test (k, l, n). Data are mean ± s.e.m. (h, i, k, l) or mean ± s.d. (n). For exact P values, see Source Data.

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Extended Data Fig. 7 Mutant crypt induces primed differentiation.

a, b, Priming scores of stem (SC) and transit-amplifying (TA) cells of mutant (a) and WT (b) crypts towards secretory and enterocyte lineages in Red2Onco and Confetti mice. Black line, 50th percentile; dashed lines, 25th and 75th percentiles. Green and black asterisks, higher and lower in Red2Onco than in Confetti, respectively. c, qPCR of lineage markers (Lgr5, ISC; Clca1, goblet cell; Fabp1, Alpi, enterocyte; Mki67, proliferation) using sorted RFP+ and YFP+ cells from Villin-CreERT2;R26R-Confetti or Red2Onco mice 2 w after tamoxifen administration. d, e, Confocal images (d) and quantification (e) of MUC2+ goblet cells. f, hj, Images (f) from RNA in situ hybridization of enterocyte marker Fabp1 and quantification in remote WT (Remote_R2KR, Remote_R2P3; h), proximate WT (Prox_R2KR, Prox_R2P3; i) and mutant crypts (MT_R2KR or MT_R2P3; j) along crypt axis. In f, Fabp1+ cells in lower crypts (below +8) marked by white arrow. g, Illustration of cellular localization along crypt axis. Position 0 is crypt base cell. k, l, UMAPs showing distributions of enrichment scores for BMP (k, left), WNT (k, right), and NOTCH (l) pathways in epithelial cells of Red2Onco and Confetti mice. Colour bars, enrichment scores. m, Fractions of ‘active’ cells with high enrichment scores for NOTCH pathway in mutant (MT) and WT epithelial cells from Red2Onco and Confetti mice. Small intestine sections from Villin-CreERT2;R26R-Confetti or Red2Onco 2 w after tamoxifen administration (d, f). WT and mutant crypts marked with white and grey dashed outlines, respectively (d, f). Remote WT in crypts separated by >3 crypt diameters from mutant crypts. Proximate WT in crypts neighbouring fixed mutant crypts. *P < 0.05, **P < 0.01, ***P < 0.0001; two-sided Kolmogorov–Smirnov test (a, b), unpaired two-tailed t-test (c, e, hj) and two-sided likelihood ratio test (m). Data are mean ± s.e.m. (c, e, m) or mean ± s.d. (hj). For exact P values, see Source Data. Scale bars, 50 μm (d, f).

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Extended Data Fig. 8 Mutation-induced environmental changes.

a, t-SNE representing immune cells from Confetti and Red2Onco mice. b, c, Heat maps representing differential expression (DE) patterns for mesenchymal (b) and immune (c) cells from Red2Onco and Confetti mice: top 300 genes or less (FDR < 0.05, pairwise t-test). Colour bar, averaged Z-scores of log2-transformed normalized UMIs. d, Secretion factor expression in stromal clusters for Confetti. In d, f, dot size denotes percentage of cells expressing gene; colour shows average expression. e, UMAPs showing expression of Bmpr1a and Fzd7 in epithelial cells. Colour bar, log2-transformed normalized UMIs. Inset from Fig. 3b. f, Dot plots showing expression of receptors upstream of BMP and WNT pathways for epithelial cells. g, h, Fractions of mesenchymal (g) and immune (h) cells in Red2Onco and Confetti mice. Data are mean ± s.e.m. Two-sided likelihood ratio test: *P < 0.05, **P < 0.01; n.s., statistically not significant (P > 0.05). i, Degree of transcriptomic change for immune cells estimated by cell-to-cell variability (PVAR) or separability of perturbed and unperturbed cells (PAUGUR). Dotted lines show −log10(0.01). Dot colour denotes cell type; dot shape shows Red2Onco. j, Enriched biological processes from gene ontology (GO) analysis of DE genes in STC2 of Red2-PIK3CAH1047R mice relative to Confetti. One-sided Fisher’s exact test. Dotted line, −log10(0.05). k, l, Heat maps representing DE genes and their numbers (in parentheses) for mesenchymal (k) and immune (l) cells in Red2Onco mice compared to Confetti. Colour bar, log2(fold change) (Supplementary Table 3). m, Volcano plot representing DE genes in STC2 of Red2-PIK3CAH1047R mice relative to Confetti. Two-sided pairwise t-test. Red dots, genes for biological processes in j. Vertical dotted lines, absolute value of log2(fold change) = 0.259; horizontal, −log10(0.05). n, Model of direct and indirect cross-talk between mutant and WT crypts in Red2Onco mice. BC, B cell; DC, dendritic cell; Mono, monocyte; MP1, 2, macrophage 1, 2; PLC, plasma cell; TC, T cell. For exact P values, see Source Data.

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Extended Data Fig. 9 Mutant clones secrete functional BMP ligands.

ad, Representative in situ hybridization images and quantification of Axin2 (a, b) and Id1 (c, d) on sections of small intestine from Villin-CreERT2;R26R-Confetti or Red2Onco mice 2 w after tamoxifen administration. Arrowheads, fixed mutant crypts. Crypts are marked with dashed outlines. e, f, qPCR analysis of Axin2 (e), and Id1 (f). g, Experimental set-up for hj. h, Bright-field images of intestinal organoids after 2 days of treatment. The number and size of crypt-like budding structures are reduced in treated organoids. i, qPCR analysis of lineage markers. j, Representative images of LGR5–EGFP organoids show that the number of LGR5+ cells decreases following treatment. k, Experimental set-up for l, m. l, m, Bright-field images (l) and quantification (m) of intestinal organoids after 6 days of culture in WENR medium. n, qPCR analysis of BMP ligands (Bmp2 and Bmp7). o, Experimental set-up for p. p, qPCR analysis of Id1 using WT organoids after the CM treatment. q, Bright-field images of intestinal organoids from Villin-CreERT2;R26R-Confetti or Red2Onco mice 1 month after tamoxifen administration. Insets, RFP expression in the mutant organoids. r, qPCR analysis of WT and mutant organoids cultured in ENR medium. In e, f, n, sorted RFP+ or YFP+ cells from Villin-CreERT2;R26R-Confetti or Red2Onco mice 2 w after tamoxifen administration (4 mg per 20 g body weight, mosaic dosage) were analysed. *P < 0.05, **P < 0.01, ***P < 0.0001; one-way ANOVA with Games-Howell’s multiple comparisons test (b, d) and unpaired two-tailed t-test (e, f, i, m, n, p, r). Quantification graphs show data from three independent experiments (i, m, p, r). Data are mean ± s.d. (b, d, i, m, p) or mean ± s.e.m. (e, f, n, r). For exact P values, see Source Data. Scale bars: 50 μm (a, c, h), 100 μm (j, q) and 500 μm (l).

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Extended Data Fig. 10 Mutant clones drive niche stromal remodelling.

a, Heat map showing marker gene expression for STC2 among mesenchymal cells. Colour bar, averaged Z-scores of log2-transformed normalized UMIs over all cells within a cell type in Confetti mice. b, c, Representative multiplexed in situ hybridization images (b) and quantification (c) of Sfrp2 in Grem1+ cells on small intestine sections from Villin-CreERT2;R26R-Confetti or Red2Onco mice 2 w after tamoxifen administration. Arrowheads, fixed mutant crypts; grey dashed outlines indicate crypts; arrows, Grem1+ STC2 cells. d, Heat map showing expression of marker genes and secreted factors in STC1, 2 from Red2Onco and Confetti mice. Colour bar, averaged Z-scores of log2-transformed normalized UMIs over all cells within a cell type and condition. e, Projection of Pdgfra expression (middle) onto UMAP from Fig. 3f (left) for comparison. Projection of Cd81 expression onto Pdgfralo cell clusters (STC1, 2) (right). Colour bar, log2-transformed normalized UMIs. f, Sorting strategy to isolate STC2 from intestinal mesenchymal cells by FACS. R1, non-immune cells (CD45); R2, mesenchymal cells (EPCAM); R3, PDGFRAlo population. g, qPCR of STC2 markers (Cd81, Grem1), STC1 marker (Frzb) and secreted WNT modulators (Rspo3, Sfrp2, Sfrp4) using sorted CD45EPCAMPDGFRAloCD81 cells (STC1) or CD45EPCAMPDGFRAloCD81+ cells (STC2) from Villin-CreERT2;R26R-Confetti or Red2Onco mice 2 w after tamoxifen administration. h, qPCR of telocyte markers (Pdgfra, Foxl1) using sorted STC1, 2 and PDGFRAhi telocytes. *P < 0.05, **P < 0.01, ***P < 0.0001; one-way ANOVA with Games-Howell’s multiple comparisons test (c) and unpaired two-tailed t-test (g, h). Data are mean ± s.d. (c, g) or mean ± s.e.m. (h). For exact P values, see Source Data. Scale bars, 25 μm (b).

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Extended Data Fig. 11 Functional validation of oncogene-driven niche remodelling.

a, b, qPCR analysis of Id1 (a), Axin2 and Lgr5 (b) after administration of indicated inhibitor. c, Fraction of monoclonal WT (YFP+) crypts remote from (Remote) or proximate to (Prox) mutant crypts in Red2Onco mice. d, Heat maps indicate relative clone fractions of the indicated sizes. Data are mean ± s.e.m. e, Fraction of monoclonal (RFP+) mutant crypts in Red2Onco mice. f, g, Representative confocal images (f) and quantification (g) of EGFP+ (LGR5+) ISCs. Images are representative of tissues quantified in g. Arrows, proximate WT crypts; arrowheads, fixed mutant crypts. h, Representative confocal images of whole-mount small intestine. Images are representative of tissues quantified in i. Arrowheads, fixed mutant crypts. i, Violin plots of proximate WT crypt size. j, k, RNA in situ hybridization (j) and quantification (k) of Bmp2. Arrowheads, fixed mutant crypts. l, m, Representative multiplexed in situ hybridization images (l) and quantification (m) of Rspo3 in Cd81+ cells. Arrow: Cd81-positive STC2 cells; Arrowheads, fixed mutant crypts. Whole mount (fi) and sections (jm) of small intestine from Lgr5-EGFP-IRES-CreERT2 control (L5), Lgr5-EGFP-IRES-CreERT2;LSL-KrasG12D (enKR) or PIK3CALat-H1047R (enP3) mice 2 w after tamoxifen administration. In ce, graphs show data collected 2 w after concomitant administration of indicated drug and tamoxifen. Crypt borders are marked by dashed outlines (f, h, j, l). In f, h, j, l, white (f, h) or red (j, l): immunostaining for mutant KRAS(G12D) in enKR, or p-AKT in enP3. *P < 0.05, **P < 0.01, ***P < 0.0001; one-way ANOVA with Games-Howell’s multiple comparisons test (k, m) and unpaired two-tailed t-test (c, e, g, i). Data are mean ± s.d. (a, b, g, k, m) or mean ± s.e.m. (c, e). For exact P values, see Source Data. Scale bars: 50 μm (f, h, j) and 25 μm (l).

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Extended Data Fig. 12 Apc mutation induces reduction of stem cells in neighbouring wild-type crypts.

a, b, Representative confocal images of small intestine from Villin-CreERT2; Apcf/f mice at 2 w after tamoxifen administration (a) and from ApcMin/+ mice at 12 weeks of age (b). Images are representative of two independent experiments. OLFM4 staining shows a reduced number of stem cells in wild-type crypts neighbouring mutant crypts. Grey dashed outlines, Apc mutant foci (Villin-CreERT2; Apcf/f) or polyps (ApcMin/+); white dashed outlines, crypt borders. Scale bars, 50 μm. c, Bright-field images of intestinal organoids after 7 days of culture in ENR medium. Images are representative of three independent experiments. Note that organoids from Villin-CreERT2; Apcf/f mice form spheroids in ENR medium. Scale bars, 500 μm. d, qPCR analysis of WNT target gene (Axin2) and secreted WNT inhibitory factors (Dkk2, Wif1 and Notum) following Apc deletion. Data are mean ± s.d. n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.0001; unpaired two-tailed t-test. e, f, Representative multiplexed in situ hybridization images of Axin2 and Wif1 (e) and Lgr5 and Notum (f) on sections of small intestine from Villin-CreERT2; Apcf/f mice 2 w after tamoxifen administration. Images are representative of two independent experiments. Axin2 (e) and Lgr5 (f) staining shows a reduced number of stem cells in wild-type crypts neighbouring Apc mutant crypts. Grey dashed outlines, Apc mutant foci (Villin-CreERT2; Apcf/f); white dashed outlines, crypt borders. Scale bars, 50 μm.

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Supplementary information

Supplementary Information

This file contains gene set enrichment analysis (GSEA) for senescence-associated secretory phenotype (SASP) and inflammation, and theory for clonal analysis, related to Figs 1–3 and Extended Data Figs 2–4 and 8. This consists of two parts: 1) Supplementary Methods describing GSEA for SASP and inflammation, and 2) Supplementary Theory explaining clonal analysis and modelling.

Reporting Summary

Peer Review File

Supplementary Table 1

Marker genes and differentially expressed genes of epithelial cells in Red2Onco models, related to Fig. 3 and Extended Data Fig. 6. This table lists marker genes for epithelial cell types and differentially expressed genes for all epithelial cells or individual cell types in Red2Onco samples compared to Confetti control.

Supplementary Table 2

Gene sets for evaluating stem cell priming and enrichment scores for signalling pathways, related to Fig. 3 and Extended Data Fig. 7. This table shows the gene sets for: 1) differentiated sub-lineages of epithelial cells; 2) BMP, Wnt and Notch signalling pathways, and 3) senescence-associated secretory phenotype (SASP) and inflammation.

Supplementary Table 3

Cell type markers and differentially expressed genes of mesenchymal and immune cells, related to Fig. 3 and Extended Data Figs 8 and 10. The table lists marker genes for mesenchymal and immune cell types and differentially expressed genes for each of their cell types in Red2Onco samples compared to Confetti control.

Supplementary Table 4

List of expressed ligands regulating BMP and Wnt signalling pathways, related to Fig. 3 and Extended Data Figs 8 and 10. This table shows the expression of ligands regulating BMP and Wnt signalling pathways that are secreted from mutant epithelial cells and mesenchymal and immune cells.

Video 1

: Whole mount z-stack imaging of Villin-CreERT2; Red2-KrasG12D small intestine, related to Figure 1 Representative z-stack imaging of Villin-CreERT2; Red2-KrasG12D small intestine 2 weeks post-tamoxifen administration. The video is representative of 3 biologically independent samples analysed.

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Yum, M.K., Han, S., Fink, J. et al. Tracing oncogene-driven remodelling of the intestinal stem cell niche. Nature 594, 442–447 (2021). https://doi.org/10.1038/s41586-021-03605-0

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