Cell clusters adopt a collective amoeboid mode of migration in confined nonadhesive environments

Abstract

Cell migration is essential to living organisms and deregulated in cancer. Single cell's migration ranges from traction-dependent mesenchymal motility to contractility-driven propulsive amoeboid locomotion, but collective cell migration has only been described as a focal adhesion-dependent and traction-dependent process. Here, we show that cancer cell clusters, from patients and cell lines, migrate without focal adhesions when confined into nonadhesive microfabricated channels. Clusters coordinate and behave like giant super cells, mobilizing their actomyosin contractility at the rear to power their migration. This polarized cortex does not sustain persistent retrograde flows, of cells or actin, like in the other modes of migration but rather harnesses fluctuating cell deformations, or jiggling. Theoretical physical modeling shows this is sufficient to create a gradient of friction forces and trigger directed cluster motion. This collective amoeboid mode of migration could foster metastatic spread by enabling cells to cross a wide spectrum of environments.

Fig. 1.. Cell clusters from patient explants migrate in confined nonadhesive environments.

(A) TSIPs (tumor spheres with inverted polarity) from a representative CRC tumor specimen (micropapillary). Low and high magnification. HES, hematoxylin/eosin/saffron; CK20, anti-cytokeratin 20. (B) Immunofluorescence of TSIP#1 after 24 hours in collagen I. The boxed region is shown at high magnification. (C) Tumor microemboli from CRC (mucinous). H&E, hematoxylin/eosin; ITGB1, anti-integrin β1. The boxed region is shown at high magnification. (D) Scheme, not to scale, of microchannels (width, w = 60 μm; height, h = 30 μm). (E) Time-lapse sequences of clusters migration from pancreatic cancer, TSIP#1, and HT29-MTX. (F) Mean migration speed of tumor clusters from patients and patient-derived xenografts (PDXs). CRC, colorectal cancer; NSGCT, nonseminomatous germ cell tumor. Log₁₀ scale. Dots, mean speed over clusters for each independent experiment; lines, means over experiments. (G) Proportion of migrating (>25 μm/day) clusters. Error bars: SEM. For (F) and (G), n = 14 to 153 clusters per cell type. (H) Maximum instantaneous (Max. instant.) speed of clusters, log₁₀ scale. (I) Representative tracks of clusters migrating in one direction (>0) or the other (<0). n = 9 to 10 clusters per cell type. (J and K) For clusters migrating substantially (>25 μm/day), duration of the longest period of consecutive migration (J) and persistence (K). n = 38 to 107 clusters. For (E) to (K), clusters migrate 1 day in PEG-coated microchannels. (F, H, J, and K) Dots, mean speed over clusters for each independent experiment; lines, means over experiments. Scale bars, 200 μm [(A), low magnification], 100 μm [(C), low magnification], 50 μm [(A) and (C), high magnification; (B), low magnification; (E)], and 10 μm [(B), high magnification].

Fig. 2.. Cluster migration in nonadhesive environments is traction independent but friction dependent.

For (A) to (G), microchannels coating; PEG, pLL-PEG; Col-I, Cy5–collagen I. (A and B) Representative images (A; scale bar, 50 μm) and maximum (Max.) contact angle of clusters (B; n = 39 to 59, Mann-Whitney test). (C to E) Representative images of Paxillin at bottom planes (C; scale bar, 10 μm), area covered by Paxillin foci (D; n = 19 to 28), and mean speed of HT29-MTX clusters (E; n = 90 to 106) (Mann-Whitney tests). (F and G) Mean speed of HT29-MTX clusters treated in PEG-coated (F) or Col-I–coated (G) microchannels. PF271, PF562271. n = 70 to 182 (one-way ANOVA). (H and I) PIV maps of TFM measurements (bead displacements following SDS-mediated relaxation) (H; scale bars, 20 μm) and orientation of forces exerted by HT29-MTX clusters (I; n = 8 to 14, Welch’s t test) on Col-I– or PEG-coated substrates. θ, angle between cluster radius and bead displacement vector (fig. S3B). On PEG, small pushing forces are probably due to agarose pad confinement. (J) Mean speed of HT29-MTX clusters. cRGD, cyclic RGD; SB, SB273005. n = 74 to 149 (Student’s t tests). (K) Mean speed of HT29-MTX clusters. Microchannels coating: PEG ± F127 and BSA at indicated concentrations. n = 78 to 151 (one-way ANOVA). (L) Mean speed of HT29-MTX clusters treated with cRGD. Microchannels coating: PEG + F127 ± BSA (300 μg/ml; cRGD + BSA). n = 67 to 82 (one-way ANOVA). n, number of clusters; ns, not significant; *P < 0.05, ***P < 0.001, and ****P < 0.0001. For each panel, SuperPlots, violin plot (all clusters); dots, mean of each experiment; same color, same independent experiment; black line, mean of individual means (see the “Data presentation, statistics, and reproducibility” section).

Fig. 3.. Focal adhesion–independent collective migration is driven by the contractility of the polarized actomyosin cortex.

(A) Median section of HT29-MTX stably expressing F-tractin–mRuby3 (top) and mTurquoise-MLC (bottom), in PEG-coated channels. Red dashed lines visually materialize the front and back of clusters for quantification of ratio in (B). Scale bars, 10 μm. (B) Polarization of clusters expressed as MLC expression ratio between rear and front of clusters migrating in PEG-coated or PEG + F127–coated microchannels, as indicated in Materials and Methods. n = 12 to 13 clusters. (C) Correlation between cluster speed and polarization of clusters; statistics for linear regression are shown on the graph. n = 23 clusters from three independent experiments. (D) Time-lapse sequences of HT29-MTX clusters migrating in PEG-coated microchannels in control condition and under Y27632 (25 μM) or blebbistatin (Bleb; 50 μM) treatments. Red dots, starting position of the clusters. Scale bars, 50 μm. (E) Representative tracks of clusters treated with Bleb or with DMSO (Ctrl). n = 10 clusters for each cell type. (F) Mean speed of clusters treated with Y27632, Bleb, or DMSO (Ctrl); log₁₀ scale. n = 106 to 205 clusters (one-way ANOVA). **P < 0.01 and ****P < 0.0001.

Fig. 4.. Polarized RhoA activation dictates the direction of migration.

(A) Schematic of the molecular effect of light activation in optoRhoA cells. (B and C) Optogenetic manipulations: experimental setup (B) and representative time-lapse sequence (C). Scale bar, 20 μm. (D) Displacement of clusters over time before (−1 hour 30 min < t < 0 hours) and after (0 hours < t < 10 hours) optogenetic activation of control and optoRhoA–stably expressing HT29-MTX cells. Bold lines are the mean displacement of clusters changing direction (mean displacement after activation, >0). Dotted lines are the mean of clusters not changing direction (mean displacement after activation, <0). Purple zone, optogenetic activation. n = 23 for control and n = 24 for optoRhoA from at least three independent experiments. P = 0.0003 (Fisher’s exact test on the proportion of turning clusters). (E) Left: Mean velocity before (average over 1 hour 30 min, x axis) and after (average over 10 hours, y axis) activation (act.) of control and optoRhoA clusters. n = 119 (control) and 62 (optoRhoA) clusters. Right: Schematic of activation protocol, showing corresponding colors on the graph shown on the left panel.

Fig. 5.. Focal adhesion–independent collective migration occurs without persistent retrograde flows.

(A) Schematic representation of cell treadmilling (left) and translation of the whole cluster (right). (B) Representative tracks of nuclei in the cluster reference frame. Median section of a histone 2B (H2B)-RFP–expressing HT29-MTX cluster, migrating to the right in a PEG-coated microchannel over 11 hours. The image is the first time point. (C) Representative example of nuclei tracks in the lab reference frame. Median section of an H2B-RFP–expressing HT29-MTX cluster, migrating in a PEG-coated microchannel over 10 hours. Scale bar, 50 μm. (D) Superimposition of the maps of individual nuclei displacements for n = 22 clusters (from seven independent experiments). Nuclei are tracked in median sections of H2B-RFP–expressing HT29-MTX clusters migrating in PEG-coated microchannels (25 min to 11 hours). Blue boxes, lateral nuclei defined for further nuclei speeds analysis (15 μm thickness at the contact with the channel walls). (E) Frequency distribution of the x component of the mean velocity of every lateral nucleus. Same clusters as in (D). (F) Median section showing instantaneous myosin flow velocity vectors superimposed on the raw image and detected by particle image velocimetry (PIV) of a representative HT29-MTX cluster–expressing mTurquoise-MLC and migrating in a PEG-coated channel. Time point, T = 58 min. Blue boxes, contact zone defined for further myosin flows analysis (2 μm thickness at the contact with the channel walls). (G) PIV map of myosin flow velocity vectors of median sections of clusters, averaged over time (25 min to 7.4 hours) and clusters (n = 10 migrating clusters from four independent experiments). (H) Frequency of velocities (x component) of myosin flow velocity vectors at the contact obtained from PIV maps of median sections. Same clusters as in (G).

Fig. 6.. Cell deformations and actomyosin polarity are the minimal components for collective amoeboid migration.

For (A), (C), and (D), myosin and nuclei are analyzed at the contact with channels walls (see Fig. 5, D and G, and Materials and Methods) in median sections of HT29-MTX clusters migrating in PEG-coated microchannels. (A) Representative kinetics of myosin flow velocity (x component, spatial average). Lab ref. frame. (B) Nuclei tracks in representative clusters, every 2 to 10 min over 8 to 11 hours. Cluster ref. frame. (C) Amplitudes of myosin flows and nuclei speeds fluctuations. (D) Amplitudes of nuclei speeds fluctuations and cluster speeds (i.e., centroid direct displacement between first and last time point over total time, as migrating clusters do not change direction). Log₂ scales. (E) Cluster migration persistence correlation with myosin (MLC) polarization. For (C) to (E), lines, linear regressions; threshold between migrating/nonmigrating clusters, 2 μm/hour. n = 27 to 28 clusters (six independent experiments). (F) Discrete, five-beads one-dimensional model of the cell cluster, averaged along the z direction and discretized along the x direction. R₀, average separation; gray springs, elastic elements; color gradient represents the gradient of active fluctuations of contractile stress (ζ_i and ∂_xζ); friction forces (Γ_i, green) depend on the local strain u = R_{i + 1} − R_i − R₀; v, cluster velocity. (G) Representative trajectory obtained from a stochastic simulation of (F). Mobility M_i = Γ_i⁻¹ = M₀ + M₁u_i; elastic potential ∑_i ku_i²/2; distance, units of R₀; time, units of 1/M₀k ≡ Γ₀/k. (H) Numerical evaluation of mean cluster velocity and ∂_xζ (see Supplementary Text and fig. S10). (I) Schematic representation of the four main different modes of cell migration.