Volker Senner

Glioblastoma cells release factors that disrupt blood-brain barrier features

The blood-brain barrier (BBB), mediated by endothelial tight junctions, is defective in malignant gliomas such as glioblastoma, resulting in cerebral edema and contrast enhancement upon neuroradiological examination. The mechanisms underlying BBB breakdown are essentially unknown. Since non-neoplastic astrocytes are required to induce BBB features of cerebral endothelial cells, it is conceivable that malignant astrocytes have lost this ability due to dedifferentiation. Alternatively, glioma cells might actively degrade previously intact BBB tight junctions. To examine the latter hypothesis, we have employed a transepithelial electrical resistance breakdown assay using monolayers of the C7 subclone of Madin-Darby canine kidney (MDCK-C7) cells forming tight junctions similar to those of BBB endothelial cells. We found that glioblastoma primary cells co-cultured with the MDCK-C7 monolayer (without direct contact of the two cell types) resulted in marked breakdown of electrical resistance, whereas primary cultures derived from low-grade gliomas (fibrillary astrocytoma, oligoastrocytoma) showed delayed or no effects. These results suggest that malignant gliomas have acquired the ability to actively degrade tight junctions by secreting soluble factors, eventually leading to BBB disruption within invaded brain tissue.

CD24 Promotes Invasion of Glioma Cells In Vivo

Based on the hypothesis that adhesion molecules expressed on the surface of glioma cells mediate brain invasion, we examined the effect of CD24 on growth and migration of gliomas in vitro and in vivo. CD24, a glycosylphosphatidylinositol anchored, highly glycosylated adhesion molecule, is expressed in hematopoietic and neural cells. We found immunohistochemical expression of CD24 in human glioblastomas. We then established a clone from C6 rat glioblastoma cells, where mouse CD24 (also called heat stable antigen) is under control of a tetracycline-responsive promoter. In the presence of tetracycline (1 microg/ml) CD24 was downregulated by 20-fold. In vitro migration assays were performed on a basement membrane preparation (matrigel) and on myelin, the main substrates of in vivo glioma migration. While the cells were more motile on matrigel as compared with myelin, no relation between CD24 expression and motility was observed. We then transplanted the C6 clone into the striatum of nude mice and regulated CD24 expression via tetracycline in the drinking water (1 mg/ml). After 3 weeks, CD24 positive tumors of mice getting no tetracycline showed diffuse invasion of tumor cells in a brain area 10-fold larger than in CD24-suppressed tumors of mice receiving tetracycline. These data show that CD24 stimulates migration of gliomas in vivo and they suggest a role for this adhesion molecule in diffuse brain invasion of human gliomas.

Serum S100β increases in marathon runners reflect extracranial release rather than glial damage

The contribution of extracranial tissue damage to serum S100β increases was examined in 18 marathon runners without clinical or laboratory signs of brain damage. Postrace serum S100β and creatine kinase (CK) concentrations increased (p < 0.001), and areas under the curve were highly correlated (p = 0.001). To conclude, serum S100β increases after running originate from extracranial sources. CK determination may improve specificity of S100β as a marker of brain tissue damage in acute trauma.

Establishment of a benign meningioma cell line by hTERT-mediated immortalization

Meningioma represents the most common intracranial tumor, but well-characterized cell lines derived from benign meningiomas are not available. A major reason for the lack of benign tumor cell lines is senescence of nonmalignant cells in vitro, while malignant cells are often immortal. We have developed a meningioma cell line by retrovirally transducing primary cells derived from a human WHO grade I meningothelial meningioma with the human telomerase reverse transcriptase (hTERT) gene, which enables bypassing cellular senescence. Five clones have been cultured for more than 21 months so far, while corresponding nontransfected cells ceased proliferation within 3 months. Quantitative RT-PCR and a telomeric repeat amplification protocol (TRAP) assay revealed high hTERT mRNA levels and high telomerase activity in all transduced populations, while nontransduced cells were negative. The average telomere size of transduced cells was considerably longer than that of parental cells and the biopsy specimen. One clone, designated Ben-Men-1, was characterized in more detail, and exhibited typical cytological, immunocytochemical, ultrastructural and genetical features of meningioma, including whorl formation, expression of epithelial membrane antigen, desmosomes and interdigitating cell processes, as well as −22q. Following subdural transplantation into nude mice, tumor tissue with typical histological features of meningothelial meningioma was found. We conclude that Ben-Men-1 represents an immortalized yet differentiated cell line useful for biological and therapeutical studies on meningioma.

Epileptiform activity preferentially arises outside tumor invasion zone in glioma xenotransplants

Seizures occur commonly with brain tumors. The underlying mechanisms are not understood. We analyzed network and cellular excitability changes in tumor-invaded and sham neocortical tissue in vitro using a rat glioblastoma model. Rat C6 glioma cells were transplanted into rat neocortex, yielding diffusely invading gliomas resembling human glioblastomas. We hypothesized that network excitability would increase in regions neighboring the tumor, and that initiation of epileptic discharges might be correlated to a higher density of intrinsically bursting neurones. Voltage-sensitive dye imaging revealed epileptic activity to be initiated in paratumoral zones (1-2 mm from main tumor mass), in contrast to control tissue, where epileptic foci appeared randomly throughout the neocortex. Neuronal firing patterns revealed significantly more intrinsically bursting neurones within these initiation zones than in regions directly adjacent to the tumor or in control tissue. We conclude that gliomas are associated with a higher density of intrinsically bursting neurones, and that these may preferentially initiate epileptiform events.

Genes associated with fast glioma cell migration in vitro and in vivo.

Identification of genes mediating glioma invasion promotes the understanding of glia motility and might result in biologically based therapeutic approaches. Most experimental studies have been performed in vitro, although glial cells typically undergo marked phenotypic change following placement into cell culture. To evaluate migration mechanisms operating in vitro versus in vivo, we used C6 rat glioblastoma cells for selecting highly migratory cells in a monolayer migration assay as well as in brains of nude mice, and analyzed in each paradigm the expression profiles of these “fast” cells versus those of the original “slow” cells using oligonucleotide microarrays comprising 8832 genes. In vitro, 516 (10.6%) of 4848 expressed genes were regulated (ie, differentially expressed in fast versus slow cells); 916 genes were expressed only in vitro, including 142 (15.5%) regulated genes. In vivo, 245 (6.1%) of 4044 expressed genes were regulated; 112 genes were expressed only in vivo, including 25 (22.3%) regulated genes, none of them having a known relation to glioma invasion. Of 730 regulated genes, only 31 (4.2%) were regulated in parallel in vitro and in vivo, most of them having a known relation to (glioma) invasion. Our data provide new molecular entry points for identifying glioma invasion genes operating exclusively in the brain. They further suggest that genes underlying glia cell motility are strikingly different in vitro and in vivo.

Rocks are expressed in brain tumors and are required for glioma‐cell migration on myelinated axons

The interactions between migrating glioma cells and myelinated fiber tracts are poorly understood. We identified that C6 glioma cells can migrate along myelinated chicken retinal axons in a novel coculture, thereby expressing small GTPases of the Rho family and serine/threonine Rho‐associated kinases (ROCKs). We found that the ROCK1 isoform is also highly expressed in native human high‐grade gliomas. Glioma cells migrated faster in vitro along myelinated axons than on laminin‐1, with the former but not the latter being specifically and reversibly blocked by the ROCK inhibitor Y27632. These data suggest that the mechanisms underlying the migration of glioma cells on myelinated axons differ from those underlying the migration on extracellular matrix molecules such as laminin‐1.

Matrix gla protein (MGP): an overexpressed and migration-promoting mesenchymal component in glioblastoma

BackgroundRecent studies have demonstrated that a molecular subtype of glioblastoma is characterized by overexpression of extracellular matrix (ECM)/mesenchymal components and shorter survival. Specifically, gene expression profiling studies revealed that matrix gla protein (MGP), whose function has traditionally been linked to inhibition of calcification of arteries and cartilage, is overexpressed in glioblastomas and associated with worse outcome.MethodsIn order to analyze the role of MGP in glioblastomas, we performed expression, migration and proliferation studies.ResultsReal-time PCR and ELISA assays confirmed overexpression of MGP in glioblastoma biopsy specimens and cell lines at mRNA and protein levels as compared to normal brain tissue. Immunohistochemistry verified positivity of glial tumor cells for MGP. RNAi-mediated knockdown of MGP in three glioma cell lines (U343MG, U373MG, H4) led to marked reduction of migration, as demonstrated by wound healing and transwell assays, while no effect on proliferation was seen.ConclusionOur data suggest that upregulation of MGP (and possibly other ECM-related components as well) results in unfavorable prognosis via increased migration.

Collagen XVI expression is upregulated in glioblastomas and promotes tumor cell adhesion

MINT‐6743179: Collagen IV (uniprotkb:P02462‐1) and Collagen XVI (uniprotkb:Q07092) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT‐6743170: GFAP (uniprotkb:P14136) and Collagen XVI (uniprotkb:Q07092) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

ECM stiffness regulates glial migration in Drosophila and mammalian glioma models

Cell migration is an important feature of glial cells. Here, we used the Drosophila eye disc to decipher the molecular network controlling glial migration. We stimulated glial motility by pan-glial PDGF receptor (PVR) activation and identified several genes acting downstream of PVR. Drosophila lox is a non-essential gene encoding a secreted protein that stiffens the extracellular matrix (ECM). Glial-specific knockdown of Integrin results in ECM softening. Moreover, we show that lox expression is regulated by Integrin signaling and vice versa, suggesting that a positive-feedback loop ensures a rigid ECM in the vicinity of migrating cells. The general implication of this model was tested in a mammalian glioma model, where a Lox-specific inhibitor unraveled a clear impact of ECM rigidity in glioma cell migration.