Monitoring E-selectin-mediated adhesion using green and red fluorescent proteins.

Abstract

Accumulating evidence suggests that E-selectin, which is physiologically involved in leukocyte recruitment during inflammation, plays an important role in the early stages of tumor cell interactions with vessel walls and contributes to the hematogenous spreading of cancer cells. Therapy designed to block this key step may provide an effective anti-inflammatory and anti-metastatic treatment. It is therefore critical to establish a safe, rapid and sensitive E-selectin adhesion assay. In this regard, we propose a simple and highly sensitive adhesion system based on CHO cells permanently co-expressing E-selectin and the enhanced green fluorescent protein EGFP or the red fluorescent protein DsRed2. This is an inverted adhesion assay in which tumor cells are maintained intact while fluorescent cells expressing E-selectin and EGFP (or DsRed2) are added to them. Adherent cells are then quantified by three different fluorescence-based techniques including spectrofluorimetry, ELISA-type cytofluorimetry and fluorescence microscopy coupled to digital image quantification. In this assay, a battery of cell lines can be analysed at once since only one cell line (fluorescent E-selectin-expressing cells) needs to be harvested. We used this approach to analyze a number of E-selectin-specific binding parameters of intestinal cancer cells in comparison with adhesion to activated endothelial cells or to plastic dishes coated with recombinant E-selectin. Besides the possibility of analyzing a battery of cell lines at once, this assay might be suitable for screening anti-metastatic compounds and could provide valuable information on the metastatic potential of human cancers.