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Sequential cancer mutations in cultured human intestinal stem cells

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Abstract

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.

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Figure 1: Inactivation of APC and P53 in human intestinal organoids.
Figure 2: KRASG12D/APCKO/P53KO/SMAD4KO organoids grow in the absence of stem-cell-niche factors in vitro.
Figure 3: Quadruple-mutant organoids grow as invasive carcinomas in vivo.
Figure 4: Progressive CIN and aneuploidy upon introduction of CRC mutations.

Accession codes

Primary accessions

European Nucleotide Archive

Data deposits

Sequencing data have been deposited in the EMBL European Nucleotide Archive under accession number ERP009240.

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Acknowledgements

We would like to thank H. M. Rodermond for help with in vivo transplantation assays and members of the contributing laboratories for support. We thank A. Pronk, W. van Houdt and J. van Gorp for facilitating human colon tissue. We are grateful for support from the following: The Netherlands Organisation for Scientific Research (NWO-ZonMw) VENI grant to J.D. (91614138); University of Amsterdam (2012-5735) and The Dutch Digestive Diseases Foundation (MLDS) (FP13-07) to C.Z. and J.P.M.; Netherlands Institute of Regenerative Medicine (N.S. and G.S.); Dutch Cancer Society (KWF) (KWF/PF-HUBR 2007-3956 for H.B.; KWF Fellowship UU2013-6070 for H.J.S.); Stand Up to Cancer/Stichting Vrienden van het Hubrecht (M.v.d.W.); NWO-ZonMw (116.005.002 for R.v.B.); and the CancerGenomics.nl (NWO Gravitation) program.

Author information

Authors and Affiliations

Authors

Contributions

J.D. and H.C. conceived the project and wrote the manuscript. J.D. engineered and characterized all mutant organoid lines. R.H.v.J., B.P., H.J.S., R.M.O. and G.J.P.L.K. designed and performed live-cell imaging experiments. C.Z. and J.P.M. performed in vivo transplantation assays. R.v.B. and E.C. performed off-target analyses. J.D. performed karyotyping. A.B. made karyograms. G.J.O. staged subcutaneous tumours. H.B. and J.K. performed immunohistochemistry. N.S. optimized matrix for organoid growth. G.S. designed APC sgRNAs. M.v.d.W. established normal human colon organoid line. M.L. helped genotype the mutant small intestinal organoids.

Corresponding author

Correspondence to Hans Clevers.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Introducing inactivating mutations in the APC and P53 genes in human intestinal organoids using CRISPR/Cas9.

a, Schematic representation of the targeted exon of the human APC (left) and P53 (right) loci and sequences of the designed sgRNAs. b, c, PCR amplification products of the mutated alleles of APC (b) and P53 (c) were obtained using primers flanking the targeted exon. Subsequent sequencing revealed indels at the expected locations. PAM sequences are underlined in red in wild-type sequences. Of note, the curved lines bridging the gaps in deleted alleles are drawn by the alignment software.

Extended Data Figure 2 KRASG12D, APCKO, P53KO and SMAD4KO mutation combinations in human intestinal organoids.

ac, PCR amplification products of the indicated genes of KRASG12D/APCKO (a), KRASG12D/APCKO/P53KO (b) and KRASG12D/APCKO/P53KO/SMAD4KO (c) organoids were obtained using primers flanking the targeted exon. Subsequent sequencing revealed indels at the expected locations. PAM sequences are underlined in red. Of note, the curved lines bridging the gaps in deleted alleles are drawn by the alignment software. d, Schematic representation of the targeted exon of the human SMAD4 locus and sequences of the designed sgRNAs. e, qRT–PCR for AXIN2 (top) and P21 (bottom) in the indicated organoid cultures. Top, the indicated organoid lines were cultured in the presence (WENR) or absence (EN) of WNT/R-spondin. Bottom, the indicated organoid lines were cultured in the presence or absence of nutlin-3 for 24 h. Expression was normalized to GAPDH. Horizontal bars represent mean of n = 3 independent experiments.

Extended Data Figure 3 Using CRISPR/Cas9-mediated genome editing to introduce APC, P53, KRASG12D and SMAD4 mutations in human colonic organoids.

ad, Using the strategies depicted in Figs 1a and 2a, d, APCKO, APCKO/P53KO (a), KRASG12D (b), KRASG12D/APCKO, KRASG12D/APCKO/P53KO, KRASG12D/APCKO/P53KO/SMAD4KO (c) and P53KO (d) mutant human colon organoids were generated. Experiment was performed at least three independent times for each mutation. e, qRT–PCR for AXIN2 in the indicated organoid lines cultured in the presence (WENR) or absence (EN) of WNT/R-spondin. Expression was normalized to GAPDH. Horizontal bars represent mean of n = 3 independent experiments. f, Western blot analysis of P53 and P21 expression in the indicated human colon organoid lines cultured in the presence or absence of nutlin-3. GAPDH, loading control. g, qRT–PCR for AXIN2 in the indicated organoid lines cultured in the presence (WENR) or absence (EN) of WNT/R-spondin. Expression was normalized to GAPDH. Horizontal bars represent mean of n = 3 independent experiments. h, Western blot analysis of SMAD4 and P53 expression in the indicated human colon organoid lines. Please note that quadruple-mutant clone 1 contains SMAD4 frameshift-inducing indels in both alleles whereas clone 2 contains a frameshift-inducing indel in one and an in-frame deletion in the other allele (reduced SMAD4 expression). GAPDH, loading control. Scale bars, 100 µm.

Extended Data Figure 4 Quadruple-mutant human intestinal organoids grow as tumours with features of invasive carcinoma in vivo.

a, Wild-type and all engineered human intestinal organoid lines were injected subcutaneously in immunodeficient mice. Mice injected with KRASG12D/APCKO/P53KO (triple) and KRASG12D/APCKO/P53KO/SMAD4KO (quadruple) organoids developed visible nodules. b, Tumour sizes were examined 8 weeks after transplantation. c, d, H&E (top left, bottom left), hKRT (top right, bottom middle) and Ki67 (bottom right) immunostainings on nodules isolated from triple- (c) and quadruple-mutant (d) injected mice. Triple-mutant organoids did engraft but remained small, showed only weak proliferation and had adenoma features (n = 3 mice). Quadruple-mutant-derived tumours were highly proliferative with features of invasive carcinoma (n = 13 mice). See Fig. 3 for more details. Scale bars, 100 µm.

Extended Data Figure 5 Histological analysis of triple- and quadruple-mutant organoids reveals morphological changes in vitro.

a, Representative H&E and Ki67 immunostainings on the indicated human small intestinal organoid lines (n = 4 independent experiments). b, Representative H&E and Ki67 immunostainings on the indicated human colon organoid lines (n = 3 independent experiments). c, Representative E-cadherin immunostainings on wild-type and quadruple-mutant human small intestinal organoids (n = 4 independent experiments). Asterisk indicates residual Matrigel. Scale bars, 100 µm.

Extended Data Figure 6 Progressive aneuploidy upon introduction of CRC mutations.

ad, Karyograms of APCKO (a), APCKO/P53KO (b), KRASG12D/APCKO/P53KO (c) and KRASG12D/APCKO/P53KO/SMAD4KO (d) organoids, showing extensive aneuploidy in organoids harbouring CRC mutations (20 spreads were analysed per line). Note the occurrence of trisomy 7 in APCKO and APCKO/P53KO (independent clones) organoids. M, marker chromosomes.

Extended Data Figure 7 Loss of both APC and P53 results in extensive CIN and aneuploidy.

a, APCKO, P53KO and APCKO/P53KO mutations were introduced in a second independent human intestinal organoid line. PCR amplification products of the mutated alleles of APC and P53 were obtained using primers flanking the targeted exon. Subsequent sequencing revealed frameshift-inducing indels at the expected locations. Left, APC genotyping; right, P53 genotyping. PAM sequences are underlined in red. Of note, the curved lines bridging the gaps in deleted alleles are drawn by the alignment software. b, Western blot analysis for P53 and P21 expression in the second human intestinal organoid line cultured in the presence or absence of nutlin-3. GAPDH, loading control. c, qRT–PCR for AXIN2 in the second human intestinal organoid line cultured in the presence (WENR) or absence (EN) of WNT/R-spondin. Expression was normalized to GAPDH. Horizontal bar represents mean of n = 3 independent experiments. d, Chromosome numbers were counted in the second human intestinal organoid lines. Graphs plot the percentage of cells with chromosome counts <44, 44–48 (normal) and >48 (at least 50 spreads were counted). e, As in d, but for indicated human colon organoid lines. f, Live-cell imaging was performed to monitor chromosome segregations in the indicated human small intestinal organoid lines. Graph shows the percentage of erroneous mitoses. Each dot represents the percentage of errors in one organoid. Horizontal bars represent median of all dots. A video is included of organoids depicted as dots with green outline (Supplementary Video 6). WT, wild type; KO, knockout. g, As in f, but for indicated human colon organoid lines. h, Western blot analysis of phospho-CHK1 and P53 expression in the indicated organoid lines treated with the DNA-damaging drug doxorubicin, or left untreated. GAPDH, loading control.

Extended Data Figure 8 Engineered mutant human colon organoids grow as invasive carcinomas in vivo.

a, Wild-type, triple- and quadruple-mutant human colon organoids were injected subcutaneously in immunodeficient mice. Nodules were counted 7 weeks after transplantation. b, Tumour sizes were examined 7 weeks after transplantation. c, Representative pictures of a ‘cystic’ triple-mutant (left) and ‘solid’ quadruple-mutant (right) tumour in immunodeficient mice. d, H&E (top left, bottom left), hKRT (top middle, bottom middle) and Ki67 (top right, bottom right) immunostainings on nodules isolated from triple-mutant-injected mice. Representative pictures of a well-differentiated carcinoma with limited invasive growth. The invasive growth has an expansive growth pattern with little tumour budding. n = 6 mice. e, As in d but for quadruple-mutant-derived tumours. Representative pictures of a poorly differentiated invasive carcinoma with frequent tumour budding at the invasive front (invasion of isolated or small aggregates of cells into the stroma is frequently observed (black arrowheads)). Invasive character is confirmed by the invasive growth into the underlying muscle tissue (asterisk, muscle tissue). n = 8 mice. Scale bars, 100 µm.

Extended Data Table 1 Introducing oncogenic mutations in human intestinal organoids using CRISPR/Cas9

Supplementary information

Supplementary Figure

This file contains Supplementary Figure 1, uncropped images of western blots. (PDF 1147 kb)

Supplementary Table 1

This table shows off-target analysis of KRAS, APC, P53 and SMAD4 sgRNAs. Off-targets were assessed by amplicon-based NGS sequencing of candidate off-target sites in the indicated organoid cultures. The columns in the table indicate the position of the candidate off-target sites (chromosome, start and end position), the alignment of the off-target sequence with the sequence of the sgRNA (indicated are bases that differ), the off-target score (a score of 1 indicates complete sequence similarity, the lower the score the more sequence differences), the mean base coverage per sample over the indicated off-target sequence, coordinates of identified small insertions and deletions (indels) in the indicated candidate off-target site and the variant allele frequencies (VAF) for the identified indels in the different samples, respectively. Only indels at the target sites for which the sgRNAs were designed (KRAS, APC, P53 and SMAD4; marked in yellow) were identified. One indel was observed at a candidate off-target site of the APC sgRNA in all samples (non-coding region). However, as the KRASG12D organoid line was not transfected with this sgRNA, this indel is not considered as an off-target effect. No additional indels were observed. (XLSX 45 kb)

Live cell imaging of chromosome segregations in human intestinal stem cell organoids

Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and RASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video. (MP4 16929 kb)

Live cell imaging of chromosome segregations in human intestinal stem cell organoids

Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video. (MP4 18060 kb)

Live cell imaging of chromosome segregations in human intestinal stem cell organoids

Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video. (MP4 15960 kb)

Live cell imaging of chromosome segregations in human intestinal stem cell organoids

Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video. (MP4 19297 kb)

Live cell imaging of chromosome segregations in human intestinal stem cell organoids

Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video. (MP4 18622 kb)

Live cell imaging of chromosome segregations in human intestinal stem cell organoids

Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video. (MP4 17866 kb)

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Drost, J., van Jaarsveld, R., Ponsioen, B. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015). https://doi.org/10.1038/nature14415

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