Abstract
Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C m = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest C m values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.
Highlights
Glioblastoma multiforme (GBM) is the most common and aggressive human brain cancer, accounting for about 15% of all intracranial tumors [1,2,3]
We explore in the present study the plasma membrane morphology in five GBM lines differing in the mutational status of PTEN and p53
Given that CC reflects the total membrane area, these findings indicate that the swelling-mediated increase in cell surface was achieved without incorporation of new material into the plasma membrane, but largely via membrane unfolding, which was detected by the marked Cm reduction mentioned above
Summary
Glioblastoma multiforme (GBM) is the most common and aggressive human brain cancer, accounting for about 15% of all intracranial tumors [1,2,3]. The unfavorable clinical outcome is largely attributed to the highly invasive nature of GBM cells [7], based on their remarkable ability to invade healthy brain tissue and migrate extensively within the CNS [8], escaping surgical removal as well as exposure to radiation and chemotherapy [6,9]. Numerous light and electron microscopic studies reveal complex surface topography of primary glioma cells and GBM cell lines, which exhibit dense microvilli, membrane folds, filopodia and other membrane protrusions [10,11,12,13]. Microvilli have been reported to protect GBM cells from being killed by cytolytic effector cells of the immune system [15,16]
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