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

Nature Cell Biology volume 19, pages 224–237 (2017)Cite this article

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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.

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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.

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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.

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Authors and 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

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  1. Anna Labernadie
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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.

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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. (c–e) 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.

(a–d) 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. (b–d) 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. (f–i) 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.

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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)

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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

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