Article | Published:

A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

Nature Cell Biology volume 19, pages 224237 (2017) | Download Citation

Abstract

Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers β-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , & Collective invasion in breast cancer requires a conserved basal epithelial program. Cell 155, 1639–1651 (2013).

  2. 2.

    , , & Classifying collective cancer cell invasion. Nat. Cell Biol. 14, 777–783 (2012).

  3. 3.

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

  4. 4.

    et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 36, 1453–1463 (2015).

  5. 5.

    et al. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat. Cell Biol. 9, 1392–1400 (2007).

  6. 6.

    et al. Real-time imaging reveals local, transient vascular permeability, and tumor cell intravasation stimulated by TIE2hi macrophage-derived VEGFA. Cancer Discov. 5, 932–943 (2015).

  7. 7.

    & Fibroblasts in cancer. Nat. Rev. Cancer 6, 392–401 (2006).

  8. 8.

    Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 59, 5002–5011 (1999).

  9. 9.

    & Why the stroma matters in breast cancer: insights into breast cancer patient outcomes through the examination of stromal biomarkers. Cell Adhes. Migr. 6, 249–260 (2012).

  10. 10.

    et al. Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell 6, 17–32 (2004).

  11. 11.

    et al. Extensive rewiring of epithelial-stromal co-expression networks in breast cancer. Genome Biol. 16, 128 (2015).

  12. 12.

    et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121, 335–348 (2005).

  13. 13.

    , , & Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting. PLoS Med. 5, e19 (2008).

  14. 14.

    & Activation of fibroblasts in cancer stroma. Exp. Cell Res. 316, 2713–2722 (2010).

  15. 15.

    et al. Competition amongst Eph receptors regulates contact inhibition of locomotion and invasiveness in prostate cancer cells. Nat. Cell Biol. 12, 1194–1204 (2010).

  16. 16.

    , , & Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation. Nat. Rev. Mol. Cell Biol. 9, 603–615 (2008).

  17. 17.

    et al. Mechanical tugging force regulates the size of cell–cell junctions. Proc. Natl Acad. Sci. USA 107, 9944–9949 (2010).

  18. 18.

    , , & Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters. eLife 3, e03282 (2014).

  19. 19.

    et al. Control of cell–cell forces and collective cell dynamics by the intercellular adhesome. Nat. Cell Biol. 17, 409–420 (2015).

  20. 20.

    et al. Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force. Science 346, 1254211 (2014).

  21. 21.

    et al. α-catenin cytomechanics–role in cadherin-dependent adhesion and mechanotransduction. J. Cell Sci. 127, 1779–1791 (2014).

  22. 22.

    Contact inhibition and malignancy. Nature 281, 259–262 (1979).

  23. 23.

    Inter-cellular forces orchestrate contact inhibition of locomotion. Cell 161, 361–373 (2015).

  24. 24.

    et al. Integrin and cadherin synergy regulates contact inhibition of migration and motile activity. J. Cell Biol. 141, 515–526 (1998).

  25. 25.

    et al. Cadherin switch during EMT in neural crest cells leads to contact inhibition of locomotion via repolarization of forces. Dev. Cell 34, 421–434 (2015).

  26. 26.

    , & Nm23-H1 regulates contact inhibition of locomotion, which is affected by ephrin-B1. J. Cell Sci. 125, 4343–4353 (2012).

  27. 27.

    , , & Cell-ECM traction force modulates endogenous tension at cell–cell contacts. Proc. Natl Acad. Sci. USA 108, 4708–4713 (2011).

  28. 28.

    et al. Collective cell guidance by cooperative intercellular forces. Nat. Mater. 10, 469–475 (2011).

  29. 29.

    et al. Physical forces during collective cell migration. Nat. Phys. 5, 426–430 (2009).

  30. 30.

    et al. Epithelial bridges maintain tissue integrity during collective cell migration. Nat. Mater. 13, 87–96 (2014).

  31. 31.

    , , & Mechanism of extracellular domain-deleted dominant negative cadherins. J. Cell Sci. 112, 1621–1632 (1999).

  32. 32.

    et al. Linking molecular affinity and cellular specificity in cadherin-mediated adhesion. Proc. Natl Acad. Sci. USA 106, 11594–11599 (2009).

  33. 33.

    et al. Structural and energetic determinants of adhesive binding specificity in type I cadherins. Proc. Natl Acad. Sci. USA 111, E4175–E4184 (2014).

  34. 34.

    et al. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res. 65, 9455–9462 (2005).

  35. 35.

    et al. Cadherin-dependent mechanotransduction depends on ligand identity but not affinity. J. Cell Sci. 125, 4362–4371 (2012).

  36. 36.

    , & A mechanoresponsive cadherin-keratin complex directs polarized protrusive behavior and collective cell migration. Dev. Cell 22, 104–115 (2012).

  37. 37.

    et al. Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II-dependent manner. J. Cell Biol. 189, 1107–1115 (2010).

  38. 38.

    , , , & α-catenin as a tension transducer that induces adherens junction development. Nat. Cell Biol. 12, 533–542 (2010).

  39. 39.

    et al. Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling. J. Cell Biol. 196, 641–652 (2012).

  40. 40.

    Vinculin-dependent cadherin mechanosensing regulates efficient epithelial barrier formation. Biol. Open 1, 1128–1140 (2012).

  41. 41.

    et al. Tage4/Nectin-like molecule-5 heterophilically trans-interacts with cell adhesion molecule Nectin-3 and enhances cell migration. J. Biol. Chem. 278, 28167–28172 (2003).

  42. 42.

    , , , & A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 392, 190–193 (1998).

  43. 43.

    , , , & Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell 66, 107–119 (1991).

  44. 44.

    , & Tumors as organs: complex tissues that interface with the entire organism. Dev. Cell 18, 884–901 (2010).

  45. 45.

    , & The role of tumour–stromal interactions in modifying drug response: challenges and opportunities. Nat. Rev. Drug Discov. 12, 217–228 (2013).

  46. 46.

    & Friends or foes—bipolar effects of the tumour stroma in cancer. Nat. Rev. Cancer 4, 839–849 (2004).

  47. 47.

    & Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 124, 263–266 (2006).

  48. 48.

    , , , & Cancer-associated fibroblasts are activated in incipient neoplasia to orchestrate tumor-promoting inflammation in an NF-κB-dependent manner. Cancer Cell 17, 135–147 (2010).

  49. 49.

    & Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell 21, 309–322 (2012).

  50. 50.

    & Cadherin-23 mediates heterotypic cell–cell adhesion between breast cancer epithelial cells and fibroblasts. PLoS ONE 7, e33289 (2012).

  51. 51.

    et al. Contact interactions between epitheliocytes and fibroblasts: formation of heterotypic cadherin-containing adhesion sites is accompanied by local cytoskeletal reorganization. Proc. Natl Acad. Sci. USA 98, 8632–8637 (2001).

  52. 52.

    , & Single-cell adhesion tests against functionalized microspheres arrayed on AFM cantilevers confirm heterophilic E- and N-cadherin binding. Biophys. J. 99, L100–L102 (2010).

  53. 53.

    et al. E-N-cadherin heterodimers define novel adherens junctions connecting endoderm-derived cells. J. Cell Biol. 195, 873–887 (2011).

  54. 54.

    , & Formation of heterotypic adherens-type junctions between L-CAM-containing liver cells and A-CAM-containing lens cells. Cell 50, 987–994 (1987).

  55. 55.

    et al. The osteogenic niche promotes early-stage bone colonization of disseminated breast cancer cells. Cancer Cell 27, 193–210 (2015).

  56. 56.

    The cellular basis of tissue separation. Development 141, 3303–3318 (2014).

  57. 57.

    , & Cell adhesion. Mechanical strain induces E-cadherin-dependent Yap1 and β-catenin activation to drive cell cycle entry. Science 348, 1024–1027 (2015).

  58. 58.

    & Observations on the social behaviour of cells in tissue culture. I. Speed of movement of chick heart fibroblasts in relation to their mutual contacts. Exp. Cell Res. 5, 111–131 (1953).

  59. 59.

    et al. Contact inhibition of locomotion in vivo controls neural crest directional migration. Nature 456, 957–961 (2008).

  60. 60.

    et al. Chase-and-run between adjacent cell populations promotes directional collective migration. Nat. Cell Biol. 15, 763–772 (2013).

  61. 61.

    et al. Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nat. Cell Biol. 15, 637–646 (2013).

  62. 62.

    et al. Matrix stiffening and β1 integrin drive subtype-specific fibroblast accumulation in lung cancer. Mol. Cancer Res. 13, 161–173 (2015).

  63. 63.

    et al. Aberrant DNA methylation in non-small cell lung cancer-associated fibroblasts. Carcinogenesis 36, 1453–1463 (2015).

  64. 64.

    et al. Mechanical waves during tissue expansion. Nat. Phys. 8, 628–634 (2012).

  65. 65.

    et al. Collective cell migration requires suppression of actomyosin at cell–cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6. Nat. Cell Biol. 13, 49–58 (2011).

  66. 66.

    , , & Clustering of α(5)β(1) integrins determines adhesion strength whereas α(v)β(3) and talin enable mechanotransduction. Proc. Natl Acad. Sci. USA 106, 16245–16250 (2009).

  67. 67.

    & High-force magnetic tweezers with force feedback for biological applications. Rev. Sci. Instrum. 78, 114301 (2007).

  68. 68.

    et al. Optimizing imaging conditions for demanding multi-color super resolution localization microscopy. PLoS ONE 11, e0158884 (2016).

  69. 69.

    et al. STRIPAK components determine mode of cancer cell migration and metastasis. Nat. Cell Biol. 17, 68–80 (2014).

  70. 70.

    et al. Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy. PLoS ONE 4, e7716 (2009).

Download references

Acknowledgements

We thank N. Castro for technical assistance, J. de Rooij (UMC Utrecht, Netherlands) for plasmids, S. Pérez-Amodio (IBEC, Spain) for dermal fibroblasts, N. Reguart (Hospital Clinic, Spain) and M. Gabasa (University of Barcelona, Spain) for lung fibroblasts, and A. Schertel (Zeiss) for assistance with the FIB-SEM. This work was supported by the Spanish Ministry of Economy and Competitiveness/FEDER (BFU2012-38146 to X.T., BFU2014-52586-REDT to P.R.-C., IJCI2014-19843 to A.L. and IJCI-2014-19156 to A.E.-A.), the Generalitat de Catalunya (2014-SGR-927 to X.T. and CERCA Programme), the European Research Council (StG-CoG-616480 to X.T.), Obra Social ‘La Caixa’, Marie-Curie action (CAFFORCE 328664 to A.L.), EMBO Long-term fellowship (EMBO ALTF 1235-2012 to A.L.), a Career Integration Grant within the seventh European Community Framework Programme (PCIG10-GA-2011-303848 to P.R.-C.), Fundació la Marató de TV3 (project 20133330 to P.R.-C.), and AXA research fund (L.A.). E.S., E.A., A.W. and S.D. are funded by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001144), the UK Medical Research Council (FC001144), and the Wellcome Trust (FC001144). T.K. is funded by Marie-Curie action (HeteroCancerInvasion no. 708651) and the Japanese Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation.

Author information

Affiliations

  1. Institute for Bioengineering of Catalonia, Barcelona 08028, Spain

    • Anna Labernadie
    • , Agustí Brugués
    • , Xavier Serra-Picamal
    • , Victor González-Tarragó
    • , Alberto Elosegui-Artola
    • , Lorenzo Albertazzi
    • , Pere Roca-Cusachs
    •  & Xavier Trepat
  2. The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK

    • Takuya Kato
    • , Stefanie Derzsi
    • , Esther Arwert
    • , Anne Weston
    •  & Erik Sahai
  3. Unitat de Biofísica i Bioenginyeria, Facultat de Medicina, Universitat de Barcelona, Barcelona 08036, Spain

    • Xavier Serra-Picamal
    • , Jordi Alcaraz
    • , Pere Roca-Cusachs
    •  & Xavier Trepat
  4. Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain

    • Xavier Trepat
  5. Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Barcelona 08028, Spain

    • Xavier Trepat

Authors

  1. Search for Anna Labernadie in:

  2. Search for Takuya Kato in:

  3. Search for Agustí Brugués in:

  4. Search for Xavier Serra-Picamal in:

  5. Search for Stefanie Derzsi in:

  6. Search for Esther Arwert in:

  7. Search for Anne Weston in:

  8. Search for Victor González-Tarragó in:

  9. Search for Alberto Elosegui-Artola in:

  10. Search for Lorenzo Albertazzi in:

  11. Search for Jordi Alcaraz in:

  12. Search for Pere Roca-Cusachs in:

  13. Search for Erik Sahai in:

  14. Search for Xavier Trepat in:

Contributions

A.L., E.S. and X.T. conceived the study and designed experiments, with additional input from T.K. A.L. performed most experiments and data analysis. T.K. performed and analysed spheroid invasion experiments and generated A431 KO cell lines. A.L., A.E.-A., V.G.-T. and P.R.-C. designed, performed and analysed magnetic cytometry assays. A.B. and X.S.-P. developed software for image analysis and force measurements. S.D. performed QRT–PCR experiments. A.L. and L.A. performed STORM imaging. J.A. contributed CAFs from patients with non-small lung cell carcinoma. E.A. performed the intravital imaging and assisted with the patient sample analysis, A.W. performed electron microscopy. A.L., E.S. and X.T. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Erik Sahai or Xavier Trepat.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

Excel files

  1. 1.

    Supplementary Table 1

    Supplementary Information

Videos

  1. 1.

    CAFs lead cancer cell strands in 3D invasion assays.

    Representative 3D rendering of a fixed spheroid containing vulval CAFs (VCAF, CAGAP-cherry) and A431 cells (green) (ratio 1:1) after 24 h embedded in an organotypic ECM. z-step, 0.25 μm.

  2. 2.

    CAFs favor expansion of cancer cell spheroids in 2D.

    Representative time-lapse of a 2D migration assay on a soft polyacrylamide substrate (Young’s modulus E = 6 kPa). White rectangles highlight three CAFs (CAGAP-mCherry) leading the expansion of the A431 spheroid (unlabeled). Images were acquired every 5 min. Scale bar, 100 μm.

  3. 3.

    CAFs lead collective migration of cancer cells in 2D.

    Representative time-lapse of one CAF (CAGAP-mCherry) leading the 2D migration of A431 cells (unlabeled) away from the spheroid edge. Cells are adhered on an elastic substrate (Young’s modulus E = 6 kPa). Images were acquired every 5 min. Scale bar, 2 μm.

  4. 4.

    FIB-SEM reveals multiple contact points at the CAF-A431 interface.

    Representative FIB-SEM z-stack sequence of areas of contact between CAFs (VCAF) and A431 cells. White arrows show the location of contact between CAF and cancer cells. Image dimensions, 10 × 12 μm, z-stack steps 50 nm.

  5. 5.

    W2A mutation in the extracellular domain of E-cadherin drastically diminishes co-localization with N-cadherin.

    Representative confocal time-lapse movie of A431-EcadWT-Ruby or A431-EcadW2A-Ruby cells co-cultured with CAFs expressing N-cadherin-GFP. Dynamics of the co-culture was recorded at 5 min intervals. Scale Bar, 20  μm.

  6. 6.

    Calcium chelation abrogates reversibly E-cadherin/N-cadherin co-localization.

    Representative confocal time-lapse movie of A431-E-cadherin-Ruby mixed with CAF-N-cadherin-GFP showing dynamics of the E-cadherin/N-cadherin contact during a calcium chelation assay. After 10 min of acquisition, the EGTA solution was added to the medium (final concentration, 4 mM). After 4 min incubation, the medium containing EGTA was washed three times with normal medium. Arrows show the formation of the E-cadherin/N-cadherin contact after washout of EGTA. Images were acquired every 2 min. Scale Bar, 20 μm.

  7. 7.

    Dynamics of the E-cadherin/N-cadherin adhesion during CAF-led cancer cell migration.

    Representative confocal time-lapse movie of A431-E-cadherin-Ruby spheroid seeded on glass and surrounded by CAF-N-cadherin-GFP. The magnified panel represents the area of contact between the leading CAF and A431 cells. White arrow shows the location of the E-cadherin/N-cadherin contact. Images were acquired every 5 min. Scale Bars, 20 μm (right panel), 10 μm (left panel).

  8. 8.

    E-cadherin and β-catenin colocalize at heterotypic contacts.

    Representative confocal time-lapse movie of A431-E-cadherin-Ruby expressing β-catenin-GFP mixed with unlabeled CAFs. The magnified panel represents the area of contact between the leading CAF and the A431 cells. The white arrow shows the localization of the contact between the A431 cells and the CAF (white asterisk). Images were acquired every 2 min. Scale Bar, 10 μm.

  9. 9.

    E-cadherin and vinculin colocalize at heterotypic contacts.

    Representative confocal time-lapse movie of A431-E-cadherin-Ruby expressing vinculin-GFP mixed with unlabeled CAFs. The white arrow shows the localization of the contact between the A431 cells and the CAF (white asterisk). Note an enrichment of E-cadherin (red) and vinculin (green) at the contact. Images were acquired every 2 min. Scale Bar, 10 μm.

  10. 10.

    CAFs exert pulling forces on cancer cells.

    Representative time-lapse of a CAF (CAGAP-mCherry) dragging A431 cells. The magnitude and direction of the force exerted by the CAF on the cancer cell is represented by the green vector. For clarity, the force vector is represented at the geometric center of the CAF. See Fig. 5 for a quantification of the force throughout the time-lapse. Scale bar, 50 μm.

  11. 11.

    E-cadherin is required for force transmission between CAFs and A431 cells.

    Representative time-lapse of a CAF contacting the edge of A431-EcadKO cells. The magnitude and direction of the force exerted by the CAF on the cancer cell is represented by the green vector. For clarity, the force vector is represented at the geometric center of the CAF. See Fig. 5 for a quantification of the force throughout the time-lapse. Scale bar, 50 μm.

  12. 12.

    ‘Leader’ versus ‘loner’ CAF phenotypes.

    Representative time-lapse of a ‘leader’ CAF (left) and a ‘loner’ CAF (right) (CAGAP-mCherry, white arrow). Images were acquired every 5 min. Scale bars, 20 μm.

  13. 13.

    The heterotypic contact regulates CAF repolarization.

    Representative time-lapse of a control CAF (left panel) or N-cadherin depleted CAF (CAF-siNcad, right panel) contacting the edge of a spheroid of A431 control cells (left and right panel) or A431-EcadKO cells (middle panel). Colored spots show the location of the CAFs. Images were acquired every 10 min. Scale bar, 50 μm.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ncb3478

Further reading