MMP-2 Promotes avb3 Mediated Tumor Cell Migration Figure 5. MMP-2 activity affects the migration of human A375 melanoma cells. (A) The effects of GM6001 on the migration of tumor cells in the agarose drop model. GM6001 were applied at different concentrations regarding its function on the different MMP individuals as described in MATERIALS AND METHODS. (B) Photomicrographs showed the A375 cell migration in the agarose drop model 48 h after seeding. The arrows point the direction and distance of cell migration. Cells were untreated (ctrl) or treated with RGD or MMP-2I. Scale bar represents 100 mm. (C) The distance of cell migration was measured. The extent of migrating cells out of the drop = [(total area/drop area)6100] - 100, and the mean values 6 SEM were obtained from at least three independent experiments. (**, P,0.05). (D) The effects of GM6001 on the tumor cell migration was measured by transwell assay. GM6001 was applied at different concentrations regarding its function on the different MMP individuals as described in MATERIALS AND METHODS. (E) Photomicrographs showed the effects of MMP-2I and RGD on the migration of A375 cell 24 h after seeding in the transwells. Scale bar = 100 mm. (F) The number of migrated cells from different groups was calculated by transwell assay (***, P,0.05). Statistical difference was determined by comparing the treated group with the normal control by t-test. doi:10.1371/journal.pone.0041591.g005 (Figure 1C). Once the cells were completely spread, both MMP-2 and avb3 integrin were strongly expressed. These results imply that MMP-2 is recruited on the surface of tumor cells before avb3 integrin. To further confirm this observation, we performed an agarose drop assay with immunofluorescence analysis. After the tumor cells were resuspended in an agarose drop, the distribution of MMP-2 and avb3 integrin was examined from 1 h to 12 h. Cells were permitted to adhere to coverslips for 1 h after seeding. We found MMP-2 displayed a cluster-like signal, which was enriched at the leading edge of the cells. However, the expression of avb3 integrin was evenly distributed on the surface of the cells. When an increasing number of cells were adhered to the coverslips at 3 h or 7 h after seeding, partial co-localization of MMP-2 and avb3 integrin was observed (Figure 2). Based on these findings, we speculated that MMP-2 may regulate avb3 integrin. To verify this hypothesis, we first tested whether MMP-2 activity was involved in tumor cell adhesion. Our static adhesion assay showed that the inhibition of MMP-2 impaired the adhesion of tumor cells (Figure 3A), suggesting that MMP-2 activity may regulate the adhesion of tumor cells. To obtain some mechanistic insights, we examined whether the MMP-2 regulated adhesion of tumor cells requires avb3 integrin. We employed human M21 (wild-type of avb3) and M21-L (avb3 defective) melanoma cells and found that tumor cell adhesion was avb3 integrin-dependent (Figure 3B). Next, we detected the effect of MMP-2 activity on cell spreading. Morphometric analysis of the tumor cells revealed the differences in the rate of cell spreading after MMP-2 inhibition (Figure 4 B), indicating that MMP-2 activity affected the spreading of tumor cells. Furthermore, we showed that MMP-2 inhibition strongly limited the tumor cell migration both in the agarose drop model and the transwell assay (Figure 5). Collectively, these results demonstrated that MMP-2 regulates avb3 integrin-dependent tumor cell migration. MMP-2 can cleave various ECM proteins [17]. However, whether these cleavage products affect avb3 integrin-mediated tumor cell migration is not well understood. We hypothesize that MMP-2 may cleave ECM and facilitate the binding of avb3 integrin to ECM. Fibronectin is a substrate of MMP-2 and a ligand of avb3 integrin. We investigated the possibility that MMP-2 cleaves fibronectin into shorter fragments to facilitate the interaction between avb3 integrein and cleaved fibronectin. Indeed, we found that wild-type avb3 integrin-expressed cells preferentially attached to the cleaved shorter fibronectin fragments (Figure 6). In contrast, avb3 integrin-mutant cells showed similar adhesive abilities before and after fibronectin digestion. We propose the following mechanism for the initial steps of invasion based on tumor cell adhesion and migration. Active MMP-2 is first recruited to the leading edge of invasive tumor cells and cleaves fibronectin into shorter fibronectin products. The fibronectin fragments promote avb3 integrin recruitment to the area of cleaved fibronectin products to facilitate tumor cell adhesion and migration. Collectively, avb3 integrin-mediated adhesion and migration of tumor cells is regulated, at least in part, by MMP-2 via fibronectin cleavage. Additionally, as we were conscious of the contribution of another integrin, b1 integrin, in the adhesion of tumor cells to fibronectin, we detected the expression of b1 on the selected cells and its effects on the initial cell adhesion on fibronectin. FACS experiments showed that all the selected cells highly expressed b1 integrin, and b1 integrin blocking with antibody affected the adhesion of the tumor cells to fibronectin. This may explain why melanoma cell adhesion to fibronectin is reduced in the avb3- deficient cells, but not equal to BSA control. Immunofluorescence experiments showed that in early phases of tumor cell adhesion, b1 integrin may form clusters earlier than avb3 integrin, suggesting that b1 integrin may play adhesion role earlier than avb3 integrin. These results implicated b1 integrin also play an important role in the initial adhesion of tumor cells (Figure S1). We also detected another human tumor cells (human DU145 prostatic cancer cell) with several assays as above. The results showed a similar tendency (Figure S2, S3), which imply that MMP-2 and avb3 integrin may regulate each other in different cells. Supporting Information Figure S1 The effects of b1 integrin expression on the adhesion of human melanoma cells (A375, M21 and M21-L). (A) The expression of b1 integrin on human melanoma cell surface was evaluated by flow cytometric analysis using anti-b1 antibodies. Isotype IgG was used as negative control. (B) Human melanoma cells were fluorescently stained and seeded on 48-well plates coated with 10 mg/ml human fibronectin (FN) or 0.5% BSA. The cells were treated with b1 antibody, RGD peptides or MMP-2I. (C) A375 cells were seeded onto coverslips coated with human fibronectin for 1 h, then labeled with anti-avb3 or anti-b1 antibodies respectively. Scale bar = 10 mm. (TIF) Figure S2 Expression and interaction of MMP-2 and avb3 integrin in tumor cells. (A) Gelatin zymography of three kinds of tumor cells. Lane 1 & lane 2: human A375 melanoma cells; lane 3 & lane 4: human M21 melanoma cells; lane 5 & lane 6: human DU145 prostatic cancer cells. All these cells expressed gelatinases. (B) Flow cytometric analysis of avb3 integrin and MMP-2 expression on the DU145 cell surface. The expressions were evaluated using anti-avb3 and anti-MMP-2 antibodies, and isotype IgG was used as negative control. (C) Coimmunoprecipita- tion assay of avb3 integrin and MMP-2 in tumor cells. Upper panel, MMP-2 was in the immunoprecipitated complex of avb3 integrin; Lower panel, avb3 integrin was in the immunoprecipi- tated complex of MMP-2. (D) DU145 cells were suspended in PBS and labeled with anti-avb3 (red) and anti-MMP-2 (green) antibodies. Scale bar = 20 mm. (TIF) PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e41591