Single-Molecule AFM-Based Force Spectroscopy Reveals VLA-4/VCAM-1 Interactions Underlying Chemokine-Independent B16 Melanoma Cell Arrest

Integrin-mediated adhesion is essential for leukocyte trafficking and contributes to hematogenous tumor metastasis. We recently showed that metastatic B16 melanoma cells undergo VLA-4-dependent rolling and rapid arrest on endothelial cells under physiological shear flow without classical chemokine-induced integrin activation. However, the molecular basis of this chemokine-independent adhesion remained unresolved. Here, atomic force microscopy (AFM)-based single-molecule force spectroscopy was used to characterize VLA-4-mediated adhesion between living B16 melanoma cells and bEnd.3 endothelial cells at the level of individual receptor–ligand interactions. Rupture-force histograms revealed a dominant population centered at approximately 33 pN, consistent with single VLA-4/VCAM-1 bonds. VLA-4 blockade reduced adhesion frequency, whereas VCAM-1 blockade shifted the rupture-force distribution toward lower forces; both effects support molecular specificity. VLA-4-directed antibody and pharmacological inhibition further indicated that regulation primarily affects bond-formation probability rather than the strength of individual receptor–ligand interactions. Homotypic VLA-4-mediated adhesion between melanoma cells was also detected. These findings provide a functional link between single-molecule receptor interactions and the rolling-to-arrest transition of metastatic melanoma cells under physiological flow. Together with our recent flow-chamber studies, they support a model in which VLA-4/VCAM-1 bond formation contributes to vascular arrest without requiring classical integrin activation, providing molecular insight into an early step of metastatic dissemination.