Primary Meningioma Cells Research Articles

Aminolevulinic acid-mediated photodynamic therapy of human meningioma: an in vitro study on primary cell lines.

Objective: Five-aminolevulinic acid (5-ALA)-induced porphyrins in malignant gliomas are potent photosensitizers. Promising results of ALA-PDT (photodynamic therapy) in recurrent glioblastomas have been published. Recently, 5-ALA-induced fluorescence was studied in meningioma surgery. Here, we present an experimental study of ALA-PDT in an in vitro model of primary meningioma cell lines. Methods: We processed native tumor material obtained intra-operatively within 24 h for cell culture. Epithelial membrane antigen (EMA) immunohistochemistry was performed after the first passage to confirm that cells were meningioma cells. For 5-ALA-PDT treatment, about 5000 cells per well were seeded in 20 wells of a blank 96-well plate. Each block of 4 wells was inoculated with 150 µL of 0, 25, 50 and 100 µg/mL 5-ALA solutions; one block was used as negative control without 5-ALA and without PDT. Following incubation for 3 h PDT was performed using a laser (635 nm, 18.75 J/cm2). The therapeutic response was analyzed by the water soluble tetrazolium salt (WST-1) cell viability assay 90 min after PDT. Results: 5-ALA-PDT was performed in 14 primary meningioma cell lines. EMA expression was verified in 10 primary cell cultures. The remaining 4 were EMA negative and PDT was without any effect in these cultures. All 10 EMA-positive cell lines showed a significant and dose-dependent decrease in viability rate (p < 0.001). Cell survival at 5-ALA concentrations of 12.5, 25, 50 and 100 μg/mL was 96.5% ± 7.6%, 67.9% ± 29.9%, 24.0% ± 16.7% and 13.8% ± 7.5%, respectively. For the negative controls (no 5-ALA/PDT and ALA/no PDT), the viability rates were 101.72% ± 3.5% and 100.17% ± 3.6%, respectively. The LD50 for 5-ALA was estimated between 25 and 50 µg/mL. Conclusion: This study reveals dose-dependent cytotoxic effects of 5-ALA-PDT on primary cell lines of meningiomas. Either 5-ALA or PDT alone did not affect cell survival. Further efforts are necessary to study the potential therapeutic effects of 5-ALA-PDT in vivo.

The transducible TAT-RIZ1-PR protein exerts histone methyltransferase activity and tumor-suppressive functions in human malignant meningiomas

Malignant meningiomas are a rare meningioma subtype and tend to have post-surgical recurrence. Significant endeavors have been taken to identify functional therapeutic targets to halt the growth of this aggressive cancer. We have recently discovered that RIZ1 is downregulated in high-grade meningiomas, and RIZ1 overexpression inhibits proliferation while promoting cell apoptosis of the IOMM-Lee malignant meningioma cell line. In this report, we show that the N-terminal PR domain of RIZ1 alone possessed growth-inhibitory activity and anticancer activity in primary human meningioma cells. Interestingly, the effects seem to be dependent on differential RIZ1 protein levels. Transducible TAT-RIZ1-PR protein could also inhibit meningioma tumor growth in nude mice models. We further demonstrate that PR protein exerts histone methyltransferase activity. A microarray analysis of TAT-RIZ1-PR-treated human malignant meningioma cells reveals 969 differentially expressed genes and 848 alternative splicing exons. Moreover, c-Myc and TXNIP, two putative downstream targets of H3K9 methylation, may be involved in regulating RIZ1 tumor-suppressive effects. The reciprocal relationship between RIZ1 and c-Myc was then validated in primary meningioma cells and human tumor samples. These findings provide insights into RIZ1 tumor suppression mechanisms and suggest that TAT-RIZ1-PR protein is a potential new epigenetic therapeutic agent for advanced meningiomas.

Bortezomib sensitizes primary meningioma cells to TRAIL-induced apoptosis by enhancing formation of the death-inducing signaling complex.

A meningioma is the most common primary intracranial tumor in adults. Here, we investigated the therapeutic potential of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in 37 meningiomas. Freshly isolated primary meningioma cells were treated with TRAIL with or without different sensitizing protocols, and apoptotic cell death was then quantified. Mechanisms of TRAIL sensitization were determined by a combination of Western blotting, flow cytometry, receptor complex immunoprecipitation, and siRNA-mediated knockdown experiments. Tumor necrosis factor-related apoptosis-inducing ligand receptor expression was analyzed using immunohistochemistry and quantified by an automated software-based algorithm. Primary tumor cells from 11 (29.7%) tumor samples were sensitive to TRAIL-induced apoptosis, 12 (32.4%) were intermediate TRAIL resistant, and 14 (37.8%) were completely TRAIL resistant. We tested synergistic apoptosis-inducing cotreatment strategies and determined that only the proteasome inhibitor bortezomib potently enhanced expression of the TRAIL receptors TRAIL-R1 and/or TRAIL-R2, the formation of the TRAIL death-inducing signaling complex, and activation of caspases; this treatment resulted in sensitization of all TRAIL-resistant meningioma samples to TRAIL-induced apoptosis. Bortezomib pretreatment induced NOXA expression and downregulated c-FLIP, neither of which caused the TRAIL-sensitizing effect. Native TRAIL receptor expression could not predict primary TRAIL sensitivity. This first report on TRAIL sensitivity of primary meningioma cells demonstrates that TRAIL/bortezomib cotreatment may represent a novel therapeutic option for meningiomas.

PM-11 * DEVELOPMENT OF A HUMAN MENINGIOMA MOUSE XENOGRAFT MODEL WITH THE SPONTANEOUSLY IMMORTAL CELL LINE KCI-MENG1

BACKGROUND: There is a paucity of effective therapies for recurrent/aggressive meningiomas. Establishment of improved in vitro and in vivo meningioma models will facilitate development and testing of novel therapeutic approaches. METHODS: A primary meningioma cell line was generated from a patient harboring a WHO grade I olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and genomic analysis. A xenograft model of human meningioma using immunocompromised SCID mice was also developed. RESULTS: Histopathology was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile. Low-passage KCI-MENG1 cells were composed of two cell types with spindle and round morphologies, showed linear growth curve, had very low telomerase activity, and were composed of two distinct unrelated clones on cytogenetic analysis. In contrast, high-passage cells were homogeneously round, rapidly growing, had high telomerase activity, and were composed of a single clone with a near triploid karyotype containing 64-66 chromosomes with numerous aberrations. Following subcutaneous transplantation of low- or high-passage cells into SCID mice, tumor tissue with typical histological features of meningothelial meningioma was found. Cultured cells isolated from the xenograft tumors also had the near-triploid karyotype and the same immunostaining profile. CONCLUSIONS: The newly-established spontaneously immortal KCI-MENG1 meningioma cell line and human meningioma mouse xenograft model will provide a biologically relevant platform from which to investigate meningioma tumor biology and disease progression as well as to develop novel therapeutic targets to improve treatment options for all grades of meningiomas.

Curcumin acts anti-proliferative and pro-apoptotic in human meningiomas

Meningiomas, the most frequent benign intracranial and intraspinal types of tumors are normally removed by surgery. Complications can occur when the tumor is critically localized and cannot be completely removed or when comorbidities of the mostly elder patients increase the general surgical risk. Thus, alternate medical treatment concepts for the therapy of meningiomas would be desirable. Curcumin, the active ingredient of the spice plant Curcuma longa has shown anti-tumorigenic actions in many different types of tumors and therefore, its effect on growth and apoptosis of meningioma cells was studied in the present paper. In vitro, treatment of the human Ben-Men-1 meningioma cell line and of a series of 21 primary human meningioma cell cultures with curcumin (1-20 μM) strongly reduced the proliferation in all cases in a dose dependent manner. Cell cycle analysis by fluorescence-activated cell sorting showed growth arrest at G2/M phase, which was confirmed by demonstrating the corresponding modulation of proteins involved in G2/M arrest by immunoblotting and/or confocal laser microscopy. High dosages (20, 50 μM) of curcumin induced a significant increase of apoptosis in Ben-Men-1 and primary meningioma cell cultures as demonstrated by morphological changes of cell nuclei, DNA fragmentation, translocation of cell membrane associated phosphatidyl serine and the induction of apoptotic-acting cleaved caspase-3. Our results suggest that the multi-targeting drug curcumin has potent anti-tumorigenic actions in meningioma cells and might therefore be a putative candidate for the pharmacological treatment of meningiomas.

Detection and quantification of farnesol-induced apoptosis in difficult primary cell cultures by TaqMan protein assay

Apoptosis can be detected reliably by assaying for cleaved caspase-3, for which active caspase-3 antibodies are used in several methods, such as immunocytochemistry, enzyme-linked immunosorbent assay, and western blot. In this study, we used TaqMan protein assay (TPA), a novel method for protein detection and quantification that detects proteins by amplification of substitute DNA templates. TPA uses antibodies and proximity ligation for quantitative real-time PCR. Meningiomas are primarily benign intracranial tumors. Primary cell cultures of meningiomas are often unsuitable for sensitive protein detection methods. We optimized a TPA to detect active caspase-3 and evaluated its ability to detect farnesol-induced apoptosis in primary meningioma cells. The specificity and sensitivity of the inactive and active caspase-3 assay were determined using recombinant caspase-3. Apoptosis was induced in meningiomas in the presence of 0.2 μM farnesol as shown by immunocytochemistry of single-stranded DNA. Also, viability decreased by over 90 % after treatment with 1.2 μM farnesol for 24 h. The TPA detected a significant increase in active caspase-3 after treatment with 2 and 4 μM farnesol for 2 h, which could not be detected using standard methods such as western blot and immunofluorescence. In addition, TPA determined that meningiomas show disparate sensitivities to low concentrations of farnesol. Caspase-3 expression fell significantly in cells that were treated with 0.25 μM farnesol for 2 h. Further, by TPA, active caspase-3 peaked after 2 h and declined with longer incubation times. This study demonstrates that cleaved caspase-3 is detected and quantified reliably in meningiomas by TPA.

CD133-Positive Cells Might Be Responsible for Efficient Proliferation of Human Meningioma Cells

Owing to lack of appropriate model systems, investigations of meningioma biology have come to a stop. In this study, we developed a comprehensive digestion method and defined a culture system. Using this method and system, primary meningioma cells in conditioned suspension medium and a hypoxic environment could be amplified in spheres and were passaged for more than ten generations. Meningioma sphere cells were positive for meningioma cell markers and negative for markers of neural cell types. Importantly, we found the cells expressed the stem cell marker, CD133, but not nestin. All of the tumor sphere cell populations showed a slower degree of cell proliferation than that of human glioma cells and fetal neural stem cells (NSCs). Further studies showed that the proliferative rate was positively correlated with CD133 expression. The higher the CD133 expression, the faster the cell proliferation. With the increase in cell generations, the cell proliferation rate gradually slowed down, and CD133 expression also decreased. Single CD133+ cells rather than CD133− cells could form spheres. Thus, the results above indicated that those cells expressing CD133 in spheres might be stem-like cells, which may be responsible for efficient amplification of human meningioma cells. Decreased expression of CD133 may lead to the failure of long-term passaging.

AR42, a novel histone deacetylase inhibitor, as a potential therapy for vestibular schwannomas and meningiomas

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disease that results in the formation of bilateral vestibular schwannomas (VSs) and multiple meningiomas. Treatment options for NF2-associated tumors are limited, and to date, no medical therapies are FDA approved. The ideal chemotherapeutic agent would inhibit both VS and meningiomas simultaneously. The objectives of this study are (1) to test the efficacy of AR42, a novel histone deacetylase inhibitor, to inhibit VS and meningioma growth and (2) to investigate this drug's mechanisms of action. Primary cultures of human VS and meningioma cells were established. Nf2-deficient mouse schwannoma and benign human meningioma Ben-Men-1 cells were also cultured. Cells were treated with AR42, and the drug's effects on proliferation and the cell cycle were analyzed using a methanethiosulfonate assay and flow cytometry, respectively. Human phospho-kinase arrays and Western blots were used to evaluate the effects of AR42 on intracellular signaling. The in vivo efficacy of AR42 was investigated using schwannoma xenografts. Tumor volumes were quantified using high-field, volumetric MRI, and molecular target analysis was performed using immunohistochemistry. AR42 inhibited the growth of primary human VS and Nf2-deficient mouse schwannoma cells with a half maximal inhibitory concentration (IC(50)) of 500 nM and 250-350 nM, respectively. AR42 also inhibited primary meningioma cells and the benign meningioma cell line, Ben-Men-1, with IC(50) values of 1.5 µM and 1.0 µM, respectively. AR42 treatment induced cell-cycle arrest at G(2) and apoptosis in both VS and meningioma cells. Also, AR42 exposure decreased phosphorylated Akt in schwannoma and meningioma cells. In vivo treatment with AR42 inhibited the growth of schwannoma xenografts, induced apoptosis, and decreased Akt activation. The potent growth inhibitory activity of AR42 in schwannoma and meningioma cells suggests that AR42 should be further evaluated as a potential treatment for NF2-associated tumors.

Meningioma mouse models

Meningiomas, although mostly benign, may sometimes present aggressive features and raise issues concerning alternative treatment options besides surgery. In order to gain new insights in meningioma biology and develop alternative treatments, several meningioma mouse models have been engineered during the past two decades. As rodents very rarely develop spontaneous meningiomas, animal models have been first developed by implanting human meningioma cells derived from a primary tumor and meningioma cell lines subcutaneously into athymic mice. Induction of de novo meningiomas in rodents with mutagens, such as nitrosourea, has also been reported. Advances in our understanding of molecular genetics of meningioma have pinpointed the central role of NF2 tumor suppressor gene in the pathogenesis of those tumors. These discoveries have led to the creation of a genetically engineered model utilizing conditional mutagenesis to specifically inactivate the mouse Nf2 gene in arachnoidal cells, resulting in the formation of intracranial meningothelial hyperplasia and meningiomas and thus reproducing the main mechanism of human meningeal tumorigenesis. This powerful new technology significantly improves on prior models and may open avenues of investigation never before possible in meningioma research. We present here a review of current meningioma mouse models used in translational therapeutics with associated imaging and pre-clinical studies.

Fibroblast growth factor receptor–3 expression in meningiomas with stimulation of proliferation by the phosphoinositide 3 kinase–Akt pathway

Fibroblast growth factor receptors (FGFRs)-1, -2, and -3 are expressed in the developing brain and may participate in CNS neoplasia. Fibroblast growth receptor-3 has not been demonstrated in the human CNS or its tumors. Nonetheless, it has been implicated in the pathogenesis of several other forms of neoplasia. Twenty-four human meningiomas were evaluated using Western blot analysis for expression of FGFR3, its ligand acidic FGF, and concomitant phosphorylation/activation of p44/42 mitogen-activated protein kinase (MAPK), Akt, and STAT3. Mutations in exons 7 and 10 of the FGFR3 gene were analyzed by polymerase chain reaction in 10 meningiomas. Primary meningioma cells cultured from 10 human meningiomas were also treated with acidic FGF and evaluated for cell proliferation or activation/phosphorylation of p44/42 MAPK, Akt, and STAT3. Immunoblotting demonstrated the presence of FGFR3 in 12 (71%) of 17 primarily fibroblastic and transitional WHO Grade I meningiomas. The FGFR3 was detected in 4 (80%) of 5 WHO Grade II, and 2 of 2 Grade III tumors. Acidic FGF was detected in 3 (18%) of 17 Grade I, 1 (20%) of 5 Grade II, and 1 (50%) of 2 Grade III meningiomas. In WHO Grade I meningiomas, 3 of 6 tumors with no detectable FGFR3 had no detectable p-STAT3. In WHO Grades II and III meningiomas, FGFR3 expression was associated with p-STAT3, p-Akt, and p-p44/42 MAPK expression. No mutations were demonstrated in exons 7 or 10 by polymerase chain reaction in any meningioma. Treatment with acidic FGF, a ligand for FGFR3, stimulated meningioma cell proliferation and activation of Akt and STAT3 in primary meningioma cell cultures. These findings suggest that FGFR3 and acidic FGF are expressed in adult human leptomeninges as well as WHO Grades I and II meningiomas. Fibroblast growth factor receptor-3 activation stimulates meningioma cell proliferation by activation of the phosphoinositide 3 kinase-Akt-PRAS40-mTOR and STAT3 pathways.

Expression of vascular endothelial growth factor-A and mRNA stability factor HuR in human meningiomas

We studied the expression of vascular endothelial growth factor-A (VEGF-A) and mRNA stability factor HuR in 40 supratentorial meningiomas using RT-PCR, ELISA and immunohistochemistry, and analyzed their associations with the clinicopathological characteristics, including microvascular density (MVD), peritumoral brain edema (PTBE), histological subtypes and grades, and the performance of preoperative arterial embolization. Furthermore, we investigated the involvement of HuR in the upregulation of VEGF-A expression using primary meningioma cell cultures. The level of VEGF-A is elevated in meningiomas with PTBE and in higher grade meningiomas. Preoperative arterial embolization did not significantly increase the level of VEGF-A, but it did increase the expression of HuR in tumor tissues. HuR expression was correlated positively with VEGF-A expression in meningioma tissues. In in vitro experiments, hypoxia induced the upregulation of VEGF-A expression and the cytoplasmic translocation of HuR protein in meningioma cells, and inhibition of the cytoplasmic translocation of HuR reduced the upregulation of VEGF-A expression in meningioma cells. These findings suggest that the expression of VEGF-A relates to the development of PTBE with meningiomas and the histological grade, and that HuR is involved in the upregulation of VEGF-A expression in human meningiomas.

Expression of survivin and p16 INK4a/Cdk6/pRB proteins and induction of apoptosis in response to radiation and cisplatin in meningioma cells

Although meningiomas represent the most common class of tumors of the central nervous system, the molecular events underlying their genesis and development are still not well defined, and therapeutic approaches based on the genetics of these tumors are currently lacking. In the present study we have used the immunoblotting technique to show that the p16 INK4A, Cdk6 and pRB proteins are differentially expressed in primary meningioma cells with 20-, 30- and 36-fold difference between the lowest and the highest levels of each protein, respectively. In addition, we present evidence that the level of the anti-apoptosis survivin protein is high in these benign tumors. Moreover, the annexin V-associated flow cytometry technique was used to show that 60% of meningioma cell cultures underwent apoptosis in response to both γ-rays and cisplatin, and 50% of these cells exhibited significant sensitivity to hydroxyurea. These agents triggered apoptosis through the mitochondrial pathway, by increasing the Bax/Bcl-2 ratio. Interestingly, the induction of apoptosis following radiation and cisplatin was significant in all cells that expressed low levels of p16 INK4A, Cdk6 and pRB proteins. These data shed more light on the molecular biology of meningioma cells and suggest that survivin and proteins of the RB pathway could play a determinant role in the development and the treatment of meningiomas.

Nelfinavir potentiation of imatinib cytotoxicity in meningioma cells via survivin inhibition

Although most meningiomas are treated surgically, it may not be possible to completely remove atypical, malignant, and surgically inaccessible meningiomas; in the majority of these cases there is tumor recurrence. The authors have already reported initial preclinical results on the efficacy of imatinib in the treatment of meningiomas; however, a recent Phase II trial of imatinib in patients with recurrent meningiomas did not demonstrate significant antitumor activity. To enhance the activity of imatinib, the authors investigated the use of a combination therapy with nelfinavir on primary meningioma cells and meningioma cell lines IOMM-Lee and CH157. Cytotoxicity was measured using methylthiotetrazole and colony formation assays. In low-dose combination therapy with imatinib, nelfinavir potentiated the antiproliferative and anti-colony formation effects of imatinib. Primary meningioma cells responded better to combination therapy than to imatinib alone. Treatment induced a dose-dependent antiproliferative effect, decreased cell survival, and inhibited colony formation. Western blotting demonstrated decreased levels of survivin protein on combination therapy. Because meningiomas have very high levels of survivin protein, survivin inhibition by nelfinavir may represent a potential mechanism for the additive effect observed with imatinib. Moreover, an increase in the proapoptotic Bax/Bcl-2 protein ratio was demonstrated with the combination of imatinib and nelfinavir. The authors propose that nelfinavir not only potentiates imatinib efficacy, it also abrogates resistance to imatinib by decreasing survivin protein levels in meningiomas. In an in vivo assay, this combination therapy was found to be more effective than imatinib alone. More preclinical work with in vivo models is needed to determine if this new combination therapy will translate into a viable future therapy for meningiomas.

Human primary brain tumor cell growth inhibition in serum-free medium optimized for neuron survival

Glioblastoma is the most common primary brain tumor in adults from which about 15,000 patients die each year in the United States. Despite aggressive surgery, radiotherapy and chemotherapy, median survival remains only 1 year. Here we evaluate growth of primary human brain tumor cells in a defined nutrient culture medium (Neuregen) that was optimized for neuron regeneration. We hypothesized that Neuregen would inhibit tumor cell growth because of its ability to inhibit gliosis in rat brain. Tumor tissue was collected from 18 patients including 10 males and 8 females (mean age 60 ± 12 years) who underwent craniotomy for newly diagnosed, histologically confirmed brain tumors. The tissue was shipped overnight in Hibernate transport medium. Tumor cells were isolated and plated in Neurobasal/serum or Neuregen on culture plastic. After 1 week, growth in Neuregen was significantly less in 9/10 glioblastoma multiforme cases, 5/5 meningioma cases and 3/3 cases of brain metastasis. Analysis of deficient formulations of Neuregen and formulations to which selected components were added back implicate no single active component. However, individual cases were sensitive to corticosterone, selenium, ethanolamine, fatty acids and/or antioxidants. Therefore, a defined culture medium that promotes neuron regeneration inhibits the growth of human primary glioblastoma, meningioma and metastatic tumor cells in culture. The possible in vivo efficacy of Neuregen for treatment of brain tumor resections remains to be determined.

The p53/p21 DNA damage-signaling pathway is defective in most meningioma cells

Although meningiomas represent the most common class of tumors of the central nervous system, the molecular events underlying their genesis and development are still not well defined. In the present study we have used the immuno-blotting technique to study the expression level of the tumor suppressor proteins p53, p21 and PTEN in primary meningioma cells. We have also studied the induction of p21 and p53 in response to both UV light and gamma-rays. We present evidence that the p53/p21-dependent gamma-ray signaling pathway is defective in 5 out of 8 (62%) of these cells. Furthermore, we have shown that the tumor suppressor p21, p53 and PTEN proteins are differently expressed in these cells, with up to 40-folds difference between the lowest and the highest levels of each protein. These results suggest that the tumor suppressors p53/p21 signaling pathway and PTEN play important roles in the development of benign meningiomas.

CXCR4 and SDF1 expression in human meningiomas: A proliferative role in tumoral meningothelial cells in vitro1

Chemokines participate in cellular processes associated with tumor proliferation, migration, and angiogenesis. We previously demonstrated that stromal cell-derived factor 1 (SDF1) exerts a mitogenic activity in glioblastomas through the activation of its receptor CXCR4. Here we studied the expression of this chemokine in human meningiomas and its possible role in cell proliferation. Reverse transcriptase-PCR analysis for CXCR4 and SDF1 was performed on 55 human meningiomas (47 WHO grade I, 5 WHO II, and 3 WHO III). Immunolabeling for CXCR4 and SDF1 was performed on paraffin-embedded sections of these tumors. [(3)H]Thymidine uptake and Western blot analyses were performed on primary meningeal cell cultures of tumors to evaluate the proliferative activity of human SDF1alpha (hSDF1alpha) in vitro and the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) activation in this process. CXCR4 mRNA was expressed by 78% of the tumor specimens and SDF1 mRNA by 53%. CXCR4 and SDF1 were often detected in the same tumor tissues and colocalized with epithelial membrane antigen immunostaining. In 9 of 12 primary cultures from meningiomas, hSDF1alpha induced significant cell proliferation that was strongly reduced by the mitogen-activated protein kinase kinase inhibitor PD98059, involving ERK1/2 activation in the proliferative signal of hSDF1alpha. In fact, CXCR4 stimulation led to ERK1/2 phosphorylation/activation. In addition, the hSDF1alpha-induced cell proliferation was significantly correlated with the MIB1 staining index in the corresponding surgical specimen. In conclusion, we found that human meningiomas express CXCR4 and SDF1 and that hSDF1alpha induces proliferation in primary meningioma cell cultures through the activation of ERK1/2.

A genetic strategy to overcome the senescence of primary meningioma cell cultures

Even though meningiomas are the second most common brain tumor in adults, little is known about the molecular basis of their growth and development. The lack of suitable cell culture model systems is an impediment to this understanding. Most studies on meningiomas rely on primary, early passage cell lines that eventually senesce or a few established cell lines that have been derived from aggressive variants of meningiomas. We have isolated three primary meningioma cell lines that are negative for telomerase activity. We can overcome the senescence of a Grade III derived meningioma cell line by expressing the telomerase catalytic subunit (hTERT), whereas Grade I meningioma cell lines require the expression of the human papillomavirus E6 and E7 oncogenes in conjunction with hTERT. Meningioma cell lines, immortalized in this manner, maintain their pre-transfection morphology and form colonies in vitro. We have confirmed the meningothelial origin of these cell lines by assessing expression of vimentin and desmoplakin, characteristic markers for meningiomas. Additionally, we have karyotyped these cell lines using array CGH and shown that they represent a spectrum of the genetic diversity seen in primary meningiomas. Thus, these cell lines represent novel cellular reagents for investigating the molecular oncogenesis of meningiomas.

Different Activation of Mitogen-Activated Protein Kinase and Akt Signaling Is Associated with Aggressive Phenotype of Human Meningiomas

Activation of intracellular signaling cascades has been implicated in the growth control of benign meningiomas, but their role for meningioma progression and outcome is unknown. Here we determined the expression and function of proteins involved in mitogen-activated protein kinase (MAPK) and phosphinositol-3 kinase (PI3K)/Akt signaling in benign, atypical, and malignant meningiomas and studied their association with clinicopathologic data including meningioma recurrence. Expression of various MAPK and PI3K signaling proteins was determined in 70 primary meningiomas and, if present, in recurrent tumors by immunohistochemistry and Western blotting. The expression patterns in primary and recurrent tumors were related to clinical data. The effect of MAPK and PI3K pathway inhibition on cell proliferation and apoptosis was determined using a primary malignant meningioma cell culture. Atypical and malignant meningiomas showed higher levels of phospho-Akt compared with benign tumors, and their proliferation could be inhibited by PI3K blocking using wortmannin. PI3K inhibition did not induce apoptosis in malignant meningioma cells. In contrast, expression of phospho-Raf and phospho-MAPK was decreased in aggressive meningiomas compared with benign tumors, but MAPK inhibition by PD98059 resulted in tumor cell apoptosis and decreased proliferation. Reduced MAPK activation was associated with meningioma recurrence, and PI3K activation was associated with poor preclinical condition and brain invasion of malignant meningiomas. Both MAPK and PI3K/Akt pathways are activated at different levels in benign and malignant meningiomas. Activation of PI3K/Akt signaling contributes to the aggressive behavior of malignant meningiomas, whereas MAPK activation is involved in both proliferation and apoptosis of malignant meningiomas.

Oncolytic Virotherapy of Meningiomas in Vitro with Replication-competent Adenovirus

To evaluate the efficacy of the conditionally replicating adenovirus (Ad) Ad.d24 for oncolysis of benign and malignant meningiomas. Primary meningioma cells and organotypic spheroids were cultured from tumor biopsies of 12 consecutive unselected patients. Four different Ads were constructed and tested on meningioma cells and spheroids: a replication-deficient Ad encoding the luciferase marker gene (Ad.Luc), a replication-competent Ad with complete E1 region (Ad.E1+), a replication-competent Ad encoding the luciferase gene in the E3 region (Ad.E1Luc), and a conditionally replicating Ad with an E1ACR2 deletion (Ad.d24). Replication of the latter is restricted to cells with a defective retinoblastoma pathway, whereas Ad.E1+ and Ad.E1Luc can replicate in all human cells like a wild-type Ad. Their oncolytic activity was compared in primary meningioma cells and spheroids by use of viability and outgrowth assays. Adenoviral penetration into organotypic meningioma spheroids was detected with the replication-competent Ad.E1Luc, whereas infection with the replication-deficient Ad.Luc was limited to the outer layer of the spheroid. Replication of the Ads and oncolysis was demonstrated in primary cell cultures of meningioma cells at high dose, i.e., greater than 50 plaque-forming units per cell. At a lower dose of 5 plaque-forming units per cell, Ad.d24 kills meningioma cells more efficiently than Ad.E1+. Infection of organotypic meningioma spheroids with Ad.d24 resulted in decreased viability and suppression of outgrowth as compared with untreated control spheroids. Infection of meningioma cells and spheroids with replication-competent Ads results in efficient oncolysis. The Ad modified to replicate selectively in retinoblast-mutant cells, Ad.d24, seemed to be an efficient oncolytic agent in benign, atypical, and malignant meningiomas.

Transforming growth factor-beta effects on meningioma cell proliferation and signal transduction pathways.

The role of transforming growth factor-beta (TGF-beta) in regulation of meningioma growth and intracellular events transducing its signals are not established. In this study, we evaluated the effects of TGF-beta1 on basal meningioma cell proliferation in 10 primary human meningioma cell cultures and whether TGF-beta's signals are transduced by the Smad 2/3, MAPK/Erk kinase-1 (MEK-1)-mitogen-activated protein kinase (MAPK), Akt-p70(S6K) or p38-JUNK pathways in 5. We also tested whether neutralizing antibodies to TGF-beta alter CSF stimulation of meningioma cell proliferation. On average, TGF-beta reduced meningioma cell [3H]-thymidine incorporation to 58% of controls at 24% and to 61% of controls at 36 h. TGF-beta inhibition of meningioma cell proliferation was associated with a suggestion increased phosphorylation of Smad 2/3 in 2 cases and high basal phosphorylation in 3 but no change in activation of the MEK-1-MAPK, Akt-p70(S6K) or p38-JUNK pathways. As shown previously, CSF stimulated meningioma cell proliferation in the 3 cultures tested. Neutralizing antibody against TGF-beta augmented this stimulation in 2 of 3 cultures. These findings suggest that TGF-beta exerts a largely inhibitory effect on basal meningioma proliferation, perhaps in part through Smad 2/3.