Abstract

Recent studies suggest that one of the most important treatment resistance mechanisms of cells with the BCR-ABL translocation could be the interaction with the hematopoietic niche. During the contact with the respective microenvironment, leukemic stem and progenitor cells acquire both a state of cell adhesion mediated drug resistance (CAM-DR) and may change their proliferation and differentiation potential.

Studies on the effect of the BCR-ABL oncogene on cell adhesion and migration have yielded conflicting results due to different cell type models (primary cells vs. transduced cell lines), non-standardized quantification methods and diverse substrates (fibronectin vs. bone marrow stromal cells) studied. In the present work, we investigated the adhesion properties of 32D cells which were stably transformed with the BCR-ABL fusion transcript in comparison to an empty vector transformed control. The commonly used panning-style adhesion assay determining the percentage of hematopoietic cells adhering to a stroma cell line (M2-10B4) clearly demonstrated higher adhesion capacity of BCR-ABL transformed cells. Accordingly, an atomic force microscopy (AFM)-based method to measure adhesion forces of a single leukemic cell over stroma cells, showed an increased ‘stickyness’ of BCR-ABL transformed cells. AFM experiments demonstrated highly different forces that were needed to retract attached 32D-vector cells as compared to 32D/BCR-ABL cells (1000 pN vs. 3500 pN; p<0.01). This higher superficial adhesion of BCR-ABL transformed cells was associated with a decreased migration and homing potential of adhering cells into the stromal layer (quantified by cell tracking movies analysis) and cobblestones formation which was analysed by confocal laser scanning microscopy. The differences found between control and BCR-ABL-expressing cells were reversible by pre-incubation with 0.5 μM Imatinib Mesylate (IM), suggesting that altered adhesion is a tyrosine kinase-activity dependent process.

To further gain insight in the mechanisms of adhesion we used a cDNA microarray (Affymetrix-Gene chip 430A 2.0) for the identification of BCR-ABL related gene expression with special focus on adhesion associated genes. P-Selectin upregulation (40 folds) in cells harbouring BCR-ABL was seen as the most dramatic change in adhesion related molecules. Consequently, a monoclonal antibody against P-selectin inhibited adhesion capacity of BCR-ABL cells on M2-10B4.

In conclusion, we describe an increased adhesion capacity of 32/BCR-ABL transformed cells which is associated with an up-regulation of P-selectin expression. Moreover, whilst 32D/BCR-ABL cells show a high potential to primarily attach to the stroma layer, physiological functions like cobblestone formation is impaired by a reduced migration and consecutively impaired homing potential of the cells into the supporting niches formed by the feeder cells.

Disclosure: No relevant conflicts of interest to declare.

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