The role of bone marrow mesenchymal stromal cells in the protection of B-cell chronic lymphocytic leukaemia from spontaneous and drug induced apoptosis
An in vitro model to mimic in vivo conditions was developed using adherent BM Mesenchymal Stromal Cells (MSCs), isolated from the iliac crest of normal donors. These "fibroblast-like" MSCs provide a supporting layer for haemopoietic differentiation. They lack expression of haemopoietic differentiation antigens and lineage markers:- CD34, CD45, HLA-DR, CD31 and CD68 but express Vimentin, alphaSmooth Muscle Actin and Ab-1 (fibroblast marker). The model was used to determine if MSCs could protect B-CLL B cells from spontaneous apoptosis and prevent the effects of the therapeutic monoclonal antibodies Rituximab (CD20) and Campath-1H (CD52), the proteasomal inhibitor MG132 and BisIX, a protein kinase C inhibitor. Levels of apoptosis were determined by flow cytometry, using Annexin V-FITC/PI. MSCs abrogated spontaneous apoptosis of B-CLL B cells in direct contact with them, over 4 days (80-90% viability, n=30). Rituximab and Campath-1H induced apoptosis in the B-CLL B cells that could not be prevented by contact with MSCs (n=6). MG132 and BisIX caused substantial apoptosis in B-CLL B cells (0-15% viability) but MSCs could protect against lower concentrations (200nM) of both drugs (70-90% viability, n=6). MSCs' protection of B-CLL B cells from spontaneous apoptosis was through the upregulation of the expression of Mcl-1 and Xiap, but not Survivin. The cell-cell interactions include the beta1, beta2, beta3 and beta4 integrins but have variable effects on B-CLL cases when bound by blocking antibodies. Therefore MSCs can maintain B-CLL viability in vitro and influence chemotherapeutic strategies. The model provides a reliable method to evaluate drug cytotoxicity and Mcl-1 offers a novel therapeutic target.