Astrocytic Tumor Cells Research Articles

Identification of Dysregulated microRNAs in Glioblastoma Stem-like Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite multimodal therapy, median survival is poor at 12-15 months. At the molecular level, radio-/chemoresistance and resulting tumor progression are attributed to a small fraction of tumor cells, termed glioblastoma stem-like cells (GSCs). These CD133-expressing, self-renewing cells display the properties of multi-lineage differentiation, resulting in the heterogenous composition of GBM. MicroRNAs (miRNAs) as regulators of gene expression at the post-transcriptional level can alter many pathways pivotal to cancer stem cell fate. This study explored changes in the miRNA expression profiles in patient-derived GSCs altered on differentiation into glial fiber acid protein (GFAP)-expressing, astrocytic tumor cells using a polymerase chain reaction (PCR) array. Initially, 22 miRNAs showed higher expression in GSCs and 9 miRNAs in differentiated cells. The two most downregulated miRNAs in differentiated GSCs were miR-17-5p and miR-425-5p, whilst the most upregulated miRNAs were miR-223-3p and let-7-5p. Among those, miR-425-5p showed the highest consistency in an upregulation in all three GSCs. By transfection of a 425-5p miRNA mimic, we demonstrated downregulation of the GFAP protein in differentiated patient-derived GBM cells, providing potential evidence for direct regulation of miRNAs in the GSC/GBM cell transition.

P10.06.A Expression of GDNF and its receptors in glioblastoma

Abstract Background Neurotrophic factors are important for brain development, but their role in glioblastoma, the most aggressive primary brain cancer, is not fully understood. We focused on glial cell line-derived neurotrophic factor (GDNF), which has been shown to play a role in growth and migration in glioblastoma. The aim of this study was to investigate the expression of GDNF and its primary receptor GFRA1 as well as GDNF family receptors GFRA2-4 and RET. Material and Methods Tissue sections from 10 glioblastoma patients were stained with antibodies against GDNF (frozen tissue) and each of the receptors (formalin-fixed paraffin-embedded tissue). Moreover, the sections were stained with an RNA Scope assay for GDNF mRNA. The area fractions of positive receptor staining were quantified with a software-based classifier. Double immunofluorescence stainings were performed for GFRA1 and the astrocytic tumor cell marker GFAP, the microglia and macrophage marker IBA1, and the tumor stem cell markers OLIG2 and SOX2, respectively, for assessment of co-expression. Results GDNF staining in a varying number of cells was seen in 5 of the 10 samples. GDNF mRNA stainings were observed in a varying number of cells in all stained tissues, including in hypoxic areas and in microvascular proliferations. Mean area fractions of the receptors were 31 % (range 7-56) for GFRA1, 3 % (0,2-7) for GFRA2, 0,4 % (0,01-2) for GFRA3, 0,1 % (0,02-0,3) for GFRA4 and 1 % (0,1-2) for RET. Co-expression of GFRA1 and GFAP was widely present. Co-expression of GFRA1 and either OLIG2 or SOX2 was partially present. Co-expression of GFRA1 and IBA1 was limited. Conclusion Our results suggest that GDNF and GFRA1 are widely expressed in glioblastoma tissue, but GFRA2-4 and RET are also expressed and may play a role in glioblastoma tumor biology. Future experimental studies are needed to clarify the functional role of GDNF and its receptors in glioblastoma biology.

The epidermal growth factor receptor variant type III mutation frequently found in gliomas induces astrogenesis in human cerebral organoids.

ObjectivesThe epidermal growth factor receptor variant type III (EGFRvIII) is the most common mutation of EGFR in glioblastoma multiforme (GBM) and is found in approximately 25% of all GBMs. Intriguingly, EGFRvIII is mostly found in GFAP+ astrocytic tumour cells in the brain, suggesting connection of EGFRvIII to astrogenesis. In this study, we explored whether EGFRvIII mutation facilitates astrogenesis in human development setting.Materials and methodsUsing CRISPR‐Cas9, we generated EGFRvIII mutations in H9‐hESCs. Wild type (wt) H9‐hESCs were used as an isogenic control. Next, we generated cerebral organoids using the wt and EGFRvIII‐hESCs and examined the astrogenic differentiation of the brain organoids.Results EGFRvIII‐organoids showed abundant astrocytes (GFAP+, S100β+), while no astrocytes were detected in wt hESC‐derived organoids at day 49. On the contrary, TUJ1+ neurons were more abundant in the wt‐organoids than the EGFRvIII‐organoids. This result suggested that constitutively active EGFRvIII promoted astrogenesis at the expense of neurogenesis. In addition, the EGFRvIII‐organoids were larger in size and retained more Ki67+ cells than wt‐organoids, indicating enhanced cell proliferation by the mutation. The EGFRvIII‐organoids displayed massive apoptotic cell death after treatment with temozolomide and hence, could be used for evaluation of anti‐GBM drugs.Conclusions EGFRvIII mutation‐induced astrogenesis and massive cell proliferation in a human brain development model. These results provide us new insights into the mechanisms relating EGFRvIII mutation‐mediated gliogenesis and gliomagenesis.

HGG-24. HIGH-GRADE GLIOMA WITH A NOVEL FUSION GENE OF VCL-ALK

A previously healthy 2-year-old boy presented with status epilepticus following intermittent vomiting. Computed tomography scan showed a 7cm mass on the left occipital lobe with midline shift, inferior cerebellar herniation, and diffuse cerebral edema. The extensive dissemination to bilateral cerebral hemispheres, brain stem, and optic nerve was also observed. He underwent brain biopsy from the lesion on his left occipital lobe. The histopathological diagnosis determined the diffuse or epithelial proliferation of astrocytic tumor cells with high mitotic rate, positive for p53 and glial fibrillary acidic protein positive staining consistent with high-grade glioma. The progressive tumor led to communicating hydrocephalus, that was favorably controlled by cerebrospinal fluid shunting. The data from the FoundationOne CDx cancer genome profile disclosed a novel VCL- anaplastic lymphoma kinase (ALK) fusion in the tumor cells of the patient. ALK rearrangement was determined to be positive for the tumor cells assessed by fluorescence in situ hybridization. Only 4 pediatric cases of glioma with ALK-rearrangement have ever been reported. All of them received subtotal or gross total resections and then survived with or without chemotherapy. This is the first case of glioma harboring VCL as a novel partner of ALK fusion gene. After the favorable response to the first-line chemotherapy, subsequent irradiation therapy has now been scheduled. The molecular classification of high-grade glioma may help to expand the targeted therapy for unresectable advanced brain tumor.

Differential somatostatin, CXCR4 chemokine and endothelin A receptor expression in WHO grade I-IV astrocytic brain tumors.

Glioblastomas represent the most common primary malignant tumor of the nervous system and the most frequent type of astrocytic tumors. Despite improved therapeutic options, prognosis has remained exceptionally poor over the last two decades. Therefore, new treatment approaches are urgently needed. An overexpression of somatostatin (SST) as well as chemokine CXCR4 and endothelin A (ETA) receptors has been shown for many types of cancer. Respective expression data for astrocytic brain tumors, however, are scarce and contradictory. SST subtype, CXCR4 and ETA expression was comparatively evaluated in a total of 57 grade I-IV astrocytic tumor samples by immunohistochemistry using well-characterized monoclonal antibodies. Overall, receptor expression on the tumor cells was only very low. SST5 was the most prominently expressed receptor, followed by SST3, ETA, SST2 and CXCR4. In contrast, tumor capillaries displayed strong SST2, SST3, SST5, CXCR4 and ETA expression. Presence of SST5, CXCR4 and ETA on tumor cells and of SST3, CXCR4 and ETA on microvessels gradually increased from grade II to grade IV tumors. Ki-67 values correlated significantly with CXCR4 expression on tumor cells and with vascular SST3, CXCR4 or ETA positivity. SST5 or CXCR4 positivity of tumor cells and vascular SST3 or CXCR4 expression negatively correlated with patient outcome. Though having some prognostic value, SST, CXCR4 or ETA expression on astrocytic tumor cells is clearly of no therapeutic relevance. Indirect targeting of these highly vascularized tumors via SST3, SST5, CXCR4 or ETA on the microvessels, in contrast, may represent a promising additional therapeutic strategy.

Saponin-rich fraction from Agave sisalana: effect against malignant astrocytic cells and its chemical characterisation by ESI-MS/MS

Astrocytic tumour cells derived from human (GL-15) and rat (C6) gliomas, as well as non-tumoural astrocytic cells, were exposed to the saponin-rich fraction (SF) from Agave sisalana waste and the cytotoxic effects were evaluated. Cytotoxicity assays revealed a reduction of cell viability that was more intensive in glioma than in non-tumoural cells. The SF induced morphological changes in C6 cells. They were characterised by cytoplasmic vacuole formation associated with increase in the formation of acidic lysosomes. The SF was subjected to purification on Sephadex LH-20, which characterised three probable steroidal saponins (sisalins) by electrospray ionisation mass spectrometry multistage (ESI-MSn). Sisalins from sisal may be responsible for the cytotoxicity, which involves cytoplasmatic vacuole formation and selective action for glioma cells.

Function of neuronal nitric oxide synthase enzyme in temozolomide-induced damage of astrocytic tumor cells.

Astrocytic tumors, including astrocytomas and glioblastomas, are the most common type of primary brain tumors. Treatment for glioblastomas includes radiotherapy, chemotherapy with temozolomide (TMZ) and surgical ablation. Despite certain therapeutic advances, the survival time of patients is no longer than 12-14 months. Cancer cells overexpress the neuronal isoform of nitric oxide synthase (nNOS). In the present study, it was examined whether the nNOS enzyme serves a role in the damage of astrocytoma (U251MG and U138MG) and glioblastoma (U87MG) cells caused by TMZ. First, TMZ (250 µM) triggered an increase in oxidative stress at 2, 48 and 72 h in the U87MG, U251MG and U138MG cell lines, as revealed by 2',7'-dichlorofluorescin-diacetate assay. The drug also reduced cell viability, as measured by MTT assay. U87MG cells presented a more linear decline in cell viability at time-points 2, 48 and 72 h, compared with the U251MG and U138MG cell lines. The peak of oxidative stress occurred at 48 h. To examine the role of NOS enzymes in the cell damage caused by TMZ, N(ω)-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI) were used. L-NAME increased the cell damage caused by TMZ while reducing the oxidative stress at 48 h. The preferential nNOS inhibitor 7-NI also improved the TMZ effects. It caused a 12.8% decrease in the viability of TMZ-injured cells. Indeed, 7-NI was more effective than L-NAME in restraining the increase in oxidative stress triggered by TMZ. Silencing nNOS with a synthetic small interfering (si)RNA (siRNAnNOShum_4400) increased by 20% the effects of 250 µM of TMZ on cell viability (P<0.05). Hoechst 33342 nuclear staining confirmed that nNOS knock-down enhanced TMZ injury. In conclusion, our data reveal that nNOS enzymes serve a role in the damage produced by TMZ on astrocytoma and glioblastoma cells. RNA interference with nNOS merits further studies in animal models to disclose its potential use in brain tumor anticancer therapy.

Diagnostic revision of 206 adult gliomas (including 40 oligoastrocytomas) based on ATRX, IDH1/2 and 1p/19q status.

The diagnosis of 206 low and high grade adult gliomas, including 40 oligoastrocytomas, was revised based on the immunohistochemical reactivity for the ATRX protein, IDH1/2 mutation status and 1p/19q chromosomal status. All oligodendrogliomas kept the initial diagnosis. Astrocytomas did not change diagnosis in 30 of 36 cases (83.3 %); four of 36 (11.1 %) cases were reclassified as oligodendroglioma, one (2.8 %) as DNT and the other (2.8 %) as reactive gliosis. Oligoastrocytomas changed diagnosis in 35 of 40 (87.5 %) cases, being reclassified 22 of 40 (55 %) as astrocytoma, 11 of 40 (27.5 %) as oligodendroglioma and two of 40 (5 %) as reactive gliosis. Four (10 %) remained unclassifiable. In one case only (2.5 %), the diagnosis of oligoastrocytoma could not be excluded since tumor astrocytes and tumor oligodendrocytes coexisted in mixed tumor areas. In the GBM tumor subgroup, GBMO disappeared because they were not substantiated by molecular genetics. Pilocytic astrocytomas retained ATRX expression. Loss of nuclear ATRX protein expression was strongly associated to IDH1/2 mutations (p = 0.0001) and mutually exclusive with total 1p/19q co-deletion (p = 0.0001). In astrocytic tumors, loss of immunoreactivity for the ATRX protein was significantly associated to the ALT phenotype (p = 0.0003). The constitutive ATRX expression in microglia/macrophages may be misleading, especially in the identification of an oligodendroglial tumor infiltration. Of paramount importance in the recognition of oligodendroglial and astrocytic tumor cells were the double immunostainings for ATRX/GFAP, ATRX/IDH1R132H, ATRX/Iba-1 and ATRX/CD68.

CPEB4 interacts with Vimentin and involves in progressive features and poor prognosis of patients with astrocytic tumors.

Cytoplasmic polyadenylation element binding protein 4 (CPEB4) is a regulator of gene transcription and has been reported to be associated with biological malignancy in cancers. However, it is unclear whether CPEB4 has any clinical significance in patients with astrocytic tumors, and mechanisms that CPEB4 contribute to progression of astrocytic tumors remain largely unknown. Here, correlation between CPEB4 expression and prognosis of patients with astrocytic tumors were explored by using qPCR, WB and IHC, and X-tile, SPSS software. Cell lines U251 MG and A172 were used to study CPEB4's function and mechanisms. Co-immunoprecipitation, mass spectrometry, immunofluorescent assay, and western blot were performed to observe the interaction between CPEB4 and Vimentin. CPEB4 mRNA and protein levels were markedly elevated in 12/12 astrocytic tumors in comparison to paratumor. High expression of CPEB4 was significantly correlated with clinical progressive futures and work as an independent adverse prognostic factor for overall survival of patients with astrocytic tumors (relative risk 4.5, 95% CI 2.1-11.2, p = 0.001). Moreover, knockdown of CPEB4 in astrocytic tumor cells inhibited their proliferation ability , clonogenicity, and invasiveness. Five candidate proteins, GRP78, Mortalin, Keratin, Vimentin, and β-actin, were identified, and the interaction between CPEB4 and Vimentin was finally confirmed. Downregulation of CPEB4 could reduce the protein expression of Vimentin. Our studies first validated that CPEB4 interacts with Vimentin and indicated that high CPEB4 expression in astrocytic tumors correlates closely with a clinically aggressive future, and that CPEB4 might represent a valuable prognostic marker for patients with astrocytic tumors.

CBIO-16NON-STEROIDAL ANTI-INFLAMMATORY DRUGS ENHANCE THE THERAPEUTIC EFFECTS OF TEMOZOLOMIDE AND RADIATION AGAINST MALIGNANT ASTROCYTIC TUMOR CELLS IN VITRO

BACKGROUND: The present study aimed to define the anti-tumor effects of aspirin (ASA) and celecoxib (CXB) alone or combined with temozolomide (TMZ) and/or irradiation (XRT), against C6, U87MG and T98G malignant astrocytic tumor cell lines in vitro. METHODS: Cell viability, cell cycles and apoptosis were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry and terminal deoxynucleotidyl transferase-mediated nick end-labeling assays, respectively. RESULTS: Aspirin and CXB exerted similar anti-tumor effects by inducing cell cycle arrest and apoptosis. Aspirin and CXB induced the G0/G1 and G2/M phases of cell cycle arrest respectively, in all three malignant astrocytic tumor cell lines, whereas ASA with TMZ induced G2/M cell cycle arrest in the C6 and U87MG lines. The viability of malignant astrocytic tumor cell lines was reduced more effectively by ASA or CXB when combined with TMZ and XRT than by any of these agents alone or by a combination of any two modalities in the C6 and T98G cell lines. CONCLUSIONS: Both ASA and CXB enhance the therapeutic effects of concomitant XRT and TMZ and thus might serve as a novel strategy for treating malignant astrocytic tumors.

Protein interacting with C kinase 1 suppresses invasion and anchorage-independent growth of astrocytic tumor cells.

Astrocytic tumors are the most common form of primary brain tumor. Astrocytic tumor cells infiltrate the surrounding CNS tissue, allowing them to evade removal upon surgical resection of the primary tumor. Dynamic changes to the actin cytoskeleton are crucial to cancer cell invasion, but the specific mechanisms that underlie the particularly invasive phenotype of astrocytic tumor cells are unclear. Protein interacting with C kinase 1 (PICK1) is a PDZ and BAR domain-containing protein that inhibits actin-related protein 2/3 (Arp2/3)-dependent actin polymerization and is involved in regulating the trafficking of a number of cell-surface receptors. Here we report that, in contrast to other cancers, PICK1 expression is down-regulated in grade IV astrocytic tumor cell lines and also in clinical cases of the disease in which grade IV tumors have progressed from lower-grade tumors. Exogenous expression of PICK1 in the grade IV astrocytic cell line U251 reduces their capacity for anchorage-independent growth, two-dimensional migration, and invasion through a three-dimensional matrix, strongly suggesting that low PICK1 expression plays an important role in astrocytic tumorigenesis. We propose that PICK1 negatively regulates neoplastic infiltration of astrocytic tumors and that manipulation of PICK1 is an attractive possibility for therapeutic intervention.

Mixed Glioma (Oligoastrocytoma) in the Brain of an African Hedgehog (Atelerix albiventris)

This report describes an oligoastrocytoma in the brain of a 3.5-year-old female pet African hedgehog (Atelerix albiventris) that showed progressive central nervous system signs for 6 months. Microscopical examination of the brain revealed a widely infiltrative, deep-seated glioma within the white matter of the cerebral hemispheres, basal nuclei, hippocampus, thalamus, midbrain, pons and the medulla of the cerebellum with extension of neoplastic cells into the cerebral cortex and overlying leptomeninges. Morphological features of the neoplastic cells, together with variable immunohistochemical expression of glial fibrillary acidic protein, Olig-2 and Nogo-A, indicated the presence of intermingled astrocytic and oligodendroglial tumour cells with an astrocytic component of approximately 40% consistent with an oligoastrocytoma. The distribution of the tumour is consistent with gliomatosis cerebri.

Sulforhodamine 101 selectively labels human astrocytoma cells in an animal model of glioblastoma

Sulforhodamine 101 (SR101) is a useful tool for immediate staining of astrocytes. We hypothesized that if the selectivity of SR101was maintained in astrocytoma cells, it could prove useful for glioma research. Cultured astrocytoma cells and acute slices from orthotopic human glioma (n=9) and lymphoma (n=6) xenografts were incubated with SR101 and imaged with confocal microscopy. A subset of slices (n=18) were counter-immunostained with glial fibrillary acidic protein and CD20 for stereological assessment of SR101 co-localization. SR101 differentiated astrocytic tumor cells from lymphoma cells. In acute slices, SR101 labeled 86.50% (±1.86; p<0.0001) of astrocytoma cells and 2.19% (±0.47; p<0.0001) of lymphoma cells. SR101-labeled astrocytoma cells had a distinct morphology when compared with in vivo astrocytes. Immediate imaging of human astrocytoma cells in vitro and in ex vivo rodent xenograft tissue labeled with SR101 can identify astrocytic tumor cells and help visualize the tumor margin. These features are useful in studying astrocytoma in the laboratory and may have clinical applications.

Rat astrocytic tumour cells are associated with an anti‐inflammatory microglial phenotype in an organotypic model

Microglia form a high proportion of cells in glial tumours but their role in supporting or inhibiting tumour growth is unclear. Here we describe the establishment of an in vitro model to investigate their role in astrocytomas. Rat hippocampal slices were prepared and, after 7 days to allow microglia to become quiescent, rat C6 astrocytic tumour cells were added. Over the following 7 days, infiltration and cell death were studied using fluorescent C6 tumour cells and confocal microscopy; immunophenotyping of microglia was performed using CD68 (phagocytosis), MHCII (antigen-presentation) and Iba1 (microglial marker regardless of functional state). Cell proliferation was assessed using Ki67 and qPCR to detect cytokine expression. Sham and control groups were included. Microscopy showed proliferation of C6 tumour cells with both infiltration of tumour cells into the hippocampal tissue and of microglia among the tumour cells. Confocal experiments confirmed increasing tumour cell infiltration into the hippocampal slice with time (P<0.001), associated with cell death (σ=0.313, P=0.022). Ki67 showed increased proliferation (P<0.001), of both tumour cells and Iba1+ microglia and increased microglial phagocytosis (CD68: P<0.001). Expression of pro-inflammatory cytokines IL1, IL6 and TNFα were downregulated with expression of the anti-inflammatory cytokine TGFβ1 maintained. This model allows study of the proliferation and infiltration of astrocytic tumour cells in central nervous system tissue and their interaction with microglia. Our data suggest that microglial function is altered in the presence of tumour cells, putatively facilitating tumour progression. Manipulation of the microglial functional state may have therapeutic value for astrocytic tumours.

Role of GalNAc4S-6ST in Astrocytic Tumor Progression

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is the sulfotransferase responsible for biosynthesis of highly sulfated chondroitin sulfate CS-E. Although involvements of CS-E in neuronal cell functions have been extensively analyzed, the role of GalNAc4S-6ST in astrocytic tumor progression remains unknown. Here, we reveal that GalNAc4S-6ST transcripts were detected in astrocytic tumors derived from all 30 patients examined using quantitative reverse transcription-PCR analysis. Patients with high GalNAc4S-6ST mRNA expression had significantly worse outcome compared with patients with low expression, and multivariate survival analysis disclosed that GalNAc4S-6ST is an independent poor prognostic factor for astrocytic tumors. We then tested whether CS-E enhanced haptotaxic migration of glioblastoma U251-MG cells that endogenously express both the CS-E’s scaffold tyrosine phosphatase ζ (PTPζ) and GalNAc4S-6ST, in the presence of CS-E’s preferred ligands, pleiotrophin (PTN) or midkine (MK), using a modified Boyden chamber method. Haptotaxic stimulation of cell migration by PTN was most robust on control siRNA-transfected U251-MG cells, while that enhancing effect was cancelled following transduction of GalNAc4S-6ST siRNA. Similar results were obtained using MK, suggesting that both PTN and MK enhance migration of U251-MG cells by binding to CS-E. We also found that PTPζ as well as PTN and MK were frequently expressed in astrocytic tumor cells. Thus, our findings indicate that GalNAc4S-6ST mRNA expressed by astrocytic tumor cells is associated with poor patient prognosis likely by enhancing CS-E-mediated tumor cell motility in the presence of PTN and/or MK.

Immunohistochemical and molecular genetics study of a granular cell astrocytoma: A case report of malignant transformation to a glioblastoma

We treated a 56-year-old woman who had a right temporal lobe tumor found by chance after a traffic accident. MRI confirmed a heterogeneously enhanced tumor in the temporal lobe with large peritumoral edema extending to the superior parietal lobe. The patient underwent tumor resection. The tumor consisted largely of distinct cells with discrete borders and granular cytoplasm. In granular cells, the accumulation of PAS-positive granules was observed. Immunohistochemical analysis demonstrated positive staining for GFAP, S-100, and oligodendrocyte transcription factor 2 and negative staining for synaptophysin. CD68 was negative in granular cells, but positive in stromal cells. Ki-67 labeling index was quite low. The tumor was diagnosed as a granular cell astrocytoma (GCA). Postoperative radiotherapy combined with temozolomide was administered. One month after chemoradiotherapy, the tumor occurred in the parietal lobe, and a tumorectomy was performed. The tumor was composed of poorly differentiated astrocytic tumor cells with prominent microvascular proliferation and necrosis. A small number of granular cells were locally observed and the tumor was diagnosed as a glioblastoma. O6-methylguanine-DNA methyltransferase promoter methylation was detected in the GCA but not in the glioblastoma. Isocitrate dehydrogenase mutations were not detected in either tumor. Comparative genomic hybridization analysis demonstrated that no chromosomal abnormality was found in the GCA; however, a gain of chromosomes 7 and 19 and a loss of chromosomes 10 and 9p21 (CDKN2A) were found in the glioblastoma. p53 was strongly expressed in both the GCA and glioblastoma. The tumor progressed despite extensive chemotherapy, and the patient died 1 year after the initial treatment. Our immunohistochemical, genetic and chromosomal analyses indicate that the glioblastoma was transformed from the GCA.

Dual role of the mitochondrial protein frataxin in astrocytic tumors

The mitochondrial protein frataxin (FXN) is known to be involved in mitochondrial iron homeostasis and iron–sulfur cluster biogenesis. It is discussed to modulate function of the electron transport chain and production of reactive oxygen species (ROS). FXN loss in neurons and heart muscle cells causes an autosomal-dominant mitochondrial disorder, Friedreich's ataxia. Recently, tumor induction after targeted FXN deletion in liver and reversal of the tumorigenic phenotype of colonic carcinoma cells following FXN overexpression were described in the literature, suggesting a tumor suppressor function. We hypothesized that a partial reversal of the malignant phenotype of glioma cells should occur after FXN transfection, if the mitochondrial protein has tumor suppressor functions in these brain tumors. In astrocytic brain tumors and tumor cell lines, we observed reduced FXN levels compared with non-neoplastic astrocytes. Mitochondrial content (citrate synthase activity) was not significantly altered in U87MG glioblastoma cells stably overexpressing FXN (U87-FXN). Surprisingly, U87-FXN cells exhibited increased cytoplasmic ROS levels, although mitochondrial ROS release was attenuated by FXN, as expected. Higher cytoplasmic ROS levels corresponded to reduced activities of glutathione peroxidase and catalase, and lower glutathione content. The defect of antioxidative capacity resulted in increased susceptibility of U87-FXN cells against oxidative stress induced by H2O2 or buthionine sulfoximine. These characteristics may explain a higher sensitivity toward staurosporine and alkylating drugs, at least in part. On the other hand, U87-FXN cells exhibited enhanced growth rates in vitro under growth factor-restricted and hypoxic conditions and in vivo using tumor xenografts in nude mice. These data contrast to a general tumor suppressor function of FXN but suggest a dual, pro-proliferative but chemosensitizing role in astrocytic tumors.

Cytomorphology of giant cell glioblastoma: Report of a case and brief review of literature

Giant cell glioblastoma is a histological variant of glioblastoma that accounts for less than 1% of intracranial tumors and to 5% of glioblastoma. They occur at any age and are likely to affect the younger as well the older age group unlike the conventional glioblastoma multiforme (GBM). They are often located subcortically in the temporal and parietal lobes. Cytological descriptions of giant cell glioblastoma are extremely rare. We describe squash cytomorphology of giant cell glioblastoma of left posterior frontal region in 35-year-old male. The squash smears were moderately cellular displaying malignant astrocytic tumor cells disposed in cohesive clusters and dispersed population on a necrotic background. Most striking feature was numerous multinucleated giant cells. We also discuss the differential diagnosis in light of relevant literature.

Long survival of primary diffuse leptomeningeal gliomatosis following radiotherapy and temozolomide: case report and literature review

ObjectivePrimary diffuse leptomeningeal gliomatosis (PDLG) is a rare neoplasm with a short survival time of a few months. there is currently no standardized therapeutic approach for PDLG.Materials and methodsWe report on a 53-year-old male patient who presented with epileptic seizures, gait disturbance, paraparesis and sensory deficits in the dermatomes T8-10.ResultsMagnetic resonance imaging (MRI) revealing numerous spinal and cranial gadolinium-enhancing nodules in the meninges and histopathology led us to diagnose primary diffuse leptomeningeal gliomatosis with WHO grade III astrocytic cells. Consecutively, the patient underwent craniospinal radiotherapy (30 Gy) and 11 sequential cycles of temozolomide. This regimen led to partial tumor regression. Thirteen months later, spinal MRI revealed tumor progression. Second-line chemotherapy with 5 cycles of irinotecan and bevacizumab did not prevent further clinical deterioration. The patient died twenty-two months after diagnosis, being the longest survival time described thus far with respect to PDLG consisting of astrocytic tumor cells.ConclusionsRadiochemotherapy including temozolomide, as established standard therapy for brain malignant astrocytomas, might be valid as a basic therapeutic strategy for this PDLG subtype.

Secreted protein, acidic and rich in cysteine (SPARC) expression in astrocytic tumour cells negatively correlates with proliferation, while vascular SPARC expression is associated with patient survival

Secreted protein, acidic and rich in cysteine (SPARC) is a regulator of cell-matrix interaction and has been associated with tumour stage and patient survival in various malignancies. As no large-scale study has yet been undertaken, we investigated human brain and astrocytomas for SPARC expression and associations with tumour grade, proliferation, vascular density and patient survival. A spectrum of 188 WHO grade I-IV astrocytic tumours and 24 autopsy cases were studied by immunohistochemistry for SPARC, MIB-1 proliferation index and CD31-positive vessels. SPARC protein expression was confirmed by quantitative real-time polymerase chain reaction and Western blot in 13 cases. In normal brain, SPARC is expressed in cortical marginal glia, cerebellar Bergmann glia and focally in white matter but is absent in neurones or vessels. High SPARC expression levels in the cytoplasm of astrocytic tumour cells decreased with the grade of malignancy but showed an increase with grade of malignancy in tumour vessels. SPARC negatively correlated with tumour proliferation but not with vascular density. While cytoplasmic SPARC staining was not associated with survival, vascular SPARC showed a significant association in the group of grade II-IV tumours (P = 0.02) and also in grade II astrocytomas alone (P = 0.01) with vascular SPARC associated with worse prognosis. SPARC is highly expressed in astrocytomas and decreases with tumour progression. We confirm an association of increased SPARC expression and decreased proliferation. While there is no association between the level of SPARC in the tumour cells and patient survival, increased tumour vascular SPARC expression is associated with decreased patient survival.