Tissue samples and cell culture
Tissue of 24 VS, surgically resected in the Department of Neurosurgery of the University Hospital Würzburg, was collected from January 2018 until July 2019. Half of each sample was embedded in paraffin for immunohistochemistry, the other half was processed for primary cell culture as described elsewhere [5]. Briefly, the fresh tumor samples were washed three times with ice-cold HBSS +/+ (Gibco, Carlsbad, CA, USA), shred into 0.2 × 0.2 cm small pieces and digested with 1.7 mg/ml dispase (PluriSTEM, Millipore, Massachusetts, USA) and 1.5 mg/ml collagenase (Sigma, Munich, Germany) in 6 ml VS-medium (DMEM supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin (all from Gibco, Carlsbad, CA, USA), 0.35% Glucose (B. Braun, Melsungen, Germany), 8 nM Heregulin (Stemcell Technologies, Cologne, Germany), 0.5 mM IBMX and 0.25 µg/ml insulin (both from Sigma, Munich, Germany)) for 17 h at 37°C and 5% CO2. Afterwards, the suspension was centrifuged for 5 min at 300 ×g, the supernatant was discharged and the pellet resuspended in 12 ml VS-medium, before plating 2 ml into each well of laminated 6-well-plates [5]. The cells were cultured in a humidified atmosphere of 5% CO2 at 37°C.
Merlin overexpression and ADAM9 knock down
95,000 cells per well of the primary cell cultures were treated with 2 ml VS-medium containing 8 µg/ml protamine (Sigma, Munich, Germany) and 100 µl NF2 transcript variant 1 (NM_000268) Human mGFP Tagged ORF Clone Particle RC205883L2 (Merlin overexpression), 57 µl ADAM9 Human shRNA Lentiviral Particle TL314947VB (Locus ID 8754) (ADAM9 knock down) or 120 µl Lenti-shRNA Control Particles TR30021V (scrambled control) (all from OriGene, Rockville, MD, USA) and incubated for 2 (Merlin overexpression) or 3 days (shRNA). On the next day the medium was replaced by 2 ml fresh VS-medium, the Merlin overexpressing cells were photographically documented and lysed as described below. The shRNA transfected cells were cultured for another 24 h. Their medium was replaced by 2 ml VS-medium containing 1 µg/ml puromycin (Gibco, Carlsbad, CA, USA) on day 5, which was exchanged every 2 days by DMEM. At day 11 five fields of view of each well were photographed through the 10× magnifying objective of the DMI 3000B fluorescence microscope and the DFC 450C camera (Leica, Wetzlar, Germany) in bright field with 35 ms exposure time and for green fluorescence using filter L5 ET and an exposure time of 900 ms. Cells were counted supported by usage of the program ImageJ (National Institutes of Health (NIH), Bethesda, MD, USA; http://imagej.nih.gov/ij/).
Western-blot
After washing the cells twice with phosphate buffered saline (PBS; Biochrom, Berlin, Germany), they were lysed with 100 µl lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycerol-bis (β-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), 1% Triton X-100, 0.5% IGEPAL CA-630, 1 mM phenylmethanesulfonylfluoride (PMSF), 10 µg/ml leupeptin and 23 µg/ml aprotinin (all from Sigma, Munich, Germany)). The extracted protein was measured with the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Polyacrylamid gel electrophoresis (PAGE) was performed as described [6] and the gel blotted for 7 min using the iBlot system (Thermo Fisher Scientific, Waltham, MA, USA) set to program 3. The membrane was blocked in TBST (Sigma, Munich, Germany) containing 5% non-fat milk powder (Roth, Karlsruhe, Germany) and probed with antibodies as described previously [6]. Antibodies NF2 B-12 sc-55575 (Santa Cruz Biotechnology, Dallas, TX, USA), ADAM9 ab186833 (Abcam, Cambridge, UK), and anti--tubulin T6557 (Sigma, Munich, Germany) were utilized diluted in TBST at 1:200, 1:1500, and 1:5000, respectively. Goat anti-rabbit IgG H&L (HRP) ab6721 (Abcam, Cambridge, UK), anti-mouse IgG HRP NA931 (GE Healthcare, Freiburg, Germany) and anti-mouse m-IgGk BP-HRP sc516102 (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used as secondary antibodies at a 1:1000 dilution in TBST. The ECL Western Blotting Analysis System (Amersham, Freiburg, Germany) was used to visualize the antibody labeled proteins.
Immunofluorescence double-staining
Immunofluorescence staining of 3 µm thick formalin-fixed paraffin sections has been described previously [6, 7]. However, blocking with 10 % goat serum (Life Technologies, Waltham, MA, USA) was performed for 2 h prior to an incubation of the slides with antibody dilution buffer (DCS, Jena, Germany) containing antibodies anti-ADAM9 ab186833 (1:100) in combination with anti-Integrin α2β1 [16B4] ab30483 (1:50) or anti-Integrin α6 [MP 4F10] ab20142 (1:50) (all from Abcam, Cambridge, UK) at 4°C overnight. Protein expression was visualized by 1 h incubation using Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (Alexa Fluor Plus 488 (A32732) and 555 (A32732); Thermo Fisher Scientific, Waltham, MA, USA), both diluted 1:400. Slides were mounted using Fluoroshield mounting medium, containing DAPI (Abcam, Cambridge, UK) and photographed with the Leica microscope DMI 3000B and the DFC 450C camera, using three different filters (Filtercubes A, L5 ET, and TXR ET) with exposure times of 77 ms, 2.5 s and 1.5 s, respectively, at both 10 and 40× objective magnification.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, USA) to determine significance using unpaired t-tests. p<0.05 was considered to be statistically significant.