Updated project metadata. In the context of breast cancer metastasis study, we have shown in an in vitro model of cell migration that IGDQ-exposing (IsoLeu-Gly-Asp-Glutamine type I Fibronectin motif) monolayers (SAMs) on gold sustain the adhesion of MDA-MB-231 cells by triggering Focal Adhesion Kinase by activating integrins. Such tunable scaffolds are used to mimic the extracellular environment, inducing and controlling cell migration towards an anisotropic surface. The observed migratory behavior induced by the IGDQ-bearing peptide gradient along the surface suggests the presence of cell subpopulations: “stationary” or a “migratory” phenotype. We focused on the integrins α5(β1) and (αv)β3, already known to be implicated in cell migration. To this aim, a whole proteomic analysis was performed in beta 3 integrin (ITGB3) or alpha 5 integrin (ITGA5) knock-down MDA-MB-231, in order to highlight the pathways implied into the integrin-dependent cell migration. Our results showed that i) ITGB3 depletion influenced ITGA5 mRNA expression, ii) ITGB3 and ITGA5 were both necessary for IGDQ-mediated directional single cell migration, iii) integrin (αv)β3 was activated by IGDQ fibronectin type I motif and iv) co-regulation of retrograde transport of ITGB3 by ITGA5 is potentially necessary for directional IGDQ-mediated cell migration.