Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Tumour spheres with inverted polarity drive the formation of peritoneal metastases in patients with hypermethylated colorectal carcinomas

Abstract

Metastases account for 90% of cancer-related deaths; thus, it is vital to understand the biology of tumour dissemination. Here, we collected and monitored >50 patient specimens ex vivo to investigate the cell biology of colorectal cancer (CRC) metastatic spread to the peritoneum. This reveals an unpredicted mode of dissemination. Large clusters of cancer epithelial cells displaying a robust outward apical pole, which we termed tumour spheres with inverted polarity (TSIPs), were observed throughout the process of dissemination. TSIPs form and propagate through the collective apical budding of hypermethylated CRCs downstream of canonical and non-canonical transforming growth factor-β signalling. TSIPs maintain their apical-out topology and use actomyosin contractility to collectively invade three-dimensional extracellular matrices. TSIPs invade paired patient peritoneum explants, initiate metastases in mice xenograft models and correlate with adverse patient prognosis. Thus, despite their epithelial architecture and inverted topology TSIPs seem to drive the metastatic spread of hypermethylated CRCs.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: TSIPs predominate in peritoneal effusions of patients with CRCs of poor prognosis.
Fig. 2: MUC CRC cell survival is sustained by cell–cell interactions.
Fig. 3: TSIPs form by collective apical budding from serrated/hypermethylated CRCs.
Fig. 4: Decreased canonical and non-canonical TGF-β signalling promotes TSIP formation by collective apical budding.
Fig. 5: TSIPs collectively invade in their inverted topology.
Fig. 6: TSIPs collective invasion is driven by ROCK and myosin-II activity downstream of the non-canonical TGF-β signalling.
Fig. 7: TSIPs are efficient initiators of metastases.

References

  1. 1.

    Bilder, D. Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors. Genes. Dev. 18, 1909–1925 (2004).

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Huang, L. & Muthuswamy, S. K. Polarity protein alterations in carcinoma: a focus on emerging roles for polarity regulators. Curr. Opin. Genet. Dev. 20, 41–50 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Muthuswamy, S. K. & Xue, B. Cell polarity as a regulator of cancer cell behavior plasticity. Annu. Rev. Cell. Dev. Biol. 28, 599–625 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Nieto, M. A., Huang, R. Y.-J., Jackson, R. A. & Thiery, J. P. EMT: 2016. Cell 166, 21–45 (2016).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Thiery, J. P., Acloque, H., Huang, R. Y.-J. & Nieto, M. A. Epithelial–mesenchymal transitions in development and disease. Cell 139, 871–890 (2009).

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Friedl, P. & Alexander, S. Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147, 992–1009 (2011).

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Liu, Y.-J. et al. Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells. Cell 160, 659–672 (2015).

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Paluch, E. K., Aspalter, I. M. & Sixt, M. Focal adhesion-independent cell migration. Annu. Rev. Cell. Dev. Biol. 32, 469–490 (2016).

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Fischer, K. R. et al. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 527, 472–476 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Zheng, X. et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 527, 525–530 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Friedl, P., Locker, J., Sahai, E. & Segall, J. E. Classifying collective cancer cell invasion. Nat. Cell. Biol. 14, 777–783 (2012).

    Article  PubMed  Google Scholar 

  12. 12.

    Cheung, K. J., Gabrielson, E., Werb, Z. & Ewald, A. J. Collective invasion in breast cancer requires a conserved basal epithelial program. Cell 155, 1639–1651 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Montell, D. J., Yoon, W. H. & Starz-Gaiano, M. Group choreography: mechanisms orchestrating the collective movement of border cells. Nat. Rev. Mol. Cell. Biol. 13, 631–645 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Friedl, P. & Gilmour, D. Collective cell migration in morphogenesis, regeneration and cancer. Nat. Rev. Mol. Cell. Biol. 10, 445–457 (2009).

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Pino, M. S. & Chung, D. C. The chromosomal instability pathway in colon cancer. Gastroenterology 138, 2059–2072 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Vogelstein, B. et al. Genetic alterations during colorectal-tumor development. N. Engl. J. Med. 319, 525–532 (1988).

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330–337 (2012).

  18. 18.

    Guinney, J. et al. The consensus molecular subtypes of colorectal cancer. Nat. Med. 21, 1350–1356 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Marisa, L. et al. Gene expression classification of colon cancer into molecular subtypes: characterization, validation, and prognostic value. PLoS. Med. 10, e1001453 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Juo, Y. Y. et al. Prognostic value of CpG island methylator phenotype among colorectal cancer patients: a systematic review and meta-analysis. Ann. Oncol. 25, 2314–2327 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Weisenberger, D. J. et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat. Genet. 38, 787–793 (2006).

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Phipps, A. I. et al. Association between molecular subtypes of colorectal cancer and patient survival. Gastroenterology 148, 77–87 (2015).

  23. 23.

    Ward, R. L. et al. Adverse prognostic effect of methylation in colorectal cancer is reversed by microsatellite instability. J. Clin. Oncol. 21, 3729–3736 (2003).

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Bettington, M. et al. The serrated pathway to colorectal carcinoma: current concepts and challenges. Histopathology 62, 367–386 (2013).

    Article  PubMed  Google Scholar 

  25. 25.

    O’Brien, M. J., Zhao, Q. & Yang, S. Colorectal serrated pathway cancers and precursors. Histopathology 66, 49–65 (2015).

    Article  PubMed  Google Scholar 

  26. 26.

    van Gestel, Y. R. B. M. et al. Patterns of metachronous metastases after curative treatment of colorectal cancer. Cancer Epidemiol. 38, 448–454 (2014).

    Article  PubMed  Google Scholar 

  27. 27.

    Segelman, J. et al. Incidence, prevalence and risk factors for peritoneal carcinomatosis from colorectal cancer. Br. J. Surg. 99, 699–705 (2012).

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Sugarbaker, P. H. Peritoneal carcinomatosis: natural history and rational therapeutic interventions using intraperitoneal chemotherapy. Cancer Treat. Res. 81, 149–168 (1996).

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Franko, J. et al. Prognosis of patients with peritoneal metastatic colorectal cancer given systemic therapy: an analysis of individual patient data from prospective randomised trials from the Analysis and Research in Cancers of the Digestive System (ARCAD) database. Lancet Oncol. 17, 1709–1719 (2016).

    Article  PubMed  Google Scholar 

  30. 30.

    Hugen, N., van de Velde, C. J. H., de Wilt, J. H. W. & Nagtegaal, I. D. Metastatic pattern in colorectal cancer is strongly influenced by histological subtype. Ann. Oncol. 25, 651–657 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Numata, M. et al. The clinicopathological features of colorectal mucinous adenocarcinoma and a therapeutic strategy for the disease. World J. Surg. Oncol. 10, 109 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Goéré, D. et al. Is there a possibility of a cure in patients with colorectal peritoneal carcinomatosis amenable to complete cytoreductive surgery and intraperitoneal chemotherapy? Ann. Surg. 257, 1065–1071 (2013).

    Article  PubMed  Google Scholar 

  33. 33.

    Mohamed, F., Cecil, T., Moran, B. & Sugarbaker, P. A new standard of care for the management of peritoneal surface malignancy. Curr. Oncol. 18, e84–e96 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Davidowitz, R. A. et al. Mesenchymal gene program-expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance. J. Clin. Invest. 124, 2611–2625 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Tom, B. H. et al. Human colonic adenocarcinoma cells. I. Establishment and description of a new line. Vitro 12, 180–191 (1976).

    CAS  Article  Google Scholar 

  37. 37.

    Julien, S. et al. Characterization of a large panel of patient-derived tumor xenografts representing the clinical heterogeneity of human colorectal cancer. Clin. Cancer Res. 18, 5314–5328 (2012).

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Laiho, P. et al. Serrated carcinomas form a subclass of colorectal cancer with distinct molecular basis. Oncogene 26, 312–320 (2007).

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    de Miranda, N. F. C. C. et al. Transforming growth factor β signaling in colorectal cancer cells with microsatellite instability despite biallelic mutations in TGFBR2. Gastroenterology 148, 1427–1437 (2015).

  41. 41.

    Brown, K. A., Pietenpol, J. A. & Moses, H. L. A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling. J. Cell. Biochem. 101, 9–33 (2007).

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Maiuri, P. et al. The first World Cell Race. Curr. Biol. 22, R673–R675 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Alexander, S., Koehl, G. E., Hirschberg, M., Geissler, E. K. & Friedl, P. Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model. Histochem. Cell. Biol. 130, 1147–1154 (2008).

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Giampieri, S., Pinner, S. & Sahai, E. Intravital imaging illuminates transforming growth factor beta signaling switches during metastasis. Cancer Res. 70, 3435–3439 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    O’Brien, L. E. et al. Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly. Nat. Cell. Biol. 3, 831–838 (2001).

    Article  PubMed  Google Scholar 

  46. 46.

    Aceto, N. et al. Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158, 1110–1122 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    McCauley, H. A. et al. TGFβ signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium. Development 141, 4628–4639 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Zhu, Y., Richardson, J. A., Parada, L. F. & Graff, J. M. Smad3 mutant mice develop metastatic colorectal cancer. Cell 94, 703–714 (1998).

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Lamouille, S., Xu, J. & Derynck, R. Molecular mechanisms of epithelial–mesenchymal transition. Nat. Rev. Mol. Cell. Biol. 15, 178–196 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Ozdamar, B. et al. Regulation of the polarity protein Par6 by TGFß receptors controls epithelial cell plasticity. Science 307, 1603–1609 (2005).

  51. 51.

    Giampieri, S. et al. Localized and reversible TGFβ signalling switches breast cancer cells from cohesive to single cell motility. Nat. Cell Biol. 11, 1287–1296 (2009).

  52. 52.

    Bilder, D., Li, M. & Perrimon, N. Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113–116 (2000).

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Zhan, L. et al. Deregulation of Scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma. Cell 135, 865–878 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Wang, X., He, L., Wu, Y. I., Hahn, K. M. & Montell, D. J. Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat. Cell. Biol. 12, 591–597 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Hegerfeldt, Y., Tusch, M., Bröcker, E.-B. & Friedl, P. Collective cell movement in primary melanoma explants: plasticity of cell–cell interaction, β1-integrin function, and migration strategies. Cancer Res. 62, 2125–2130 (2002).

  56. 56.

    Callan-Jones, A. C. & Voituriez, R. Actin flows in cell migration: from locomotion and polarity to trajectories. Curr. Opin. Cell. Biol. 38, 12–17 (2016).

    CAS  Article  PubMed  Google Scholar 

  57. 57.

    Fellmann, C. et al. An optimized microRNA backbone for effective single-copy RNAi. Cell. Rep. 5, 1704–1713 (2013).

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Rahman, M. et al. Alternative preprocessing of RNA-sequencing data in The Cancer Genome Atlas leads to improved analysis results. Bioinformatics 31, 3666–3672 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: Precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank all the patients who participated in this study and the medical staff for their assistance with the acquisition of primary human specimens. We also thank the members of the Jaulin Lab and the Digestive Cancer Unit for discussion, B. Baum for mentoring and support, and S. Deborde, B. Goud and G. Kreitzer for critical reading of the manuscript. We thank D. Vignjevic, S Guilmeau, the Plateforme Anticorps Recombinant (Curie Institute), DSHB (University of Iowa) and J. Zuber for reagents and the technical services provided by PFIC core facility, module HCP (F. Drusch and V. Marty), O. Bawa, V. Roufiac, S. Piterboth and I. Villa. This work was supported by the CNRS and INSERM (the ATIP-AVENIR program), the Gustave Roussy Foundation (Roulons pour le colon, Natixis), Canceropole (Emergence) Taxe d’apprentissage Gustave Roussy (2016 to C.C.J.).

Author information

Affiliations

Authors

Contributions

O.Z., J.R., F.L., D.S., S.S., G.P., I.M., P.R., C.B., P.A. and J.-L.P. designed the research, performed the experiments and analysed the data. L.B., M. Polrot and P.G. carried out the mice experiments. C.L. analysed the microarray and RNA-sequencing data. D.E., D.G., C.E., M. Pocard, M.D. and D.M. provided clinical samples. P.D. and J.-Y.S. performed the histological analyses. F.J. conceived the project, designed the research and wrote the manuscript. All authors provided intellectual input.

Corresponding author

Correspondence to Fanny Jaulin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figures 1–8 and legends for Supplementary Tables and Supplementary Videos.

Life Sciences Reporting Summary

Supplementary Table 1

Patient data

Supplementary Table 2

Signalling pathways

Supplementary Table 3

Pathway mutations

Supplementary Table 4

Antibody information

Supplementary Video 1

Supplementary Video 2

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zajac, O., Raingeaud, J., Libanje, F. et al. Tumour spheres with inverted polarity drive the formation of peritoneal metastases in patients with hypermethylated colorectal carcinomas. Nat Cell Biol 20, 296–306 (2018). https://doi.org/10.1038/s41556-017-0027-6

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing