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Apc-mutant cells act as supercompetitors in intestinal tumour initiation

Abstract

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3β. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals.

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Fig. 1: Apc-mutant cells actively impair outgrowth of WT organoids.
Fig. 2: Apc mutants induce differentiation in adjacent WT cells.
Fig. 3: Apc-mutant cells secrete Wnt antagonists.
Fig. 4: LiCl neutralizes biased drift and reduces adenoma formation in Apc−/− mice.

Data availability

The sequence libraries generated in this study are publicly available through the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under accession GSE144325. Other data sets used in this study are also publicly available via the NCBI GEO under accession numbers GSE145308, GSE65461 and GSE8671. For information regarding stem cell drift modelling, contact E.M. (edward.morrissey@imm.ox.ac.uk). All source data can be explored via the online data sharing platform Figlinq: https://create.figlinq.com/~vermeulen.lab/272. Source data are provided with this paper.

Code availability

The clone data were modelled using an R package implementing the model and are available at https://github.com/MorrisseyLab/CryptDriftR.

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Acknowledgements

S.M.v.N. is supported by a NWO OOA PhD scholarship (022.005.002). This work is supported by The New York Stem Cell Foundation and grants from KWF (UVA2014-7245), the Maurits en Anna de Kock Stichting (2015-2), Worldwide Cancer Research (14-1164), the Maag Lever Darm Stichting (MLDS-CDG 14-03), the European Research Council (ERG-StG 638193) and ZonMw (Vidi 016.156.308) to L.V. L.V. is a New York Stem Cell Foundation–Robertson Investigator. We thank the AMC laboratory for clinical chemistry (LAKC), the mouse breeding and research facilities, and the core facilities for genomics, cellular imaging and pathology for their technical support.

Author information

Affiliations

Authors

Contributions

S.M.v.N. and L.V. conceptualized the project. S.M.v.N. and L.V. designed the experiments. S.M.v.N., N.E.d.G., L.E.N., M.S.v.D. and D.R.S. performed the in vitro organoid (co-)culture experiments, the DNA, RNA and protein assays, stainings and RNA-ISH. V.K. performed the in vitro organoid recombination assays. F.A.V.B. assisted with the FACS assays. P.R. and A.S.A. assisted with the human organoid cultures and the FAP adenoma data. M.F.v.B. generated the fluorescent organoid cultures. N.L. designed and generated the overexpression constructs. N.E.d.G., L.E.N., M.S.v.D. and M.C.L. performed the in vivo experiments and tissue processing. D.O.W. developed the CRISPR strategies. L.F.M. and S.t.H. generated the clone size plots. P.M.K. generated the spider plots and helped with visualization of the data. L.K. and E.D. provided human materials. B.P.S. and J.K. analysed the RNA sequencing data. E.M. designed and performed the mathematical modelling. S.M.v.N. and L.V. wrote the manuscript, with help from B.P.S., J.K., E.M. and N.L. J.P.M., D.J.W. and M.F.B. advised on the project.

Corresponding author

Correspondence to Louis Vermeulen.

Ethics declarations

Competing interests

L.V. received consultancy fees from Bayer, MSD, Genentech, Servier and Pierre Fabre, but these had no relation to the content of this publication.

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Peer review information Nature thanks Hans Clevers, James DeGregori and Toshiro Sato for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Apc−/− cells actively impair outgrowth of Apc+/− organoids.

a, Schematic workflow for in vitro co-cultures. b, Representative images of full wells containing WT/Apc+/− co-cultures; scale bar, 1 mm. c, Relative surface contribution in WT/Apc+/− co-cultures (P = 0.3771, day 1–day 7, two-tailed paired t-test). d, Organoid expansion in WT/Apc+/− co-cultures. n = 4 independent experiments. e, Representative images of full wells containing Apc+/−/Apc−/− co-cultures; scale bar, 1 mm. f, Reduction in surface contribution of Apc+/− and Apc−/− organoids in Apc+/−/Apc−/− co-culture (P = 0.0012, day 1–day 4; P = 0.0016, day 1–day 7, two-tailed paired t-test). g, Organoid expansion of Apc+/− and Apc−/− organoids in Apc+/−/Apc−/− co-culture. h, Full well images of Apc+/− organoids with Apc+/− or Apc−/− CM at day 7; scale bar, 1 mm. Zoom panel right, 250 μm. i, Apc+/− organoid expansion in CM (P = 0.0322, day 4; P = 0.0006, day 7). Data are mean ± s.d., n = 3 independent experiments, analysed using two-sided Student’s t-test, unless otherwise specified. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns, not significant.

Source data

Extended Data Fig. 2 Apc mutants induce differentiation in adjacent WT cells through Wnt inhibition.

ad, Signature scores for Wnt signatures (a, b) and stem cell signatures (c, d) for WT organoids treated with CM for 2 or 4 days. Signature scores were calculated by summing the standardized expression of the genes within each signature. Box plots are minimum to maximum values, the box shows the 25th to the 75th percentiles, the median is indicated with a line; n = 3 biological replicates. eh, Effect of 10x concentrated WT or Apc−/− CM on WT organoid growth (e; scale bar, 250 μm), Lgr5 expression (P = 0.0033; data are mean ± s.e.m.) (f), the percentage of Lgr5–GFPhigh cells (P = 0.0371; data are mean ± s.e.m.) (g) and the clonogenicity (P = 0.0044) of WT organoids (h). n = 4 independent experiments. i, Schematic illustration of the TOP-GFP construct. j, FACS histograms showing TOP-GFP expression in mouse embryonic fibroblasts (MEFs) in the absence (unstimulated) or presence of Wnt3a. km, Mean fluorescent intensity (MFI) of TOP-GFP upon upstream stimulation with Wnt3a (WT CM versus Apc−/− CM, P < 0.0001) (k), or upon downstream pathway activation with 5 mM LiCl (WT CM versus Apc−/− CM, P = 0.9812) (l) or 2.5 μM CHIR99021 (WT CM versus Apc−/− CM, P = 0.8082) (m); n = 4 independent experiments. Data are mean ± s.d.; n = 3 independent experiments, analysed using two-sided Student’s t-test, unless otherwise specified. FACS gating can be found in Supplementary File 2. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, ns, not significant.

Source data

Extended Data Fig. 3 Apc-mutant cells secrete Wnt antagonists.

a, mRNA expression of Wnt antagonists in a time course following tamoxifen-mediated recombination. n = 3 technical replicates from a representative experiment performed 3 times (P = 0.0039 (Notum), P = 0.0115 (Wif1), P = 0.0252 (Dkk2), 72 h versus control). b, Protein levels of NOTUM and WIF1 (P < 0.0001) detected in CM of WT or Apc−/− organoids. c, Volcano plot for significantly upregulated Wnt antagonists in pooled normal or adenoma murine tissue (GSE65461; 2,483 DEGs). d, Expression of Wnt antagonists Notum, Wif1 and Dkk2 in mouse adenoma tissue by RNA-ISH. Scale bar, 100 μm. n = 3 mice per ISH probe. e, Volcano plot for significantly upregulated Wnt antagonists in human matched normal or adenoma tissue (GSE8671; 9,478 DEGs). f, NOTUM expression in FAP adenomas. Scale bar, 100 μm. g, APC-mutant crypts, marked with asterisk. Scale bar, 100 μm. APC-mutant crypts are recognized as low-grade dysplasia by their enlarged pencillate nuclei (H&E staining, right panel). Scale bar, 50 μm. h, Protein levels of NOTUM in CM of WT or APC-mutant organoids (P = 0.0291). Data are mean ± s.e.m., n = 2 WT organoid lines, n = 6 APC-mutant organoid lines. Data are mean ± s.d., n = 3 independent experiments, analysed using two-sided Student’s t-test, unless otherwise specified. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001.

Source data

Extended Data Fig. 4 Characterization of the role of individual Wnt antagonists.

a, Schematic illustration of overexpression (OE) constructs for Notum, Wif1 and Dkk2. b, mRNA expression of Wnt antagonists in OE lines; n = 3 technical replicates. c, Protein concentration in CM of OE lines; n = 3 technical replicates. df, Fluorescent images (d), relative organoid expansion (e) and clonogenic potential (f) of WT organoids incubated with recombinant NOTUM (2 μg/ml), WIF1 (5 μg/ml) and DKK2 (1 μg/ml) protein. Scale bar, 250 μm. P = 0.0011 (rNotum), P = 0.0006 (rWif1), P = 0.0144 (rDkk2) and P = 0.0003 (combination) all relative to the control. Data are mean ± s.e.m. g, Representative image of WT/Apc−/−Notum KO co-culture at day 4. Scale bar, 1 mm. h, Relative expansion of WT organoids in co-culture with Apc−/− organoids that contain CRISPR-based modifications in Wnt antagonist genes Notum, Wif1 or Dkk2. n = 2 single-cell Apc−/− KO clones per Wnt antagonist. i, MFI for TOP-GFP expression in the presence of Wnt3a and Apc−/− KO CM (P = 0.3606, one-way ANOVA, between all Apc-mutant conditions). Data are mean ± s.e.m., each dot represents a single-cell Apc−/− KO clone. j, Clonogenic potential of WT organoids that are incubated with a titration of Apc−/− KO CM; n = 2 independent experiments. k, l, Phase images (k), and clonogenicity (l) of WT human organoids incubated with recombinant NOTUM (P = 0.0113 (1:200, 0.5 μg/ml), P = 0.0059 (1:100, 1 μg/ml)) Data are mean ± s.e.m. All data are mean ± s.d., n = 3 independent experiments, analysed using two-sided Student’s t-test, unless otherwise specified. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns, not significant.

Source data

Extended Data Fig. 5 Downstream activation of the Wnt pathway rescues the Apc-mutant supercompetitor phenotype in vitro.

a, Relative clonogenic potential of WT organoids incubated with Apc−/− CM in the absence or presence of 2.5 μM CHIR (P = 0.0040, P3). b, Validation of overexpression of a non-degradable variant of β-catenin, Ctnnbs, on mRNA (P < 0.0001, for Ctnnb1 versus WT and Ctnnb1 vs Apc−/−; n = 3 biological replicates; data are mean ± s.e.m.) and protein level. Full western blot images can be found in Supplementary File 1. c, d, Fluorescent image (c) and relative surface contribution (d) of co-culture between Ctnnbs (purple) and Apc−/− (green) (P = 0.4604 day 1–day 4, P = 0.2734 day 1–day 7; two-tailed paired t-test). Scale bar, 500 μm. e, Relative LGR5 expression of human colon organoids incubated with CM in the absence or presence of LiCl (P = 0.0012, FAP CM ± LiCl). Data are mean ± s.e.m. f, Relative clonogenic potential of human colon organoids incubated with WT or FAP CM in the absence or presence of LiCl (n = 4 biological replicates; P < 0.0001, one-way ANOVA). Data are mean ± s.d., n = 3 independent experiments, analysed using two-sided Student’s t-test, unless otherwise specified. **P ≤ 0.01, ****P ≤ 0.0001, ns, not significant.

Source data

Extended Data Fig. 6 Biallelic Apc mutants exclusively express Wnt antagonists.

a, mRNA expression of Wnt antagonists Notum, Wif1 and Dkk2 in Apc+/+, Apc+/− and Apc−/− organoids. P < 0.0001 (Notum), P = 0.006 (Wif1) and P = 0.0004 (Dkk2). Data are mean ± s.e.m., n = 3 biological replicates, two-sided Student’s t-test. b, RNA-ISH on consecutive tissue slices for detection of recombined Apc alleles (ApcE14-16) and Notum in Apc+/+, Apc+/− and Apc−/− tissues. Scale bar, 50 μm for Apc+/+ and Apc+/− crypt base images, and 100 μm for Apc−/− adenoma. c, Expression of Notum in ageing Paneth cells is not detected in young mice (upper panel, <100 days old). Notum+ Paneth cells are observed in old mice (middle panel, >800 days old); positive cells are marked with arrowheads. Notum+ Paneth cells do not interfere with Notum+/Apc−/− clonal analysis and are not detected in Apc−/− mice (lower panel, <100 days old). Scale bar, 10 μm. All RNA-ISH has been performed on n = 3 mice per condition. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Source data

Extended Data Fig. 7 Effects of LiCl on the WT mouse intestine.

a, Detection of lithium (Li+) levels in mouse serum; n = 4 mice per condition, data are mean ± s.d. b, mRNA expression of Wnt target gene Lgr5 in isolated crypts of control (n = 8) or LiCl-treated (n = 10) mice (P = 0.0037). c, Percentage of Lgr5–GFP expressing cells in isolated crypts from control (n = 11) or LiCl-treated (n = 12) mice, as measured by FACS (P = 0.0140). d, Fluorescent images of Lgr5–GFP+ ISCs in crypt bases of control or LiCl-treated mice, adjacent quantification is the frequency distribution of Lgr5–GFP+ cells per half (2D) crypt; n = 125 crypts per condition, each data point is a crypt base. Scale bar, 50 μm. e, Schematic illustration of the in vivo treatment scheme with tamoxifen and LiCl in Lgr5-CreErt2;Rosa26mTmG (WT) mice. f, Box plot for Cre-reporter activity as measured by the percentage of induced crypts at day 4 (n = 5 mice, P = 0.3322). g, Scatter dot plots for tdTomatoneg/GFPpos clone induction per intestinal region as measured by FACS (P = 0.6335 (proximal SI), 0.5171 (distal SI) and 0.7804 (colon)). h, Fluorescent images of representative clone sizes of WT crypts of mice treated with or without LiCl, mTmGFP+ clones are visualized in white. Scale bar, 20 μm. i, Respective box plots of clone size distributions of WT mice in the presence or absence of LiCl; data points indicate fractional crypt sizes per time point, with random x and y jitter added for visualization. The mean is indicated with a dashed line. j, k, Relative clone sizes (P = 0.7749, day 21) (j) and the relative amount of fixed clones (k) remain unaffected by LiCl (P = 0.8668, day 21). l, No effect of LiCl on the probability of replacement for WT drift. All box plots are minimum to maximum, the box shows the 25th to the 75th percentiles, and the median is indicated with a line. Data are mean ± s.e.m.; n = 3 control mice, n = 2 LiCl mice, unless otherwise specified. All data are analysed using two-sided Student’s t-test. For FACS gating data, see Supplementary File 2. *P ≤ 0.05, **P ≤ 0.01, ns, not significant.

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Extended Data Fig. 8 Notum influences Lgr5 expression in adjacent crypt bases.

a, b, Duplex RNA-ISH of Lgr5 (magenta) and Notum (blue) in crypt bases (scale bar, 50 μm) (a) and relative Lgr5 expression in crypts adjacent to Notumpos crypts (b) (P < 0.0001, one-way ANOVA). c, d, Duplex RNA-ISH of Lgr5 (magenta) and Notum (blue) in crypt bases in the presence of LiCl (scale bar, 50 μm) (c) and relative Lgr5 expression in crypts adjacent to Notumpos crypts in the presence of LiCl (d) (P = 0.4032, one-way ANOVA). Box plots are the minimum to maximum values, the box shows the 25th to the 75th percentiles, and the median is indicated with a line; each data point represents a crypt; n = 3 mice per condition. ****P ≤ 0.0001, ns, not significant.

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Extended Data Fig. 9 LiCl influences stem cell dynamics and reduces adenoma formation.

a, b, The effect of LiCl on WT stem cell dynamics based on the inferred replacement probability (PR) (a) or when the number of WT stem cells (NWT) is determined (b). c, d, Fits of clone size distributions for the adapted stem cells model (NWT) for WT and Apc−/− clone dynamics in the absence (c) or presence (d) of LiCl. Each data point indicates the average clone size proportion of that particular time point, and the error bars are the 95% credible interval for the proportion. Modelling is based on crypt data from n = 12 mice for both the control group and the LiCl-treated group. e, The amount of adenomas counted per intestinal region in the absence or presence of LiCl (P = 0.0037 (proximal SI), P < 0.0001 (distal SI) and P = 0.0077 (colon)); n = 9 (control) and n = 12 (LiCl). The box plot is the minimum to maximum values, the box shows the 25th to the 75th percentiles, and the median is indicated with a line; each data point represents a mouse. All data are analysed using two-sided Student’s t-test. **P ≤ 0.01, ****P ≤ 0.0001.

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Extended Data Fig. 10 LiCl does not influence KrasG12D stem cell dynamics.

a, Schematic illustration of PCR strategy to detect WT (KrasWT) and mutant (KrasG12D) alleles. b, Successful recombination of KrasG12D organoids after tamoxifen administration results in loss of Lox-Stop-Lox band, which means transcription of KrasG12D. ce, Fluorescent images (c), relative Lgr5 expression (P = 0.0013) (d) and clonogenicity (P = 0.0007, data are mean ± s.d.) (e) of KrasG12D organoids incubated in the absence or presence of 5 mM LiCl. Scale bar, 250 um. n = 3 independent experiments. f, Schematic illustration of in vivo treatment scheme with tamoxifen and LiCl in Lgr5-CreErt2; Rosa26mTmG;KrasG12D mice. g, Sorting strategy of crypts isolated from KrasG12D mice 7 days after tamoxifen administration for KrasWT (tdTom+) and KrasG12D (GFP+) cells. h, Validation of recombination ( = loss of LSL-site) of the KrasG12D locus shows complete recombination in the GFP+-sorted fraction. i, Representative clone sizes of KrasG12D mice treated with or without LiCl; mTmGFP+ clones are visualized in white. Scale bar, 20 μm. j, Respective box plots of clone size distributions of KrasG12D mice in the presence orabsence of LiCl. The box plot is the minimum to maximum values, the box shows the 25th to the 75th percentiles, and the median/mean is indicated with a dashed/straight line respectively; the data points indicate fractional crypt sizes per time point, with random x and y jitter added for visualization. The mean is indicated with a dashed line. n = 2 mice per time point. k, l, Relative clone sizes (P = 0.5861, day 21, n = 2 mice per time point) (k) and the relative number of fixed clones remain unaffected by LiCl (P = 0.6718, day 21, n = 2 mice per time point). m, Modelling the probability of replacement (PR) of KrasG12D LiCl mice (versus WT LiCl mice) compared to untreated KrasG12D mice (versus WT control mice). Data are mean ± s.e.m.; n = 3 independent experiments, analysed using two-sided Student’s t-test, unless otherwise specified. PCRs on gel (b, h) have been repeated three times. For gel source data, see Supplementary File 1. For FACS gating data, see Supplementary File 2. **P ≤ 0.01, ***P ≤ 0.001, ns, not significant.

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van Neerven, S.M., de Groot, N.E., Nijman, L.E. et al. Apc-mutant cells act as supercompetitors in intestinal tumour initiation. Nature 594, 436–441 (2021). https://doi.org/10.1038/s41586-021-03558-4

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