A NEW HISTOBIOCHEMICAL METHOD TO ANALYZE SIALYLATION ON CELL-SURFACE GLYCOPROTEINS OF HEAD AND NECK SQUAMOUS-CELL CARCINOMAS

Oncogenic transformation is often accompanied by alterations of glycos ylation on a tumor cell's surface, which may contribute to uncontrolle d cell growth. The sialoglycans and degree of sialylation on the cell surface are of increasing interest because of their possible role in m etastasis and tissue invasion. Since primary tumors and metastases may differ in the degree of sialylation, we examined the expression of si alic acid as a terminal constituent of lactosaminyl glycans on the cel l surfaces of 30 cervical lymph-node metastases and 30 squamous-cell c arcinomas of the oropharynx and oral cavity. Cell-surface sialylation was determined by a new histobiochemical assay on cryostat sections an d was based on the enzymatic introduction of a fluorescence-labelled s ialic acid into lactosaminyl type (Gal-beta 1-4 GlcNAc) oligosaccharid e chains of cell-surface-expressed glycoproteins. To this end, tissues were incubated in the presence of 5-acetamido-9-deoxy-9-fluoresceinyl -thioureido neuraminic acid (CMP-9-fluoresceinyl-NeuAc) and alpha-2,6- sialyltransferase. In order to compare the degree of sialylation with the potential total amount of sialylation sites, pretreatment with sia lidase for desialylation was required. We observed a significantly hig her amount of lactosaminyl-type binding sites for sialic acid on metas tases compared to the primary tumors (P = 0.001), indicating a lower d egree of sialylation in metastases. In primary tumors no correlation w as seen between the amount of binding sites and tumor localization, TN M stage or histologic grading. Pretreatment of specimens with sialidas e demonstrated a significant degree of sialylation on both primary tum ors and lymph-node metastases, but no difference between primary tumor s and metastases. When tumor stroma of primary tumors and metastases w as compared, tumor cells showed a higher degree of free binding sites for sialic acid, but a low degree of sialylation. Our results suggest that differences in the degree of sialylation of glycoconjugates on a tumor cell's surface may play an important role in the process of cell metastasis. Our histobiochemical method turned out to be very reliabl e, effective and readily performed.