Adhesion of Head and Neck Squamous Cell Carcinoma to Endothelial Cells: The Missing Links

Abstract

To examine the direct adhesion of head and neck squamous cell carcinoma (HNSCC) cells to basal and cytokine-activated human endothelial cells and to determine the cell adhesion molecules (CAMs) that mediate binding under these two conditions. Using an established model of tumor metastasis, the adhesion of four HNSCC cell lines to human umbilical vein endothelial cell (HUVEC) monolayers was examined, with and without pretreatment of HUVEC with tumor necrosis factor alpha (TNF-alpha). Surface CAM expression of HNSCC and HUVEC was determined by flow cytometry, and the results were used to direct studies of adhesion blocking using monoclonal antibodies. The contribution of various CAM to HNSCC binding of basal and cytokine-activated human endothelial cells in vitro was established. Adhesion of HNSCC to HUVEC monolayers was determined by a sodium chromate Cr 51-labeling assay in the presence or absence of monoclonal antibodies directed against specific CAMs. Four HNSCC cell lines were shown by flow cytometry to constitutively express the following CAMs: intercellular CAM-1, CD44, lymphocyte function-associated antigen-3, integrin chains alpha 6 and beta 1, and sialyl Lewis(x). No cell lines expressed lymphocyte function-associated antigen-1 or the integrin subunit alpha 4. Adhesion of JHU-011-SCC to TNF-alpha-activated HUVEC was enhanced above the untreated level in a time-dependent manner, with maximal adhesion at 12 hours. This increase correlated with endothelial-selectin expression by HUVEC and HNSCC expression of its ligand sialyl Lewis(x). Monoclonal antibody to sialyl Lewis(x) blocked the increased adhesion to TNF-alpha-activated HUVEC in two of four HNSCC cell lines. Monoclonal antibody to the alpha 6 integrin reduced binding to TNF-alpha-activated and non-activated HUVEC and to subendothelial matrix, but not to fibronectin. Studies of four HNSCC cell lines disclosed a consistent and distinctive pattern of adhesion molecule expression. The alpha 6 integrin subunit may be involved in direct adhesion to nonactivate and cytokine-activated endothelial cells or to laminin present on the endothelial surface. Sialyl Lewis(x) was more specifically involved in the increased adhesion to cytokine-activated HUVEC. This suggests that the sialyl Lewis(x)-endothelial-selectin ligand interaction may be important in facilitating HNSCC adhesion during metastasis to sites of active or chronic inflammation in vivo.