Neuron-glial antigen 2 (NG2, also known as CSPG4) and hyaluronic acid receptor CD44 are chondroitin sulphate proteoglycans actively involved in brain development and its malignant transformation. Here, we aimed to compare prognostic significances of NG2, CD44 and Ki-67 expression in glioblastoma multiforme patients. Totally, 45 tissue samples and 83 paraffin-embedded tissues for 75 patients were analysed. The prognostic values of the genes were analysed using Kaplan-Meier survival curves. Grade III gliomas showed 2-fold difference in NG2 expression between anaplastic astrocytoma and oligoastrocytoma (10.1 ± 3.5 and 25.5 ± 14.5, respectively). For grade IV gliomas, upregulated NG2 expression (21.0 ± 6.8) was associated with poor glioblastoma multiforme prognosis (overall survival < 12 months) compared with glioblastoma multiforme patients with good prognosis (4.4 ± 3.2; overall survival > 12 months). Multivariate survival analysis using Cox proportional hazards model confirmed that high NG2 expression was associated with low survival of the patients (hazard ratio: 3.43; 95% confidence interval: 1.18-9.93; p = 0.02), whereas age (hazard ratio: 1.02; 95% confidence interval: 0.96-1.09; p = 0.42), tumour resection (hazard ratio: 1.03; 95% confidence interval: 0.98-1.08; p = 0.25) and sex (hazard ratio: 0.62; 95% confidence interval: 0.21-1.86; p = 0.40) did not show significant association with prognosis. Although the positive correlation was shown for NG2 and CD44 expression in the glioblastomas (Pearson coefficient = 0.954), Kaplan-Meier and multivariate survival analyses did not revealed a significant association of the increased CD44 expression (hazard ratio: 2.18; 95% confidence interval: 0.50-9.43; p = 0.30) or high Ki-67 proliferation index (hazard ratio: 1.10; 95% confidence interval: 1.02-1.20; p = 0.02) with the disease prognosis. The results suggest that upregulation of NG2/CSPG4 rather than changes in CD44 or Ki-67 expression is associated with low overall survival in glioblastoma multiforme patients, supporting NG2/CSPG4 as a potential prognostic marker in glioblastoma.

Heparan sulfate (HS) is an important component of the extracellular matrix and cell surface, which plays a key role in cell–cell and cell–matrix interactions. Functional activity of HS directly depends on its structure, which determined by a complex system of HS biosynthetic enzymes. During malignant transformation, the system can undergo significant changes, but for glioma, HS biosynthesis has not been studied in detail. In this study, we performed a comparative analysis of the HS biosynthetic system in human gliomas of different grades. RT-PCR analysis showed that the overall transcriptional activity of the main HS biosynthesis-involved genes (EXT1, EXT2, NDST1, NDST2, GLCE, HS2ST1, HS3ST1, HS3ST2, HS6ST1, HS6ST2, SULF1, SULF2, HPSE) was decreased by 1.5–2-fold in Grade II-III glioma (p < 0.01) and by 3-fold in Grade IV glioma (glioblastoma multiforme, GBM) (p < 0.05), as compared with the para-tumourous tissue. The inhibition was mainly due to the elongation (a decrease in EXT1/2 expression by 3–4-fold) and 6-O-sulfation steps (a decrease in 6OST1/2 expression by 2–5-fold) of the HS biosynthesis. Heparanase (HPSE) expression was identified in 50% of GBM tumours by immunostaining, and was characterised by a high intratumoural heterogeneity of the presence of the HPSE protein. The detected disorganisation of the HS biosynthetic system in gliomas might be a potential molecular mechanism for the changes of HS structure and content in tumour microenvironments, contributing to the invasion of glioma cells and the development of the disease.

Glycosaminoglycans are major components of brain extracellular matrix (ECM), although heparan sulfate (HS) contribution in brain physiology and carcinogenesis remains underinvestigated. This study examined HS content and distribution in glioblastoma multiforme (GBM) tissues in the context of potential molecular mechanisms underlying its deregulation in brain tumours. Totally, 42 tissue samples and paraffin-embedded tissues for 31 patients with different prognosis were investigated. HS expression was demonstrated in 50-55% of the GBM tumours by immunohistochemistry (IHC), while almost no HS content was detected in the surrounding paratumourous brain tissues. Heterogeneous HS distribution in the HS-positive tumours was more related to the necrosis or glandular-like brain zones rather than glioma cells with high or low Ki-67 index. According the Kaplan-Meier curves, HS accumulation in glioma cells was associated with low relapse-free survival (RS) of the GBM patients (p < 0.05) and was likely to be due to the increased transcriptional activity of HSPG core proteins (syndecan-1, 2-3 fold; glypican-1, 2,5 fold; perlecan/HSPG2, 13-14 fold). Activation of perlecan/HSPG2 expression correlated with the patients' survival according Kaplan-Meier (p = 0.0243) and Cox proportional-hazards regression (HR = 3.1; P(Y) = 0.03) analyses, while up-regulation of syndecan-1 and glypican-1 was not associated with the patients survival. Taken together, the results indicate that increase of HS content and up-regulation of perlecan/HSPG2 expression in glioblastoma tissues contribute to tumour development through the transformation of brain extracellular matrix into tumour microenvironment, and represent negative prognostic factors for glioblastoma progression.

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Abstract:Here we present the analysis of alternative splicing events on an example of glioblastoma cell culture samples using a set of computer tools in combination with database integration. The gene expression profiles of glioblastoma were obtained from cell culture samples of primary glioblastoma which were isolated and processed for RNA extraction. Transcriptome profiling of normal brain samples and glioblastoma were done by Illumina sequencing. The significant differentially expressed exon-level probes and their corresponding genes were identified using a combination of the splicing index method. Previous studies indicated that tumor-specific alternative splicing is important in the regulation of gene expression and corresponding protein functions during cancer development. Multiple alternative splicing transcripts have been identified as progression markers, including generalized splicing abnormalities and tumor-and stage-specific events. We used a set of computer tools which were recently applied to analysis of gene expression in laboratory animals to study differential splicing events. We found 69 transcripts that are differentially alternatively spliced. Three cancer-associated genes were considered in detail, in particular: APP (amyloid beta precursor protein), CASC4 (cancer susceptibility candidate 4) and TP53. Such alternative splicing opens new perspectives for cancer research.

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Boron neutron capture therapy (BN T) is characterized by a selective effect directly on the cells of malignant tumors. The carried out research showed the perspective of the given kind of therapy concerning malignant tumors of the brain. However, the introduction of BNCT into clinical practice is hampered by the lack of a single protocol for the treatment of patients and the difficulty in using nuclear reactors to produce a neutron beam. This problem can be solved by using a compact accelerator as a source of neutrons, with the possibility of installation in a medical institution. Such a neutron accelerator for BN T was developed at Budker Institute of Nuclear Physics, Novosibirsk. A neutron beam was obtained on this accelerator, which fully complies with the requirements of BN T, as confirmed by studies on cell cultures and experiments with laboratory animals. The conducted experiments showed the relative safety of the method with the absence of negative effects on cell cultures and living organisms, and also confirmed the effectiveness of BN T for malignant brain tumors.