A photoactivatable nanopatterned substrate for analyzing collective cell migration with precisely tuned cell-extracellular matrix ligand interactions

Collective cell migration is involved in many biological and pathological processes. Various factors have been shown to regulate the decision to migrate collectively or individually, but the impact of cell-extracellular matrix (ECM) interactions is still debated. Here, we developed a method for analyzing collective cell migration by precisely tuning the interactions between cells and ECM ligands. Gold nanoparticles are arrayed on a glass substrate with a defined nanometer spacing by block copolymer micellar nanolithography (BCML), and photocleavable poly(ethylene glycol) (Mw = 12 kDa, PEG12K) and a cyclic RGD peptide, as an ECM ligand, are immobilized on this substrate. The remaining glass regions are passivated with PEG2K-silane to make cells interact with the surface via the nanoperiodically presented cyclic RGD ligands upon the photocleavage of PEG12K. On this nanostructured substrate, HeLa cells are first patterned in photo-illuminated regions, and cell migration is induced by a second photocleavage of the surrounding PEG12K. The HeLa cells gradually lose their cell-cell contacts and become disconnected on the nanopatterned substrate with 10-nm particles and 57-nm spacing, in contrast to their behavior on the homogenous substrate. Interestingly, the relationship between the observed migration collectivity and the cell-ECM ligand interactions is the opposite of that expected based on conventional soft matter models. It is likely that the reduced phosphorylation at tyrosine-861 of focal adhesion kinase (FAK) on the nanopatterned surface is responsible for this unique migration behavior. These results demonstrate the usefulness of the presented method in understanding the process of determining collective and non-collective migration features in defined micro- and nano-environments and resolving the crosstalk between cell-cell and cell-ECM adhesions.