MMP-2 Promotes avb3 Mediated Tumor Cell Migration Figure 2. Distribution of MMP-2 and avb3 integrin in migrating human A375 melanoma cells. (A) human A375 melanoma cells in agarose drops were seeded onto coverslips coated with human fibronectin. The cells were labeled with anti-avb3 (green) and anti-MMP-2 (red) antibodies and observed under a confocal microscope at 1 h, 3 h or 7 h after seeding. The second row of each panel represents the blowups of the first row at the designated regions. The shorter arrows indicate the direction of cell migration, and the longer arrows indicate the co-localization of MMP-2 and avb3 integrin. Scale bar = 10 mm. (B) The percentages of adherent cells were calculated at different time points (1 h, 3 h, 5 h, 7 h, 9 h, 12 h). doi:10.1371/journal.pone.0041591.g002 MMP-2 is Recruited to the Leading Edge of Invasive Human A375 Melanoma Cells before avb3 Integrin during Migration To further study the interaction between MMP-2 and avb3 integrin, we cultured the human A375 melanoma cells using an agarose drop model with the following steps: 2 ml drops of cells (56106 cells/ml) in the agarose were seeded onto fibronectin- coated coverslips in a 24-well plate. The agarose drop model was used to demonstrate the direction of cell migration by forcing cells to move from a high cell density area to a low cell density area. The coverslips were then examined by using immunofluorescence method at different time points. As shown in Figure 2A and 2B, approximately 12% of cells adhered to the fibronectin-coated coverslip at 1 h after seeding. avb3 integrin was evenly distributed on the cells and the cluster-like signal of MMP-2 appeared at the invasive leading edge of the cells (Figure 2A, upper panel). Co- localization of the two molecules was not obviously observed on this time point. At 3 h after seeding (Figure 2A, middle panel), approximately 23% of cells adhered to the coverslip, and a partial co-localization of MMP-2 and avb3 integrin at the invasive front of the cells was observed. The co-localization of MMP-2 and avb3 integrin in a large number of adherent cells (approximately 47%) was observed at 7 h after seeding (Figure 2A, lower panel). The results indicated that MMP-2 was recruited to the leading edge of invasive tumor cells before avb3 integrin during migration, implying that MMP-2 may regulate avb3 integrin recruitment. MMP-2 Activity Regulates avb3 Integrin-Mediated the Adhesion of Human A375 Melanoma Cell on Fibronectin Based on the observation that MMP-2 is recruited to the leading edge of invasive A375 cells before avb3 integrin during migration (Figure 2), we hypothesized that MMP-2 may regulate avb3 integrin-mediated A375 cell adhesion. A static adhesion assay was employed to detect the effect of MMP-2 on the adhesion of A375 cells mediated by avb3 integrin. We first assessed the effects of synthetic inhibitor GM6001 for MMPs (IC50 values have been reported as follows: 0.4 nM for MMP-1; 0.5 nM for MMP-2; 27 nM for MMP-3; 0.1 nM for MMP-8 and 0.2 nM for MMP-9) [45]. As shown in Figure 3, we treated the human A375 melanoma cells with different concentration of GM6001, and we found that the cell adhesion was efficiently inhibited by GM6001 at concentration of 0.5 nM, implying an inhibition on MMP-2 as reported [45] (Figure 3A). Higher or lower concentrations of GM6001 did not provide further inhibition. The MMP-2 in- hibition (MMP-2I) significantly reduced the adhesion of human A375 cells by approximately 37% when compared with that of control cells (Figure 3B). To determine whether the MMP-2I- reduced adhesion was mediated by avb3 integrin, human M21 and M21-L melanoma cells were used in the static adhesion assay (Figure 3C). M21-L cells were selected in the present study because they weakly express avb3 integrin and served as a negative control. We found that MMP-2I significantly reduced the adhesion of M21 cells by approximately 30% when compared with that of control cells, but MMP-2I did not induce significant changes in M21-L cells. RGD peptides were used as another control to block integrin-mediated cell-fibronectin interactions. The results demonstrated that MMP-2 activity was essential for avb3 integrin-dependent adhesion of human melanoma cells. Inhibition of MMP-2 Activity Affects the Spreading of Human A375 Melanoma Cells The morphology of migrating cells is an important parameter and reflects the ability of cells in migration. Therefore, we investigated whether MMP-2 activity affects the spreading of human A375 melanoma cells. The cells treated with MMP-2I were seeded onto fibronectin-coated coverslips, and the spreading cells were analyzed and quantified at different time points after seeding (Figure 4). Spreading cells were divided into three groups according to their morphology as follows (Figure 4A): round (the edge of cell is round and smooth), partially spread (the lamellipodia of cell were partially stretched) and completely spread (the lamellipodia of cell were completely stretched). We found almost all of the cells were round at 3 h after seeding (Figure 4B). Six hours later, approximately 39% of cells were completely spread and 29% were partially spread in the untreated group, whereas only 8% of cells were completely spread and 16% were partially spread in the MMP-2I group. However, no significant difference was observed between the groups with or without MMP-2I at 9 h. These data demonstrated that MMP-2 activity affected the spreading of human A375 melanoma cells. MMP-2 Activity Regulates avb3 Integrin-Mediated Migration of Human A375 Melanoma Cells on Fibronectin Base on the findings of the static adhesion assay and spreading analysis, we further queried whether MMP-2 activity regulates avb3 integrin-mediated migration of human A375 melanoma on fibronectin. In the agarose drop model, A375 cells migrated on fibronectin-coated coverslips, and a linear expansion of corona- like region consisting of migrating cells around the drop was observed over 48 h, and the cells in the migrated area were measured and statistically analyzed (Figure 5). Different concen- tration of GM6001 was used, and 0.5 nM GM6001 (MMP-2I) was found to efficiently inhibit cell migration (Figure 5A). The extension of the corona-like area of MMP-2I treated cells was considerably diminished compared with that of untreated control cells, and RGD peptide dramatically blocked the outward dissemination of cells from the drop (Figure 5B). We measured the drop area and total area (area of the drop + area of migrating cells). Cell migration was expressed as the extent of migrating cells out of the drop = [(total area/drop area)6100] - 100. As shown in Figure 5C, RGD peptides reduced the cell migration by more than 90%, and the extension of the area of MMP-2I treated group was approximately 85% less than that of the control group, indicating that MMP-2 activity was required for the A375 cell migration. To further understand the role of MMP-2 activity in A375 cell migration, a transwell assay was performed. Different concentra- tions of GM6001 were also applied, and MMP-2I (0.5 nM GM6001) was found to efficiently inhibit cell migration (Figure 5D). MMP-2I treatment dramatically inhibited cell migration through the fibronectin-coated polycarbonate mem- brane compared with the untreated group (Figure 5E). The number of migrated cell in the MMP-2I treated group was PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41591