Cell detachment and migration from the endothelium occurs during vasculogenesis and also in pathological states. Here, we use a novel approach to trigger single cell release from an endothelial monolayer by in-situ opening of adhesive, fibril-like environment using light-responsive ligands and scanning lasers. Cell escapes from the monolayer were observed on the fibril-like adhesive tracks with 3-15 μm width. The frequency of endothelial cell escapes increased monotonically with the fibril width and with the density of the light-activated adhesive ligand. Interestingly, treatment with VEGF induced cohesiveness within the cell layer, preventing cell leaks. When migrating through the tracks, cells presented body lateral reduction and nuclear deformation imposed by the line width and dependent on myosin contractility. Cell migration mode changed from mesenchymal to amoeboid-like when the adhesive tracks narrowed (≤5 μm). Moreover, cell nucleus was shrunk showing packed DNA on lines narrower than the nuclear dimensions in a mechanisms intimately associated with the stress fibers. This platform allows the detailed study of escapes and migratory transitions of cohesive cells, which are relevant processes in development and during diseases such as organ fibrosis and carcinomas.