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Self-generated gradients steer collective migration on viscoelastic collagen networks

Nature Materials volume 21, pages 1200–1210 (2022)Cite this article

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Abstract

Growing evidence suggests that the physical properties of the cellular microenvironment influence cell migration. However, it is not currently understood how active physical remodelling by cells affects migration dynamics. Here we report that cell clusters seeded on deformable collagen-I networks display persistent collective migration despite not showing any apparent intrinsic polarity. Clusters generate transient gradients in collagen density and alignment due to viscoelastic relaxation of the collagen networks. Combining theory and experiments, we show that crosslinking collagen networks or reducing cell cluster size results in reduced network deformation, shorter viscoelastic relaxation time and smaller gradients, leading to lower migration persistence. Traction force and Brillouin microscopy reveal asymmetries in force distributions and collagen stiffness during migration, providing evidence of mechanical cross-talk between cells and their substrate during migration. This physical model provides a mechanism for self-generated directional migration on viscoelastic substrates in the absence of internal biochemical polarity cues.

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Fig. 1: Cell clusters migrate persistently on collagen networks.
Fig. 2: Local collagen topology is asymmetric during collective migration and relaxes viscoelastically.
Fig. 3: Theoretical model of persistent migration on a viscoelastic substrate.
Fig. 4: Collagen crosslinking decreases viscoelastic relaxation time and reduces migration persistence.
Fig. 5: Collagen deformation and migration persistence depend on cluster size.
Fig. 6: Persistent migration is associated with asymmetries in traction force distribution and collagen stiffness gradients.

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Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information files and from the corresponding authors upon reasonable request. A minimum data set has been uploaded to the following public repository: https://doi.org/10.5281/zenodo.6390650.

Code availability

Custom software used to analyse images and data will be made available upon reasonable request.

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Acknowledgements

We acknowledge the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infractucture France-BioImaging (ANR10-INBS-04). We thank T. Kato and E. Sahai for the stable MLC-GFP and Raichu-Rac A431 cell lines and V. Marthiens and R. Basto for the pericentrin antibody. We thank J. Barbazan, O. Zajac and R. Bouras for assistance making the stable LifeAct-mCherry, paxillin-GFP and zyxin-mCherry lines. We also thank O. Zajac for designing and sharing the protocol for generating thin, aligned collagen networks. We thank N. Elkhatib and G. Montagnac for sharing reagents and protocols. We thank C. Bevilacqua and R. Prevedel, who developed the custom Brillouin microscope utilized in this work. We acknowledge M. Gómez-González and E. Latorre for developing the 3D PIV and traction force microscopy code. We thank H. Mohammadi, E. Sahai and all members of the Vignjevic lab for helpful discussions and comments on the manuscript. A.G.C. was supported by the European Moleclular Biology Organization (ALTF 1582-2014 to A.G.C.). This project also received funding from the European Research Council under the European Union Horizon 2020 research and innovation programme (grant agreement no. 772487 to D.M.V.) and Institut National du Cancer (PLBIO18-087 to D.M.V.). A.M. and R.V. were funded by PHYMAX and POLCAM grants. A.M. thanks a Talent fellowship awarded by the CY Cergy Paris University. M.B. and A.D.-M. were funded by the European Molecular Biology Laboratory and the Deutsche Forschungsgemeinschaft (DI2205/2-1 to A.D.-M.). X.T. was funded by Spanish Ministry for Science, Innovation and Universities MICCINN/FEDER (PGC2018-099645-B-I00, Severo Ochoa Award of Excellence), the Generalitat de Catalunya/CERCA programme (SGR-2017-01602), Fundació la Marató de TV3, Obra Social 'La Caixa', the European Research Council (Adv-883739) and the European Commission (H2020-FETPROACT-01-2016-731957.

Author information

Author notes

  1. Andrew G. Clark

    Present address: Institute of Cell Biology and Immunology, Stuttgart Research Center Systems Biology, University of Stuttgart, Stuttgart, Germany

  2. Andrew G. Clark

    Present address: Center for Personalized Medicine, University of Tübingen, Tübingen, Germany

  3. These authors contributed equally: Raphaël Voituriez, Danijela Matic Vignjevic.

Authors and Affiliations

  1. Cell Biology and Cancer Unit, Institut Curie, PSL Research University, CNRS, Paris, France

    Andrew G. Clark, Cécile Jacques, Carlos Pérez-González, Anthony Simon, Luc Lederer & Danijela Matic Vignjevic

  2. Laboratoire Jean Perrin, Sorbonne Université and CNRS, Paris, France

    Ananyo Maitra

  3. Laboratoire de Physique Théorique et Modélisation, CNRS, CY Cergy Paris Université, Cergy-Pontoise Cedex, France

    Ananyo Maitra & Raphaël Voituriez

  4. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Martin Bergert & Alba Diz-Muñoz

  5. Institute for Bioengineering of Catalonia, The Barcelona Institute for Science and Technology (BIST), Barcelona, Spain

    Xavier Trepat

  6. Facultat de Medicina, University of Barcelona, Barcelona, Spain

    Xavier Trepat

  7. Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

    Xavier Trepat

  8. Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Barcelona, Spain

    Xavier Trepat

  9. Laboratoire de Physique Théorique de la Matière Condensée, Sorbonne Université and CNRS, Paris, France

    Raphaël Voituriez

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Contributions

A.G.C., A.M., R.V. and D.M.V. designed the research and wrote the manuscript; A.G.C. carried out most of the experiments and image analysis; A.M. and R.V. created the theoretical model; C.J. performed and analysed some migration and 2D traction force microscopy experiments and performed some staining experiments; A.S. performed preliminary experiments; L.L. prepared and performed some staining, migration and micropatterning experiments; M.B. and A.D.-M. performed and helped analyse the BM experiments; C.P.-G. and X.T. provided reagents, software and technical assistance for the traction force microscopy and 3D displacement experiments; C.P.-G. performed and analysed the results of the experiment measuring different monomeric collagen coatings; D.M.V. prepared and performed some staining, migration and micropatterning experiments; and all authors discussed the results and manuscript.

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Correspondence to Andrew G. Clark or Ananyo Maitra.

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Nature Materials thanks M. Lisa Manning, Giorgio Scita and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Analysis of cell rearrangement and MSD.

a. Montage from live imaging of stable A431 cells expressing GFP-CAAX (Plasma Membrane marker) and mCherry-H2B (DNA marker). Colored lines are trajectories of tracked nuclei over the course of 17 hours of imaging. Representative image from n=43 clusters imaged over N=2 independent experiments. Scale Bar: 100μm. b. Plot of the relative nuclear movements from a, registered to the migration of the whole cluster over time. c. Plot of the MSD curves from all trajectories of clusters migrating on PAA gels + collagen networks (red) or PAA gels + monomeric collagen (black). Individual trajectories are shown in translucent colors. Solid colored lines reflect mean MSD curves. Blue lines indicate slopes for power laws with exponents of α = 2 or α = 1. d. Crossover time from fits of individual MSD curves. For c,d, data from n=28,59 clusters, N=2,3 independent experiments. ***p < 0.001 for Tukey HSD test (p=0.0319).

Extended Data Fig. 2 Analysis of PAA gel stiffness and collagen concentration.

a. Representative micrographs from PAA gel coated with different concentrations of fluorescently-labeled monomeric collagen or 2mg/ml collagen networks. Scale bar: 30μm. b. Boxplot of mean collagen intensity for conditions shown in a. Each point represents one imaging field, relative to background fluorescence in areas with no coating. For a,b, images/data from n=5 positions per condition from one representative experiment, N=3 independent experiments. One-way ANOVA p=2.10e-11. *p < 0.05, **p < 0.01, ***p < 0.001 for Tukey HSD post-hoc test (p=0.00199, < 0.001, 0.0101, 0.00162, < 0.001, 0.0305, 0.679, 0.00717, < 0.001, 0.121, 0.00186, < 0.001, 0.00674, < 0.001, 0.248). c. Example micrographs of cell clusters plated on PAA gels with different concentrations of monomeric collagen. Scale bar: 50μm. d. Boxplots of mean instantaneous speed and coefficient of persistence for conditions in c. Data represents n=68, 72, 97, 56, 52, 46 clusters, N=3, 3, 3, 3, 2, 2 independent experiments. Data for collagen network and 100μg/ml monomeric collagen is the same data as presented in Fig. 1f. One-way ANOVA p=1.68e-20, 2.42e-25. *p < 0.05, **p < 0.01 for Tukey HSD post-hoc test (p=0.0304, 0.0368, 0.00153, < 0.001, 0.00795, 0.659, 0.0547, < 0.001, < 0.001, 0.0145, < 0.001, < 0.001, < 0.001, < 0.001, < 0.001; p=0.855, 0.773, 0.0490, < 0.001, < 0.001, < 0.900, 0.0518, < 0.001, < 0.001, 0.0383, < 0.001, < 0.001, 0.0253, < 0.001, < 0.001). e. Example micrographs of cell clusters plated on PAA gels of different stiffness (100μg/ml monomeric collagen). Scale bar: 50μm. f. Boxplots of mean instantaneous speed and coefficient of persistence for conditions in e. Data represents n=72, 62, 57, 69, 46, 46, 114 clusters, N=3, 3, 3, 3, 3, 2, 6 independent experiments. Data for collagen network and 100μg/ml monomeric collagen is the same data as presented in Fig. 1f. One-way ANOVA p=6.26e-13, 6.25e-30. *p < 0.05, **p < 0.01, ***p < 0.001 for Tukey HSD post-hoc test (p < 0.001, 0.00688, 0.238, 0.00704, < 0.001, < 0.001, < 0.001, < 0.001, 0.0122, 0.889, 0.564, 0.137, 0.504, < 0.001, < 0.001, 0.0758, < 0.001, < 0.001, 0.0143, 0.00996, 0.700; p= < 0.001, < 0.001, 0.728, 0.850, 0.233, < 0.001, < 0.001, 0.00300, < 0.001, 0.0327, < 0.001, < 0.001, 0.589,0.427, < 0.001, < 0.001, 0.158, < 0.001, < 0.001, < 0.001, < 0.001, 0.0763). g. Representative micrographs of fluorescently-labeled collagen networks with different collagen concentration or polymerization temperature. Scale bar: 100μm. h. Boxplot of mean collagen intensity for conditions shown in g. For g,h, representative images/data from n=4, 3, 3, 3, 3 positions, N=1 experiment. One-way ANOVA p=7.74e-6. i. Boxplots of mean instantaneous speed and coefficient of persistence for cluster migration on collagen network conditions in g. Data represents n=37, 114, 47, 85, 43 clusters, N=2, 3, 6, 3, 2 independent experiments. Data for 2mg/ml 37∘ C is the same data as presented in Fig. 1f. One-way ANOVA p=6.56e-12, 0.000609. *p < 0.05, **p < 0.01, ***p < 0.001 for Tukey HSD post-hoc test (p < 0.001, 0.00918, 0.175, 0.121, < 0.001, < 0.001, < 0.001,0.111,0.206,0.793; p=0.0134, 0.58, 0.812, 0.893, 0.0217, 0.00226, 0.00612, 0.775, 0.468, 0.705).

Extended Data Fig. 3 Clusters lack front-back polarity during migration.

a. Montage from time-lapse of a myosin light chain (MLC)-GFP expressing A431 cell cluster. MLC-GFP intensity was measured in the peripheral cortical region (segmentation in magenta) around the perimeter of the cluster. See also Supplementary Video 3. At each time point, the relative cortical intensity was measured for different angles around the perimeter (blue bars) as well as the cluster trajectory (red arrow). Lower panel: The relative cortical intensity of myosin (mean ± SD), averaged over all time points for n=39 clusters, N=3 independent experiments. Scale bar: 25μm. p-value reflects Rayleigh test of uniformity. b. Fixed cell clusters plated on a collagen network and immunostained for Rac1 GTPase with DAPI (DNA) and Phalloidin (F-Actin). Shown are two representative examples from n=21 clusters and N=3 independent experiments. Scale bar: 25μm. c.Upper panels: Single time point from a live imaging experiment using Raichu-Rac1 showing YFP:CFP ratio. Middle panels: Micrographs of YFP:CFP ratio for two successive time points with segmentation of the cortical region (magenta lines). Lower panel: The cortical YFP:CFP ratio for Raichu-Rac1 (mean ± SD) with respect to the migration direction (red arrow), averaged over all time points for n=8 clusters, N=3 independent experiments. Scale bar: 25μm. d. Boxplots of mean instantaneous speed and coefficient of persistence for cluster migration on collagen network conditions with the Rac1 inhibitor NSC23766. Data represents n=114, 57, 37, 26, 65, 23, 24 clusters, N=6, 3, 2, 1, 3, 1, 1 independent experiments. Control data is the same data as presented in Fig. 1f. For left panel, one-way ANOVA p=9.58e-9. *p < 0.05, ***p < 0.001 for Tukey HSD post-hoc test (p=0.243, < 0.001, 0.893, 0.0858, 0.0508, 0.1155, 0.00143, 0.430, 0.00304, 0.00205, 0.00844, < 0.001, < 0.001, < 0.001, < 0.001, 0.0949, 0.0103, 0.0497, 0.238, 0.552, 0.549).

Extended Data Fig. 4 Analysis of centrosome positioning and shape symmetry.

a. Single z-slice and 3D projection of a cell cluster on a collagen network with pericentrin staining. Scale bar: 20μm. Right: Rendering of the 3D segmented nuclei (multicolor), cluster (gray) and paired centrosomes (red). Black arrows: centrosome orientation with respect to paired nucleus. Representative example from n=7 clusters, N=1 independent experiment. b. Shape asymmetry quantification. A center line (dotted black line) is drawn along the cluster length passing through the center of mass and parallel with the migration direction. At every pixel along the contour (red dot, for example), the distance x from the center of mass projected onto the center line is determined. The shape asymmetry index is calculated for each time frame as the sum of x3 over all contour points. Lower panel: histogram of the shape asymmetry from n=34 cells, N=5 independent experiments. c. Aspect ratio quantification. Using the Hull convex of the contour (blue dotted line) to minimize shape irregularities, the major axis x1 (dotted black line) is taken as the cluster diameter along the migration direction and passing through the center of mass (black dot). The minor axis x2 (dotted red line) is taken as the cluster diameter perpendicular to the migration direction and passing through the contour center of mass. The aspect ratio is x1/x2. Lower panel: histogram of the aspect ratio from n=34 clusters, N=5 independent experiments.

Extended Data Fig. 5 Analysis of focal adhesions and integrins during migration.

a. Fixed cell clusters plated on glass or on a collagen network and immunostained for Paxillin with DAPI (DNA) and Phalloidin (F-Actin) counterstaining. Representative examples, N=2 independent experiments. Scale bars: 50μm. b. Single time points from live imaging of A431 cells stably expressing Paxillin-GFP or Zyxin-mCherry, plated on glass or collagen networks. Representative examples, N=3 independent experiments. Scale bar: 50μm. c. Boxplots of mean instantaneous speed and coefficient of persistence for cluster migration on collagen networks with the integrin binding inhibitors Cilengitide and AIIB2 and MMP inhibitors GM6001 and BB94. Data represents n=146, 57, 72, 31, 26, 17, 14 clusters, N=8, 3, 4, 2, 2, 1, 1 independent experiments. Control data is pooled from data presented in Fig. 1f. One-way ANOVAs: p=4.36e-6, 1.76e-18. **p < 0.01, ***p < 0.001 for Tukey HSD post-hoc test (p=0.268, 0.126, < 0.001, 0.00492, 0.390, > 0.9, 0.832, < 0.001, < 0.001, 0.865, 0.447, < 0.001, < 0.001, > 0.9, 0.256, 0.537, < 0.001, < 0.001, < 0.001, < 0.001, 0.243; p=0.0788, 0.0195, < 0.001, < 0.001, 0.88, 0.25, 0.689, < 0.001, < 0.001, 0.130, 0.016, < 0.001, < 0.001, 0.061, 0.00522, 0.268, < 0.001, 0.0148, < 0.001, 0.00246, 0.354). d. Example micrograph from a cell cluster on a collagen network following treatment with the Integrin-β1 functional antibody AIIB2 (10μg/ml). Scale bar: 100μm. HH:MM. See also Supplementary Video 4. Representative image series from n=26 clusters, N=2 independent experiments.

Extended Data Fig. 6 Cell clusters do not deposit additional ECM during migration.

a. Collagen networks containing no cells or A431 clusters fixed and immunostained for Fibronectin, with additional DAPI (DNA) and Phalloidin (F-Actin) staining. The Fibronectin panel represents a brightest point projection over 25μm in the z-axis to capture the network in the region directly under the cluster. Representative image from n=23 clusters, N=3 independent experiment. Scale bar: 50μm. b. Average radial linescan (median ± SD) of fibronectin intensity from the cluster center to the periphery from data represented in e. At all distances, p > 0.05 for a two-tailed t-test with μ0 = 1. c. Collagen networks with A431 clusters seeded on top were fixed and immunostained for Collagen-IV and Laminin, with additional DAPI (DNA) and Phalloidin (F-Actin) staining. Representative image from n=5 clusters, N=1 independent experiments. Scale bar: 50μm. d. Fluorescent TAMRA-collagen networks with A431 clusters plated on top were fixed and immunostained for Collagen-I. Panels reflect single confocal slices at different z-steps. Colocalization between the TAMRA-Collagen and Collagen-I Antibody signal was R = 0.895 ± 0.040 (mean ± SD; p < 0.001) from n=15 clusters, N=2 independent experiments. Scale bar: 50μm. e. A431 cluster plated on a collagen network, fixed and immunostained with a Collagen-I antibody plus Phalloidin and imaged by two-photon microscopy. Representative image from n=9 clusters, N=2 experiments. Horizontal scale bars: 50μm, 25μm. Vertical scale bar: 10μm.

Extended Data Fig. 7 Analysis of collagen network alignment during collective migration.

a. Montage of a cluster on a fluorescent collagen network (single z-slice on network surface). Scale Bar: 50μm. HH:MM. b. Montage of cluster in a overlaid with the local collagen alignment. Rods: local nematic order (orientation indicates mean collagen fiber orientation, length indicates order parameter magnitude S, color indicates orientation with respect to cluster center of mass (90∘ is oriented toward the cluster, 0∘ is oriented perpendicular to the cluster). Scale rod: S = 1. Image length scale as for a. c. Polar plot of nematic order within 50μm of the cluster boundary (mean ± SD over all time points). P-value: Rayleigh test of uniformity. For a-c, representative images/data from n=28 cells, N=3 independent experiments. d. Live imaging of clusters using AiryScan super-resolution microscopy. Upper Panels: Brightfield images with segmentation and front-to-back regions in the direction of migration. Lower Panels: AiryScan images of TAMRA-labeled collagen at different z-positions. e. Box plot of the collagen fiber nematic order from AiryScan images. Dots represent individual clusters. For d,e, representative images/data from n=11 clusters, N=3 independent experiments. f,g. Montages of clusters migrating on thick deformable (f) or thin non-deformable (g) aligned collagen networks. Lower Panels: Overlaid cluster migration trajectories, adjusted to start at the origin (0,0) and rotated with respect to collagen alignment direction. Scale Bars: 100μm. HH:MM. Representative images from n=66, 86 clusters, N=4, 4 independent experiments h. Boxplot of orientational index along the collagen alignment direction for conditions in f,g. One-way ANOVA: p=0.00113. *p < 0.05, ***p < 0.001 for Tukey HSD post-hoc test (p=0.340, < 0.001, 0.0184). i. Boxplots of mean instantaneous speed and coefficient of persistence for conditions in f,g. For h,i, data from n=114, 66, 86 clusters, N=6, 4, 4 independent experiments. Thick isotropic data is the same as Fig. 1f. One-way ANOVAs: p=0.0180, 7.83e-6. *p < 0.05, **p < 0.01, ***p < 0.001 for Tukey HSD post-hoc test (p=0.567, 0.00919, 0.0393; p=0.0413, < 0.001, 0.0190).

Extended Data Fig. 8 Role of crosslinking on different migration modes.

a. Montages from wound healing assays using A431 cells plated using stencils on collagen networks pre-treated with PBS or the crosslinker glutaraldehyde. Red lines indicate the leading edge. Horizontal Scale Bar: 500μm. HH:MM. Lower Panels: Kymograph showing progression of the leading edge over time. Red lines indicate the velocity of wound closure on each side. Vertical Scale Bar: 20 hours. b. Boxplot of wound closure speed on control and crosslinked collagen gels. For a,b, representative images/data from n=7, 7 collagen gels, N=3,3 independent experiments. **p < 0.01 for Welch’s t-test (p=0.00258). c. Boxplots of relative collagen density and relaxation time (τr) following cluster removal using Trypsin/NH4OH for collagen networks treated with Lysyl Oxidase (LOX). Each dot represents one cluster that was rapidly removed. Data represents n=40, 25 clusters, N=5, 3 independent experiments. ns:p > 0.05, ***p < 0.001 for Welch’s t-test (p=0.717, 0.000550). d. Boxplots of mean instantaneous speed and coefficient of persistence for cluster migration on LOX-treated collagen networks. Data represents n=114, 42 clusters, N=6, 2 independent experiments. Control data is the same data as presented in Fig. 1f. *p < 0.05, ***p < 0.001 for Welch’s t-test (p=5.707e-6, 0.0472). e. Boxplots of relative collagen density (left) and relaxation time (τr, right) following cluster removal using Trypsin/NH4OH for collagen networks using different collagen concentration or polymerization temperature. Each dot represents one cluster that was rapidly removed. Data represents n=30, 40, 23, 18, 30 clusters, N=3, 5, 3, 2, 3 independent experiments. One-way ANOVA p=8.30e-5, 5.22e-5. **p < 0.01 for Tukey HSD post-hoc test (p=0.375, < 0.001,0.001665, < 0.001, 0.0332, 0.176,0.01, 0.539, 0.204, 0.195; p=0.27, 0.506, 0.159, < 0.001, 0.0579, 0.373, < 0.001, 0.0497, < 0.001, 0.00683).

Extended Data Fig. 9 Single cell collagen deformation and migration.

a. Micrographs of a primary human cancer associated fibroblast (CAF) on a fluorescent collagen network. Scale bar: 50μm. Scale vector: 0.5μm/min. HH:MM after addition of Trypsin/NH4OH. Representative image from n=18 CAFs, N=2 independent experiments. b. Boxplots comparing relative collagen density before and after CAF removal. c. Boxplot of viscoelastic relaxation time (τr) following CAF removal. For b,c: each dot represents one cluster. Data from n=39, 18 clusters/CAFs, N=5 2 independent experiments. For b,c, *p < 0.01, ***p < 0.001 for Welch’s t-test (p=6.65e-6, 1.19e-6, 0.0260). d. Micrographs from CaCo2 single cell or cluster on collagen networks. Red: migration trajectories. Scale Bar: 50μm. HH:MM. e. Overlaid migration trajectories for CaCo2 single cells and clusters, adjusted to start at the origin (0,0). f. Boxplots of mean instantaneous speed and coefficient of persistence for A431/CaCo2 clusters/single cells. For c-e, representative examples/data from n=114, 89, 69, 27 clusters/single cells, N=6, 3, 2, 2 independent experiments. A431 cluster data is same as in Fig. 1f. *p < 0.05, ***p < 0.001 for Welch’s t-test (p=2.57e-9, 3.44e-15, 5.25e-13, 0.0185). g. Overlaid migration trajectories for clusters and single cells on thin isotropic or aligned collagen networks, adjusted to start at the origin (0, 0) and rotated with respect to the collagen alignment direction. h. Boxplot of orientational index along collagen alignment direction. i. Boxplots of mean instantaneous speed and coefficient of persistence. For g-i, Data represents n=56, 86, 103, 45 clusters, N=2, 4, 2, 2 independent experiments. Data for cluster migration on thin aligned networks is same as Extended Data Fig. 5g-i. For h,i, ns=p > 0.05, ***p < 0.001 for Welch’s t-test (p=3.89e-9, 4.28e-9, 0.209, 2.30e-5, 8.74e-6, 0.000379).

Extended Data Fig. 10 Traction force distributions are asymmetric and require myosin-2.

a. Integrated 2D tractions from clusters on PAA + thin collagen network or monomeric collagen, n=54, 69 clusters, N=3, 3 independent experiments. ***p < 0.001 for Welch’s t-test. b. Integrated traction force vs. cluster area on PAA + collagen network or monomeric collagen. Solid lines: power-law fits. c. 3D displacements of a cluster on PAA gel + thin collagen network with blebbistatin. Black arrows: xy displacements. Color scale: z displacements. d. Mean radial traction linescans from c. e. Summed displacements before/after blebbistatin treatment, n=11 clusters, N=1 independent experiment. ***p < 0.001 for Welch’s t-test. f. Micrographs of clusters following blebbistatin treatment. Scale Bar: 100μm. HH:MM. g. Micrographs of mCherry-LifeAct A431 cells. Red arrow: migration direction. Lower Panel: PIV vectors of actin flows in the cortical region. Scale bar: 20μm. Scale vector: 0.05μm/min. MM:SS. See also Supplementary Video 22. h. Mean actin flow speeds with respect to migration direction for n=10 clusters, N=3 independent experiments. ***p < 0.001 for Welch’s t-test. i. Radial histogram of summed PIV vector angle with respect to the migration direction (red arrow). P-value: Rayleigh test of uniformity. j. Plot of traction peak asymmetry (Front/Back). Solid circles: all binned data (mean ± SEM). Line: linear fit (raw data). Data represents n=54, 104 clusters/single cells, N=3, 3 independent experiments. k,l. Traction force magnitudes (total (k) or projected along migration axis (l)) for the cluster in Fig. 6a. Lower panels: Unfolded radial linescans with respect to the cluster front. Scale bars: 20μm. m,n. Linescans of tractions along migration axis (absolute values in n). Dots: raw values. Lines: smoothed data.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Note 1, Discussion, video captions and references.

Reporting Summary

Supplementary Video 1

A431 cluster migrating on a 0.5 kPa PAA gel coated with a thin collagen-I network. Scale bar, 100 μm. Time in hours:minutes.

Supplementary Video 2

A431 cluster migrating on a 0.5 kPa PAA gel coated with 100 μg ml–1 monomeric collagen-I. Scale bar, 100 μm. Time in hours:minutes.

Supplementary Video 3

A431 MLC-GFP clusters migrating on collagen-I networks. Magenta lines indicate region used for segmentation of the cluster cortex. Scale bar, 50 μm. Time in hours:minutes.

Supplementary Video 4

A431 cluster detaching from a collagen-I network following treatment with 10 μg ml–1 AIIB2. Scale bar, 100 μm. Time in hours:minutes.

Supplementary Video 5

A431 cluster migrating on a fluorescently labelled collagen network. Left: the fluorescent collagen is shown in pseudocolour, and the cell outline is shown in white. The white dot represents the cluster centre of mass. Scale bar, 50 μm. Time in hours:minutes. Right: separation of the segmentation into equal-length segments from front (yellow) to rear (purple).

Supplementary Video 6

Rapid removal of an A431 cell cluster on a fluorescent collagen network. Left: bright field. Middle: collagen in greyscale with cluster segmentation in magenta. Right: collagen in greyscale with vectors from PIV shown in colours according to angle. Time 0:00 refers to the frame prior to the addition of trypsin/NH4OH. Subsequent timestamps refer to the time after trypsin/NH4OH treatment. Scale bar, 50 μm. Scale vector, 0.5 μm min–1. Time in hours:minutes.

Supplementary Video 7

The 3D displacement microscopy experiment for an A431 cell cluster on a collagen network and rapidly removed using trypsin/NH4OH. Black arrows are xy displacement vectors. Colour scale shows z displacement vectors (negative values indicate displacement down towards the substrate). Time 0:00 refers to the frame prior to the addition of trypsin/NH4OH. Subsequent timestamps refer to the time after trypsin/NH4OH treatment. Scale bar, 50 μm. Scale vector, 50 μm. Time in hours:minutes.

Supplementary Video 8

Rapid removal of an A431 cell cluster on a fluorescent collagen network crosslinked with 1 mM threose. Left: bright field. Middle: collagen in greyscale with cluster segmentation in magenta. Right: collagen in greyscale with vectors from PIV shown in colours according to angle. Time 0:00 refers to the frame prior to the addition of trypsin/NH4OH. Subsequent timestamps refer to the time after trypsin/NH4OH treatment. Scale bar, 50 μm. Scale vector, 0.5 μm min–1. Time in hours:minutes.

Supplementary Video 9

Rapid removal of an A431 cell cluster on a fluorescent collagen network crosslinked with 10 mM threose. Left: bright field. Middle: collagen in greyscale with cluster segmentation in magenta. Right: collagen in greyscale with vectors from PIV shown in colours according to angle. Time 0:00 refers to the frame prior to the addition of trypsin/NH4OH. Subsequent timestamps refer to the time after trypsin/NH4OH treatment. Scale bar, 50 μm. Scale vector, 0.5 μm min–1. Time in hours:minutes.

Supplementary Video 10

Rapid removal of an A431 cell cluster on a fluorescent collagen network crosslinked with 0.05% glutaraldehyde. Left: bright field. Middle: collagen in greyscale with cluster segmentation in magenta. Right: collagen in greyscale with vectors from PIV shown in colours according to angle. Time 0:00 refers to the frame prior to the addition of trypsin/NH4OH. Subsequent timestamps refer to the time after trypsin/NH4OH treatment. Scale bar, 50 μm. Scale vector, 0.5 μm min–1. Time in hours:minutes.

Supplementary Video 11

A431 cluster migrating on a fluorescently labelled collagen network crosslinked with 1 mM threose. Left: the fluorescent collagen is shown in pseudocolour, and the cell outline is shown in white. The white dot represents the cluster centre of mass. Scale bar, 50 μm. Time in hours:minutes. Right: separation of the segmentation into equal-length segments from front (yellow) to rear (purple).

Supplementary Video 12

A431 cluster migrating on a fluorescently labelled collagen network crosslinked with 10 mM threose. Left: the fluorescent collagen is shown in pseudocolour, and the cell outline is shown in white. The white dot represents the cluster centre of mass. Scale bar, 50 μm. Time in hours:minutes. Right: Separation of the segmentation into equal-length segments from front (yellow) to rear (purple).

Supplementary Video 13

A431 cluster migrating on a fluorescently labelled collagen network crosslinked with 0.05% glutaraldehyde. Left: the fluorescent collagen is shown in pseudocolour, and the cell outline is shown in white. The white dot represents the cluster centre of mass. Scale bar, 50 μm. Time in hours:minutes. Right: separation of the segmentation into equal-length segments from front (yellow) to rear (purple).

Supplementary Video 14

A431 cluster migrating on a collagen-I network. Scale bar, 100 μm. Time in hours:minutes.

Supplementary Video 15

A431 cluster migrating on a collagen-I network crosslinked with 1 mM threose. Scale bar, 100 μm. Time in hours:minutes.

Supplementary Video 16

A431 cluster migrating on a collagen-I network crosslinked with 10 mM threose. Scale bar, 100 μm. Time in hours:minutes.

Supplementary Video 17

A431 cluster migrating on a collagen-I network crosslinked with 0.05% glutaraldehyde. Scale bar, 100 μm. Time in hours:minutes.

Supplementary Video 18

Rapid removal of an individual A431 cell on a fluorescent collagen network. Left: bright field. Middle: collagen in greyscale with cluster segmentation in magenta. Right: collagen in greyscale with vectors from PIV shown in colours according to angle. Time 0:00 refers to the frame prior to the addition of trypsin/NH4OH. Subsequent timestamps refer to the time after trypsin/NH4OH treatment. Scale bar, 50 μm. Scale vector, 0.5 μm min–1. Time in hours:minutes.

Supplementary Video 19

A431 single cell migrating on a fluorescently labelled collagen network. Left: the fluorescent collagen is shown in pseudocolour, and the cell outline is shown in white. The white dot represents the cluster centre of mass. Scale bar, 50 μm. Time in hours:minutes. Right: separation of the segmentation into equal-length segments from front (yellow) to rear (purple).

Supplementary Video 20

A431 single cell migrating on a collagen-I network. Scale bar, 50 μm. Time in hours:minutes.

Supplementary Video 21

A431 clusters migrating on a 0.5 kPa PAA gel coated with a thin, collagen-I network analysed by traction force microscopy. Arrows indicate xy traction stresses on the substrate. Scale bar, 100 μm. Scale vector, 50 Pa. Time in hours:minutes.

Supplementary Video 22

A431 cells stably expressing mCherry-LifeAct overlaid with vectors from PIV analysis of actin flows in the peripheral cortical region. Scale bar, 20 μm. Scale vector, 0.05 μm min–1. Time in minutes:seconds.

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Clark, A.G., Maitra, A., Jacques, C. et al. Self-generated gradients steer collective migration on viscoelastic collagen networks. Nat. Mater. 21, 1200–1210 (2022). https://doi.org/10.1038/s41563-022-01259-5

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