Microparticle (MP) biogenesis occurs following cellular activation and follows loss of membrane phospholipid asymmetry and activation of calcium dependent cytosolic cysteine protease activity. MPs confer the transfer and acquisition of cell phenotypes through the intercellular transfer of bioactive molecules. In the context of cancer, Bebawy et al. (2009) discovered that MPs provide a ‘non-genetic’ mechanism for the acquisition of multidrug resistance and increased metastatic capacity in cancer cell populations.
The aim of this study was to define the biogenic pathways involved in MP vesiculation in malignant and non-malignant cells using high resolution biological Atomic Force Microscopy (AFM) (Nanowizard, JPK Instruments, Germany). Identification and elucidation of cancer specific biogenic pathways would provide novel therapeutic targets and strategies to circumvent deleterious traits acquired through MPs in cancer.
A comparative analysis was performed using non-malignant human brain endothelial cells (HBEC-D3), human mammary epithelial cells (MBE-F), and drug sensitive and resistant human breast adenocarcinoma cells, MCF-7 and MCF-7/Dx respectively. Vesiculation of resting cells and cells activated with calcium ionophore, A23187, were studied +/- calpain inhibitor II (ALLM). Cell surface topography and the extent of vesiculation was determined using contact mode methodology. At rest, malignant cells exhibit an intrinsically higher degree of vesiculation relative to non-malignant cells. In the presence of ALLM, vesiculation was inhibited in malignant cells whilst non-malignant cells exhibited enhanced vesiculation. The latter supports the presence of a calpain independent pathway for vesiculation of normal cells at rest. Increasing intracellular calcium release with A23187 resulted in an increase in vesiculation across all cell types; however this was especially pronounced in non-malignant cells. We conclude that vesiculation at rest in malignant breast cells is driven by a calcium-calpain dependent pathway, whereas, an alternative pathway governs MP biogenesis in resting normal cells. These results support therapeutic approaches to selectively target malignant cells.