Clinical samples
Meningioma (MN) specimens were collected following the national ethical approvals (REC No: 14/SW/0119; IRAS project ID: 153351) (Plymouth Hospitals NHS Trust: R&D No: 14/P/056 and North Bristol NHS Trust: R&D No: 3458), receiving a unique MN number. Blood was collected prior to surgery. ‘J’ specimens were collected via UK-Brain-Archive Information-Network (BRAIN UK; Ref no: 15/011; REC no: 14/SC/0098). Clinical and histopathological data for all samples used in this study are detailed in table 1.
Table 1. Clinical characteristics of the tumor samples used (n=130).
Clinical features
|
Group
|
Patients
|
n
|
(%)
|
Sex
|
Female
|
74
|
66.7
|
Male
|
37
|
33.3
|
WHO grade
|
WHO I
|
81
|
62.4
|
WHO II
|
37
|
28.4
|
WHO III
|
12
|
9.2
|
Primary or recurrent
|
Primary
|
117
|
94.4
|
Recurrent
|
7
|
5.6
|
Age at 1st diagnosis
|
Median
Range
|
61
20-87
|
Cell culture
Human meningeal cells (HMC, cat# 1400) were obtained from Sciencell™, and maintained following the manufacturer’s protocol. The malignant meningioma cell line KT21-MG1 (RRID:CVCL_M429) was cultured as previously reported (20). The benign meningioma cell line Ben Men-1 (RRID:CVCL_1959), and the malignant meningioma cell line IOMM-Lee (RRID:CVCL_5779) were both maintained as in (21). WHO I primary meningioma cells were obtained as reported in (22), and WHO II primary meningioma cells were isolated from resected tumors following the same protocol, but maintained in Dulbecco’s Modified Eagle Medium F-12 Nutrient Mixture (Ham) (DMEM/F-12 (1:1)(1X) + GlutaMAX™-I; Thermo Fisher Scientific, Loughborough, UK) supplemented with 20% FBS (Sigma Aldrich, Gillingham, UK), 1% D-(+)-glucose (Sigma Aldrich, Gillingham, UK) and 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific, Loughborough, UK).
RNA isolation, miRNA profiling, data mining tools, and gene expression
Total RNA was extracted using the Qiazol® reagent (Qiagen UK), following the manufacturer’s protocol. RNA quality, integrity, and concentration were established using the NanoDrop ND-2000 (ThermoFisher Scientific UK).
Quantimir™ Cancer MicroRNA qPCR Array (Cambridge Biosciences) was performed according to the manufacturer’s protocol. The bioinformatics analysis of the 96-miRNA profiling was performed using the NormFinder software (23) on an Excel 2016 platform (Microsoft, USA). Statistically significant miRNA candidates were identified by applying a Student’s t-Test, assuming both equal and unequal variances (p<0.01). We selected only those candidates statistically significant in both groups, applying Venn analysis (Venny 2.1.0; http://bioinfogp.cnb.csic.es/tools/venny/index.html). Results were visualized by hierarchical clustering using the software Morpheus®, applying the Euclidean distance metrics (https://software.broadinstitute.org/morpheus/index.html).
The RT² Profiler™ PCR Array Human EGF/PDGF Signaling Pathway and the RT² Profiler™ PCR Array Human miR-9 Targets (Qiagen) were performed according to the manufacturer’s recommendations on a LightCycler® 480 II System (Roche).
RT-PCR was performed using 1 μg of total RNA using the TaqMan® MicroRNA Reverse Transcription Kit or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific UK), accordingly. Real Time PCR (qPCR) was conducted using the TaqMan® Fast Advanced Master Mix supplemented with TaqMan® assays (ThermoFisher Scientific UK) on a LightCycler® 480 II System (Roche), in triplicate, employing the following assays (ThermoFisher Scientific UK): hsa-mir-9* (ID 002231), hsa-mir-134-3p (ID 466606_mat), hsa-mir-145* (ID 002149), hsa-mir-10b* (ID 002315), hsa-mir-126 (ID 002228), FOS (Hs04194186_s1), CDH1 (Hs01023895_m1). As internal controls, we used RNU6B (ID 001093) or GAPDH (Hs02786624_g1), accordingly.
Gene expression levels were computed using the quantitative 2-(ΔΔCt) method, employing the HMC as calibrator, in triplicate (24).
Proliferation and viability assays
Malignant meningioma KT21-MG1 and IOMM-Lee cells were plated in 96-well culture plates (3000 cell/well) for the proliferation assay, and in 6-well culture plates (3×105 cells/well) for the viability assay. Cells were allowed to adhere overnight (18 hours). The following day, medium was replaced with fresh warmed medium, and Afatinib was administered for 24 hours. Cell proliferation was assayed using the ‘CellTiter-Glo® Luminescent Cell Viability Assay’ as recommended by the supplier (Promega). Cell viability was determined by hemocytometer counting chamber using the trypan blue staining (Gibco).
Western blotting
Protein immunoblot was conducted as reported in (25). Briefly, tissues and cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (Roche and Santa Cruz Biotechnology, respectively). Protein concentration was estimated using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) following the instructions of the supplier on a FLUOstar Omega microplate reader (BMG Labtech, Germany). Proteins were separated on a Laemmli SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Immun-Blot® PVDF Membrane, Bio-Rad). Membrane blocking, antibody incubation, and washes were performed as previously described (21).
The following primary antibodies were from Cell Signaling Technology: anti-c-FOS (#4384, RRID:AB_2106617), anti-CD9 (#13174, RRID:AB_2798139), anti-p-EGFR (#3777), anti-EGFR (#4267), anti-p-HER2/ErbB2 (#2247), anti-HER2/ErbB2 (#4290), anti-p-AKT (#4060), anti-AKT (#9272, RRID:AB_329827), anti-E-cadherin (#14472, RRID:AB_2728770) and anti-N-cadherin (#14215). Anti-CD63 (sc-59286, RRID:AB_784278) and anti-Calnexin (sc-46669, RRID:AB_626784) were purchased from Santa Cruz Biotechnology (USA), while anti-GM130 (clone 35, RRID:AB_398142) and GAPDH (#MAB374, RRID:AB_2107445) were from BD Biosciences (UK) and Millipore (USA), respectively. Detection was achieved using the ECL or ECL Plus Western Blotting substrate (Pierce). Membranes were exposed to Amersham Hyperfilm ECL (GE Healthcare Life Sciences). Immunoreactive bands were acquired at a resolution of 600 dpi, quantified using the ImageJ software, and each band was normalized vs. the corresponding GAPDH value.
Lentiviral-mediated transduction
KT21-MG1and IOMM-Lee cells were plated in 6-well plates at 1.5×105/well and left adhering O/N. Sub-confluent cells were washed once with PBS, and medium was replaced with complete medium supplemented with 8% protamine sulphate (Sigma-Aldrich). C-Fos shRNA (h) lentiviral particles (sc-29221-V, Santa Cruz Biotechnology) and Control shRNA Lentiviral Particles-A (sc-108080, Santa Cruz Biotechnology) were added according to the manifacturer’s protocol. After 48h of incubation, medium containing lentiviral particles was removed, cells were washed once with PBS, and transduced KT21-MG1 and IOMM-Lee cells were selected for 4 days using 10 μg/mL puromycin (Thermo Fisher Scientific), before being assayed.
Exosome isolation
Ben-Men-1, KT21-MG1, and WHO I primary meningioma cells were cultured in medium supplemented with Exo-FBS™ Exosome-depleted FBS (System Biosciences). Briefly, cells were plated in 10-cm dishes at 1×106/well, and left adhering O/N. Then, medium was replaced, and cells were cultured for three days (see Fig. S7a). Supernatant was collected from confluent cells and exosomes were harvested using the Total Exosome Isolation (from cell culture media) reagent (Invitrogen, UK), following manufacturer’s recommendations.
After whole blood collection in gold Vacutainer® tubes (Becton Dickinson, UK), specimens were allowed to clot undisturbed at room temperature for 30 minutes and serum was obtained by centrifuging at 2400 x g for 10 minutes at 4 °C. Exosome isolation from patients’ serum samples was performed using the Total Exosome Isolation (from serum) Reagent (Invitrogen, UK) following manufacturer’s instructions. Exosome enrichment was assessed by immunoblotting, performed using 40 µL of the exosomes resuspension, according to the Total Exosome RNA & Protein Isolation Kit (Invitrogen, UK) protocol.
Statistical analysis
Statistical and receiver operating characteristic (ROC) analyses were performed using the GraphPad Prism software. The unpaired Student’s t-Test was applied in experiments with two different groups and the one-way ANOVA in experiments with three or more different groups, using the Tukey’s multiple comparison as post-test. Data are expressed as mean ± SEM.