Collective and individual migration following the epithelial-mesenchymal transition

Abstract

During cancer progression, malignant cells in the tumour invade surrounding tissues. This transformation of adherent cells to a motile phenotype has been associated with the epithelial-mesenchymal transition (EMT). Here, we show that EMT-activated cells migrate through micropillar arrays as a collectively advancing front that scatters individual cells. Individual cells with few neighbours dispersed with fast, straight trajectories, whereas cells that encountered many neighbours migrated collectively with epithelial biomarkers. We modelled these emergent dynamics using a physical analogy to phase transitions during binary-mixture solidification, and validated it using drug perturbations, which revealed that individually migrating cells exhibit diminished chemosensitivity. Our measurements also indicate a degree of phenotypic plasticity as cells interconvert between individual and collective migration. The study of multicellular behaviours with single-cell resolution should enable further quantitative insights into heterogeneous tumour invasion.

Fig. 1. Epithelial and mesenchymal cells migrate collectively and individually within enclosed micropillar arrays

(A) Cells invaded an enclosed array of fibronectin-coated PDMS micropillars with height, diameter and spacing of 10 μm. (B) Cell migration was automatically tracked from time-lapse microscopy by segmenting fluorescently labelled nuclei. (C) A representative metric for collective or individual migration is based on the lifetime-average number of nearest neighbours within one pillar spacing. Immunofluorescent staining reveals biomarker expression associated with (D) epithelial phenotype (E-cadherin, green) in MCF-10A, (E) mesenchymal phenotype (vimentin, red) in MDA-MB-231, (F) both epithelial (E-cadherin, green) and mesenchymal phenotypes (vimentin, red) in MCF-10A Snail. Histograms of the number of lifetime averaged nearest neighbours per cell indicate (G) collective migration in MCF-10A, (H) individual migration in MDA-MB-231, and (I) collective and individual migration in MCF-10A Snail.

Fig. 2. Differences in migratory behaviour associated with collective or individual migration phenotypes were classified using a Gaussian mixture model

The lifetime-averaged nearest neighbours of the migrating cells are compared to (A) the final Y position in the device, (B) averaged velocity, and (C) path straightness. Overall, at the completion of the experiment, individually migrating cells are observed to the front with collectively migrating cells at the rear. (D) Single cell tracking reveals that individually migrating cells scatter effectively due to increases in speed and straighter trajectories, relative to collectively migrating cells.

Fig. 3. The dynamics of individual scattering from a collectively migrating front can be understood as a dispersion phenomenon from a moving interface

(A) Immunofluorescent staining of the pillar region (0 < y) reveals individually migrating mesenchymal cells detaching from a collectively migrating epithelial front. Cells in the rear loading region (y < 0) exhibit phenotypic plasticity and undergo a mesenchymal to epithelial transition (MET) over 24 h. (B) The measured spatial distributions of individual and collectively migrating cells are plotted as a function of time, showing an interface that propagates outward as the square root of time. (C) A solidification model for binary mixtures shows quantitative agreement with experimental data.

Fig. 4. Perturbation of invasion behaviours in different cell lines with different Rsk pathway inhibitors

The individually migrating subpopulation of MCF-10A Snail displays low sensitivity to generic Rsk inhibitors, whereas the collectively migrating subpopulation of MCF-10A Snail shows higher sensitivity. Both subpopulations display sensitivity to FMKMEA. Unlike MCF-10A Snail, MCF-10A and MDA-MB-231 cell lines display sensitivity to all Rsk inhibitors tested. (A) Relative invasion based on based on best fits to the solidification model (dispersion coefficients). (B) Relative proliferation based on differences in subpopulation size. Error bars are standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 10^-3. (C) Combined scores incorporating invasion and proliferation. (D) Immunostaining of induced EMT population (MCF-10A Snail) indicates heterogeneous sensitivity of collective and individually invading subpopulations.