Part 1: identifying meningioma-specific candidate biomarkers
Specimens of previously untreated, primary intracranial meningiomas resected between January 2006 and December 2010 were retrospectively analysed for the expression of four potential biomarkers (i.e. EMA, PDGF-β, VEGF-α and SSTR-2). A total number of 148 meningioma specimens were available for analysis. All patient data were anonymized according to the regulations of the Medical Ethical Research Committee of the University Medical Center Groningen.
Meningioma samples were examined using tissue microarrays (TMAs). TMA sections were deparaffinised with xylene, rehydrated in ethanol, and rinsed in distilled water. After antigen retrieval with a Tris-EDTA or Tris-HCl buffer, endogenous peroxidase was blocked for 30 min using a 0.1% H2O2 PBS solution. Respective antibody staining was performed at room temperature. The selected primary antibodies of interest were anti-EMA (mouse monoclonal, Dako), anti-PDGFR-β (rabbit polyclonal, Santa Cruz), anti-VEGF-α (rabbit polyclonal, Santa Cruz) and anti-SSTR-2 (rabbit monoclonal, Epitomics). We used normal cerebellar and anterior pituitary tissue as positive controls. Additionally, we omitted the primary antibody and used IgG controls. Secondary and tertiary antibody staining was performed for 30 min. All sections were subjected to a 3,3-diaminobenzidine solution for 10 min and finally counterstained with haematoxylin for 2 min, dehydrated in ethanol, cleared, mounted and cover slipped.
For immunohistochemical (IHC) evaluation, TMA sections were scanned with an ultra-resolution digital scanner ScanScope CS®, Aperio® with × 20 image magnification and evaluated with Aperio ImageScope® software. Each tissue core of the TMA section was scored using the following scoring method: negative, (0); weak/focal staining, (1); moderate/diffuse staining, (2); strong/diffuse staining, (3). Two authors (AM and WFD) independently evaluated tissue cores and in case of discrepant scores, consensus was reached by way of discussion between both evaluators. Cores were regarded as non-informative and consequently dismissed when > 50% tissue was lost or presented inappropriate amounts of collagen staining. Tumour specimens which were represented by less than two complete cores were excluded. A mean score was calculated for each specimen and specimens with an average score of 1 were considered positive, whereas specimens with a mean score of ≥ 2 were summarized as “high score”.
Statistical analysis
All statistical analyses were performed using IBM® SPSS® Statistics 20. Spearman rank-order correlation was used to find correlation between targets and WHO classifications. All reported P values were two sided and a value of P ≤ 0.05 was considered as statistically significant.
Part 3: confirming expression in in vitro cultures
Meningioma 3D cell culture models were established to provide an accurate model for the disease [
13,
24]. Surgical leftover fresh tumour tissue was washed with ice-cold PBS and mechanically dissociated. After adding 15 to 20 ml accutase, tissue incubated for 30 min at room temperature. The suspension was passed through a 70-μm cell strainer to procure single cells and pelleted. Cells were seeded in T75 flasks with medium containing DMEM/F12 supplemented with 2% B27, 20 ng/ml EGF, 20 ng/ml bFGF [
22], and 2% pen/strep.
For IHC analyses, 3D cultures were dissociated with accutase and cytospun. Subsequently, cells were fixated with 4% formaldehyde, washed with PBS and underwent a blocking step with 1% H2O2 in PBS for 10 min. Cells were then incubated with anti-SSTR-2 (1:100; MAB4224, R&D systems) for 1 h, followed by incubation of the corresponding secondary and tertiary antibodies diluted at 1:50 in PBS with 1% BSA and 1% AB serum for 30 min. Lastly, cells were incubated with 5% 3-amino-9-ethylcarbazole diluted in acetate buffer with 0.1% hydrogen peroxide for 10 min, counterstained with haematoxylin for 2 min, and mounted with Kaiser’s glycerin for microscopic examination using a Leica DM 3000 microscope.