Abstract
Oncogenic mutations that drive colorectal cancer can be present in healthy intestines for long periods without overt consequence1,2. Mutation of Apc, the most common initiating event in conventional adenomas3, activates Wnt signalling, thus conferring fitness on mutant intestinal stem cells (ISCs)4,5. Apc mutations may occur in ISCs that arise by routine self-renewal or by dedifferentiation of their progeny. Although ISCs of these different origins are fundamentally similar6,7, it is unclear whether both generate tumours equally well in uninjured intestines. It is also unknown whether cis-regulatory elements are substantively modulated upon Wnt hyperactivation or as a feature of subsequent tumours. Here we show in two mouse models that adenomas are not an obligatory outcome of Apc deletion in either ISC source, but require proximity of mutant intestinal crypts. Reduced crypt density abrogates, and aggregation of mutant colonic crypts augments, adenoma formation. Moreover, adenoma-resident ISCs open chromatin at thousands of enhancers that are inaccessible in Apc-null ISCs that are not associated with adenomas. These cis elements explain adenoma-selective gene activity and persist, with little further expansion of the repertoire, as other oncogenic mutations accumulate. Thus, cooperativity between neighbouring mutant crypts and new accessibility at specific enhancers are key steps early in intestinal tumorigenesis.
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Data availability
Sequencing data, deposited in the Gene Expression Omnibus (GEO) under accession numbers GSE241384 and GSE281056, are available without restriction. Sources of published data for epigenome comparisons (Fig. 4) are listed in the Methods. Source data are provided with this paper.
Code availability
The script for Monte Carlo simulations of crypt clustering is included among the source data.
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Acknowledgements
This work was supported by the Claudia Adams Barr programme in basic research at Dana-Farber Cancer Institute, NIH grants R01DK081113 and R01 DK082889 (R.A.S.), and generous gifts from the Lind family. The authors thank J. Kraiczy and F. de Sousa e Melo for helpful discussions, and flow cytometry and organoid facilities at Dana-Farber Cancer Institute and Harvard Digestive Disease Center (P30DK034854).
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Contributions
L.G., H.S. and R.A.S. conceived and designed the study. L.G. (Figs. 1a–h, 2a–c,e–g,i, 3a–f,h,i and 4a–e and Extended Data Figs. 1e,f, 2a,b,d,e, 3a,e,g, 4, 5, 6b–e and 8b–d), H.S. (Figs. 1f,g, 2b and 4b,d and Extended Data Fig. 8b–d), G.T. (Figs. 1f,g and 4d and Extended Data Figs. 2g, 3a, 6a and 8a), K.B. (Figs. 1i, 2d and 3g and Extended Data Figs. 2c,f,g, 3b,d,g and 8a), K.M. (Extended Data Fig. 1a,b), S.B. (Extended Data Fig. 1a–d) and U.J. (Figs. 1f,g and 4b) acquired and analysed data. L.G. (Figs. 1f,g, 2b,f,g, 3b,h and 4b–d and Extended Data Figs. 3c,f, 4b–d, 5b,c, 6b,c, 7a,e–g and 8b–f), H.S. (Fig. 4b,c and Extended Data Figs. 3c and 7a), S.B. (Extended Data Fig. 1c,d) and U.J. (Fig. 4b) performed computational analyses. S.M. supported all computational analyses. A.M. and F.J.d.S. generated the mutant organoid series (Extended Data Fig. 8d). M.H. provided Atoh1creERT2 mice. L.G. and R.A.S. wrote the manuscript, with input and approval from all authors.
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Extended data figures and tables
Extended Data Fig. 1 Atoh1Cre(ER) (Sec-Cre) mice capture routine Sec dedifferentiation into conventional homeostatic Lgr5+ ISCs.
a, Crypt-villus units from a representative Sec-Cre;R26RTom (Apc+/+) ileum 1 day PCI (N = 8 mice), showing labelled villus Sec and crypt base Paneth cells. Ten days PCI, nearly every villus carried scattered goblet cells and strips of consecutive tdTom+ enterocytes, which represent the progeny of dedifferentiated Atoh1Cre labelled ISCs competing neutrally with unlabelled ISCs (N = 3 mice). Three weeks PCI, tdTom label persisted only in long-lived Paneth cells and occasional whole crypt-villus units (N = 5 mice), reflecting clonal fixation of dedifferentiated Atoh1Cre-marked ISCs. Scale bars 50 μm. Created with BioRender.com. b, Labelled clonal ribbons were present 100 days PCI, indicating that dedifferentiated and clonally fixed ISCs are long-lived. N = 3 mice. Scale bars: left 1 mm, right 50 μm. c-d, Single-cell RNA analysis of ileal Sec-Cre;R26RTom crypt cells captured by tdTom flow cytometry 0.5 and 1.5 days (combined) PCI, showing a substantial fraction of tdTom+ cells that uniquely express classic ISC markers: Lgr5, Axin2, Olfm4, and others shared with replicating Atoh1+ Sec progenitors (Sec-pro): Top2a and Mki67. The findings confirm that Sec-cell dedifferentiation is a routine homeostatic event. Ent-pro, enterocyte progenitors; Gob/Pan (GP), goblet/Paneth precursors; EE, enteroendocrine. e, Higher labelling of whole crypts in ISC-Cre (N = 4 mice) than in Sec-Cre (N = 6 mice) Apc+/+ (WT) ilea. Bars, mean +SD; unpaired t test with Welch’s correction, one-tailed). f, Adenomas carpeted ISC-Cre duodenum 3 weeks PCI, necessitating euthanasia (N = 12 mice). Scale bar 1 mm.
Extended Data Fig. 2 Different tumour loads in ISC-Cre and Sec-Cre ApcFl/Fl ileum.
a, Distributions of clonal events in ISC-Cre and Sec-Cre Apc+/+ and ApcFl/Fl ilea. N, mouse numbers. P values from comparisons between ApcFl/Fl ilea in ISC-Cre vs. Sec-Cre derived using unpaired t test with Welch’s correction (one-tailed). b, Photomicrograph of an Apc−/− Sec-Cre ileum 16 weeks PI, showing a single diminutive adenoma (dashed rectangle, magnified in inset). Scale bar 1 mm, inset 100 µm. c, ISC-Cre Apc−/− ileal adenoma showing that S-phase (EdU+) cells are not confined to crypts, as in neighboring Apc+/+ tissue, but present throughout the tumour. N = 4 mice, scale bar 50 μm. d, Illustrative tdTomato- microadenoma (dashed oval, 1 example from N = 2 mice) in Sec-Cre ileum, reflecting Cre-induced recombination at the Apc but occasionally not at the Rosa26 locus. Bar 50 μm. e, PCR genotyping of genomic DNA for Apc exon 14 shows the excised (floxed, 240 bp) product in FACS-purified ISCs (Tom+ cells) from ISC-Cre or Sec-Cre mice, but not in Tom- cells. A larger non-specific PCR product is amplified in all samples. N = 2 independent isolates. Whole gel is shown. f, Axin2 RNA in situ hybridization shows expression as a marker of active Wnt signaling in proliferative normal crypt cells and elevated levels in scattered cystic structures (arrows) in Sec-Cre;Apcfl/fl ileum. N = 3 intestines. Scale bar 1 mm; boxed area is magnified below, scale bar 50 μm. g, PCR genotyping of genomic DNA for KrasG12D (top, N = 3 mice) and null Smad4 (right, N = 2 mice) alleles verifies Cre-mediated recombination in FACS-purified ISCs (Tom+ cells) but not in DNA extracted from toes (top N = 3 mice, bottom N = 2 mice), which lack Cre recombinase. Source gels shown in Supplementary Fig. 1.
Extended Data Fig. 3 Gene deregulation in Apc-null ISCs and organoids.
a, Representative FACS plots for purification of Apc−/− ISCs (GFP+ Tom+) from ISC-Cre;R26RTom mice (left, n > 10) and from Sec-Cre;R26RTom mice that also carry the Lgr5Dtr-Gfp allele44 for fluorescent ISC marking (right, n > 10). ISCs reflecting dedifferentiation of Atoh1Cre labelled Sec cells represent 1.7% of viable cells in Sec-Cre mice 6 weeks PCI. Supplementary Fig. 2 shows the FACS gating strategy. b, Apc-null adenomas in ISC-Cre contain a high proportion of GFP+ (Lgr5+) ISCs, extending well beyond the crypt base. N = 8 mice. Left, merged fluorescent signals; right, isolated GFP (Lgr5) signal. Scale bar 50 μm. c, Aberrant expression of 3,286 genes (RNA-seq, DESeq2 analysis, two-tailed Wald test corrected for multiple comparisons, q < 0.01, log2 fold-change >2, base mean >20 counts) in Apc−/− ISCs isolated from ISC-Cre mice compared to Apc+/+ (WT, from mice lacking Cre) ISCs, both isolated 14 days PCI. d, Additional example of Notum expression (RNA in situ hybridization) in Sec-Cre ileal clones (N = 4 mice, scale bar 50 μm). e, Wild-type (Apc+/+) organoids are budding structures, distinct from the spheroidal morphology observed in Apc-null organoids. N = 5 independent cultures, scale bar 1 mm. f, Aberrant gene expression in Apc−/− organoids in vitro overlaps with genes dysregulated in Apc−/− ISCs isolated from ISC-Cre mice. Table shows examples of genes dysregulated (average DESeq read counts from N = 2 mice each) in ISCs from ISC-Cre mice and their relative expression in ISCs isolated from WT mice, Apc−/− ISCs isolated from Sec-Cre mice (in vivo), and organoids cultured from each source of small intestine crypts (N = 2 independent cultures from each). g, RNA in situ hybridization shows Notum (top, N = 2 mice) and Spock2 (bottom, N = 2 mice) expression predominantly in ISC-Cre adenomas. Scale bars 50 μm; dashed lines demarcate crypt bottoms. h, Context-dependent consequences of Apc loss. In gene activity and absence of adenomatous features, ISCs from Sec-Cre behave like WT ISCs in vivo but their expansion in organoid medium lacking RSPO phenocopies their ISC-Cre counterparts. Created with BioRender.com.
Extended Data Fig. 4 Monte Carlo simulations of crypt clustering.
a, Variegated Lgr5Cre expression in ISC-Cre mice and sporadic dedifferentiation in ISC-Cre mice result in areas with variably dense (top, 5 days PCI, N = 6 mice) and sparse (bottom, N = 15 mice) tdTom labelling, respectively. Scale bars 50 μm. b, Top: the relation between crypt clustering (y-axis, values +SD) and basal rates of crypt labelling (x-axis) is non-linear. Bottom: derivatives of the clustering rates. The largest change in clustering rate occurs between the labelling rates observed in ISC-Cre and Sec-Cre ileum. c, Simulation of crypt clustering in silico. Using normal distributions fit to the observed rates of crypt labelling, sequential crypts with randomly selected cells were represented by vectors, given the prescribed rate of labelling, and simulated in silico. Distributions of clustered crypts were generated from 100,000 iterations for each dataset. d, The cluster distribution of Sec-Cre crypts (top) fell at the low end of the range expected from random labelling. In contrast, ISC-Cre mouse ilea (bottom) showed more clustering than predicted from random basal crypt labelling, as expected from the known patchiness of variegated Lgr5Cre expression. Dark grey: 5th to 95th percentile range, light grey: maximum range for the simulated data. Cumulative frequencies displayed together for comparison in Fig. 2f. e, Whole mount fluorescence micrograph of the largest crypt cluster detected in a Sec-Cre ileum (14 weeks PCI, example from N = 4) and showing adenomatous features: extension beyond the tissue plane and effaced crypt boundaries. Scattered isolated crypts appear on the periphery. Whole-mount fluorescence image from ApcFl/Fl ISC-Cre ileum 16 days PCI shows an adenoma (dashed yellow structure: extra-planar growth, fused dysmorphic crypts) among variably sized patches of untransformed Apc-null crypts with distinct borders. N = 4 mice, scale bars 1 mm.
Extended Data Fig. 5 Perturbations of crypt clustering in Sec-Cre mice.
a, Whole-mount images of representative ISC-Cre ilea 5 days and 15 days PCI demonstrate dense, patchy labelling correlated with adenomas (15 days PCI), compared to diffusely scattered singletons observed in Sec-Cre as late as 14 weeks PCI. Bars 1 cm. Below, tdTom fluorescence signals in ISC-Cre whole mounts (5 days PCI, N = 4 ilea) are greatest in the distal 2 cm, correlating with tumour size and abundance at euthanasia (2-3 weeks PCI, N = 4 ilea). b, Increased rates of clonal crypt marking in Sec-Cre;ApcFl/Fl mice with 12 injections of Tam or by ablating Lgr5DTR-Gfp ISCs with Diphtheria toxin (DT), hence increasing Sec cell dedifferentiation. N, mouse numbers; first two columns repeated from Fig. 1d; unpaired t test with Welch’s correction, one-tailed. c, Upon elevation of basal crypt labelling with 12 doses of Tam (top) or DT treatment (bottom), Monte Carlo simulations showed more doublets than expected from random crypt distributions (dark dots in right graph), but larger clusters remained rare. Cumulative frequencies displayed together for comparisons in Fig. 3b; dark grey shading: 5th to 95th percentile range, light grey: maximum range for simulated data. d, High TdTom labelling in Sec-Cre colon, owing to large number of native Sec cells available to dedifferentiate. N = 6 colons, scale bar 1 mm. e, High adenoma burden after 12X Tam in a representative Sec-Cre;ApcFl/Fl colon from N = 8 mice. Scale bar 1 mm, boxed area magnified below (scale bar 150 μm).
Extended Data Fig. 6 Perturbation of crypt clustering in ISC-Cre mice.
a, PCR genotyping of genomic DNA for Apc exon 14 shows the excised product (240 bp) in FACS-purified ISCs (Tom+ cells) from ISC-Cre mice treated with 0.1 mg Tam (N = 2 mice) but not in ISCs purified from animals that did not receive Tam (N = 2 mice). Purified Tom- and Tom+ crypt cell fractions from mice treated with 1 mg Tam (Extended Data Fig. 2g) serve as negative and positive controls. Whole gel is shown; a larger non-specific PCR product is amplified in all samples. b, In situ hybridization revealed Notum expression in clonal structures in ISC-Cre;Apcfl/fl ileum after 2 doses of 0.1 mg Tam (N = 2 mice). Top: low magnification, bottom (2 images): high magnification; all scale bars 50 μm. c, Monte Carlo simulations revealed high clustering after ISC-Cre mice were treated with 0.1 mg Tam, as expected from the underlying patchy variegation, but cluster sizes were substantially reduced compared to mice treated with 1 mg Tam. Cumulative frequencies compared with others in Fig. 3g; dark grey: 5th to 95th percentile range, light grey: maximum range for simulated data. Note different y-axis ranges for 1 mg and 0.1 mg treatments; circled dots: measured clustering frequencies. d, Sparse crypt labelling detected in ISC-Cre ApcFl/Fl ileum whole mounts 5 days PCI with 0.1 mg Tam. N = 3 mice, scale bar 1 cm. e, Whole ISC-Cre;ApcFl/Fl ileum 8 weeks (N = 4 mice) and 23 weeks (N = 2 mice) PCI after 0.1 mg Tam, showing small discrete adenomas (dashed ovals) in one of the latter ilea. Scale bars 1 cm.
Extended Data Fig. 7 Altered enhancer accessibility in adenoma-resident Apc-null ISCs.
a, Aberrant accessibility at 7,298 enhancers (DESeq2 analysis, log2 fold-change >2, two-tailed Wald test corrected for multiple comparisons, q < 0.01) in Apc−/− duodenal ISCs from ISC-Cre mice, compared to Apc+/+ (WT, from mice lacking Cre) ISCs, both isolated 14 days PCI. N = 2 mice each. b, DNA at enhancers newly accessible in ISCs from adenoma-bearing ISC-Cre mice are hypomethylated (range 0% to 100% methylation in sites containing >5 CpGs) relative to the same sites in Apc+/+ ISCs. Signals representing the full spectrum of DNA methylation are shown for comparison: at 5,000 arbitrary active H3K27ac+ enhancers (unmethylated in ISCs), at all CpGs (largely methylated), and at 5,000 neuronal enhancers37 (unmethylated in neuronal cells but methylated in WT and Apc−/− ISCs). c, DNA sequence motifs appreciably enriched among enhancers newly accessible in Apc−/− ISCs match transcription factors from the AP-1, TCF/LEF and FOX0 families. P values derived using the Fisher Exact test (one-tailed, corrected for multiple comparisons). d, Additional representative PyGenome tracks illustrate newly accessible enhancers (asterisks) in a locus (Spock2) with elevated RNA expression in Apc−/− ISCs. e, Top: differential analysis of enhancers in Apc−/− ileal ISCs from ISC-Cre mice treated with 1 mg or 0.1 mg Tam, showing differences similar to those across ~100,000 called ATAC-seq peaks (DEseq2, two-tailed Wald test corrected for multiple comparisons, q < 0.01, log2 fold-change ≥2) between WT and adenoma-resident duodenal ISCs. Bottom, alternative display of volcano plot shown in panel a. f, Pearson correlations among called peaks in ATAC-seq analysis of WT or adenoma-resident Apc−/− duodenal ISCs and of WT or Apc−/− ileal ISCs from Sec-Cre mice and from ISC-Cre mice treated with 1 mg or 0.1 mg Tam. Black boxes demarcate like samples: WT ISCs (bottom left, showing regional differences); adenoma-resident ISCs (top right, duodenal and ileal); ISCs not associated with adenomas (center, Sec-Cre mice treated with 1 mg Tam and ISC-Cre mice treated with 0.1 mg Tam). g, Differential analysis of enhancers in Apc−/− ISCs from Sec-Cre mice treated with 1 mg Tam and from ISC-Cre mice treated with 0.1 mg Tam, showing few differences across ~100,000 called ATAC-seq peaks, in contrast to the modulated chromatin access in adenoma-resident Apc−/− ISCs from ISC-Cre mice (1 mg Tam, panel e). h, Additional PyGenome tracks from ATAC-seq samples represented in Fig. 4d, showing sites selectively accessible (shaded boxes) in adenoma-resident ISCs.
Extended Data Fig. 8 Lack of appreciable additional enhancer accessibility when oncogenic mutations are superimposed on Apc-null ISCs.
a, PCR genotyping of genomic DNA for Trp53 deletion reveals the 612-bp product (verified by DNA sequence) expected from Cre-mediated recombination in FACS-purified ISCs (Tom+ cells, N = 3 independent isolates) but not in DNA extracted from toes (N = 3 mice), which lack Cre recombinase. A non-specific PCR product (uninterpretable DNA sequence) was amplified in all samples. Source gel shown in Supplementary Fig. 1. b, Minimal expansion of the enhancer repertoire (519 additional accessible sites, log2 fold-change ≥2, DESeq2, two-tailed Wald test corrected for multiple comparisons, q <0.01), in adenoma-associated Apc−/− ISCs upon addition of KrasG12D mutation (N = 4 mice) and no further expansion upon Trp53 loss (N = 2 mice) in vivo. Two samples shown for each genotype. c, Principal component (PC) analysis of open chromatin (MACS2 called peaks) in WT (N = 4 independent organoid cultures), Apc−/− (A, N = 3 cultures), Apc−/−;KrasG12D (AK, N = 4 cultures), Apc−/−;KrasG12D;Trp53−/− (AKP, N = 2 cultures), and Apc−/−;KrasG12D;Trp53−/−;Smad4−/− (AKPS, N = 3 cultures) intestinal organoids. The largest distinction (PC1) is between WT and all Apc-null organoids. d, K-means clustering (k = 3) of 7,574 sites with objectively differential chromatin access in the mutational series compared to WT ISCs. All mutant organoids gave consistent signals across strong (cluster 1: n = 701) and moderate (cluster 2: n = 2,376) sites. The volcano plot shows that 4,497 sites (cluster 3) with ostensibly enriched access in AKPS organoids fail stringent thresholds for significance (two-tailed Wald test corrected for multiple comparisons). Control sites are 4,000 arbitrary enhancers accessible in all organoids, including WT. e, Significant overlap between enhancers differentially accessible in Apc-null ISCs in vivo and Apc−/− organoid cultures. Sites are clustered by whether peaks were called only in vivo, only in organoids, or in both. At sites called only in one, signals are evident in the other, but not in WT ISCs or organoids. Drawings in b–e were created with BioRender.com. f, PyGenome tracks for ATAC-seq signals at the Sox17 locus in Apc−/− ISCs isolated from ISC-Cre crypts in vivo and in organoids cultured from WT or Apc−/− intestinal crypts. Boxed areas are magnified below to highlight chromatin access in those locations.
Supplementary information
Supplementary Information
This file contains Supplementary Figs. 1 and 2 and Suppleemntary Tables 1. Supplementary Fig. 1: Uncropped gels from genotyping analyses shown in extended data. Supplementary Fig. 2: FACS gating strategy to sort GFP+ tdTom+ intestinal stem cells. Supplementary Table 1: Summary of all statistical tests used in the study.
Supplementary Data
Scripts for Monte Carlo simulations.
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Gaynor, L., Singh, H., Tie, G. et al. Crypt density and recruited enhancers underlie intestinal tumour initiation. Nature 640, 231–239 (2025). https://doi.org/10.1038/s41586-024-08573-9
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DOI: https://doi.org/10.1038/s41586-024-08573-9
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