Integrin VLA-4 enhances sialyl-Lewisx/a-negative melanoma adhesion to and extravasation through the endothelium under low flow conditions

Abstract

During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells. The capacity of tumor cells to form metastasis is related to their ability to interact with and extravasate through endothelial cell layers, which involves multiple adhesive interactions between tumor cells and endothelium (EC). Thus it is essential to identify the adhesive receptors on the endothelial and melanoma surface that mediate those specific adhesive interactions. P-selectin and E-selectin have been reported as adhesion molecules that mediate the cell-cell interaction of endothelial cells and melanoma cells. However, not all melanoma cells express ligands for selectins. In this study, we elucidated the molecular constituents involved in the endothelial adhesion and extravasation of sialyl-Lewis(x/a)-negative melanoma cell lines under flow in the presence and absence of polymorphonuclear neutrophils (PMNs). Results show the interactions of alpha(4)beta(1) (VLA-4) on sialyl-Lewis(x/a)-negative melanoma cells and vascular adhesion molecule (VCAM-1) on inflamed EC supported melanoma adhesion to and subsequent extravasation through the EC in low shear flow. These findings provide clear evidence for a direct role of the VLA-4/VCAM-1 pathway in melanoma cell adhesion to and extravasation through the vascular endothelium in a shear flow. PMNs facilitated melanoma cell extravasation under both low and high shear conditions via the involvement of distinct molecular mechanisms. In the low shear regime, beta(2)-integrins were sufficient to enhance melanoma cell extravasation, whereas in the high shear regime, selectin ligands and beta(2)-integrins on PMNs were necessary for facilitating the melanoma extravasation process.

Fig. 1.

VLA-4/VCAM-1 interactions support melanoma adhesion to the endothelial cells (EC) under low shear flow. HPMEC was either untreated as a control or treated with tumor necrosis factor-α (TNF-α) for 4 or 24 h, respectively. WM9 cells were injected into the parallel plate chamber, and cell tethering on the HPMEC undergoing different treatments was measured. In some cases, antibodies (anti-α₄-subunit of VLA-4) were used to block the VLA-4 functions on WM9 cells, and their tethering on the HPMEC was then measured. *, #, **P < 0.05 compared with cases where HPMEC was either nonstimulated or stimulated with TNF-α for 4 h. Values were means ± SE for N ≥ 3.

Fig. 2.

Resistance to detachment of melanoma cells from the EC. Melanoma cells were injected into the parallel plate flow chamber at 0.5 dyn/cm² for 5 min to allow the accumulation of adhered melanoma cells. The shear stress was then step increased in twofold increments for every 20-s interval. The fraction of the initial number of cells that remained bound at the end of each interval was determined. Values were means ± SE for N ≥ 3.

Fig. 3.

The extravasation of melanoma cells under different shear stress. WM9 cells or C8161 cells (5 × 10⁵ cells each) were injected into the flow migration chamber, and the extravasation through the HPMEC with various treatments were measured under the shear stresses at 0.5 dyn/cm². For some cases, VLA-4 on melanoma cells was functionally blocked using antibodies against its α₄-subunit, and the extravasation of melanoma cells was measured afterwards. The 24-h TNF-α stimulation of HPMEC significantly increased melanoma cell extravasation compared with nonstimulated controls under a low shear stress at 0.5 dyn/cm². Values were means ± SE for N ≥ 3.

Fig. 4.

Melanoma extravasation mediated by polymorphonuclear neutrophils (PMNs) was enhanced by interactions between VLA-4 and VCAM-1. WM9 cells and PMNs (5 × 10⁵ cells each) were injected into the flow migration chamber, and the extravasation of WM9 cells through the 24-h-stimulated HPMEC was measured under low and high shear conditions in the presence of PMNs. For some cases, VLA-4 on melanoma cells was functionally blocked using antibodies against its α₄-subunit, and the extravasation of WM9 cells was measured afterwards in the presence of PMNs. A: under low shear stress 0.6 dyn/cm², WM9 cell alone could resist shear flow and migrate through HPMEC monolayer. The presence of PMNs increased WM9 extravasation significantly. Blocking β₂-integrins reduced WM9 extravasation significantly; however, the treatment of neuraminidase on PMNs did not significantly affect WM9 extravasation. VLA-4 blocking reduced WM9 extravasation significantly even in the presence of PMNs. In the case where PMNs were treated with antibody against β₂-integrins, blocking VLA-4 further brought down WM9 extravasation, which did not happen in the case where PMNs were treated with neuraminidase. Values were means ± SE for N ≥ 3. B: under high shear stress 2 dyn/cm², there was no extravasation of WM9 cells by themselves through the EC. In the presence of PMN, WM9 cell migration dramatically increased. Blocking of VLA-4 on WM9 did not affect WM9 cell extravasation mediated by PMNs. β₂-Integrins blocking and neuraminidase treatment on PMNs both reduced WM9 extravasation significantly.