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Cell–matrix interface regulates dormancy in human colon cancer stem cells

Nature volume 608, pages 784–794 (2022)Cite this article

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Abstract

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1—a cell-adhesion molecule that strengthens hemidesmosomes—is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell–matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK–YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.

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Fig. 1: In vivo live lineage tracing of human LGR5+ CRC stem cells.
Fig. 2: Characterization of dormant LGR5+ cells.
Fig. 3: Spatial and temporal analysis of slow-cycling CSCs using 4D imaging.
Fig. 4: COL17A1 regulates the dormancy of LGR5+p27+ CSCs.
Fig. 5: The COL17A1–FAK–YAP axis mediates tumour regrowth from LGR5+p27+ cells.
Fig. 6: YAP inhibition delays xenograft regrowth after cytotoxic chemotherapy.

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Data availability

All data relevant to this study are available from the corresponding author upon reasonable request. RNA-sequencing data were deposited in the DNA Data Bank of Japan (DDBJ) database under the accession number JGAS000350 (study) and JGAD000464 (dataset) (https://humandbs.biosciencedbc.jp/en/hum0201-v5). The human genome hg38 was obtained from GENCODE, and the YAP target genes were derived from previously reported YAP–TAZ–TEAD direct targets43. Informed consent for depositing genomic sequencing data could not be obtained from the patients with IDs CCO7 and CCO20, and the data are therefore provided as raw counts in Supplementary Table 4. Source data are provided with this paper.

References

  1. Shen, S., Vagner, S. & Robert, C. Persistent cancer cells: the deadly survivors. Cell 183, 860–874 (2020).

    Article  CAS  PubMed  Google Scholar 

  2. Sharma, S. V. et al. A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 141, 69–80 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ramirez, M. et al. Diverse drug-resistance mechanisms can emerge from drug-tolerant cancer persister cells. Nat. Commun. 7, 10690 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Russo, M. et al. Adaptive mutability of colorectal cancers in response to targeted therapies. Science 366, 1473–1480 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Hong, S. P. et al. Single-cell transcriptomics reveals multi-step adaptations to endocrine therapy. Nat. Commun. 10, 3840 (2019).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  6. Maynard, A. et al. Therapy-induced evolution of human lung cancer revealed by single-cell RNA sequencing. Cell 182, 1232–1251 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dhimolea, E. et al. An embryonic diapause-like adaptation with suppressed Myc activity enables tumor treatment persistence. Cancer Cell 39, 240–256 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Echeverria, G. V. et al. Resistance to neoadjuvant chemotherapy in triple-negative breast cancer mediated by a reversible drug-tolerant state. Sci. Transl. Med. 11, eaav0936 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Merino, D. et al. Barcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer. Nat. Commun. 10, 766 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rehman, S. K. et al. Colorectal cancer cells enter a diapause-like DTP state to survive chemotherapy. Cell 184, 226–242 (2021).

    Article  CAS  PubMed  Google Scholar 

  11. Batlle, E. & Clevers, H. Cancer stem cells revisited. Nat. Med. 23, 1124–1134 (2017).

    Article  CAS  PubMed  Google Scholar 

  12. Shimokawa, M. et al. Visualization and targeting of LGR5+ human colon cancer stem cells. Nature 545, 187–192 (2017).

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Cortina, C. et al. A genome editing approach to study cancer stem cells in human tumors. EMBO Mol. Med. 9, 869–879 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ikeda, W., Sasai, K. & Akagi, T. Imaging window device for subcutaneous implantation tumor. Methods Mol. Biol. 1763, 153–163 (2018).

    Article  PubMed  Google Scholar 

  15. Lopez-Garcia, C., Klein, A. M., Simons, B. D. & Winton, D. J. Intestinal stem cell replacement follows a pattern of neutral drift. Science 330, 822–825 (2010).

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Oki, T. et al. A novel cell-cycle-indicator, mVenus-p27K−, identifies quiescent cells and visualizes G0–G1 transition. Sci Rep. 4, 4012 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Lenos, K. J. et al. Stem cell functionality is microenvironmentally defined during tumour expansion and therapy response in colon cancer. Nat. Cell Biol. 20, 1193–1202 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sakaue-Sawano, A. et al. Genetically encoded tools for optical dissection of the mammalian cell cycle. Mol. Cell 68, 626–640 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Liu, N. et al. Stem cell competition orchestrates skin homeostasis and ageing. Nature 568, 344–350 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Wang, W. et al. Hemidesmosomes modulate force generation via focal adhesions. J. Cell Biol. 219, e201904137 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Owen, G. R., Meredith, D. O., ap Gwynn, I. & Richards, R. G. Focal adhesion quantification—a new assay of material biocompatibility? Review. Eur. Cell Mater. 9, 85–96 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Kurppa, K. J. et al. Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway. Cancer Cell 37, 104–122 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kreso, A. et al. Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer. Science 339, 543–548 (2013).

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Nassar, D. & Blanpain, C. Cancer stem cells: basic concepts and therapeutic implications. Annu. Rev. Pathol. 11, 47–76 (2016).

    Article  CAS  PubMed  Google Scholar 

  26. Phan, T. G. & Croucher, P. I. The dormant cancer cell life cycle. Nat. Rev. Cancer 20, 398–411 (2020).

    Article  CAS  PubMed  Google Scholar 

  27. Giancotti, F. G. Mechanisms governing metastatic dormancy and reactivation. Cell 155, 750–764 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hadfield, G. The dormant cancer cell. Br. Med. J. 2, 607–610 (1954).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Bacterial persistence as a phenotypic switch. Science 305, 1622–1625 (2004).

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Fumagalli, A. et al. Plasticity of Lgr5-negative cancer cells drives metastasis in colorectal cancer. Cell Stem Cell 26, 569–578 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Basak, O. et al. Induced quiescence of Lgr5+ stem cells in intestinal organoids enables differentiation of hormone-producing enteroendocrine cells. Cell Stem Cell 20, 177–190 (2017).

    Article  CAS  PubMed  Google Scholar 

  32. Watanabe, M. et al. Type XVII collagen coordinates proliferation in the interfollicular epidermis. eLife 6, e26635 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Moilanen, J. M. et al. Collagen XVII expression correlates with the invasion and metastasis of colorectal cancer. Hum. Pathol. 46, 434–442 (2015).

    Article  CAS  PubMed  Google Scholar 

  34. Liu, C. C. et al. Suspension survival mediated by PP2A-STAT3-Col XVII determines tumour initiation and metastasis in cancer stem cells. Nat. Commun. 7, 11798 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Sugimoto, S. et al. Reconstruction of the human colon epithelium in vivo. Cell Stem Cell 22, 171–176 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Puig, I. et al. TET2 controls chemoresistant slow-cycling cancer cell survival and tumor recurrence. J. Clin. Invest. 128, 3887–3905 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Sanchez-Danes, A. et al. A slow-cycling LGR5 tumour population mediates basal cell carcinoma relapse after therapy. Nature 562, 434–438 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Cao, W. et al. LGR5 marks targetable tumor-initiating cells in mouse liver cancer. Nat. Commun. 11, 1961 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  39. Touil, Y. et al. Colon cancer cells escape 5FU chemotherapy-induced cell death by entering stemness and quiescence associated with the c-Yes/YAP axis. Clin. Cancer Res. 20, 837–846 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Nguyen, C. D. K. & Yi, C. YAP/TAZ signaling and resistance to cancer therapy. Trends Cancer 5, 283–296 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gregorieff, A., Liu, Y., Inanlou, M. R., Khomchuk, Y. & Wrana, J. L. Yap-dependent reprogramming of Lgr5+ stem cells drives intestinal regeneration and cancer. Nature 526, 715–718 (2015).

    Article  ADS  CAS  PubMed  Google Scholar 

  42. Yui, S. et al. YAP/TAZ-dependent reprogramming of colonic epithelium links ECM remodeling to tissue regeneration. Cell Stem Cell 22, 35–49 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zanconato, F. et al. Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth. Nat. Cell Biol. 17, 1218–1227 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Barry, E. R. et al. Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature 493, 106–110 (2013).

    Article  ADS  PubMed  Google Scholar 

  45. Cheung, P. et al. Regenerative reprogramming of the intestinal stem cell state via Hippo signaling suppresses metastatic colorectal cancer. Cell Stem Cell 27, 590–604 (2020).

    Article  CAS  PubMed  Google Scholar 

  46. Kroemer, G., Galluzzi, L., Kepp, O. & Zitvogel, L. Immunogenic cell death in cancer therapy. Annu. Rev. Immunol. 31, 51–72 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Fujii, M. et al. A colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis. Cell Stem Cell 18, 827–838 (2016).

    Article  CAS  PubMed  Google Scholar 

  48. Toshimitsu, K. et al. Organoid screening reveals epigenetic vulnerabilities in human colorectal cancer. Nat. Chem. Biol. 18, 605–614 (2022).

    Article  CAS  PubMed  Google Scholar 

  49. Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474–1485 (2015).

    Article  CAS  PubMed  Google Scholar 

  50. Filonov, G. S. et al. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat. Biotechnol. 29, 757–761 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    Article  ADS  CAS  PubMed  Google Scholar 

  52. Ohgushi, M., Minaguchi, M. & Sasai, Y. Rho-signaling-directed YAP/TAZ activity underlies the long-term survival and expansion of human embryonic stem cells. Cell Stem Cell 17, 448–461 (2015).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Project for Cancer Research and Therapeutic Evolution (P-CREATE) from the Japan Agency for Medical Research and Development (AMED) (grant number 19cm0106206h0004), JSPS KAKENHI (grant number 20K17030), AMED-CREST (grant number JP20gm1210001), JST Moonshot R&D (grant number JPMJMS2022), Keio University Academic Development Funds and a Grant-in-Aid for Scientific Research on Innovative Areas 'Fluorescence Live Imaging' of the Ministry of Education, Culture, Sports, Science, and Technology (Japan) and Stand Up To Cancer (SU2C Convergence 3.1416) (USA). We thank the Collaborative Research Resources, School of Medicine, Keio University for technical assistance and JSR–Keio University Medical and Chemical Innovation Center (JKiC, JSR Corporation) for assistance with in vivo imaging. We also thank T. Kitamura for providing the p27-mVenus vector, and T. Yano, R. Kawakami and T. Saito for discussions.

Author information

Author notes

  1. These authors contributed equally: Yuki Ohta, Masayuki Fujii

Authors and Affiliations

  1. Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan

    Yuki Ohta, Masayuki Fujii, Sirirat Takahashi, Ai Takano, Kosaku Nanki, Mami Matano, Hikaru Hanyu, Megumu Saito, Mariko Shimokawa, Yoshiko Hatano & Toshiro Sato

  2. Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan

    Kosaku Nanki

  3. Fujii Memorial Research Institute, Otsuka Pharmaceutical Company, Otsu, Japan

    Megumu Saito & Shingo Nishikori

  4. Department of Biostatistics, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

    Ryota Ishii

  5. Center for Integrated Medical Research, School of Medicine, Keio University, Tokyo, Japan

    Kazuaki Sawada

  6. KAN Research Institute, Kobe, Japan

    Akihito Machinaga & Wataru Ikeda

  7. Department of Molecular Medicine for Pathogenesis, Ehime University Graduate School of Medicine, Matsuyama, Japan

    Takeshi Imamura

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Contributions

Y.O., H.H., K.S., W.I. and T.I. performed in vivo imaging experiments. Y.O., M.F., A.T., M.M., H.H., M. Saito, M. Shimokawa, S.N., Y.H., and A.M. performed in vitro experiments. Y.O. and S.T. performed immunostaining. Y.O., A.T. and K.N. performed animal experiments. M.F. and R.I. performed statistical analysis for in vivo imaging experiments. Y.O. and T.S. conceived the project and designed experiments. Y.O., M.F. and T.S. wrote the manuscript.

Corresponding author

Correspondence to Toshiro Sato.

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Competing interests

T.S. is an inventor on several patents related to organoid culture.

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Nature thanks Silvia Fre, Johanna Ivaska, Vivian Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Lineage tracing of human LGR5+ CRC stem cells.

a. Representative images of ImageJ-based 3D tracking analysis in in vivo live lineage-tracing of LGR5-CreER/Rainbow CCO12 xenografts. 3D projection images at the indicated time points (top, RFP-reporter; red and nGFP; green), automatically recognized RFP+ objects (middle, magenta spots) and the tracked path (bottom, yellow lines). RFP signals were distinguished from auto-fluorescence which was present in non-tumour regions depicted by second-harmonic generation signal. b. The size transition of all LGR5+ cell-derived clones shown in Fig. 1c,e,g. c–e. Higher magnification images of the RFP+ clones shown in Fig. 1b (c), Fig. 1d (d), Fig. 1f (e). Scale bars: 20 μm (c, d, e)

Source data

Extended Data Fig. 2 Visualization and characterization of LGR5+p27+ CRC cells.

a. Ki67 immunostaining (green) and LGR5 expression in CCO7 xenograft tumours (top) and the parental patient tissue (bottom). The expression of LGR5 is shown by the LGR5 reporter (LGR5-tdT; red) in xenografts and by LGR5 ISH (red) in clinical sections. Nuclear counterstaining (white). Higher magnification images of the regions bounded by white box are shown on the right. Yellow arrowheads and white arrowheads indicate LGR5+Ki67+ cells and LGR5+Ki67– cells, respectively. b. The expression of cyclin-dependent kinase CDK inhibitors (CDKis; p21, p57 and p27, green) and Ki67 (red) in clinical samples. Nuclear counterstaining, white. The proportion of CDKi-positive cells in Ki67+ or Ki67– cells (mean ± s.e.m.) in CCO12 clinical samples (bottom right). Each dot represents one image. p27 marks nearly all Ki67– cells and does not overlap with Ki67. c. Immunostaining of p27 (green) and Ki67 (red) in CCO7 and CCO20 clinical samples. Nuclear counterstaining, white. The proportions of the indicated cell populations in clinical samples (right). d. The expression of LGR5-tdTomato (red) and p27-mVenus (green) reporters in CCO20 and CCO32 organoids. e. The expression of LGR5-tdTomato (red) and p27-mVeuns (green) reporters in CCO7 organoids cultured in the indicated conditions for 6 days. The percentage of the LGR5+p27+ area per LGR5+ area (mean ± s.e.m.). Each dot represents one organoid. Statistics, two-sided Welch’s t-test. From the left, n = 9, 10, 10 and 10 organoids. f. Flow cytometry dot plots of CCO32 organoids with the indicated reporter(s), which shows appropriate compensation. Representative confocal images of sorted LGR5+p27+ and LGR5+p27– cells are shown on the right. g, h. The number (g) and size (h) of organoid colonies formed from sorted LGR5+p27+ (green) and LGR5+p27– cells (grey). 1,000 cells were sorted from each population and the images on day 9 (CCO20, CCO7), day 7 (CCO12) or day 8 (CCO32) were analysed. Data are shown as mean ± s.e.m. Each dot indicates one well. Statistics, two-sided Welch’s t-test. The p-value for CCO20 in (h) is p = 4.7 x 10–07. i. Time-lapse live imaging of LGR5-tdTomato (red) and p27-mVenus (green) reporter organoid derived from sorted CCO32 LGR5+p27+ or LGR5+p27– single cells. j. Proportion of LGR5+p27+ or LGR5+p27− cells per all cells (mean ± s.e.m.) in organoids derived from sorted CCO32 LGR5+p27+ or LGR5+p27– single cells at day 10 after sorting. Statistics, two-sided Welch’s t-test. Scale bars: 25 μm (i), 50 μm (d, e), 100 μm (a, b, c)

Source data

Extended Data Fig. 3 Cell-cycle analysis after chemotherapy in vivo.

a. Schedule for EdU labelling in LGR5-tdTomato/p27-mVenus CCO12 and CCO32 xenografts. b. The expression of p27-mVenus (green) and LGR5-tdTomato (red, top), and EdU incorporation (red, bottom) in an LGR5-tdTomato/p27-mVenus CCO12 xenograft tumour. Nuclear counterstaining, white. c. Representative images of EdU signals in selected LGR5+p27+ (right) or LGR5+p27– (left) cells. d. The percentage of EdU+ cells in LGR5+p27+ (green) or LGR5+p27– (grey) cells (mean ± s.e.m.) in EdU-pulsed CCO12 or CCO32 xenografts. Each dot indicates one image. Statistics, two-sided Welch’s t-test. From the left, p < 2 x 10–16 and p < 2 x 10–16. e. Representative images showing an enrichment of LGR5+p27+ cells in xenograft tumours after chemotherapy. Tumours were collected 2 days after a treatment with the indicated regimens. f. The percentage of LGR5+p27+ cells in all LGR5+ cells in vehicle- or chemotherapy-treated xenograft tumours. Chemotherapy refers to the regimens shown in (e). Each dot shows one images. Statistics, two-sided Welch’s t-test. p = 3.9 x 10–9 (CCO7). Centre line; median, box limits; upper and lower quartiles, whiskers; 1.5 x interquartile range. Scale bars: 5 μm (c), 100 μm (b, e)

Source data

Extended Data Fig. 4 Cell-cycle analysis after chemotherapy in vitro.

a. The expression of TagBFP2-hGeminin (1/110) (blue), iRFP-hCdt1(1/100)Cy(-) (red) and p27-mVenus (green) in LGR5-tdTomato/p27-mVenus/Fucci(CA) CCO32 organoids following the indicated treatments. 5-AZA (100 nM) and 5-FU (50 μM) induces G0 arrest, and Aphidicolin (1 μM) induces G2/M arrest. b. The transition of the proportion of LGR5+p27+ and LGR5+BFP+ cells in CCO7 organoids following CPT treatment (50 nM, 48 hr). Data are shown as mean ± s.e.m. c. Cell-cycle transition of single LGR5+ p27+ cells (top) and LGR5+p27– cells (bottom) in CCO7 organoids following CPT treatment (50 nM, 48 hr). Each row shows a single LGR5+p27+ cell, or an LGR5+p27– cell in the G1 phase at 0 hr. d. A modified Fucci construct (Fucci-iRFP) and design for long-term time-lapse live imaging. iRFP marks the cells in a non-G0 state. e. A representative image of a CCO32 organoid expressing LGR5-tdTomato (red), p27-mVenus (green) and Fucci-iRFP (white) reporters. f. Representative serial images of 72-hr time-lapse imaging of p27+ cells that entered cell cycle. The green and magenta square shows the timing of the p27+-to-p27– transition and mitosis entry, respectively. The area outlined with white box in (e) is shown. g. The transition of the p27 expression (G0 status) in each clone traced from single LGR5+p27+ (top) and LGR5+p27– (bottom) cells with (right) or without (left) CPT treatment (10 nM, 24 hr). Each row corresponds to a single LGR5+p27+ or LGR5+p27– cell in LGR5-tdTomato/p27-mVenus/Fucci-iRFP CCO32 organoids. Magenta bars show the timing of mitosis entry, and the bars are coloured in light magenta afterwards. h. Representative images of organoids derived from sorted LGR5+p27+ cells and treated with or without CPT (10 nM) after plating (left). 1,000 cells were plated per well and were treated with CPT from day 0 to day 2. The images were captured on day 10. The number of organoid colonies (mean ± s.e.m.) on day 10 (right). Each dot represents one well. p = 1.9 x 10–7, two-sided Welch’s t-test. Scale bars: 50 μm (a, e), 1 mm (h)

Source data

Extended Data Fig. 5 In vivo 4D live imaging of LGR5+p27+ and LGR5+p27− cells.

a. Representative images from LGR5 lineage tracing in LGR5-CreER/LGR5-tdTomato/p27-mVenus/tdiRFP-BFP CCO32 xenogfrafts. Both LGR5+p27+ and LGR5+p27– cells stochastically convert their colour from tdiRFP to TagBFP2 (BFP (tr), blue) by day 4. Nuclear counterstaining (DRAQ5) is shown in white. b. Percentage of p27+ cells (mean ± s.e.m.) in BFP+ clones on day 5 post-tamoxifen treatment (left), and in LGR5+ cells in organoids without tamoxifen treatment (right). Each dot indicates one mouse. Statistics, two-sided Welch's t-test. c. Optical sections of LGR5+p27+BFP+ cells in untreated CCO20 xenografts (from Fig. 3c, bottom) that remained as single cells over the observation period (left). Representative clone expansion from an LGR5+p27+ cell-derived BFP+ clone (from Fig. 3f, bottom) following IRI treatment (right). The clones of interest are framed with white dotted line. d. The size transition of clones traced from individual BFP+ single LGR5+p27+ (green) or LGR5+p27– (grey) cells in CCO20 or CCO32 xenografts. Each line represents one clone. e. The survival of the clones traced from single LGR5+p27+ (green line) or LGR5+p27– (black dotted line) CCO32 cells with (bottom) or without (top) IRI treatment. p = 2.7 x 10–10, log-rank test. f. Cleaved caspase-3 (green, left) and TUNEL (green, right) staining, and LGR5-tdTomato expression in untreated CCO32 xenografts. Nuclear counterstaining (white). Insets show higher magnification. g. The proportion of apoptotic cells (mean ± s.e.m.) marked by cleaved caspase-3 or TUNEL in LGR5+ cells (red) or LGR5- cells (grey) in untreated CCO32 xenografts. Each dot represents one image. Statistics, two-sided Welch’s t-test. p = 4.5 x 10–06 (TUNEL). h. The transition of the proportion of LGR5+ cells in traced clones analysed by in vivo live imaging. Clones that expanded over time are shown in red, and those that diminished are shown in blue. Regression lines were made using local polynomial regression, and the blue or red area shows the 95% confidence interval. Scale bars: 100 μm (a: left, g), 20 μm (a: right, c, d)

Source data

Extended Data Fig. 6 Association between ECM and dormancy in LGR5+p27+ CSCs.

a. The positivity of p27 in sorted LGR5+p27+ CCO32 cells at the indicated time points after sorting. Organoids were dissociated into single cell, sorted using a cell sorter and embedded in Matrigel. b. Cumulative probability of a cell division event in single LGR5+p27+ cells, or cell doublets that contain p27+ cells. Statistics, log-rank test. c. Gene ontology analysis of the genes differentially expressed between LGR5+p27+ cells and LGR5+p27– cells in CCO7, 12, 20 and 32. Genes with FDR < 0.05 and fold change value > 2 or < –2 were used. The ontology terms related to ECM are highlighted in dark green. d. Comparative gene expression analysis of extracellular matrix-related genes in LGR5+p27+ versus LGR5+p27– cells. The transcriptomes of sorted CCO7, 12, 20 and 32 were analysed using edgeR. The genes in the gene ontology terms highlighted in (c) are listed. The overall fold changes analysed using all lines are shown on the left. Laminin genes and genes with an overall fold change value > 2 and FDR < 0.05 are highlighted. e. The proportions of Ki67+ cells in COL17A1-negative (white) or -positive (cyan) cells in the indicated samples. Each dot shows one image. Data are shown as mean ± s.e.m. *p < 0.0001 (from the left, p = 2 x 10–12, p = 6.6 x 10–6, p = 3.0 x 10–4, p = 1.0 x 10–4, p = 2.8 x 10–8), two-sided Welch’s t-test. f. Immunostaining of Laminin-5 (cyan) superimposed on the endogenous expression of LGR5-tdTomato (red) and p27-mVenus (green) reporters in a CCO32 xenograft sample. g. The distance from the LGR5+p27+ (green) or LGR5+p27– (grey) nucleus to laminin-5. Each dot represents one cell. p = 1.7 x 10–14, two-sided Welch’s t-test. Centre line; median, box limits; upper and lower quartiles, whiskers; 1.5 x interquartile range. Scale bars: 100 μm (e)

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Extended Data Fig. 7 COL17A1 regulates dormancy in LGR5+p27+ CSCs.

a. Whole-mount immunostaining of COL17A1 (cyan), and endogenous expression of p27-mVenus (green) and LGR5-tdTomato (red) in CCO32 (left) and CCO7 (right) organoids with the indicated COL17A1 genotypes. b. The expression of LGR5-tdTomato (red) and p27-mVenus (green) reporters before (left) and after COL17A1 KO (centre, right) in CCO12 organoids. c, d. The number (c) and average size (d) of organoid colonies formed from COL17A1 WT or two independent COL17A1 KO clones of CCO32 organoids. Each dot shows one well. Data are shown as mean ± s.e.m. Statistics, two-sided Welch’s t-test. e. Representative confocal images of Ki67 or EdU staining (green) in COL17A1 WT and KO CCO32 organoids (left). The proportion of Ki67 or EdU+ per all cells (mean ± s.e.m.) in COL17A1 WT and KO CCO32 organoids (right). Each dot represents one organoid. From the left, p = 8.2 x 10–11 and 7.0 x 10–4, two-sided Welch’s t-test. f. Representative confocal images of cleaved caspase-3 staining (green) in COL17A1 WT and KO, CCO7 and CCO32 organoids (left). The ratio of cleaved caspase-3-positive area to nuclei area (mean ± s.e.m.) in COL17A1 WT and KO, CCO7 and CCO32 organoids. Each dot shows one organoid. Statistics, two-sided Welch’s t-test. g. The construct of Dox-inducible COL17A1. h. Confirmation of COL17A1 overexpression using capillary-based immunoassay. COL17A1 expression was induced in CCO7 and CCO32 organoids by a 2-day treatment with 500 ng/ml Dox. i. The viability of CCO32 organoids with the indicated genotypes following CPT treatment. The viability was measured using an ATP luminescence assay and shown in relative light unit (RLU). Data are presented as mean ± s.e.m. j. The schedule for treating COL17A1 WT and KO CCO7 xenografts with IRI. k. The growth of vehicle- or IRI-treated COL17A1 WT and KO CCO7 xenografts. Tumour sizes are shown as the relative values compared to the day 0 volume and as mean ± s.e.m. *p < 0.05 in a mixed effect model with repeated measurement, followed by Šidák correction. Thick lines show model-predicted values, and the areas show 95% upper and lower prediction intervals. Scale bars: 50 μm (a, e, f), 100 μm (b)

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Extended Data Fig. 8 Activation of FAK–YAP signalling after chemotherapy.

a. Gene set enrichment analysis of COL17A1-overexpressed (OE) versus KO CCO32 organoids using YAP target genes. The YAP target genes derive from ChIP-seq-validated YAP/TAZ targets in Zanconato F., et al43. NES; normalized enrichment score. p = 8.3 x 10–14. b. Gene set enrichment analysis of LGR5+p27+ versus LGR5+p27– cells sorted from CCO7, CCO12, CCO20 and CCO32 using the same YAP target genes. p = 1.1 x 10–42, FDR = 6.1 x 10–42. c. Relative expression of YAP/TAZ/TEAD target genes (LATS2, CTGF and CYR61) (mean ± s.e.m.) in TEADi-treated CCO32 organoids compared to the control. The organoids were cultured with or without a TEADi (MYF-01-37, 20μM) for 2 days. Statistics, two-sided Welch’s t-test. The exact p-value for LATS2 is 2.1 x 10–6. d. The expression of the COL17A1 protein in COL17A1-OE CCO7 and CCO32 organoids after treatment with CPT (50 nM for CCO7, 10 nM for CCO32). COL17A1-rescued, COL17A1 KO organoids (COL17A1 KO + OE) were used. COL17A1 was induced by a 2-day treatment with 500 ng/ml Dox. Organoid lysates were collected 6, 24 and 48 hr after a treatment with CPT. The figures on the bottom of the images show the protein areas analysed by the Compass software. The values were scaled to the Dox+ condition. e. The expression of COL17A1, phospho-FAK (pFAK) and total FAK (tFAK) protein in CCO7 and CCO32 organoids after treatment with 5-FU (30 μM) or oxaliplatin (30 μM for CCO32, 50 μM for CCO7). Organoid lysates were collected 6, 24 and 48 hr after a treatment with 5-FU or oxaliplatin. f. The expression of MMP genes in CPT-treated CCO32 organoids. MMP genes with at least a transcript count > 1 in more than a half of the samples are shown. g. Capillary-based immunoassays of COL17A1 using CCO32 organoids with or without CPT treatment (10 nM). CPT-treated organoids were treated with vehicle or two different MMP inhibitor (llomastat (10 μM) or Marimastat (3 μM)). Organoid lysates were collected 48 h after treatment. h. Whole-mount immunostaining of phosho-FAK (green) in CCO7 organoids with or without CPT (50 nM, 48 hr) and FAKi (PF573228, 10 μM, 48 hr) treatments. i. The ratio of phosho-FAK-positive area to nuclei area (mean ± s.e.m.) in CCO7 organoids with or without the indicated treatments. Each dot shows one organoid. Statistics, two-sided Welch’s t-test. j. Whole-mount immunostaining of YAP (magenta) in CCO32 organoids with or without CPT (10 nM, 48 hr) and FAKi (10 μM, 48 hr) treatments. k. The proportion of nuclear YAP-positive cells (mean ± s.e.m.) in CCO7 and CCO32 organoids with or without CPT (50 nM, 48 hr for CCO7 and 10 nM, 48 hr for CCO32) and FAKi (10 μM, 48 hr) treatments. Each dot represents one organoid. From the left, p = 1.5 x 10−6, p = 3.9 x 10–9, p = 9.1 x 10–11, two-sided Welch’s t-test. l. Representative confocal images of YAP staining (magenta, left) with p27-mVenus (p27mV) reporter (green, left) and phospho-FAK (green, right) expressions in COL17A1-KO or OE CCO7 with or without CPT treatment (50 nM, 48 hr). COL17A1 was induced by a 2-day pre-treatment with 500 ng/ml Dox. m. The proportion of nuclear YAP- or pFAK positive cells per nuclei (mean ± s.e.m.) in COL17A1-KO or OE CCO7 organoids with or without the indicated treatments. Each dot shows one organoid. Statistics, two-sided Welch’s t-test. Scale bars: 25 μm (l), 50 μm (h, j)

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Extended Data Fig. 9 Perturbation of FAK and YAP after chemotherapy.

a. (left) Time-point live imaging of LGR5-tdTomato/p27-mVenus CCO32 organoids following treatment with CPT (10 nM, 24 hr) treatment alone or in combination with FAKi (10 μM, continuous) (left). LGR5-tdTomato; red and p27-mVenus; green. The experimental timeline is shown on the top. The transition of the percentage of LGR5+p27+ in all cells (mean ± s.e.m.) in CCO32 organoids following the indicated treatments (right). *p < 0.05, linear model, followed by Tukey adjustment (control vs CPT + FAKi; p = 0.0020, CPT vs CPT + FAKi; p = 0.013, FAKi vs CPT + FAKi; p = 0.013). b. Representative YAP staining (magenta) and p27-mVenus (green) expressions in sorted LGR5+p27+ cells at the indicated time points after sorting (left). Cells were treated with CPT (10 nM, 24 hr) before sorting. Representative confocal images of short-cultured LGR5+p27+ CCO32 cells with EGFR inhibition (1 μM) at the indicated time points after single-cell sorting (right). LGR5 (red), p27 (green), YAP staining (magenta). c. The cell counts in cells/clusters derived from sorted single LGR5+ p27+ cells at the indicated time points after sorting. The organoids were treated with CPT (10 nM, 24 hr) before sorting. Each cell is coloured based on the YAP and p27 status. d. The distribution of the cell counts in cells/clusters derived from sorted single LGR5+ p27+ cells on day 3 after sorting. The organoids were treated with CPT (10 nM, 24 hr) before sorting, and with or without 20 μM TEADi after sorting. Red points and lines indicate mean ± sd. p = 1.7 x 10–6, two-sided Wilcoxon rank sum test. Scale bars: 10 μm (b), 100 μm (a)

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Extended Data Fig. 10 FAK inhibition attenuates tumour regrowth after chemotherapy.

a. The in vitro organoid relapse model. RFP+ CCOs were treated with the indicated conditions to simulate cancer cell death and regrowth. b. Representative images demonstrating the regrowth of CCO7, CCO20 and CCO32 organoids following CPT treatment (CCO7; 50nM, 24 hr, CCO20 and CCO32; 10 nM, 24 hr) with or without FAKi (CCO7 and CCO32; 10 μM, CCO20; 5 μM) (left). The total area of RFP+ organoids per well (mean ± s.e.m.) in the indicated conditions (right). For each condition, 1,000 cells were plated, and the images were captured on day 10 (CCO7), day 7 (CCO20) or day 9 (CCO32) after CPT treatment. Each dot shows one well. Statistics, two-sided Welch’s t-test. c. Representative images of organoid growth (CCO7, CCO20 and CCO32) with or without FAKi (5 μM for CCO20, and 10 μM for CCO7 and CCO32) (left). Organoid areas following FAKi treatment versus control (mean ± s.e.m.) are shown (right). Each dot shows one well Statistics, two-sided Welch’s t-test. d. Representative confocal images of cleaved caspase-3 staining (green) with or without CPT (10 nM) and FAKi (10 μM) in CCO32 organoids (left). The ratio of cleaved caspase-3 positive area to nuclei area (mean ± s.e.m.) in CCO32 organoids with or without CPT and FAKi (right). Each dot indicates one organoid. Statistics, two-sided Welch’s t-test

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Extended Data Fig. 11 YAP inhibition suppresses post-chemotherapy regrowth.

a–c. CCO32 organoids were cultured in the indicated conditions with or without CPT (10 nM) and TEADi (20 μM) (left). Organoid area per well (mean ± s.e.m.) are shown on the right (a, b). Total DAPI-positive area to total organoid area (mean ± s.e.m.) is shown (c). two-sided Welch’s t-test. d. The constructs of the Dox-inducible shYAP/TAZ cassettes. e. Knockdown efficiencies of YAP1 (encoding YAP) and WWTR1 (encoding TAZ) in the indicated CCO lines. shYAP/TAZ lentivirus-infected organoids were cultured with or without 500 ng/ml Dox for 2 days. Data show mean values. f. The expression of YAP/TAZ/TEAD target genes (LATS2, CTGF and CYR61) (mean ± s.e.m.) relative to the control in shYAP/TAZ lentivirus-infected CCO32 organoids. Organoids were cultured with or without 500 ng/ml Dox for 2 days. From the left, p = 5.7 x 10–07, p = 7.5 x 10–05, two-sided Welch's t-test. g. The growth of shYAP/TAZ CCO7, CCO20 and CCO32 organoids treated with or without 500 ng/ml Dox (left). The fluorescence of mRFP1 in the shTAZ construct was captured. l. Colony formation efficiency of Dox-treated (500 ng/ml) shYAP/TAZ organoids relative to the untreated control (right). Each dot shows one well. Statistics, two-sided Welch’s t-test. h. Representative images demonstrating the regrowth of YAP/TAZ-knockdown CCO7, CCO20 and CCO32 organoids following CPT treatment (CCO7; 50nM, 24 hr, CCO20 and CCO32; 10 nM, 24 hr) with or without Dox (500 ng/ml) (left). The total area of organoids per well (mean ± s.e.m.) in the indicated conditions (right). For each condition, 1,000 cells were plated, and the images were captured on day 7 (CCO7, CCO32) or day 10 (CCO20) after CPT treatment. Each dot shows one well. Statistics, two-sided Welch’s t-test. Scale bars: 50 μm (c), 1 mm (a, k)

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Extended Data Fig. 12 YAP suppression represses tumour regrowth after chemotherapy in vivo.

a. Immunostaining of YAP (brown) in CCO xenografts on day 2 after treatment with vehicle (top) or with the indicated chemotherapy regimens (bottom) b. The proportion of cells with YAP nuclear localization (mean ± s.e.m.) in CCO xenografts on day 2 after treatment with vehicle or with the same chemotherapy regimens as in (a). Each dot represents one image. From the left, p = 6.7 x 10–16, p = 1.1 x 10–7, p = 1.1 x 10–6, two-sided Welch’s t-test. c. Validation of YAP/TAZ knockdown in vivo. For knockdown, mice bearing shYAP/TAZ-infected CCO32 xenografts were treated with 1 mg/ml Dox in drinking water for 3 days. The expression values were calculated as the expression relative to ACTB, and then scaled to the mean value of the untreated controls. Each dot shows one tumour. The mean values are shown. d. The proportion of cells with YAP nuclear localization (mean ± s.e.m.) in shYAP/TAZ CCO32 xenografts on day 3 after the indicated treatments. Each dot indicates one image. From the left, p = 1.0 x 10–8, p < 2 x 10–16, two-sided Welch’s t-test. e. In situ hybridization of LGR5 mRNA (red, top), immunostaining of p27 (green, bottom) and EdU incorporation (red, bottom) on day 3 or day 7 after the indicated treatments. EdU was administered immediately before tumour collection. Nuclear counterstaining, white. f–i. The proportion of EdU (f, g), p27 (h) and LGR5 (i) -positive cells (mean ± s.e.m.) on day 3, 7 and 10 after the indicated treatments. *p < 0.05, two-sided Welch’s t-test (f, h, i). From the left, p = 0.02574, p = 0.02542, p = 3.2 x 10–10, p = 1.9 x 10–15, p = 0.029 for (f), p = 0.0154, p = 0.0049, p = 0.0053, p = 2.7 x 10–8, p = 3.5 x 10–5 for (h), and p = 0.0002, p = 5.5 x 10–11, p < 2 x 10–16, p = 1.8 x 10–6, p = 0.00029 for (i). The difference in the EdU incorporation between IRI and IRI + Dox groups was compared using a linear model (g). *p = 6.2 x 10–14. Each dot shows one image. Scale bars: 50 μm (a), 100 μm (e). j. The growth curves of shYAP/TAZ CCO32 xenografts during control or Dox treatment. Data are shown as the tumour volume relative to the day 0 volume. Thin lines represent individual tumours. Thick lines show model-fitted values, and the light green or purple area shows the 95% upper and lower prediction intervals. k. The expression of YAP/TAZ/TEAD target genes (LATS2, CTGF and CYR61) (mean ± s.e.m.) CCO32 xenografts following YAP/TAZ knockdown or TEADi treated. For knockdown, mice bearing shYAP/TAZ-infected CCO32 xenografts received 1 mg/ml Dox in drinking water for 3 days. For TEAD inhibition, the mice received MYF-01-37 (100 mg/kg) by daily intraperitoneal injection for 3 days. The expression values were calculated as the expression relative to ACTB, and then scaled to the mean value of the untreated controls. Each dot shows one tumour. The mean values are shown. Statistics, two-sided Welch's t-test. l. The growth curves of CCO32 xenografts treated with the indicated regiments. Data are shown as the tumour volume relative to the day 0 volume (bottom). *p < 0.05, mixed effect model with repeated measures. Thin lines represent individual tumours. Thick lines show model-predicted values, and the light red or blue area shows the 95% upper and lower prediction intervals

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Supplementary information

Supplementary Information

This file contains Supplementary Methods: Data Reproducibility; Supplementary Data 1: Uncropped pseudo-blot images; Supplementary Data 2: Confirmation of reporter knock-in by genomic PCR; Supplementary Data 3: Sanger sequencing of COL17A1 knockout organoid clones; Supplementary Table 1: Sequences of primers and sgRNA targets used in the study; and Supplementary Table 2: Clinical information of the CCOs used in the study.

Reporting Summary

Supplementary Table 3

Genetic mutations in CCO32.

Supplementary Table 4

RNA-sequencing count data for CCO7 and CCO20.

Supplementary Video 1

Time-lapse live imaging of LGR5-tdTomato/p27-mVenus/Fucci-iRFP CCO20 organoids in a routine culture condition (Supplementary Video 1). 4D (x-y-z-time) images were acquired with a 1-hour interval and max-intensity projection images were converted to a 15 frames per second (fps) mov file. The quantification data is shown in Fig. 2j.

Supplementary Video 2

Time-lapse live imaging of LGR5-tdTomato/p27-mVenus/Fucci-iRFP CCO32 organoids with or without CPT treatment (10 nM, 24 h). 4D (x-y-z-t) images were acquired with a 1-hour interval, and max-intensity projection images were converted to a 15-fps movie. The quantification data is shown in Extended Data Fig. 4g.

Supplementary Video 3

Time-lapse live imaging of LGR5-tdTomato/p27-mVenus/Fucci-iRFP CCO32 organoids with or without CPT (10 nM, 24 h) and FAK inhibitor (PF573228; 10 μM, from 0 h to the last time point) treatment. 4D (x-y-z-t) images were acquired with a 1-hour interval and max-intensity projection images were converted to a 15-fps movie. The signals of LGR5-tdTomato are p27-mVenus are shown. The quantification data and representative cropped images are in Fig. 5i and Extended Data Fig. 9a.

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Ohta, Y., Fujii, M., Takahashi, S. et al. Cell–matrix interface regulates dormancy in human colon cancer stem cells. Nature 608, 784–794 (2022). https://doi.org/10.1038/s41586-022-05043-y

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