Clinical data and human specimens
Clinical data and Tumor material from 96 meningioma patients were collected and analyzed (study was approved by the local ethical committee (Application No. 03–170)).
Statistical analysis was performed using SPSS, release 22 and GraphPad Prism 7. ANOVA, t-test, Chi-square and Fisher’s exact where used for gaussian distributed data, Mann-Whitney-U and Kruskal-Wallis when data did not meet the normality assumption. Tests were performed two-tailed. R-values were calculated via Spearman-Correlation. Log-rank (Mantel-Cox) test was used for comparison of survival curves. Significance levels: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, Confidence Interval: 95%. Error bars in figures represent SD.
Targeted sanger sequencing
Targeted Sanger sequencing of the region encompassing codon 409 of the human KLF4 gene was performed after PCR amplification. To monitor AKT1 mutations we also performed targeted Sanger sequencing of AKT1 (codon 17). For Primers and further details see supplementary materials.
Tissue microarray (TMA)
Using 27 FFPE-embedded skull base WHO I° meningioma samples, a tissue microarray (TMA) was constructed as previously reported . From each FFPE block, three separate samples were spotted on the TMA. Immunohistochemistry was performed from 4 μm thick paraffin sections. Antibodies used are detailed in supplementary materials.
RNA-Seq and analysis of RNA-Seq data
Sample preparation and isolation of RNA for RNA sequencing
RNA was extracted from frozen tumor samples and sequenced by Novogene. (See supplementary materials for detailed protocol) RNA sequencing was performed on Illumina HiSeq X platform.
RNA sequencing data analysis
For RNA-seq data analyses low quality read ends as well as remaining parts of sequencing adapters were clipped off using Cutadapt (v 1.14). Subsequently, reads were aligned to the human genome (UCSC GRCh38) using HiSat2 (v 2.1.0) . FeatureCounts (v 1.5.3)  was used for summarizing gene-mapped reads. Ensembl (GRCh38.89)  was used for annotating genes. Differentially expressed genes (DEGs) were determined by utilizing the R package edgeR (v 3.18.1)  using TMM normalization.
The cell lines used were generated from the malignant meningioma cell line IOMM-Lee and cultured as previously described . HEK293T cells were purchased from DSMZ (Braunschweig, Germany).
HEK293T cells were lentivirally transfected using FuGene® HD transfection reagent (Promega, Mannheim, Germany) with pLV[Exp]-Bsd-EF1A > hKLF4[NM_004235.4] or pLV[Exp]-Bsd-EF1A > hKLF4[NM_004235.4]*(K409Q) constructs (VectorBuilder) in combination with lentiviral packaging plasmid mix pC-Pack 2 (Cellecta). After 48 h the supernatants were harvested, filtered and used to infect IOMM-Lee cells. Infected cells were finally selected with Blasticidin (Corning).
Real-time reverse transcription (RT)-qPCR
RNA from adherent cells was isolated with TRIzol reagent (ThermoFischerScientific, Dreieich, Germany) or RNeasy Mini kit (QIAGEN) as described earlier . Briefly, isolated RNA was reverse transcribed into cDNA using random hexamer primers and used for real-time PCR to determine the relative gene expression, normalized to a housekeeping gene (HPRT or β2-microglobulin). For genes analyzed, the primer sequences and systems used see supplementary materials. The relative mRNA expression was calculated through 1/2∆Ct, ∆Ct representing the difference of Ct values between gene of interest and housekeeping gene. For experiments involving hypoxic conditions Cells were cultured under hypoxia (1% O2) for 1 day before total RNA isolation.
To determine cell proliferation, the Cell Proliferation ELISA (BrdU) from Roche was used. Ten thousand cells were seeded into 96-well plate (Sarstedt, Nümbrecht, Germany) and incubated at 37 °C, 5% CO2. Upon 48 h cells were stained according to manufacturer’s information and analyzed using Tecan-Reader (Infinite 200).
Cell viability assay
The number of viable cells was determined by quantification of ATP in the cell culture using CellTiter-Glo® luminescent cell viability assay (Promega, Mannheim, Germany) according the manufacturer’s instruction. (Detailed protocol in Supplementary Materials).
Colony formation assay
Ten thousand cells in 10 ml culture medium were seeded onto 10 cm culture dishes (TPP, Switzerland). After 4 days of cultivation, cells were washed with phosphate buffered saline (PBS; PAN Biotech, Aidenbach, Germany) and stained with 0,5% crystal violet solution containing 20% methanol for 15 min. Finally, the colony sizes were determined under a microscope with AxioVision Rel. 4.8 software.
In vitro treatment of cells
0.1 × 106 cells were seeded into cell culture flasks (25 cm2, Greiner bio-one) for gene expression studies, while 10,000 cells were seeded into 10 cm dishes for colony formation assays (CFA). Drugs were added after 24 h and remained on the cells for 24 h prior to protein or RNA extraction and 4 days prior to measuring of colonies in CFA. Stocks of Temsirolimus (Sigma) in ethanol and Simvastatin (Sigma) in DMSO were prepared. Final solvent concentrations after treatment was limited to 0.1% (vol/vol). Controls were treated with the solvent equivalent of the highest drug concentration.
Cells or tissues were lysed in lysis buffer (see supplementary materials). In hypoxia experiments cells were treated with hypoxia (1% O2) or Temsirolimus (0.5 μM) for 24 h prior to lysis. Twenty micrograms of protein samples were separated on SDS polyacrylamide gels and transferred to a nitrocellulose membrane. Membranes were incubated with primary antibodies at 4 °C overnight and the protein expression was detected using horseradish peroxidase (HRP)-conjugated secondary antibodies and the chemiluminescent substrate. β-actin or Tubulin were used as loading controls. Blots were quantified using Kodak 1D3.6 software. (For antibodies and dilutions used see supplementary materials).
HEK293T cells were transfected with GFP-KLF4-wt-pLVX or GFP-KLF4-mut(K409Q)-pLVX and selected with puromycin to generate a stable overexpression. Protein translation was blocked with Emetin [100 μM] at indicated time points and protein abundance was measured via immunoblot.
Luciferase reporter assay
Cells were transiently transfected with constructs expressing firefly luciferase fused to HIF1α-ODD (Addgene plasmid #18965) together with an SV40-Renilla luciferase construct (Promega) used for normalization of transfection efficiency. Cells were cultured for 18 h under hypoxia and assayed for luciferase activity with the Dual-Luciferase Reporter-Assay System (Promega).
2.5*105 cells were cultured under hypoxia (1% O2) for 4 or 18 h. In the last 3 h before harvesting, cells were treated with MG132 (10 μM, Merck). Hydroxylation of HIF-1α on proline 564 was detected by blotting Hydroxy-HIF-1α (Pro564) antibody (Cell Signaling, 3434).
Orthotopic xenograft mouse models
Convexity meningioma model
Young Swiss Nude mice (Charles River, France), > 9 weeks old were anesthetized intraperitoneally (i.p.) with Rompun (Bayer Vital GmbH Leverkusen, Germany) /Ketamin (Bremer Pharma GmbH, Warburg, Germany) mixture and fixed in the stereotactic head frame. After a longitudinal incision two holes were drilled 2 mm anterior of the bregma and 1.5 mm right and left from the sagittal suture. Using Hamilton syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland) 2.5 × 105 of KLF4wt or KLF4K40Q transfected IOMM-Lee meningioma cells in 2.5 μl PBS (PBS; PAN Biotech, Aidenbach, Germany) were applied 1.5 mm deep in each hole. The skin was sealed with Histoacryl (B Braun Surgical, S.A., Rubi, Spain).
Skull base meningioma model
Young Swiss Nude mice, > 9 weeks old, were used. A single hole was drilled 1.5 mm anterior of the bregma and 2 mm on the right side from the sagittal suture and 0.5 × 105 transfected IOMM-Lee cells (KLFwt or KLFK409Q) in 0.5 μl PBS were deposited above the skull base bone (about 7.5 mm in depth). At day three after tumor inoculation, daily treatment with Temsirolimus i.p. (20 mg/kg) was started for 11 days. At day 14 after tumor inoculation, tumor growth was analyzed by MRI. Tumor area was determined by tracing the largest tumor cross section on a given MRI slice.
For actin staining 10,000 cells were grown in culture medium on non-treated cover slips at 37 °C, 5% CO2 overnight, fixed in 4% paraformaldehyde (PFA)/PBS for 10 min, permeabilized with 1% Triton X-100/PBS for 3–5 min and blocked with 1% BSA for 30 min. Actin was stained with Phalloidin Alexa Fluor 546 (Invitrogen, USA)/ 40x in PBS for 30 min. Cover slips were mounted on slides and imaged under a confocal microscope TCSSL (Leica, Germany).
Study of human tumor specimens was performed after written consent was received from individual patients. The study was approved by the local ethics board of University Hospital of Cologne, (Application No. 03–170). Animal studies were approved by the local state department: Landesverwaltungsamt Sachsen Anhalt, Referat Verbraucherschutz, Veterinärangelegenheiten; Licence number 42502–2-1550 UniMD.