MMP-2 Promotes avb3 Mediated Tumor Cell Migration Figure 4. MMP-2 activity affects the spreading of human A375 melanoma cells. (A) The morphology of A375 cell spreading at different time points were designated as round, partially spread and completely spread. (B) Cells were seeded onto coverslips coated with fibronectin, treated with MMP-2I at the concentrations of 0.5 nM at different time points after seeding, and assessed under the microscope. The cells without MMP-2I were defined as control. The differences of cell morphology at 3 h, 6 h and 9 h after seeding were observed and counted (n$400). doi:10.1371/journal.pone.0041591.g004 We found that A375 cells preferentially bound to 120-kDa and 70- kDa bands, which are known as MMP-2–cleaved fibronectin fragments. To test whether the MMP-2-cleaved 120-kDa and 70- kDa bands are functional for enhancing the adhesion of melanoma mediated by avb3 integrin, we retrieved the fragments from the native gel and coated them on 24-well flat-bottom plates (Figure 6D). An adhesion assay was performed using human A375 melanoma cells, and the results showed that approximately more than 39% of cells adhered to the wells coated with fragments compared with those coated with retrieved full-length fibronectin. To further certify the participation of avb3 integrin in the process showed above, adhesion assay were performed again using human M21 and M21-L melanoma cells (Figure 6E). Strikingly, approximately more than 34% of adherent cells were detected with the M21 cells, whereas, no obvious changes were detected with the M21-L cells.These results suggested that avb3 integrin is indispensible for the cell adhesion facilitated by MMP2-mediated fibronectin cleavage, and that MMP-2–cleaved fibronectin pro- mote the adhesion of tumor cells. Discussion Invasive migration of tumor cells in the basement membrane is dependent on cell adhesion and migration as well as proteolysis of the extracellular matrix, and is the initial step of tumor metastasis. The proteolytic activation of MMP-2 and the adhesive function of avb3 integrin are involved in the process [10]. MMP-2 directly binds to avb3 integrin via the C-terminus of active MMP-2 [41]. Previous studies have mainly focused on a avb3-MMP-2 mechanism to demonstrate how avb3 integrin regulates MMP-2. However, it is not clear whether MMP-2 reciprocally contributes to avb3 integrin regulation. In present work, we demonstrated that MMP-2 may promote the recruitment of avb3 integrin and regulate avb3 integrin-mediated tumor cell migration. We investigated the spatiotemporal relationship between MMP- 2 and avb3 integrin expression. We showed that MMP-2 was detected on the surface of tumor cells after the cells initially attached to matrix, nevertheless, the signal of co-localization between MMP-2 and avb3 integrin was barely detectable PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e41591