Assay: Role of Endo- Colon Carcinoma Cells to Liver Tissue in a Frozen Section Involvement of Cell Surface Glycans in Adhesion of Human

Adhesion of human colon carcinoma variant cell lines expressing different levels of the cell surface sialyl Lewis X (sLe) antigen to frozen sections of mouse liver was examined. KM12-HX cells that bound the monoclonal antibody (mAb) FH6 (anti-sLe) and thus expressed a high level of sLe demonstrated a greater degree of adhesion to liver sections than their low-binding counterparts, KM12-LX cells. The adhesion of KM12-HX cells to liver sections was partially blocked by mAb FH6, but not by another anti-sLe mAb, KM93. The adhesion was Ca dependent but was not inhibited by anti-E-selectin. Endo-b-galactosidase treatment significantly reduced adhesion and resulted in the loss of cell surface binding sites for mAb FH6. O-linked oligosaccharides from KM12-HX cells incubated in the presence of p-nitrophenyl-N-acetylgalactosaminide were fractionated by a combination of gel filtration, anion exchange chromatography, and normal phase high-performance liquid chromatography. The structure of a mAb FH6-reactive and endo-b-galactosidasesensitive glycan was estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in a post source decay mode and by glycosidase digestions to be NeuAca2–3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4(6Fuca1-3)GlcNAcb1-6(NeuAca2-3Galb1-3)GalNAcpNP. Mild detergent lysates of mouse liver surface-labeled with sulfo-NHS biotin were incubated with glutaraldehyde-fixed monolayers of KM12-HX cells, and bound components were isolated after EDTA treatment. A Mr 49,000 component that bound only to KM12-HX cells and not to KM12-LX cells was identified.