Cell invasion and metastasis is a multi-step process, initiated by the acquisition of a migratory phenotype and the ability to move through differing and complex 3D extracellular environments. In this study we set out to identify the parameters required for invasive cell migration in 3D environments. Cells interact with the extracellular matrix via transmembrane-spanning integrin adhesion complexes. We establish a technique to determine the composition of cell-matrix adhesion complexes in invasive breast cancer cells in 3D matrices and on 2D surfaces and identify an interaction complex that is enriched in 3D adhesion sites and required for invasive migration.