Substrate area confinement is a key determinant of cell velocity in collective migration

Collective cell migration is fundamental throughout development, during wound healing and in many diseases. Although much effort has focused on cell-cell junctions, a role for physical confinement in collective cell migration remains unclear. Here, we used adhesive microstripes of varying widths to mimic the spatial confinement experienced by follower cells within epithelial tissues. Our results reveal that the substrate area confinement is sufficient to modulate the three-dimensional cellular morphology without the need for intercellular adhesive cues. Our findings show a direct correlation between the migration velocity of confined cells and their cell-substrate adhesive area. Closer examination revealed that adhesive area confinement reduces lamellipodial protrusive forces, decreases the number of focal complexes at the leading edge and prevents the maturation of focal adhesions at the trailing edge, together leading to less effective forward propelling forces. The release of follower confinement required for the emergence of leader cells is associated with a threefold increase in contractile stress and a tenfold increase in protrusive forces, together providing a sufficient stress to generate highly motile mesenchymal cells. These findings demonstrate that epithelial confinement alone can induce follower-like behaviours and identify substrate adhesive area confinement as a key determinant of cell velocity in collective migration.