Breast cancer cells (BCC) and macrophages are known to interact via epidermal growth factor (EGF) produced by macrophages and colony stimulating factor-1 (CSF-1) produced by BCC. Despite contradictory findings, this interaction is perceived as a paracrine loop. Yet, the underlying mechanism of interaction remains unclear. Here, we investigated interactions of BCC with macrophages in 2D and 3D. BCC did not show chemotaxis to macrophages in custom designed 3D cell-on-a-chip devices, which was in agreement with ELISA results showing that macrophage-derived-EGF was not secreted into macrophage-conditioned-medium. Live cell imaging of BCC in the presence and absence of iressa showed that macrophages but not macrophage-derivedmatrix modulated adhesion and motility of BCC in 2D. 3D co-culture experiments in matrigel and collagen showed that BCC changed their multicellular organization in the presence of macrophages. In custom designed 3D co-culture cell-on-a-chip devices, macrophages reduced and promoted migration of BCC in matrigel and collagen, respectively. Furthermore, adherent but not suspended BCC endocytosed EGFR when in contact with macrophages. Collectively, our data revealed that macrophages showed chemotaxis towards BCC-derived-CSF-1 whereas BCC required direct contact to interact with macrophage-derived-EGF. We propose that the interaction between cancer cells and macrophages is a paracrine-juxtacrine loop of CSF-1 and EGF, respectively. Keywords and Phrases: breast cancer, epidermal growth factor, macrophage colony stimulating factor-1, paracrine signaling, juxtacrine signaling, chemotaxis, lab-on-achip, three dimensional cell culture