Molecular predictors for decitabine efficacy in meningiomas - a pilot study.

PURPOSE: Effective chemotherapeutical agents for the treatment of meningiomas are still lacking. Previous in-vitro analyses revealed efficacy of decitabine (DCT), a DNA methyltransferase (DNMT) inhibitor established in the treatment of leukemia, in a yet undefined subgroup of meningiomas. METHODS: Effects of DCT on proliferation and viability was analyzed in primary meningioma cells by immunofluorescence and MTT assays, and cases were classified as drug responders and non-responders. Molecular preconditions for efficacy were analyzed using immunofluorescence for Ki67, DNMT1, and five oncogenes (TRIM58, FAM84B, ELOVL2, MAL2, LMO3) previously found to be differentially methylated after DCT exposition, as well as by genome-wide DNA methylation analyses. RESULTS: Efficacy of DCT (10µM) was found in eight (62%) of 13 meningioma cell lines 48 h after drug exposition (p < .05). DCT significantly reduced DNMT1 expression in all but two cell lines, and median ΔDNMT1 reduction 48 h after drug exposition was lower in DCT-resistant (-11.1%) than in DCT-sensitive (-50.5%, p = .030) cells. Rates of cell lines responsive to DCT exposition distinctly decreased to 25% after 72 h. No significant correlation of the patients´ age, sex, histological subtype, location of the paternal tumor, expression of Ki67, DNMT1 or the analyzed oncogenes with treatment response was found (p > .05, each). DCT efficacy was further independent of the methylation class and global DNA methylation of the paternal tumor. CONCLUSION: Early effects of DCT in meningiomas are strongly related with DNMT1 expression, while clinical, histological, and molecular predictors for efficacy are sparse. Kinetics of drug efficacy might indicate necessity of repeated exposition and encourage further analyses.

Protoporphyrin IX (PpIX) Fluorescence during Meningioma Surgery: Correlations with Histological Findings and Expression of Heme Pathway Molecules.

Background: The usefulness of 5-ALA-mediated fluorescence-guided resection (FGR) in meningiomas is controversial, and information on the molecular background of fluorescence is sparse. Methods: Specimens obtained during 44 FGRs of intracranial meningiomas were analyzed for the presence of tumor tissue and fluorescence. Protein/mRNA expression of key transmembrane transporters/enzymes involved in PpIX metabolism (ABCB6, ABCG2, FECH, CPOX) were investigated using immunohistochemistry/qPCR. Results: Intraoperative fluorescence was observed in 70 of 111 specimens (63%). No correlation was found between fluorescence and the WHO grade (p = 0.403). FGR enabled the identification of neoplastic tissue (sensitivity 84%, specificity 67%, positive and negative predictive value of 86% and 63%, respectively, AUC: 0.75, p < 0.001), and was improved in subgroup analyses excluding dura specimens (86%, 88%, 96%, 63% and 0.87, respectively; p < 0.001). No correlation was found between cortical fluorescence and tumor invasion (p = 0.351). Protein expression of ABCB6, ABCG2, FECH and CPOX was found in meningioma tissue and was correlated with fluorescence (p < 0.05, each), whereas this was not confirmed for mRNA expression. Aberrant expression was observed in the CNS. Conclusion: FGR enables the intraoperative identification of meningioma tissue with limitations concerning dura invasion and due to ectopic expression in the CNS. ABCB6, ABCG2, FECH and CPOX are expressed in meningioma tissue and are related to fluorescence.

Expression of decitabine-targeted oncogenes in meningiomas in vivo.

Treatment of meningiomas refractory to surgery and irradiation is challenging and effective chemotherapies are still lacking. Recently, in vitro analyses revealed decitabine (DCT, 5-aza-2'-deoxycytidine) to be effective in high-grade meningiomas and, moreover, to induce hypomethylation of distinct oncogenes only sparsely described in meningiomas in vivo yet.Expression of the corresponding onco- and tumor suppressor genes TRIM58, FAM84B, ELOVL2, MAL2, LMO3, and DIO3 were analyzed and scored by immunohistochemical staining and RT-PCR in samples of 111 meningioma patients. Correlations with clinical and histological variables and prognosis were analyzed in uni- and multivariate analyses.All analyzed oncogenes were highly expressed in meningiomas. Expression scores of TRIM58 tended to be higher in benign than in high-grade tumors 20 vs 16 (p = .002) and all 9 samples lacking TRIM58 expression displayed WHO grade II/III histology. In contrast, median expression scores for both FAM84B (6 vs 4, p ≤ .001) and ELOVL2 (9 vs 6, p < .001) were increased in high-grade as compared to benign meningiomas. DIO3 expression was distinctly higher in all analyzed samples as compared to the reference decitabine-resistant Ben-Men 1 cell line. Increased ELOVL2 expression (score ≥ 8) correlated with tumor relapse in both uni- (HR: 2.42, 95%CI 1.18-4.94; p = .015) and multivariate (HR: 2.09, 95%CI 1.01-4.44; p = .046) analyses.All oncogenes involved in DCT efficacy in vitro are also widely expressed in vivo, and expression is partially associated with histology and prognosis. These results strongly encourage further analyses of DCT efficiency in meningiomas in vitro and in situ.

5-ALA kinetics in meningiomas: analysis of tumor fluorescence and PpIX metabolism in vitro and comparative analyses with high-grade gliomas.

INTRODUCTION: Although the utility 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery (FGS) in meningiomas is increasingly discussed, data about the kinetics of protoporphyrin IX (PpIX) and tumor fluorescence are sparse. METHODS: PpIX kinetics after exposition to varying 5-ALA doses (12.5-150 µg/ml) was analyzed in two immortalized as well as primary WHO grade I and II meningioma and U87 high-grade glioma cell lines. Expression of FECH, ABCB6 and ABCG2 was investigated by quantitative real-time PCR. RESULTS: Fluorescence in Ben-Men 1 and primary WHO grade I/II meningioma increased with rising 5-ALA doses up to 100 µg/ml but then showed a saturation effect. However, decrease of fluorescence was slower after 150 than after 100 µg/ml 5-ALA. Fluorescence in U87 cells marginally increased with rising 5-ALA doses. Kinetics of the fluorescence in Ben-Men 1 cells did not differ from primary meningioma cells after 25-150 µg/ml 5-ALA (p > .05, each). No difference was found when comparing the fluorescence between primary grade I and II meningiomas after any 5-ALA dosage (p > .05, each). No relevant fluorescence was found in IOMM-Lee cells. Expression of FECH, ABCB6 and ABCG2 as well as PpIX export differed between all analyzed cell lines but were not connected to fluorescence. CONCLUSIONS: Eligibility of established meningioma cell lines for in-vitro analyzes of tumor fluorescence significantly differs. Fluorescence in Ben-Men 1 and primary meningioma cell lines but less in IOMM Lee cells is 5-ALA dose-dependent, encouraging in-situ trials to encounter currently discussed shortcomings of FGS in meningiomas. Fluorescence is not related to expression of FECH, ABCB6 and ABCG2.

Efficacy of decitabine in malignant meningioma cells: relation to promoter demethylation of distinct tumor suppressor and oncogenes and independence from TERT.

OBJECTIVE: Chemotherapeutic options for meningiomas refractory to surgery or irradiation are largely unknown. Human telomerase reverse transcriptase (hTERT) promoter methylation with subsequent TERT expression and telomerase activity, key features in oncogenesis, are found in most high-grade meningiomas. Therefore, the authors investigated the impact of the demethylating agent decitabine (5-aza-2'-deoxycytidine) on survival and DNA methylation in meningioma cells. METHODS: hTERT promoter methylation, telomerase activity, TERT expression, and cell viability and proliferation were investigated prior to and after incubation with decitabine in two benign (HBL-52 and Ben-Men 1) and one malignant (IOMM-Lee) meningioma cell line. The global effects of decitabine on DNA methylation were additionally explored with DNA methylation profiling. RESULTS: High levels of TERT expression, telomerase activity, and hTERT promoter methylation were found in IOMM-Lee and Ben-Men 1 but not in HBL-52 cells. Decitabine induced a dose-dependent significant decrease of proliferation and viability after incubation with doses from 1 to 10 µM in IOMM-Lee but not in HBL-52 or Ben-Men 1 cells. However, effects in IOMM-Lee cells were not related to TERT expression, telomerase activity, or hTERT promoter methylation. Genome-wide methylation analyses revealed distinct demethylation of 14 DNA regions after drug administration in the decitabine-sensitive IOMM-Lee but not in the decitabine-resistant HBL-52 cells. Differentially methylated regions covered promoter regions of 11 genes, including several oncogenes and tumor suppressor genes that to the authors' knowledge have not yet been described in meningiomas. CONCLUSIONS: Decitabine decreases proliferation and viability in high-grade but not in benign meningioma cell lines. The effects of decitabine are TERT independent but related to DNA methylation changes of promoters of distinct tumor suppressor genes and oncogenes.

Real-time in vivo kinetics of protoporphyrin IX after administration of 5-aminolevulinic acid in meningiomas and comparative analyses with glioblastomas.

BACKGROUND: The usefulness of 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery (FGS) in meningiomas is intensely discussed. However, data about kinetics of 5-ALA and protoporphyrin (Pp) IX in meningiomas are lacking. METHODS: As the first study so far, we performed longitudinal intraoperative real-time ex situ measurements of fluorescence intensity and PpIX concentrations during FGS of ten benign and two atypical meningiomas. Kinetics were subsequently compared with data from 229 glioblastomas. RESULTS: Spectroscopy revealed fluorescence (median 2945.65 a.u.) and PpIX accumulation (median 18.31 µg/ml) in all 43 analyzed samples. Fluorescence intensity (2961.50 a.u. vs 118.41 a.u.; p < .001) and PpIX concentrations (18.72 µg/ml vs .98 µg/ml; p < .001) were higher in samples with (N = 30) than without (N = 2) visible intraoperative tumor fluorescence. ROC curve analyses revealed a PpIX cut-off concentration of 3.85 µg/ml (AUC = .992, p = .005) and a quantitative fluorescence cut-off intensity of 286.73 a.u. (AUC = .983, p = .006) for intraoperative visible tumor fluorescence. Neither fluorescence intensity (p = .356) nor PpIX (p = .631) differed between atypical and benign meningiomas. Fluorescence and PpIX peaked 7-8 h following administration of 5-ALA. Meningiomas displayed a higher fluorescence intensity (p = .012) and PpIX concentration (p = .005) than glioblastomas 5-6 h after administration of 5-ALA. Although fluorescence was basically maintained, PpIX appeared to be cleared faster in meningiomas than in glioblastomas. CONCLUSIONS: Kinetics of PpIX and fluorescence intensity differ between meningiomas and glioblastomas in the early phase after 5-ALA administration. Modification of the timing of drug administration might impact visibility of intraoperative fluorescence and helpfulness of FGS and should be investigated in future analyses.

Telomerase activity, TERT expression, hTERT promoter alterations, and alternative lengthening of the telomeres (ALT) in meningiomas - a systematic review.

Telomerase activity and (human) Telomerase Reverse Transcriptase (hTERT) expression are considered hallmarks in oncogenesis of neoplasms and are upregulated by alterations of the hTERT promoter. In meningiomas, numerous studies investigated hTERT expression, telomerase activity, promoter mutations, and methylations. Moreover, reports about hTERT-targeted chemotherapy in meningiomas have recently been published. We provide a systematic review of the literature about the role of hTERT in meningiomas. TERT expression and telomerase activity is found in benign and high-grade meningiomas and increase with WHO grade. Remarkably, rates of TERT expression/telomerase activity usually exceed mutation frequency and both telomerase activity and TERT expression have also been found in hTERT promoter wildtype meningiomas, indicating further mechanisms of TERT upregulation. Although hTERT promoter methylation has been reported in the vast majority of meningiomas, correlation with TERT expression remains controversial. Rates of promoter mutations, and methylation were shown to increase with rising WHO grade. Moreover, promoter methylation and mutations strongly correlate with prognosis. Although mutations predicted malignant progression, de novo mutations in high-grade recurrences of former benign lesions were also observed. Retroviral transduction of the TERT gene enabled immortalization in several grade I-III meningioma cell lines. In vitro analyses revealed significant effects on viability in hTERT-mutated meningioma cells after targeted treatment. Alternative mechanisms of telomere lengthening are usually absent in meningiomas. TERT and hTERT promoter alterations play a major role during oncogenesis of meningiomas with implications for prognosis and potentially treatment.

Testing ATRA and MEK inhibitor PD0325901 effectiveness in a nude mouse model for human MPNST xenografts.

OBJECTIVE: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas characterized by high recurrence rates and early metastases. These tumors arise more frequently within neurofibromatosis type 1 (NF1) and present with resistance during standard chemotherapy leading to increased mortality and morbidity in those patients. In vitro all-trans retinoic acid (ATRA) and MEK inhibitors (MEKi) were shown to inhibit tumor proliferation, especially when applied in combination. Therefore, we established a nude mouse model to investigate if treatment of xenografts derived from NF1 associated S462 and T265 MPNST cells respond to ATRA and the MEKi PD0325901. RESULTS: We demonstrated that human NF1 associated MPNST derived from S462 but not T265 cells form solid subcutaneous tumors in Foxn1 nude mice but not in Balb/c, SHO or Shorn mice. We verified a characteristic staining pattern of human MPNST xenografts by immunohistochemistry. Therapeutic effects of ATRA and/or MEKi PD0325901 on growth of S462 MPNST xenografts in Foxn1 nude mice were not demonstrated in vitro, as we did not observe significant suppression of MPNST growth compared with placebo treatment.

In-Vitro Use of 5-ALA for Photodynamic Therapy in Pediatric Brain Tumors.

BACKGROUND: Light irradiation (635 nm) of cells containing protoporphyrin IX (PPIX) after 5- aminolevulinic acid (5-ALA) pretreatment causes cell death via different pathways including apoptosis and necrosis, as previously demonstrated for malignant glioma cells. OBJECTIVE: To elucidate whether various malignant pediatric brain tumors, which have been shown to accumulate PPIX, would also be susceptible to photodynamic therapy (PDT). METHODS: Medulloblastoma (DAOY, UW228), pNET (PFSK-1), and rhabdoid tumor (BT16) cell lines were incubated with 5-ALA in variable concentrations for 4 h. Consequently, cells were irradiated by 635 nm diode laser light. After 12 h, cell viability was measured by WST-1 testing and these results were compared to control cells incubated with 5-ALA without irradiation or irradiation only without prior incubation with 5-ALA. RESULTS: We demonstrated significant cell death in malignant pediatric tumor cells after incubation with 5-ALA and laser irradiation in comparison to control groups. In all cell lines, we noticed significant cell death above a 5-ALA concentration of 50 µg/ml (P < .05). Neither 5-ALA incubation alone nor irradiation alone caused cell death. DAOY and PFSK cell lines were more susceptible than UW228 and BT16 cells. CONCLUSION: We conclude that PDT causes cell death with higher PPIX concentrations after exposure to 5-ALA in vitro in accordance to similar studies with glioma cells. This indicates that PDT might be feasible for eliminating brain tumor cells in malignant pediatric brain tumors. Additionally, we noticed a dependency between fluorescence intensity and death rates.

Visualizing protoporphyrin IX formation in the dura tail of meningiomas by mass spectrometry imaging.

BACKGROUND: The advantages of 5-aminolevulinacid (5-ALA)-induced fluorescence-guided surgery in meningiomas are increasingly discussed. In this context, despite detectable tumor tissue in histopathologial analyses, no fluorescence was shown at the dura tail using the standard operating microscope. Thus, 5-ALA metabolism in this surgically important site remains unknown but needs to be elucidated when further evaluating indications of fluorescence-guided surgery in meningiomas. METHOD: We here present the spatially resolved identification of protoporphyrin IX (PpIX) in sphenoid ridge meningioma cryosections from a patient who underwent fluorescence-guided microsurgery using molecular imaging analysis by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). RESULTS: Despite a strong fluorescence of the main tumor, no fluorescence could be detected at the dura tail using the standard operating microscope (blue-light, 405 nm). However, histopathological analyses clearly showed meningioma tissue. Remarkably, MALDI-MS/MS analysis revealed PpIX formation also at the non-fluorescing dura tail. However, no PpIX was detected in the tumor free dura mater. CONCLUSION: MALDI-MS/MS visualized a selective accumulation of PpIX within the tumor tissue including the dura tail. Thus, absence of fluorescence in the dura tail as visualized by the operating microscope is not caused by the lack of PpIX formation.

Molecular Analysis of Hybrid Neurofibroma/Schwannoma Identifies Common Monosomy 22 and α-T-Catenin/CTNNA3 as a Novel Candidate Tumor Suppressor.

Neurofibromas and schwannomas are benign Schwann cell-derived peripheral nerve sheath tumors arising sporadically and within neurofibromatoses. Multiple tumors are a hallmark of neurofibromatosis type 1 (NF1) and type 2 (NF2) and schwannomatosis. Neurofibromas in NF1 and schwannomas in NF2 or schwannomatosis are defined by distinctive molecular hits. Among these, multiple hybrid neurofibromas/schwannomas may also appear, not yet being defined by a molecular background. We therefore performed molecular analysis of 22 hybrid neurofibromas/schwannomas using array comparative genomic hybridization, immunohistochemistry, quantitative RT-PCR, and functional analyses of cultured Schwann cells. Furthermore, we analyzed SMARCB1 by fluorescence in situ hybridization and multiplex ligation-dependent probe. Monosomy 22 was identified in 44% of tumors of tested patients with hybrid neurofibromas/schwannomas. In addition, in a single case, we detected focal deletion of the α-T-catenin/CTNNA3 gene (10q21.3). To further characterize this candidate, transient knockdown of α-T-catenin in Schwann cells was performed. CTNNA3 depleted cells showed cytoskeletal abnormalities and reduced E-cadherin expression, indicating epithelial-mesenchymal transition-like abnormalities. To conclude, we uncovered loss of chromosome 22 in almost half of all cases with hybrid neurofibromas/schwannomas of patients with multiple peripheral nerve sheath tumors. We tagged α-T-catenin/CTNNA3 as a novel candidate gene. Our functional investigations might indicate involvement of α-T-catenin/CTNNA3 in the biology of peripheral nerve sheath tumors.

5-ALA Fluorescence in Native Pituitary Adenoma Cell Lines: Resection Control and Basis for Photodynamic Therapy (PDT)?

OBJECTIVE: Pituitary adenomas (PA), especially invasive ones, are often not completely resectable. Usage of 5-aminolevulinic acid (5-ALA) for fluorescence guided surgery could improve the rate of total resection and, additionally, open the doors for photodynamic therapy (PDT) in case of unresectable or partially resected PAs. The aim of this study was to investigate the uptake of 5-ALA and the effect of 5-ALA based PDT in cell lines. METHODS: GH3 and AtT-20 cell lines were incubated with different concentrations of 5-ALA, protoporphyrin IX (PPIX) fluorescence was measured by flow cytometry and fluorescencespectrometry. WST-1 assays were performed to determine the surviving fraction of cells after PDT. PPIX fluorescence intensities and PDT effect of the pituitary adenoma cells were compared to U373MG, a well-known glioblastoma cell line. RESULTS: Both cell lines showed a 5-ALA dependent intracellular PPIX fluorescence. Significant differences after 24hrs of incubation were observed in AtT-20 cells in comparison to GH3. Regardless of the incubation or metabolism time, there was a proliferation inhibiting effect after PDT, with no statistical significance. CONCLUSION: Since GH3 cells showed a heterogenous uptake of 5-ALA in the flow cytometry profile, but not constantly high concentrations they might have a 5-ALA efflux mechanism, which still needs to be determined. In the case of AtT-20, the cells might need a longer time for the uptake due to their size or slow metabolism. We showed that the different cell lines have different uptake and metabolism mechanisms, which needs to be further investigated. The general uptake of 5-ALA allows the possibility of resection control and PDT for pituitary adenomas. But, the role of PDT for unresectable pituitary adenomas deserves further investigations.

hTERT promoter methylation in meningiomas and central nervous hemangiopericytomas.

In meningiomas, prognostic impact of mutations in the human telomerase reverse transcriptase (hTERT) promoter region was recently shown, while studies of promoter methylation and analyses of hemangiopericytomas are lacking. hTERT promoter methylation was analyzed in 78 meningioma and 38 meningeal hemangiopericytoma samples by methylation-specific polymerase chain reaction (MS-PCR) and compared with histopathological and clinical variables and with immunohistochemical hTERT expression. Promoter methylation was found in 62 samples (53 %) and tended to be higher in meningiomas (N = 19/41, 46 %) than in hemangiopericytomas (N = 8/33, 24 %, p = .057). In meningiomas, methylation was 16, 60 and 77 % in grade I, II and III tumors (p < .001) and higher in recurrent (N = 33/37, 89 %) than in primary diagnosed (N = 19/41, 46 %) tumors (OR 5.14, 95 % CI 1.34-19.71, p = .017). Univariate analyses showed shorter mean progression free and overall survival in methylated than in unmethylated individuals (26 vs. 100 months; p = .045 and 110 vs. 113 months; p = .025, respectively). Moreover, hTERT expression was found in 70 % (N = 53) and was more frequent in methylated than in unmethylated samples (78 vs. 52 %, OR 3.36, 95 % CI 1.20-9.40, p = .021). In hemangiopericytomas, methylation was similar in grade II (24 %) and III (25 %, p > .05) and in primary (24 %) and recurrent tumors (40 %, p > .05). hTERT expression was similar as compared to meningiomas (74 %, N = 28, p > .05) but was independent of promoter methylation (OR 4.26, 95 % CI 0.47-39.0, p = .199). In meningeal tumors, hTERT promoter methylation is more common than mutations and in meningiomas but not in hemangiopericytomas positively correlated with WHO grade and hTERT expression.

Comparative transcriptome analysis in induced neural stem cells reveals defined neural cell identities in vitro and after transplantation into the adult rodent brain.

Reprogramming technology enables the production of neural progenitor cells (NPCs) from somatic cells by direct transdifferentiation. However, little is known on how neural programs in these induced neural stem cells (iNSCs) differ from those of alternative stem cell populations in vitro and in vivo. Here, we performed transcriptome analyses on murine iNSCs in comparison to brain-derived neural stem cells (NSCs) and pluripotent stem cell-derived NPCs, which revealed distinct global, neural, metabolic and cell cycle-associated marks in these populations. iNSCs carried a hindbrain/posterior cell identity, which could be shifted towards caudal, partially to rostral but not towards ventral fates in vitro. iNSCs survived after transplantation into the rodent brain and exhibited in vivo-characteristics, neural and metabolic programs similar to transplanted NSCs. However, iNSCs vastly retained caudal identities demonstrating cell-autonomy of regional programs in vivo. These data could have significant implications for a variety of in vitro- and in vivo-applications using iNSCs.

Fluorescence in neurosurgery: Its diagnostic and therapeutic use. Review of the literature.

Fluorescent agents, e.g. 5-aminolevulinic acid (5-ALA), fluorescein and indocyanine green (ICG) are in common use in neurosurgery for tumor resection and neurovascular surgery. Protoporphyrine IX (PPIX) as major metabolite of 5-ALA is a strong fluorescent substance accumulated within malignant glioma tissue and a very sensitive and specific tool for visualizing high grade glioma tissue during surgery. Furthermore, 5-ALA or rather PPIX also offers an intratumoral therapeutic option stimulated by laser light in specific wavelength. Fluorescein was demonstrated to show similar fluorescent reactions in neurosurgery, but is controversial in its use, especially in high grade tumor surgery. Intraoperative angiography during resection of arterio-venous malformations, extracranial-intracranial-bypass or aneurysm surgery is supported by ICG fluorescence. Generally ICG will provide beneficial information for both, exposure of the pathology and illustration of healthy structures. This manuscript shows an overview of the literature focussing fluorescence in neurosurgery.

Glioma tissue obtained by modern ultrasonic aspiration with a simple sterile suction trap for primary cell culture and pathological evaluation.

OBJECTIVES: Ultrasonic aspiration is widely used in the resection of brain tumors. Nevertheless, tumor tissue fragments obtained by ultrasonic aspiration are usually discarded. In this study, we demonstrate that these fragments are possible sources of material for histopathological study and tissue culture and compare their microscopic features and viability in tissue culture of cavitron ultrasonic surgical aspirator tissue fragments. METHODS: Brain tumor tissue collected by ultrasonic aspiration (CUSA EXcel®; Integra Radionics Inc.) in a simple sterile suction trap during resection was processed for primary cell culture. Cell viability and immunohistological markers were measured by the WST-1 test, microscopy and immunofluorescent evaluation. RESULTS: Six gliomas are presented to demonstrate that these tissue fragments show good preservation of histological detail and tissue viability in culture. CONCLUSION: Utilization of this material may facilitate pathological interpretation by providing a more representative sample of tumor histology as well as an adequate and sterile biosource of material for tissue culture studies.

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.

Histone deacetylase inhibitor AR-42 differentially affects cell-cycle transit in meningeal and meningioma cells, potently inhibiting NF2-deficient meningioma growth.

Meningiomas constitute about 34% of primary intracranial tumors and are associated with increased mortality in patients with neurofibromatosis type 2 (NF2). To evaluate potential medical therapies for these tumors, we have established a quantifiable orthotopic model for NF2-deficient meningiomas. We showed that telomerase-immortalized Ben-Men-1 benign meningioma cells harbored a single nucleotide deletion in NF2 exon 7 and did not express the NF2 protein, merlin. We also showed that AR-42, a pan-histone deacetylase inhibitor, inhibited proliferation of both Ben-Men-1 and normal meningeal cells by increasing expression of p16(INK4A), p21(CIP1/WAF1), and p27(KIP1). In addition, AR-42 increased proapoptotic Bim expression and decreased anti-apoptotic Bcl(XL) levels. However, AR-42 predominantly arrested Ben-Men-1 cells at G(2)-M whereas it induced cell-cycle arrest at G(1) in meningeal cells. Consistently, AR-42 substantially decreased the levels of cyclin D1, E, and A, and proliferating cell nuclear antigen in meningeal cells while significantly reducing the expression of cyclin B, important for progression through G(2), in Ben-Men-1 cells. In addition, AR-42 decreased Aurora A and B expression. To compare the in vivo efficacies of AR-42 and AR-12, a PDK1 inhibitor, we generated and used luciferase-expressing Ben-Men-1-LucB cells to establish intracranial xenografts that grew over time. While AR-12 treatment moderately slowed tumor growth, AR-42 caused regression of Ben-Men-1-LucB tumors. Importantly, AR-42-treated tumors showed minimal regrowth when xenograft-bearing mice were switched to normal diet. Together, these results suggest that AR-42 is a potential therapy for meningiomas. The differential effect of AR-42 on cell-cycle progression of normal meningeal and meningioma cells may have implications for why AR-42 is well-tolerated while it potently inhibits tumor growth.

Inhibition of collagen XVI expression reduces glioma cell invasiveness.

Glioblastomas are characterized by an intense local invasiveness that limits surgical resection. One mechanism by which glioma cells enforce their migration into brain tissue is reorganization of tumour associated extracellular matrix (ECM). Collagen XVI is a minor component of connective tissues. However, in glioblastoma tissue it is dramatically upregulated compared to the ECM of normal cortex. The aim of this study is to delineate tumour cell invasion and underlying mechanisms involving collagen XVI by using a siRNA mediated collagen XVI knockdown model in U87MG human glioblastoma cells. Knockdown of collagen XVI resulted in decreased invasiveness in Boyden chamber assays, and in a reduction of focal adhesion contact numbers per cell. Gene expression was upregulated for protocadherin 18 and downregulated for kindlin-1 and -2. Proliferation was not affected while flow cytometric analysis demonstrated reduced ß1-integrin activation in collagen XVI knockdown cells. We suggest that in glioblastoma tissue collagen XVI may impair the cell-cell interaction in favour of enhancement of invasion. The modification of the ß1-integrin activation pattern through collagen XVI might be a molecular mechanism to further augment the invasive phenotype of glioma cells. Elucidating the underlying mechanisms of glioma cell invasion promoted by collagen XVI may provide novel cancer therapeutic approaches in neurooncology.