Prognosis for patients suffering from malignant glioma has not substantially improved. Specific immunotherapy as a novel treatment concept critically depends on target antigens, which are highly overexpressed in the majority of gliomas, but the number of such antigens is still very limited. SOX2 was identified by screening an expression database for transcripts that are overexpressed in malignant glioma, but display minimal expression in normal tissues. Expression of SOX2 mRNA was further investigated in tumour and normal tissues by real-time PCR. Compared to cDNA from pooled normal brain, SOX2 was overexpressed in almost all (9 out of 10) malignant glioma samples, whereas expression in other, non-malignant tissues was almost negligible. SOX2 protein expression in glioma cell lines and tumour tissues was verified by Western blot and immunofluorescence. Immunohistochemistry demonstrated SOX2 protein expression in all malignant glioma tissues investigated ranging from 6 to 66% stained tumour cells. Human leucocyte antigen-A*0201-restricted SOX2-derived peptides were tested for the activation of glioma-reactive CD8 þ cytotoxic T lymphocytes (CTLs). Specific CTLs were raised against the peptide TLMKKDKYTL and were capable of lysing glioma cells. The abundant and glioma-restricted overexpression of SOX2 and the generation of SOX2-specific and tumour-reactive CTLs may recommend this antigen as target for T-cell-based immunotherapy of glioma.

The contribution of extracranial tissue damage to serum S100beta increases was examined in 18 marathon runners without clinical or laboratory signs of brain damage. Postrace serum S100beta and creatine kinase (CK) concentrations increased (p < 0.001), and areas under the curve were highly correlated (p = 0.001). To conclude, serum S100beta increases after running originate from extracranial sources. CK determination may improve specificity of S100beta as a marker of brain tissue damage in acute trauma.

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.

Slit and Robo proteins are evolutionarily conserved molecules whose interaction underlies axon guidance and neuronal precursor cell migration. During development secreted Slit proteins mediate chemorepulsive signals on cells expressing Robo receptors. Because similar molecular mechanisms may be utilized in glioma cell invasion and neuroblast migration, we studied the expression of Slit2 and its transmembrane receptor Robo1 as well as their functional role in migration in glioma cells. qRT-PCR and immunohistochemistry of human specimens revealed that Slit2 was distinctly expressed by non-neoplastic neurons, but at only very low levels in fibrillary astrocytoma and glioblastoma. Robo1 also was mainly restricted to neurons in the normal brain, whereas astrocytic tumor cells in situ as well as glioblastoma cell lines overexpressed Robo1 at mRNA and protein levels. Recombinant human Slit2 in a concentration of 0.45 nM was repulsive for glioma cell lines in a modified Boyden chamber assay. RNAi-mediated knockdown of Robo1 in glioma cell lines neutralized the repulsive effect of Slit2, demonstrating that Robo1 served as the major Slit2 receptor. Our findings suggest that a chemorepulsive effect mediated by interaction of Slit2 and Robo1 participates in glioma cell guidance in the brain.

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.