A novel flow cytometric assay for the quantification of adhesion of subsets within a heterogeneous cell population; analysis of lymphocyte function-associated antigen-1 (LFA-l)-mediated binding of bone marrow-derived primary tumour cells of patients with multiple myeloma

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Abstract
SUMMARY: In a previous study the expression of the adhesion molecule LFA-1 on tumour cells in patients suffering from multiple myeloma (MM) was correlated with growth of the malignant plasma cells in vivo. Here we describe a novel in vitro flow cytometric adhesion assay (FCAA) which, based on scatter and fluorescence properties, was used to analyse the contribution of the LFA-1/intercellular adhesion molecule-1 (ICAM-1) adhesion pathway in the binding of bone marrow (BM)-dcrived LFA-1-positive primary tumour cells of patients with MM to interferon-gamma (IFN-γ)-activated, ICAM-1-positive, human venous umbilical endothclial cells (huVEC) in vitro. To validate the FCAA. cells from different myeloma cell lines were labelled with the fluorescent dye CFDA or stained for CD38 expression, and LFA-1-mediated adhesion to IFN-γ-activated endothclial cells was quantified. Results obtained with the FCAA were compared with a conventional adhesion assay employing 51Cr-labelled cells. Statistical analysis revealed that both assays gave similar results. This allowed analysis of the contribution of LFA-1 to the adhesive potential of malignant plasma cells in bone marrow mononuclcar cells (BMMC) from MM patients to IFN-γ-activated endothclial cells. The results prove that LFA-1 expressed on bone marrow-derived plasma cells from MM patients can be used for cellular adhesion to ICAM-1 expressed on adherent growing cells, and are suggestive fora role of the LFA-1/ICAM-1 adhesion pathway in the pathophysiology of MM. The FCAA described in this study is a generally applicable assay, allowing analysis of the interaction of distinct subpopulations with in vitro grown adherent cells of different origin.