Flow cytometric assay for quantitative and qualitative evaluation of adhesive interactions of tumor cells with endothelial cells

The purpose of the study was to develop a flow cytometric assay for quantitative determination of adhesive interactions of human endothelial cells (ECs) with tumor cells. EC lines established from human lymph node, appendix, lung, skin and intestine microvessels, labeled with PKH26-GL fluorescent dye, were grown to confluency in 24-well TC plates. Human colon adenocarcinoma cell suspension was overlaid onto labeled ECs, and allowed to adhere for 20 min at 4 degrees C under static conditions. Non-adhering cells were collected first, and adhering tumor cells together with ECs were detached from the culture plate. Collected cell fractions were evaluated by flow cytometry. Results were re-calculated as a ratio (R) of adhering colon carcinoma cells per one EC. We demonstrated that immortalized human microvascular ECs preserved their organ specificity. Colon carcinoma cells adhere preferentially to ECs of intestine origin. The immunofluorescent staining of adhering and non-adhering cancer cell subpopulations has revealed an augmented level of Lewis(x) antigen on adhering cancer cells. The organ specificity of endothelial cell interactions with colon carcinoma cells demonstrated in static conditions was verified and confirmed with flow adhesion assay. The method elaborated is suitable for quantifying of tumor cells adhering to ECs, with simultaneous evaluation of cell surface phenotypic markers of both partner cells participating in adhesive interactions. Validated by comparison to dynamic shear stress adhesion assay in blood flow reconstituted conditions this assay greatly facilitates evaluation of tumor cell-endothelial cell mutual interactions taking place during metastatic process.