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.

  • Subscribe to Nature Cell Biology for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Additional information

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


  1. 1.

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

  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).

  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).

  4. 4.

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

  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).

  6. 6.

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

  7. 7.

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

  8. 8.

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

  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).

  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).

  11. 11.

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

  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).

  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).

  14. 14.

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

  15. 15.

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

  16. 16.

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

  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).

  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).

  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).

  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).

  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).

  24. 24.

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

  25. 25.

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

  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).

  27. 27.

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

  28. 28.

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

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  36. 36.

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

  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).

  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).

  39. 39.

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

  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).

  42. 42.

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

  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).

  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).

  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).

  46. 46.

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

  47. 47.

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

  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).

  49. 49.

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

  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).

  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).

  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).

  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).

  57. 57.

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

  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).

  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).

Download references


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

Author notes

    • Olivier Zajac

    Present address: Department of Translational Research, Institut Curie, Paris, France


  1. U-981, Gustave Roussy, Villejuif, France

    • Olivier Zajac
    • , Joel Raingeaud
    • , Fotine Libanje
    • , Celine Lefebvre
    • , Dora Sabino
    • , Pétronille Roy
    • , Clara Benatar
    • , Charlotte Canet-Jourdan
    • , Paula Azorin
    • , Damien Nowak
    • , Ludovic Bigot
    •  & Fanny Jaulin
  2. Université Paris Saclay, Villejuif, France

    • Isabelle Martins
    •  & Jean-Luc Perfettini
  3. U-1030, Gustave Roussy, Villejuif, France

    • Isabelle Martins
    •  & Jean-Luc Perfettini
  4. Plateforme d’Evaluation Préclinique, AMMICA UMS 3655/ US 23, Gustave Roussy, Villejuif, France

    • Mélanie Polrot
    •  & Patrick Gonin
  5. UMR-1170, Gustave Roussy, Villejuif, France

    • Salima Benbarche
    •  & Camille Lobry
  6. UMR-9196, Gustave Roussy, Villejuif, France

    • Sylvie Souquere
    •  & Gerard Pierron
  7. Digestive Cancer Unit, Gustave Roussy, Villejuif, France

    • Michel Ducreux
    • , David Malka
    • , Dominique Elias
    •  & Diane Goéré
  8. Pathology Department, Gustave Roussy, Villejuif, France

    • Jean-Yves Scoazec
    •  & Peggy Dartigues
  9. UMR-965, Lariboisière Hospital, Paris, France

    • Clarisse Eveno
    •  & Marc Pocard


  1. Search for Olivier Zajac in:

  2. Search for Joel Raingeaud in:

  3. Search for Fotine Libanje in:

  4. Search for Celine Lefebvre in:

  5. Search for Dora Sabino in:

  6. Search for Isabelle Martins in:

  7. Search for Pétronille Roy in:

  8. Search for Clara Benatar in:

  9. Search for Charlotte Canet-Jourdan in:

  10. Search for Paula Azorin in:

  11. Search for Mélanie Polrot in:

  12. Search for Patrick Gonin in:

  13. Search for Salima Benbarche in:

  14. Search for Sylvie Souquere in:

  15. Search for Gerard Pierron in:

  16. Search for Damien Nowak in:

  17. Search for Ludovic Bigot in:

  18. Search for Michel Ducreux in:

  19. Search for David Malka in:

  20. Search for Camille Lobry in:

  21. Search for Jean-Yves Scoazec in:

  22. Search for Clarisse Eveno in:

  23. Search for Marc Pocard in:

  24. Search for Jean-Luc Perfettini in:

  25. Search for Dominique Elias in:

  26. Search for Peggy Dartigues in:

  27. Search for Diane Goéré in:

  28. Search for Fanny Jaulin in:


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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Fanny Jaulin.

Supplementary information

  1. Supplementary Information

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

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Patient data

  4. Supplementary Table 2

    Signalling pathways

  5. Supplementary Table 3

    Pathway mutations

  6. Supplementary Table 4

    Antibody information

  7. Supplementary Video 1

  8. Supplementary Video 2