Silencing of CD44 in Glioma Leads to Changes in Cytoskeletal Protein Expression and Cellular Biomechanical Deformation Properties as Measured by AFM Nanoindentation

CD44, a transmembrane glycoprotein receptor for extracellular matrix molecules such as hyaluronic acid and osteopontin, is involved in glioma cellular signalling, adhesion and invasion. Although a great deal is known concerning the molecular players in adhesion, migration and invasion, little is known relating to how these invasive and migratory-promoting proteins influence biomechanical properties of glioma cells. Herein, we extend previous CD44 blocking experiments to examine effects of CD44 knock-down on expression of cytoskeletal proteins and cellular stiffness. An atomic force microscope (AFM) nanoindentation method was used to measure deformation or cellular stiffness (Young’s modulus, E ) in real time, at the single-cell level over nuclear and cytoplasmic regions. A glioblastoma cell line (SNB-19) was transfected with either CD44 small interfering RNA (siRNA), scrambled siRNA or a non-related gene siRNA. In SNB-19 CD44 knock-down cells, levels of microtubule, vimentin and glial fibrillary acidic protein (GFAP) proteins were lower compared to cells transfected with scrambled siRNA. Functionally, CD44 knock-down cells were less migratory compared to controls. AFM nanoindentation results show that the areas over the nuclei of both knock-down and parental control cells examined were significantly more compliant than their cytoplasmic regions ( p < 0.001). The most striking difference was seen when comparing nuclear regions of parental control cells versus CD44 knock-down cells. CD44 knock-down SNB-19 cells ( E = 0.56 ± 0.50 kPa) were less stiff than parental cells ( E = 1.93 ± 2.86 kPa; p < 0.001). Based on these results, we hypothesise that CD44 signalling via cytoskeletal proteins such as vimentin may influence the ability of glioma cells to respond to host tumour-derived mechanical pressures.