Involvement of integrin alpha(v)beta(3) and cell adhesion molecule L1 in transendothelial migration of melanoma cells

Abstract

Tumor metastasis involves many stage-specific adhesive interactions. The expression of several cell adhesion molecules, notably the integrin alpha(v)beta(3), has been associated with the metastatic potential of tumor cells. In this study, we used a novel in vitro assay to examine the role of alpha(v)beta(3) in the transmigration of melanoma cells through a monolayer of human lung microvascular endothelial cells. Confocal microscopy revealed the presence of the integrin alpha(v)beta(3) on melanoma membrane protrusions and pseudopods penetrating the endothelial junction. alpha(v)beta(3) was also enriched in heterotypic contacts between endothelial cells and melanoma cells. Transendothelial migration of melanoma cells was inhibited by either a cyclic Arg-Gly-Asp peptide or the anti-alpha(v)beta(3) monoclonal antibody LM609. Although both platelet endothelial cell adhesion molecule-1 and L1 are known to bind integrin alpha(v)beta(3), only L1 serves as a potential ligand for alpha(v)beta(3) during melanoma transendothelial migration. Also, polyclonal antibodies against L1 partially inhibited the transendothelial migration of melanoma cells. However, addition of both L1 and alpha(v)beta(3) antibodies did not show additive effects, suggesting that they are components of the same adhesion system. Together, the data suggest that interactions between the integrin alpha(v)beta(3) on melanoma cells and L1 on endothelial cells play an important role in the transendothelial migration of melanoma cells.

Figure 1

Confocal images showing the distribution of α_vβ₃ in HMVEC and WM239 melanoma cells. Cells were fixed with cold methanol and immunofluorescence staining was carried out with the use of mAb LM609 directed against α_vβ₃ integrin. (a) α_vβ₃ staining of a monolayer of HMVECs cultured on Matrigel. (b) WM239 cells showing an enrichment of α_vβ₃ staining at the cell-cell contact region (arrowheads). Bars, 10 μm.

Figure 2

Confocal series showing an enrichment of α_vβ₃ on membrane protrusions of melanoma cells and in heterotypic contacts during transendothelial migration. DiI-labeled melanoma cells were seeded on top of an HMVEC monolayer and fixed at different times of coculture. Coverslips were stained with the use of mAb LM609 and serial optical images were taken at 1-μm thickness. A schematic drawing is shown at the top of each series. Individual images are shown in an apical-to-basal direction and labeled with its distance from the bottom of the endothelium. (A) An optical series showing a melanoma cell at the initial stage of invasion through the endothelium. Membrane protrusions sent from the basolateral surfaces of the melanoma cells were labeled with α_vβ₃ (arrowheads). (B) An optical series showing a spindle-shaped melanoma cell intercalated between two endothelial cells. The heterotypic contacts were enriched in α_vβ₃, especially at the leading edge of an endothelial cell spreading on top of the melanoma cell (arrowheads). (C) An optical series showing a transmigrated melanoma cell spreading on the Matrigel under the endothelium. An enrichment of α_vβ₃ persisted in the heterophilic contact regions in all the X/Y sections (arrowheads). In comparison, the endothelial junctions were only weakly stained (arrows). Bars, 10 μm.

Figure 3

Inhibition of melanoma transmigration through HMVECs by mAb LM609 directed against α_vβ₃. (A) DiI-labeled WM239 melanoma cells were seeded on HMVEC monolayers and transmigration was allowed to occur in the presence of LM609 IgG (40 μg/ml). Coverslips were fixed at 1, 3, and 5 h of coculture. The number of transmigrated cells was scored as described in MATERIALS AND METHODS. Cocultures were incubated in the absence of antibody (▵), or in the presence of mAb LM609 against α_vβ₃ (○) or mAb P2B1 against PECAM-1 (●). (B) Effects of preincubating cells with mAb LM609 on the transendothelial migration of melanoma cells. Either WM239 cells or HMVECs were preincubated with LM609 (40 μg/ml) for 30 min and the unbound antibody was removed. Melanoma cells were then added to the HMVEC monolayer and cocultured for 5 h. Assays were carried out with cells either precoated with mAb (solid bars) or without prior precoating (shaded bars). Data represent the mean ± SD (n = 9). The asterisk indicates a statistically significant reduction in the percentage of transmigrated cells compared with the control (Student's t test, p < 0.01).

Figure 4

Transendothelial migration of the M21 melanoma cells through HMVEC monolayers. The percentages of transmigrated cells were scored at different times of coculture for M21 cells (α_v+) (●), M21-L cells (α_v−) (○), M21-L4 cells (α_v+, M21-L cells transfected with α_v cDNA (▴) and M21-L12 cells (α_v−, M21-L mock transfectant) (▵). Data represent the mean ± SD (n = 9).

Figure 5

Expression of L1 in HMVEC and WM239 cells. (a) Protein blots stained with anti-L1 antibody (1:500 dilution): i) total cell protein of WM239 cells, ii) total cell protein of HMVECs, iii) WM239 conditioned medium, and iv) HMVEC conditioned medium. Cell were also fixed with paraformaldehyde and stained with anti-L1 antibody: HMVEC (b), WM239 cells (c). Although endothelial junctions were clearly stained with L1, the plasma membrane of melanoma cells was only weakly stained. Granules filled with L1 were occasionally observed at the cell membrane (arrowhead). Double immunostaining was carried out to examine the localization of L1 (d and f) and α_vβ₃ (e and g) during transendothelial migration of melanoma cells. Melanoma cells were preloaded with Hoechst dye for identification. A round WM239 cell attached on the endothelium is shown in d and e. Arrows indicate a higher concentration of L1 and α_vβ₃ associated with the lamellipodial structure. The staining of L1 and α_vβ₃ was more intense at the heterotypic contacts (arrow) than the homotypic endothelial contacts (arrowheads). A melanoma cell intercalated among endothelium cells is shown in f and g. Bars, 10 μm.

Figure 6

Inhibition of melanoma transmigration through HMVECs by RGD peptides. (A) Cocultures of WM239 cells and HMVECs were carried out in the presence or absence of different concentrations of synthetic peptides: linear RGD (▪), linear RAD (□), cyclic RGD (●), and cyclic RAD (○). The percentage of transmigrated cells was determined at 5 h and then normalized to the minus-peptide control. (B) Time course experiment for the transmigration of WM239 melanoma cells in the presence of 90 μM cyclic RGD peptide (solid bars). Control assays (stippled bars) were carried out in the absence of the cyclic peptide. Data represent the mean ± SD (n = 9). The asterisks indicate a statistically significant reduction in the percentage of transmigrated cells (Student's t test, p < 0.01).

Figure 7

Effects of anti-L1 antibodies on the transendothelial migration of melanoma cells. (A) WM239 melanoma cells were added to HMVEC monolayers for transmigration in the absence of antibody (●) or in the presence of anti-L1-Ig1–3 antibody (□) or anti-L1-Ig4–6 antibody (○). (B) Effects of preincubation of cells with antibodies on WM239 cell transmigration. WM239 melanoma cells or HMVECs were preincubated with the anti-L1-Ig4–6 antibody (1:10 dilution) for 30 min at 37°C. Unbound antibodies were removed by washing before the coculture assay. The percentages of transmigrated cells were scored at 5 h. Data represent the mean ± SD (n = 9; *p < 0.05).

Figure 8

Inhibition of transmigration by a combination of antibodies against α_vβ₃ and L1. The transmigration assay was carried out in the presence of mAb LM609 (40 μg/ml) and anti-L1-Ig4–6 antibody (1:10 dilution), either singly or combined. Controls were carried out in either mAb P2B1 or rabbit preimmune serum. All values were normalized to that of the minus-antibody control. Data represent the mean ± SD (n = 9; *p < 0.01).