Primary Meningioma Cells Research Articles

DDEL-12. LOCAL DELIVERY OF CHEMOTHERAPIES VIA BIORESORBABLE IMPLANT IN MENINGIOMAS

Abstract Many meningiomas recur despite repeated surgical resections and radiation, and patients must undergo progressively morbid surgical procedures due to recurrent or progressive disease. However, unlike most gliomas, meningiomas do not diffusely infiltrate throughout the brain on a single-cell basis, but instead recur and invade locally. Thus, they may be particularly vulnerable to localized chemotherapy. To investigate this, we screened 13 patient-derived primary meningioma cell cultures and two commercially available meningioma cell lines using a library of 147 FDA-approved compounds. We identified drugs with elevated potency across all meningiomas, including bortezomib (mean IC50 = 7nM) and romidepsin (mean IC50 = 18nM), as well as drugs with selective potency against specific samples, such as docetaxel (IC50 range 6nM to >10uM). We reasoned that delivery of a multi-drug cocktail with independent anti-neoplastic mechanisms would provide the strongest therapeutic effect in meningiomas. As systemic treatment with combination agents resulted in severe toxicity in mice, we performed local injection of bortezomib / romidepsin / docetaxel (BRD) triple therapy over 24 days in flank xenografts of IOMM-LEE and CH157 meningiomas. Tumor growth was reduced by 71-88% relative to vehicle (p<0.01), and mice showed no systemic toxicity with local therapy. Notably, the addition of radiotherapy did not further enhance the effect of BRD. To optimize local delivery, we developed a polyanhydride bioresorbable wafer designed to fit in a meningioma resection cavity, analogous to carmustine wafers used in gliomas. Intracranial implantation of wafers caused no local toxicity or inflammation in mice. Docetaxel-loaded wafers prevented the growth of flank-injected IOMM-LEE cells relative to wafers without drug (0 mm3 vs. 230 mm3 at 3 weeks post-injection, N=5/group, p<0.01). Our results demonstrate that bioresorbable wafers containing a cocktail of potent chemotherapies may be an effective new strategy against locally aggressive meningiomas.

Altered O-linked glycosylation in benign and malignant meningiomas.

Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 β1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 β-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.

P18.05.A THE GROWTH PATTERN OF MENINGIOMA CELLS IN CELL CULTURE UNDER DIFFERENT CONDITIONS

Abstract BACKGROUND Cell cultures is an established method in tumor research. Thereby, the growth rate is important for the results. Depending on the experimental and clinical setting, the used tissue is exposed to different conditions. Interestingely, the different cell and tissue conditions are not well reported yet. Although, it is likely that the different conditions influence the growth pattern of cell cultures in primary cell lines. Here, the authors present their experimental analysis of cell culture of primary meningioma cells after different initial tissue conditions. MATERIAL AND METHODS From June to December 2022 ten primary human meningiomas were collected after surgery at the Neurosurgical Department of the Saarland University. Primary cell cultures (Passage 0 = P0) were set up on the day of surgery and one day postoperatively. These tissues were kept overnight in nutrient solution in the refrigerator. They were splitted in two P1 cultures (Passage 1). The termination of the cell culture occured when the flasks were fully grown. One P1 culture was used for Fluorescence in situ hybridization. The other one was frozen on liquid nitrogen. The latter is thawed after six months and cultivated again. Additionally, swab preparations were made for Fluorescence in situ hybridization. The frozen tissue of these meningiomas was also used for further cell cultures. RESULTS Eight of ten cases (80%) of the meningioma cell cultures (from the day of surgery and one day postoperatively) have grown. In one case only the cell culture from the day of surgery has grown and in another one there were only single cells, no growth out of this tissue. The cell cultures prepared from frozen tissues have not shown any culture growth. There were not any significant differences between the growth of the cell culture from the day of surgery and of the cell culture from one day after surgery. The results of the Fluorescence in situ hybridization have shown a loss of 1p in one meningioma, a loss of 22q in three meningiomas, a loss of 1p and 22q in three meningiomas and a normal chromosome set in three tumours. Further, there were not any differences between the evaluation of swab preparations and drop preparations. Until now, six out of six thawed cell cultures of the first three meningiomas can be cultured again after six months in liquid nitrogen. CONCLUSION It has been shown that the growth pattern of meningioma cell cultures is independent of the set up at the same day or one day after surgery if there are enough living cells in the tissue. On the other hand, meningioma cell culturing of frozen tissues is not possible in the same setting.

Coexistence of primary pulmonary meningioma and metastatic papillary renal cell carcinoma of the lung: A rare case report with review of the literature

Primary pulmonary meningioma (PPM) is extremely rare tumor and only a few reports have been reported to date. PPM may be overlooked when it coexists with other types of tumors in the lung. It is essential to have a knowledge of the clinicopathological features of PPM and to recognize this rare coexistence. A 57-year-old male underwent surgery for papillary renal cell carcinoma, when 2 pulmonary nodules were detected using chest computed tomography. The coexistence of benign PPM and metastatic papillary renal cell carcinoma was histologically confirmed. A lobectomy was performed. The patient recovered well after surgery and was discharged on postoperative day 4. Duo to the rarity of PPM, it is easily overlooked, especially when it coexists with other tumors in the lung. The possibility of PPM needs to be taken into account when diagnosing pulmonary nodules in clinical practice.

PATH-37. SPATIAL GENE EXPRESSION PROGRAMS AND PROTEIN SIGNALING MECHANISMS DRIVE MENINGIOMA EVOLUTION AND THERAPEUTIC VULNERABILITY

Abstract Intratumor heterogeneity drives cancer evolution and resistance to therapy, but unbiased approaches to elucidate mechanisms driving intratumor heterogeneity have been lacking. Here, we integrate spatial gene expression programs and protein signaling mechanisms across 16 meningioma samples to define how intratumor heterogeneity drives molecular classification, temporal evolution, or spatial evolution of the most common primary intracranial tumor. Spatial transcriptomic analysis was performed on 38,718 regions and spatial profiling of 72 proteins comprising proliferation, stress, microenvironment, immune, or signaling modules was performed on 82 regions. DNA methylation, copy number variant, targeted gene expression profiling, targeted DNA sequencing, histologic, or immunohistochemical analyses (Ki67, H3K27me3, p16) were performed on all meningiomas to study intratumor heterogeneity in the context of pre-existing classification schemes. Primary meningioma cells, CRISPR interference, pharmacology, and 3D co-culture models were used for mechanistic and functional validation. Spatial analyses revealed significant intratumor or intertumor heterogeneity irrespective of meningioma histologic subtype or grade, DNA methylation group (Merlin-intact, Immune-enriched, Hypermitotic), copy number variant (1p loss, 1q gain, 6p gain, 9p loss, 14q loss, 22q loss), gene expression risk score, or driver mutation (NF2, CDKN2A/B, TERT promoter, BAP1, SMARCB1, ARID1A). Spatial analyses on paired histologically or molecularly distinct regions from individual meningiomas (n=4), or on paired primary and recurrent meningioma samples (n=9), revealed conserved proliferation, immune, or signaling mechanisms underlying meningioma evolution. Mechanistic and functional studies validated therapeutic vulnerabilities across spatial clusters to combinations of FDA-approved small molecules inhibiting the cell cycle (abemaciclib), epigenetic regulators (vorinostat), the DNA damage response (nariparib), MAPK signaling (erlotinib, selumetinib), or PI3K-AKT signaling (copanlisib). In summary, these data define spatial gene expression programs and protein signaling mechanisms driving meningioma evolution and therapeutic vulnerability, shedding light on past clinical failures and elucidating novel combinations of concurrent or sequential molecular therapies to treat meningiomas that are resistant to standard interventions.

Multi-center, single arm phase II study of the dual mTORC1/mTORC2 inhibitor vistusertib for patients with recurrent or progressive grade II-III meningiomas.

2024 Background: Grade II/III meningiomas represent about 20% of tumors and have increased rates of recurrence with no approved medical therapies. Historically, the progression-free survival at 6 months (PFS-6) for these tumors is 25%. The Response Assessment in Neuro-Oncology (RANO) group identified a PFS-6 rate of > 35% to be of interest for trials of grade II/III meningioma. Methods : NF2 gene inactivation occurs in the majority of meningiomas and is associated with mTORC1 activation. Human studies of everolimus for neurofibromatosis 2 patients documented growth arrest in only a minority of tumors. Based on our studies showing mTORC2/SGK1 pathway activation in NF2-deficient meningiomas and the known paradoxical activation of the mTORC2/AKT pathway in meningiomas, we hypothesized that dual inhibition of mTORC1/2 would be superior in meningiomas. Treatment of primary meningioma cells with vistusertib led to decreased cell proliferation and showed greater efficacy than rapamycin, regardless of NF2 expression. We studied the effect of vistusertib in patients with progressive or recurrent grade II/III meningiomas (NCT03071874). Vistusertib was administered orally at 125mg twice daily on two consecutive days each week. MRIs were obtained every 2 cycles (1 cycle = 28 days). Tumor size was defined as the largest cross-sectional area. Progression was defined as ≥25% increase in the sum of products of all measurable lesions over smallest sum observed. The primary endpoint was PFS-6. Secondary endpoints included toxicity, radiographic response, and correlative studies including immunohistochemistry for mTORC1/2 pathway activation and genetic biomarkers. Results: Twenty-eight patients (13 female), with a median age of 58 years (range, 32 to 77 years), were enrolled in this multicenter study. The median Karnofsky performance status was 80. Twenty-five patients have been followed to six months or to tumor progression. The median duration of treatment was 6.5 month (range, 1-18 months). Four patients chose to discontinue treatment, 1 withdrew to intercurrent illness, and 1 was withdrawn due to non-compliance. PFS-6 is 51.5% (CI, 29.3% - 70.0%). Adverse events at least possibly related to vistusertib with frequency > 10% include nausea (54%); fatigue (36%); hypophosphatemia (29%); diarrhea, anorexia, dry mouth, and hypertriglyceridemia (all 14%); hypertension, vomiting, increased ALT, constipation, and weight loss (all 11%). Conclusions: Vistusertib treatment was associated with a PFS-6 rate that exceeds the RANO target of 35% for recurrent high-grade meningioma. The follow-up data continue to mature. Adverse events were tolerable in this patient population. Correlative studies to identify biological factors that correlate with response are under way. These data support the initiation of larger randomized studies of vistusertib in this setting. Clinical trial information: NCT03071874.

EPCO-36. GENOMIC INSTABILITY AND TRANSCRIPTOMIC SIGNATURES UNDERLYING EPIGENETIC MENINGIOMA SUBGROUPS REVEALS MECHANISMS OF IMMUNE INFILTRATION AND THERAPEUTIC VULNERABILITIES

Abstract BACKGROUND Meningioma treatments are limited due to incomplete understanding of meningioma biology. To address this, we performed multiplatform molecular profiling on 565 meningiomas with comprehensive clinical data to define genomic drivers and identify therapeutic vulnerabilities. METHODS DNA methylation profiling was performed on meningiomas from UCSF (n=200, discovery) and Hong Kong University (n=365, validation). Median follow-up was 5.6 years, and there were 388/142/35 WHO grade I/II/III meningiomas. Copy number variants (CNVs) were calculated for all meningiomas, and RNA sequencing was performed on UCSF meningiomas. Cell type deconvolution, metagenomics, CRISPR, and pharmacology were used for mechanistic and functional validation. RESULTS Unsupervised hierarchical clustering of differentially methylated DNA probes revealed that meningiomas were comprised of 3 epigenetic subgroups associated with good, intermediate, and poor outcomes, with representation from all WHO grades in each subgroup. Meningiomas from the subgroup with the best outcomes (52% WHO grade I) were distinguished by recurrent gain of Chr5. Meningiomas from the subgroup with intermediate outcomes (31% WHO grade II) were distinguished by genomic stability, enrichment of innate immune genes, and immune infiltration in the setting of endogenous retroviral gene re-expression, a mechanism of immune recruitment. The most aggressive subgroup of meningiomas (57% WHO grade III) was distinguished by genomic instability, including recurrent loss of Chr22q harboring NF2, and decreased immune infiltration. Consistently, NF2 suppression in primary meningioma cells derived from immunogenic meningiomas decreased expression of innate immune genes critical for immune recruitment, suggesting a novel immunostimulatory function of NF2. The most aggressive subgroup of meningiomas were further distinguished by activation of the mitogenic FOXM1 transcriptional program, and recurrent loss of Chr9p harboring CDKN2A/B, which rendered primary meningioma cells from this subgroup susceptible to CDK4/6 inhibitors. CONCLUSIONS Meningiomas are comprised of 3 epigenetic subgroups defined by genetic mechanisms driving immune infiltration in the tumor microenvironment and meningioma cell proliferation.

Abstract 6384: TEAD inhibition reduces tumor proliferation in Merlin null meningioma and schwannoma

Abstract Introduction: Meningiomas are the most common intracranial tumor and arise from the arachnoidal cell layer of the meninges. Schwannomas develop from Schwann cells which provide myelin and trophic support in the peripheral nerves. The tumor suppressor Merlin is deleted in approximately 50-60% of meningiomas and 70% of schwannomas. Loss of Merlin results in dysregulation of the Hippo pathway and consequent aberrant activation of the transcriptional coactivators YAP and TAZ. Nuclear localisation of YAP and TAZ has been shown to drive tumor phenotypes in many cancers. The TEAD family of transcription factors are major targets of YAP and TAZ-dependent signaling. TEAD family members undergo autopalmitoylation and this modification is required for transcriptional activation by YAP or TAZ. Vivace therapeutics have identified small molecule inhibitors of autopalmitoylation that block the interaction of TEAD family members with YAP and TAZ. This study seeks to establish the efficacy of small molecule inhibitors of TEAD activity in meningioma and schwannoma tumor cells. Experimental Procedures: Meningioma cell lines, primary human meningioma and schwannoma cells, as well as a schwannoma mouse model (PerisotinCRE /NF2 fl/fl) were treated with Vivace inhibitors to assess their effects upon tumor cell proliferation and TEAD driven transcription. Results: TEAD inhibition significantly reduced the proliferation of the BenMen-1 meningioma cell line, as well as in primary human meningioma cells. In schwannoma, inhibitors reduced proliferation and also reduced the expression of CTGF, a target of TEAD activation in both primary human meningioma and schwannoma cells. These novel inhibitors are also currently being tested in the Periostin CRE-NF2fl/fl schwannoma mouse model to evaluate their efficacy in vivo. Conclusions: The use of these novel compounds to inhibit TEAD function shows great potential as an effective treatment for reducing the rate of cell proliferation in meningioma and schwannoma, two clinically important tumor types. Citation Format: Liyam Laraba, Julio Grimm de Guibert, Tracy T. Tang, Leonard Post, David B. Parkinson. TEAD inhibition reduces tumor proliferation in Merlin null meningioma and schwannoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6384.

Characterization and comparison of genomic profiles between primary cancer cell lines and parent atypical meningioma tumors

BackgroundMeningiomas are the second most common primary tumors of the central nervous system. However, there is a paucity of data on meningioma biology due to the lack of suitable preclinical in vitro and in vivo models. In this study, we report the establishment and characterization of patient-derived, spontaneously immortalized cancer cell lines derived from World Health Organization (WHO) grade I and atypical WHO grade II meningiomas.MethodsWe evaluated high-resolution 3T MRI neuroimaging findings in meningioma patients which were followed by histological analysis. RT-qPCR and immunostaining analyses were performed to determine the expression levels of meningioma-related factors. Additionally, flow cytometry and sorting assays were conducted to investigate and isolate the CD133 and CD44 positive cells from primary atypical meningioma cells. Further, we compared the gene expression profiles of meningiomas and cell lines derived from them by performing whole-exome sequencing of the blood and tumor samples from the patients, and the primary cancer cell lines established from the meningioma tumor.ResultsOur results were consistent with earlier studies that reported mutations in NF2, SMO, and AKT1 genes in atypical meningiomas, and we also observed mutations in MYBL2, a gene that was recently discovered. Significantly, the genomic signature was consistent between the atypical meningioma cancer cell lines and the tumor and blood samples from the patient.ConclusionOur results lead us to conclude that established meningioma cell lines with a genomic signature identical to tumors might be a valuable tool for understanding meningioma tumor biology, and for screening therapeutic agents to treat recurrent meningiomas.

Meningioma cells express primary cilia but do not transduce ciliary Hedgehog signals

Meningiomas are the most common primary intracranial tumors, but treatment options for meningioma patients are limited due to incomplete understanding of tumor biology. A small percentage of meningiomas harbor somatic variants in the Hedgehog pathway, a conserved gene expression program that is essential for development and adult stem cell homeostasis. Hedgehog signals are transduced through primary cilia, and misactivation of the Hedgehog pathway is known to underlie cancer. Nevertheless, the mechanisms of Hedgehog signaling in meningioma are unknown. Here, we investigate mechanisms of ciliary Hedgehog signaling in meningioma using tissue microarrays containing 154 human meningioma samples, NanoString transcriptional profiling, primary meningioma cells, pharmacology, and CRISPR interference. Our results reveal that meningiomas of all grades can express primary cilia, but that cilia are less prevalent among anaplastic tumors. Moreover, we find that expression of Smoothened alleles that are oncogenic in other contexts fail to activate the Hedgehog transcriptional program or promote proliferation in primary meningioma cells. These data reveal that meningiomas can express the subcellular structure necessary for canonical Hedgehog signaling, but suggest that they do not transduce ciliary Hedgehog signals.

The clinical characteristics and molecular mechanism of pituitary adenoma associated with meningioma

BackgroundPituitary adenoma and meningioma are the most common benign tumors in the central nervous system. Pituitary adenoma associated with meningioma (PAM) is a rare disease and the clinical features and mechanisms of PAM are unclear.MethodsWe summarized the clinical data of 57 PAM patients and compared with sporadic pituitary adenoma (SPA) and sporadic meningioma (SM). 5 pituitary adenomas of PAM and 5 SPAs were performed ceRNA microarray. qRT-PCR, Western Blot, siMEN1 and rapamycin inhibition experiment were validated for ceRNA microarray.ResultsClinical variable analyses revealed that significant correlations between PAM and female sex as well as older age when compared with SPA and significant correlations between PAM and transitional meningioma as well as older age when compared with SM. Additionally, the characteristics of PAM were significantly different for MEN1 patients. Functional experiments showed lower expression of MEN1 can upregulate mTOR signaling, in accordance with the result of ceRNA microarray. Rapamycin treatment promotes apoptosis in primary pituitary adenoma and meningioma cells of PAM.ConclusionsMEN1 plays an important role in PAM by upregulating mTOR signaling pathway. Rapamycin represents a potential therapeutic strategy for PAM in the future.

Fluorescence imaging of meningioma cells with somatostatin receptor ligands: an in vitro study.

The use of five-aminolevulinic acid (5-ALA) in the staining of malignant glioma cells has significantly improved intraoperative radicality in the resection of gliomas in the last decade. Currently, there is no comparable selective fluorescent substance available for meningiomas. There is however a demand for intraoperative fluorescent identification of, e.g., invasive skull base meningiomas to help improve safe radical resection. Meningiomas show high expression of the somatostatin receptor type 2, offering the possibility of receptor-targeted imaging. The authors used a somatostatin receptor-labeled fluorescence dye in the identification of meningiomas in vitro. The aim of this study was to evaluate the possibility of selective identification of meningioma cells with fluorescent techniques. Twenty-four primary human meningioma cell cultures were analyzed. The tumor cells were incubated with FAM-TOC (5,6-Carboxyfluoresceine-Tyr3-Octreotide). As a negative control, four human dura tissues were cultured as well as a mixed cell culture in vitro and incubated with the same somatostatin receptor-labeled fluorescence substance. After incubation, fluorescence signal and intensity in all cell cultures were analyzed at three different time points using a fluorescence microscope with 488nm epi-illumination. Sixteen WHO I, six WHO II, two WHO III meningioma primary cell cultures, and four dura cell cultures were analyzed. Fluorescence was detected in all meningioma cell cultures (22 cell culture stained strongly, 2 cell cultures moderately) directly after incubation up until 4h later. There were no differences in the quality and quantity of fluorescence signal between the various meningioma grades. The fluorescence signal persisted unchanged during the analyzed period. In the negative control, dura cell cultures remained unstained. This study demonstrates the use of FAM-TOC in the selective fluorescent identification of meningioma cells in vitro. Further evaluation of the chemical kinetics of the applied somatostatin receptor ligand and fluorescence dye is warranted. As a next step, an experimental animal model is needed to evaluate these promising results in vivo.

PO-162 The transcription factor GATA4 is overexpressed in malignant meningioma enhancing proliferation via downregulation of miR-15 family members

IntroductionMeningioma is the most common primary brain tumour and is classified as benign (WHO I, 80%), atypical (WHO II, 15%–20%) and anaplastic (WHO III, 1%–3%). The 3 year recurrence rate in WHO I meningioma is ~50% and it is much greater in WHO II and III. Recent studies showed that cyclin D1 and E1 positively correlated with meningioma grade and higher recurrence rates, suggesting them as potential prognostic markers.Here, we show that cyclin D1-D2 and E1 overexpression in malignant meningioma is driven by downregulation of the miR-15 family members via GATA4, a transcription factor overexpressed in WHO III meningioma cells and tissues. Therefore, we propose GATA4 as a novel possible biomarker for monitoring meningioma progression.Material and methodsMeningioma (MN) specimens were collected from consented patients according to the ethical approval for this study. All cell lines and primary MN cells were isolated from tumour specimens and cultured following recommended conditions. Real Time PCR was conducted using TaqMan reagents (Applied Biosystems), according to the manufacturer’s instructions following the 2-ΔΔCT method. In silico studies were conducted using TargetScanHuman7.1 to search for putative miRNA targets. P-values were calculated using the Student’s t-Test or the ANOVA one-way analysis of variance, led by the GraphPad Prism 5.01 and MS Excel 2016 software (p values<0.05±SEM).Results and discussionsProteomic analysis showed an increase of cyclin D1 in Ben Men-1, primary WHO I and KT21-MG1 cells, and an increase of cyclin D2 in KT21-MG1 cells only. In silico studies identified cyclin D1-D2-D3 and E1 as targets of the miR-15 family members. QPCR showed that miR-195 and −497 are downregulated in WHO II and III samples compared to WHO I (3.38 and 6.02 folds, respectively; p=0.02); these results were consistent in cell culture exosomes.Analysis of GATA4 revealed that the protein is highly overexpressed in KT21-MG1 cells but not in Ben Men-1 and primary meningioma cells (17940.19 and 4899.34 folds, respectively, p=0.03). These results were consistent in tissues (2640.53 folds).ConclusionOur data show that members of the miR-15 family are downregulated in WHO III meningioma cells and tissues, suggesting their contribution to control tumour progression. In addition, in WHO III cells and tissues the transcription factor GATA4, involved in miR-15 family regulation, is overexpressed. Ongoing studies will address GATA4 role in the biological progression of meningioma. (DB and COH contributed equally).

Primary culture of human malignant meningioma cells and its intracranial orthotopic transplantation in nude mice

To obtain stable primary cultures of human malignant meningioma cells and establish an intracranial in-situ tumor model in nude mice. Ten surgical specimens of highly suspected malignant meningioma were obtained with postoperative pathological confirmation. Primary malignant meningioma cells were cultured from the tissues using a modified method and passaged. After identification with cell immunofluorescence, the cultured cells were inoculated into the right parietal lobe of 6 nude mice using stereotaxic apparatus and also transplanted subcutaneously in another 6 nude mice. The nude mice were executed after 6 weeks, and HE staining and immunohistochmistry were used to detect tumor growth and the invasion of the adjacent brain tissues. The primary malignant meningioma cells were cultured successfully, and postoperative pathology reported anaplastic malignant meningioma. Cell immunofluorescence revealed positivity for vimentin and EMA in the cells, which showed a S-shaped growth curve in culture. Flow cytometry revealed a cell percentage in the Q3 area of (95.99∓2.58)%. Six weeks after transplantation, tumor nodules occurred in the subcutaneous tumor group, and the nude mice bearing the in situ tumor showed obvious body weight loss. The xenografts in both groups contained a mean of (36∓5.35)% cells expressing Ki-67, and the intracranial in situ tumor showed obvious invasion of the adjacent peripheral brain tissues. We obtained stable primary cultures of malignant meningioma cells and successfully established a nude mouse model bearing in situ human malignant meningioma.

Comprehensive Molecular Profiling Identifies FOXM1 as a Key Transcription Factor for Meningioma Proliferation

SUMMARYMeningioma is the most common primary intracranial tumor, but the molecular drivers of aggressive meningioma are incompletely understood. Using 280 human meningioma samples and RNA sequencing, immunohistochemistry, whole-exome sequencing, DNA methylation arrays, and targeted gene expression profiling, we comprehensively define the molecular profile of aggressive meningioma. Transcriptomic analyses identify FOXM1 as a key transcription factor for meningioma proliferation and a marker of poor clinical outcomes. Consistently, we discover genomic and epigenomic factors associated with FOXM1 activation in aggressive meningiomas. Finally, we define a FOXM1/Wnt signaling axis in meningioma that is associated with a mitotic gene expression program, poor clinical outcomes, and proliferation of primary meningioma cells. In summary, we find that multiple molecular mechanisms converge on a FOXM1/Wnt signaling axis in aggressive meningioma.

370 Hypoxic Conditions and Hypoxia-Inducible Factor 1-Alpha are Critical in the Radioresistance of Meningioma

Abstract INTRODUCTION Meningiomas are common benign intracranial neoplasms treated with a combination of surgical resection, radiotherapy, and chemotherapy. Hypoxia-inducible factor 1 alpha (HIF1A) can predict disease aggressiveness, response to oxygen tension and regulate meningioma pathogenesis. The role of HIF1A and hypoxia was explored in the radiation resistance of meningioma, hypothesizing that HIF1A confers resistance to radiotherapy in meningiomas and that oxygen tension is key to radiation responsiveness. METHODS Various primary meningioma cell lines were studied (IOMM-LE, JEN, GAR, SAM). GAR primary cell lines were utilized in addition to a shRNA-HIF1A knockdown (GAR1589). Cell proliferation, viability, and apoptosis were evaluated in response to radiation in both normoxia (21% O2) and hypoxia (1% O2). RESULTS &gt;Doubling times of the various meningioma cell lines ranged from 19.5 to 213.2 hours. Cell density impacted responsiveness to radiation, with confluent cells showing greater dose tolerance before reduced proliferation. In normoxic environments, HIF1A knockout resulted in increased susceptibility to single radiation fractions of 2 Gy (P = 0.05) and 10 Gy (P = 0.02); however, both cells had strong susceptibility at 20 Gy. Hypoxic environments reduced the viability differences between GAR and GAR1589 cells and slightly increased GAR cell viability at 2 Gy (P &lt; 0.05). Fractionated radiotherapy (2 Gy) daily showed reduced GAR and GAR1589 cell viability in normoxia and hypoxia. Cell viability for GAR1589 at 3 fractions of 2-Gy radiation was reduced, but this effect was eliminated in hypoxic environments. Apoptosis was increased in GAR1589 cells at baseline and with increased fractions of radiation; however, this effect was reduced in hypoxic conditions. CONCLUSION These results support important findings for meningioma response to radiation, including 1) importance of cell density, 2) dependence on HIF1A mutation, and 3) a critical role for oxygen tension. These results may be helpful in understanding the mechanisms of meningioma resistance to radiation and serve as a model to trial novel adjuvant therapies and treatment types.

Pleomorphism and drug resistant cancer stem cells are characteristic of aggressive primary meningioma cell lines

BackgroundMeningioma tumors arise in arachnoid membranes, and are the most reported central nervous system (CNS) tumors worldwide. Up to 20% of grade I meningioma tumors reoccur and currently predictive cancer stem cells (CSCs) markers for aggressive and drug resistant meningiomas are scarce.MethodsMeningioma tissues and primary cell lines were investigated using whole transcriptome microarray analysis, immunofluorescence staining of CSCs markers (including CD133, Sox2, Nestin, and Frizzled 9), and drug treatment with cisplatin or etoposide.ResultsUnsupervised hierarchical clustering of six meningioma samples separated tissues into two groups. Analysis identified stem cells related pathways to be differential between the two groups and indicated the de-regulation of the stem cell associated genes Reelin (RELN), Calbindin 1 (CALB1) and Anterior Gradient 2 Homolog (AGR2). Immunofluorescence staining for four tissues confirmed stemness variation in situ. Biological characterization of fifteen meningioma primary cell lines concordantly separated cells into two functionally distinct sub-groups. Pleomorphic cell lines (NG type) grew significantly faster than monomorphic cell lines (G type), had a higher number of cells that express Ki67, and were able to migrate aggressively in vitro. In addition, NG type cell lines had a lower expression of nuclear Caspase-3, and had a significantly higher number of CSCs co-positive for CD133+ Sox2+ or AGR2+ BMI1+. Importantly, these cells were more tolerant to cisplatin and etoposide treatment, showed a lower level of nuclear Caspase-3 in treated cells and harbored drug resistant CSCs.ConclusionCollectively, analyses of tissues and primary cell lines revealed stem cell associated genes as potential targets for aggressive and drug resistant meningiomas.

Altered expression of E-Cadherin-related transcription factors indicates partial epithelial-mesenchymal transition in aggressive meningiomas

E-Cadherin has been suggested to be involved in meningioma progression but is also known as a key player of epithelial to mesenchymal transition (EMT). We wondered whether the adherens junction protein E-Cadherin, the tight junction protein Zo-1, and transcription factors suppressing E-Cadherin expression (Slug, Snail, Twist, Zeb-1) are differentially expressed between histopathological subtypes of meningioma, and if the expression of these factors is related to biological features of meningiomas. Analyzing 85 meningiomas of various histopathological subtypes and grades of malignancy by immunohistochemistry and 50 of them in addition by real-Time-PCR, we observed significantly reduced expression of Zeb-1, Twist and Slug, together with slightly increased expression levels for E-Cadherin and Zo- 1 in fibroblastic WHO-grade I tumors compared to meningothelial WHO grade I tumors, contradicting the hypothesis of EMT in the fibroblastic meningiomas characterized by mesenchymal appearance. However, comparing aggressive WHO grade II or III meningiomas with WHO-grade I tumors, we observed altered expression levels (loss of E-Cadherin and Zo-1, increased expression of Zeb-1 and Slug) indicating molecular features of EMT in aggressive meningiomas. This was supported by reduced E-Cadherin and increased Slug levels in recurrent compared to non-recurrent meningiomas. The expression levels of E-cadherin and Zo-1 were positively correlated with expression of NF2 mRNA. In primary meningioma cultures and IOMM-Lee meningioma cells, EMT induction by TGF-ß resulted in altered morphology and increased expression of EMT associated transcription factors. Meningioma cells with allelic losses of NF2 showed generally higher levels of various EMT relevant proteins, but were unresponsive to TGF-ß treatment. Our data indicate that aggressive meningiomas of WHO grade II/III are characterized by molecular alterations indicating partial EMT. This might contribute to the aggressive biology of these tumors.

The role and regulatory mechanism of IL-1β on the methylation of the NF2 gene in benign meningiomas and leptomeninges.

Methylation of the neurofibromatosis type 2 (NF2) gene in low-grade meningioma (WHO grade I) has crucial roles in tumorigenesis and development. Meningioma formation might also occur in the setting of an inflammatory microenvironment. However, the association between inflammation and the methylation of NF2 remains unclear. The present study investigates the role and regulatory mechanism of IL-1β, one of the most important pro-inflammatory cytokines, in the methylation of NF2 in benign meningioma. Three primary low-grade meningioma cells and leptomeningeal cells were cultured. CCK-8 and BrdU assays demonstrated that proliferation of meningioma/leptomeningeal cells treated with IL-1β occurred in a dose- and time-dependent manner. Methylation-specific PCR verified that IL-1β induced methylation of the NF2 promoter and decreased NF2/merlin expression in meningioma/leptomeningeal cells. Real-time PCR, western blotting, and immunofluorescence showed that IL-1β up-regulated DNMT1 in meningioma cells and DNMT1/3b in leptomeningeal cells but did not up-regulate DNMT3a. After co-treatment with the DNMT inhibitor 5-Aza-2'-deoxycytidine and DNMT siRNA, methylation of NF2 induced by IL-1β was attenuated and merlin expression was restored. Furthermore, we showed that DNMT1 in meningiomas and DNMT1/3b in leptomeninges were regulated via activation of the MAPK (p38, ERK, JNK) and NF-κB pathways. These results suggest that IL-1β induces methylation of NF2 by up-regulating DNMT1 in benign meningioma cells and DNMT1/3b in leptomeningeal cells via MAPK and NF-κB pathways. Therefore, NF2 methylation is a linker between IL-1β and tumor development, and DNMTs might be potential therapeutic targets in meningioma for regulating NF2 and inhibiting tumor development. © 2016 Wiley Periodicals, Inc.

Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

BackgroundThere 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.MethodsA primary meningioma cell line was generated from a patient with an olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and comparative genomic hybridization. Xenograft models using immunocompromised SCID mice were also developed.ResultsHistopathology of the patient tumor was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and occasional psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile consistent with low-grade, good prognosis meningioma. 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 and orthotopic transplantation of low passage cells into SCID mice, firm tumors positive for vimentin and progesterone receptor (PR) formed, while subcutaneous implant of high passage cells yielded vimentin-positive, PR-negative tumors, concordant with a high-grade meningioma.ConclusionsAlthough derived from a benign meningioma specimen, the newly-established spontaneously immortal KCI-MENG1 meningioma cell line can be utilized to generate xenograft tumor models with either low- or high-grade features, dependent on the cell passage number (likely due to the relative abundance of the round, near-triploid cells). These human meningioma mouse xenograft models will provide biologically relevant platforms from which to investigate differences in low- vs. high-grade meningioma tumor biology and disease progression as well as to develop novel therapies to improve treatment options for poor prognosis or recurrent meningiomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0596-8) contains supplementary material, which is available to authorized users.