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

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 options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: CAFs exert pulling forces on cancer cells.
Figure 2: CAFs and A431 cells form heterophilic E-cadherin/N-cadherin junctions.
Figure 3: Evidence of E-cadherin/N-cadherin junctions in lung adenocarcinoma and vulval squamous cell carcinoma.
Figure 4: Heterophilic E-cadherin/N-cadherin junctions withstand forces and trigger mechanotransduction.
Figure 5: E-cadherin is required for force transmission between CAFs and A431 cells.
Figure 6: A mechanically active heterotypic adhesion regulates cell trajectories, leader/follower patterns, and CAF polarization.
Figure 7: Afadin and nectins 2 and 3 are required for CAF-led migration of cancer cells and for CAF polarization.
Figure 8: The E-cadherin/N-cadherin junction enables collective cancer cell invasion in 3D.

References

  1. 1

    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  PubMed  PubMed Central  Google Scholar 

  2. 2

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

    PubMed  Google Scholar 

  3. 3

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

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

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

    Google Scholar 

  5. 5

    Gaggioli, C. 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).

    CAS  PubMed  Google Scholar 

  6. 6

    Harney, A. S. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

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

    CAS  PubMed  Google Scholar 

  8. 8

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

    CAS  PubMed  Google Scholar 

  9. 9

    Conklin, M. W. & Keely, P. J. 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).

    Google Scholar 

  10. 10

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

    CAS  PubMed  Google Scholar 

  11. 11

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

    PubMed  PubMed Central  Google Scholar 

  12. 12

    Orimo, A. 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).

    CAS  PubMed  Google Scholar 

  13. 13

    Pietras, K., Pahler, J., Bergers, G. & Hanahan, D. Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting. PLoS Med. 5, e19 (2008).

    PubMed  PubMed Central  Google Scholar 

  14. 14

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

    PubMed  Google Scholar 

  15. 15

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

    CAS  PubMed  Google Scholar 

  16. 16

    Takai, Y., Miyoshi, J., Ikeda, W. & Ogita, H. Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation. Nat. Rev. Mol. Cell Biol. 9, 603–615 (2008).

    CAS  PubMed  Google Scholar 

  17. 17

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

    CAS  PubMed  Google Scholar 

  18. 18

    Ng, M. R., Besser, A., Brugge, J. S. & Danuser, G. Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters. eLife 3, e03282 (2014).

    PubMed  PubMed Central  Google Scholar 

  19. 19

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

    PubMed  PubMed Central  Google Scholar 

  20. 20

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

    PubMed  PubMed Central  Google Scholar 

  21. 21

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

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

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

    CAS  PubMed  Google Scholar 

  23. 23

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

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

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

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Scarpa, E. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

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

    CAS  PubMed  Google Scholar 

  27. 27

    Maruthamuthu, V., Sabass, B., Schwarz, U. S. & Gardel, M. L. Cell-ECM traction force modulates endogenous tension at cell–cell contacts. Proc. Natl Acad. Sci. USA 108, 4708–4713 (2011).

    CAS  PubMed  Google Scholar 

  28. 28

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

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

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

    CAS  Google Scholar 

  30. 30

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

    CAS  PubMed  Google Scholar 

  31. 31

    Nieman, M. T., Kim, J. B., Johnson, K. R. & Wheelock, M. J. Mechanism of extracellular domain-deleted dominant negative cadherins. J. Cell Sci. 112, 1621–1632 (1999).

    CAS  PubMed  Google Scholar 

  32. 32

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

    CAS  PubMed  Google Scholar 

  33. 33

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

    CAS  PubMed  Google Scholar 

  34. 34

    Thomson, S. 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).

    CAS  PubMed  Google Scholar 

  35. 35

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

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Weber, G. F., Bjerke, M. A. & DeSimone, D. W. A mechanoresponsive cadherin-keratin complex directs polarized protrusive behavior and collective cell migration. Dev. Cell 22, 104–115 (2012).

    CAS  PubMed  Google Scholar 

  37. 37

    Le Duc, Q. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Yonemura, S., Wada, Y., Watanabe, T., Nagafuchi, A. & Shibata, M. α-catenin as a tension transducer that induces adherens junction development. Nat. Cell Biol. 12, 533–542 (2010).

    CAS  PubMed  Google Scholar 

  39. 39

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

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

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

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Ikeda, W. 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).

    CAS  PubMed  Google Scholar 

  42. 42

    Perl, A.-K., Wilgenbus, P., Dahl, U., Semb, H. & Christofori, G. A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 392, 190–193 (1998).

    CAS  Google Scholar 

  43. 43

    Vleminckx, K., Vakaet, L., Mareel, M., Fiers, W. & Van Roy, F. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell 66, 107–119 (1991).

    CAS  Google Scholar 

  44. 44

    Egeblad, M., Nakasone, E. S. & Werb, Z. Tumors as organs: complex tissues that interface with the entire organism. Dev. Cell 18, 884–901 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    McMillin, D. W., Negri, J. M. & Mitsiades, C. S. The role of tumour–stromal interactions in modifying drug response: challenges and opportunities. Nat. Rev. Drug Discov. 12, 217–228 (2013).

    CAS  PubMed  Google Scholar 

  46. 46

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

    CAS  PubMed  Google Scholar 

  47. 47

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

    CAS  Google Scholar 

  48. 48

    Erez, N., Truitt, M., Olson, P., Arron, S. T. & Hanahan, D. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

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

    CAS  PubMed  Google Scholar 

  50. 50

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

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Omelchenko, T. 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).

    CAS  PubMed  Google Scholar 

  52. 52

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

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

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

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

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

    CAS  PubMed  Google Scholar 

  55. 55

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

    PubMed  PubMed Central  Google Scholar 

  56. 56

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

    CAS  PubMed  Google Scholar 

  57. 57

    Benham-Pyle, B. W., Pruitt, B. L. & Nelson, W. J. Cell adhesion. Mechanical strain induces E-cadherin-dependent Yap1 and β-catenin activation to drive cell cycle entry. Science 348, 1024–1027 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Abercrombie, M. & Heaysman, J. E. 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).

    CAS  PubMed  Google Scholar 

  59. 59

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

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

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

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Calvo, F. 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).

    CAS  PubMed  Google Scholar 

  62. 62

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

    CAS  PubMed  Google Scholar 

  63. 63

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

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

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

    CAS  Google Scholar 

  65. 65

    Hidalgo-Carcedo, C. 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).

    CAS  PubMed  Google Scholar 

  66. 66

    Roca-Cusachs, P., Gauthier, N. C., Del Rio, A. & Sheetz, M. P. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67

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

    PubMed  Google Scholar 

  68. 68

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

    PubMed  PubMed Central  Google Scholar 

  69. 69

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

    PubMed  PubMed Central  Google Scholar 

  70. 70

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

    PubMed  PubMed Central  Google Scholar 

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

Authors

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.

Corresponding authors

Correspondence to Erik Sahai or Xavier Trepat.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 CAFs and A431 cells form heterophilic E-cadherin/N-cadherin junctions.

(a,b) Fluorescence images of a co-culture of CAFs (CAGAP-mCherry) and A431 cells stained for N-cadherin (far-red) and E-cadherin (green). Scale bars, 20 μm. (ce) 3 magnified views of the regions highlighted by white rectangles in a. Images representative of 3 samples. Scale bars, 10 μm. (f) SIM immunofluorescence images of A431 cells contacting a CAF, N-cadherin (green), F-actin (magenta). Images representative of 15 samples. Scale bar, 5 μm. (g) Fluorescence images of a co-culture of CAFs (CAGAP-mCherry) and A431 cells stained for N-cadherin (far-red) and P-cadherin (green). Images representative of 3 samples. Scale bars, 20 μm.

Supplementary Figure 2 Heterophilic E-cadherin/N-cadherin junctions between CAFs and A431 cells colocalize with β-catenin, α-catenin, vinculin, and F-actin.

(ad) Fluorescence images of a co-culture of CAFs expressing N-cadherin-GFP and A431 cells expressing E-cadherin-Ruby stained for β-catenin (a), α-catenin (b), vinculin (c), F-actin (far red) (d). Images representative of 3 samples. Scale bars, 5 μm. (e) Representative fluorescence images of a co-culture of CAFs (CAGAP-mCherry) and A431 cells expressing α-catenin-GFP stained for N-cadherin (red) and β-catenin (blue). Images representative of 2 samples. Scale bars, 5 μm.

Supplementary Figure 3 Heterophilic E-cadherin/N-cadherin junctions between CAFs and A431 cells in the 2D spheroid assay.

(a) Fluorescence images of a CAF expressing N-cadherin-GFP (green) contacting a spheroid of A431 cells expressing E-cadherin-Ruby (red) in the 2D spheroid assay on 6 kPa gels. Scale bar, 20 μm. White arrows show the presence of the E-cadherin/N-cadherin contact. (bd) Magnified views of the region marked by a dashed box in a. Images representative of >4 samples. Scale bars, 5 μm. (e) Fluorescence images of a CAF expressing N-cadherin-GFP (green) contacting a spheroid of A431 cells expressing E-cadherin-Ruby (red) in the 2D spheroid assay on 6 kPa gels and stained for β-catenin (far-red). Scale bar, 20 μm. White arrows point at the E-cadherin/N-cadherin contact. (fi) Magnified views of the region marked by a dashed box in e. Images representative of >3 samples. Scale bars, 10 μm.

Supplementary Figure 4 Characterization of A431 cells types and CAFs.

(a) Western blot of E-cadherin, P-cadherin and β-tubulin for A431 control cells (CT), A431-EcadKO cells (EKO), and A431-PcadKO cells (PKO). Image representative of 3 experiments. (b,c) Densitometric quantification of western blot bands relative to the loading (β-tubulin) of E-cadherin and P-Cadherin, respectively, for A431 control cells (CT), A431-EcadKO cells (EKO), and A431-PcadKO cells (PKO). The error bars represent mean +/− s.d. (n = 3 experiments). (d, e) Quantification of spheroid edge curvature and cancer cell velocity at the spheroid edge for control A431cells and A431-EcadKO cells in the absence of CAFs. No significant differences in cell velocity (P = 0.993) and spheroid edge curvature (P = 0.119) were observed between control cells and A431-EcadKO cells. For spheroid curvature, n = 120 measurements from 3 independent experiments for A431 control cells, and n = 265 measurements from 3 independent experiments for A431-EcadKO cells. For cell velocity, n = 3 independent experiments (control, n = 300 measurements; A431-EcadKO, n = 400 measurements). The error bars represent s.e.m., n.s. indicates not significantly different, t-test. (f) Western blot of E-cadherin, N-cadherin, β-catenin and β-tubulin for A431 control cells (A431, column 1), A431-EcadKO (EKO, column 2), control vulval CAFs (CAF, column 3), vulval CAFs transfected with siRNA control (CAF-siCT, column 4), vulval CAF-siNcad (CAF-siN, column 5), normal lung fibroblasts (NF, column 6), normal dermal fibroblasts (NF, column 7), H1437 lung cancer cells (H1437, column 8), lung CAFs (CAF, column 9). Additional western blots are shown in figure (o) for the columns marked with asterisks. Image representative of 3 experiments. (g) Western blot of E-cadherin, P-cadherin, α-catenin and β-tubulin for A431 control cells (CT), A431-EcadKO cells (EKO), A431-EcadWT (rescue control) cells, and A431- EcadW2A cells. Image representative of 2 experiments. (h) Western blot of N-cadherin and α-tubulin for vulval CAFs-siCT and CAFs-siNcad. Image representative of 3 experiments. (i) Densitometric quantification of western blot bands relative to the loading (α-tubulin) of N-Cadherin for CAFs-siCT and CAFs-siNcad (n = 3 experiments). The error bars represent s.d. (j, k) Representative fluorescence images of CAFs (CAGAP-mCherry) plated overnight on glass coverslips 3 days after siRNA transfection and fixed and stained for N-cadherin (green), and nucleus (blue). Image representative of 3 samples. Scale bars, 20 μm. (l) Western blot of E-cadherin, α-catenin and β-tubulin for A431 control cells (A431, column 1), A431-αcatKO (column 2), A431-αcatWT (rescue control, column 3), A431-αcatΔVBS (column 4). Image representative of 2 experiments. (m) Quantification of the fraction ‘leaders’ or ‘loners’ in vulval CAFs. No significant differences were found between CAFs-siCT and CAFs-WT. siCT, n = 86 from 4 independent experiments; WT, n = 57 from 3 independent experiments; n.s. indicates not significantly different (P = 0.838), Mann-Whitney test. Error bars represent mean +/− s.e.m. (n) Quantification of velocity of isolated CAFs plated on fibronectin coated-6 kPa gels transfected with siRNA Control (siCT, n = 60) or si-Ncad (siNcad, n = 63). Data were obtained from 3 independent experiments. n.s. indicates not significantly different (P = 0.736), Mann-Whitney test. Error bars represent mean +/− s.e.m. (o)Western blot of N-cadherin (Ncad) and α-tubulin (α-tub) for CAFs and normal fibroblasts (NF) from skin and lung tissue. Image representative of 3 experiments. (p) Collagen gel contraction assay for vulval CAFs (CAF, n = 10 measurements) and normal dermal fibroblasts (NF, n = 10 measurements). The percentage of gel contraction was measured immediately after gel polymerization (D0), 24 and 48 h after gel polymerization, respectively (D1 and D2). Data were obtained from 3 independent experiments over at least 3 gels per condition per experiment. Error bars represent s.e.m., *** indicates P < 0.0001, Mann-Whitney test. (q) Quantification of velocity in 3D ECM of isolated CAFs transfected with siRNA Control (siCT, n = 158 cells) or si-N-cadherin (siNcad, n = 143),P = 0.788, and of isolated A431 control cells (CT, n = 111) and A431-EcadKO cells (EKO, n = 118), P = 0.655. Data were obtained from 3 independent experiments. n.s. indicates not significantly different, Mann-Whitney test.

Supplementary Figure 5 The E-cadherin/N-cadherin junction is observed in vitro and in vivo.

(a) Co-culture of CAFs from one patient with lung adenocarcinoma and H1437 cells show E-cadherin/N-cadherin junctions. Image representative of 2 samples. Scale bars, 5 μm.

Supplementary Figure 6 Cadherin 11 is dispensable for CAF-led migration.

(a) Fluorescence images of vulval CAFs plated overnight on glass coverslips 3 days after siRNA transfection and fixed and stained for cadherin-11. Images representative of 2 experiments. Scale bar, 20 μm. (b) Quantification of the fraction of ‘leaders’ or ‘loners’ CAFs for CAFs transfected with siRNA-control (siCT) and CAF transfected with siRNA-cadherin11 (siCad11). No significant differences were found between the CAF-siCT and CAF-siCad11. siCT, n = 147 CAFs, from 3 independent experiments; siCad11, n = 201 CAFs, from 3 independent experiments. Error bars represent mean +/− s.e.m, n.s. indicates not significantly different, unpaired two-tailed t-test, P = 0.584. (c) Western blot of cadherin-11 and α-tubulin for CAFs siCT and CAFs siCad11, Image representative of 3 experiments.

Supplementary Figure 7 The E/N-cadherin contact enables collective cancer cell invasion in 3D.

(a, b) Fluorescence images of spheroids containing different mixtures of CAFs and A431 cells after 60 h of invasion in an organotypic ECM. (a) 1:1 mixture of A431-YPet (control) and control CAFs (KEIMA). (b) 1:1 mixture of A431-EPcadKO (mCherry) and control CAFs (KEIMA). (c) 1:1 mixture of A431-PcadKO (mCherry) and control CAFs (KEIMA). Images representative of >3 experiments. Scale bars, 100 μm. (d) Pie chart representation of the relative percentage of the 3 main modes of 3D invasion in our assays: strands led by CAFs (black), strands without CAFs (grey), and single cancer cells (white). The area of the circles is proportional to the total number of invasion events. A431, n = 7, average number of total invasion events = 6.29, A431 P-cadherin KO (PKO), n = 18, average number of total invasion events = 6.61, A431 E-cadherin KO, n = 18, average number of total invasion events = 1.78, A431 P-cadherin/E-cadherin double KO, n = 8, average number of total invasion events = 13.38. 3 independent experiments. Chi-squared test, *** indicates P < 0.0001.

Supplementary Figure 8 Coexistence of heterotypic and homotypic adhesion.

(a) Images show both homophilic N-cadherin junctions (yellow arrows) and heterophilic E-cadherin/N-cadherin junctions in co-cultures of cancer cells with variable endogenous N-cadherin levels. Left hand panel shows vulval SCC cells and CAFs isolated from the same patient and right hand panel shows FaDU SCC cells and oral SCC CAF (OCAF2). N-cadherin staining in green, E-cadherin in red, and F-actin in blue. Scale bar is 10 μm. (b) Schematic representation of the role of cell–cell contacts in fibroblast-led cancer cell invasion. CAFs (elongated light red cells) engage extensively with the ECM and make heterophilic E-cadherin/N-cadherin junctions with cancer cells (light green cells). Heterophilic contacts and nectin/afadin complexes re-polarize CAFs to migrate away from the contact site (yellow arrows indicate directional cue). However, mechanical coupling via E-cadherin/N-cadherin and α-catenin/vinculin engagement leads to the dragging of cancer cells behind the CAF (white arrows with borders). This long-lived contact continually promotes CAF migration. Af, afadin; v, vinculin; αcat, α-catenin;βcat, β-catenin; N-E, N-cadherin/E-cadherin junction; E-E, E-cadherin/E-cadherin junction; Int, integrin.

Supplementary information

Supplementary Information

Supplementary Information (PDF 69000 kb)

Supplementary Table 1

Supplementary Information (XLSX 11 kb)

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. (AVI 870 kb)

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. (AVI 27926 kb)

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. (AVI 3147 kb)

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. (AVI 35809 kb)

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. (AVI 1352 kb)

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. (AVI 21225 kb)

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). (AVI 1236 kb)

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. (AVI 20221 kb)

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. (AVI 9496 kb)

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. (AVI 3105 kb)

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. (AVI 1673 kb)

‘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. (AVI 2040 kb)

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. (AVI 3170 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Labernadie, A., Kato, T., Brugués, A. et al. A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion. Nat Cell Biol 19, 224–237 (2017). https://doi.org/10.1038/ncb3478

Download citation

Further reading

Search

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing