Cell culture and small molecule treatments
The primary human meningioma M10G cell line was derived from a fresh meningioma resection (WHO grade I, NF2 wild type, Chr22q loss of 1 copy, convexity) by mechanically mincing approximately 100 mg of tumor tissue in Hanks’ Balanced Salt Solution (HBSS) and then plating in media with a 1:1 ratio of Dulbecco’s Modified Eagle Medium (DMEM) and F12 (Life Technologies, #10565) and Neurobasal medium (Life Technologies, #21103), supplemented with 5% fetal bovine serum (FBS) (Life Technologies, #16141), B-27 supplement without vitamin A (Life Technologies, #12587), N-2 supplement (Life Technologies, #17502), 1X GlutaMAX (Life Technologies, #35050), 1 mM NEAA (Life Technologies, #11140), 100 U/mL Anti-Anti (Life Technologies, #15240), 20 ng/mL EGF (R&D systems, Minneapolis, MN, #236-EG), and 20 ng/mL FGF2 (Peprotech, Rocky Hill, NJ, #100-18C).
The primary human meningioma BenMen cell line (WHO grade I, NF2-mutant, falx) was originally derived from a meningothelial meningioma and transfected with hTERT to achieve immortalization [17]. Cells were cultured in DMEM (10313021, Life Technologies) supplemented with 10% FBS and 1X GlutaMAX, which was also used to culture NIH3T3 (CRL-1658, ATCC) and HEK293T (CRL-3216, ATCC) cells.
For ciliation and Hedgehog signaling assays, NIH3T3, BenMen and M10G cultures were transitioned to OptiMEM (31985062, Thermo Fisher Scientific) and treated with recombinant Sonic Hedgehog 1 μg/ml (1845, R&D Systems), Smoothened agonist 100 nM (566660, Calbiochem) or vehicle control, for 24 h.
Cell transfection
SMO, the oncogenic allele SMOW535L, and the somatic variant SMOL412F were cloned into the pEGFP vector, and constitutively active GLI2 (GLI2CLEG) was cloned into pCMV vector. According to the manufacturer’s instructions, Fugene HD reagent (Promega, E2311) was used for transfection of constructs into NIH3T3, BenMen and M10G cells. Cells were harvested for experimentation 72 h after transfection.
CRISPR interference
Lentiviral particles containing pMH0001 (UCOE-SFFV-dCas9-BFP-KRAB, Addgene #85969) were produced by transfecting HEK293T cells with standard packaging vectors using the TransIT-Lenti Transfection Reagent (Mirus Bio, MIR 6605). M10G cells were stably transduced with lentiviral particles to generate M10GdCas9-KRAB cells by isolating BFP-expressing cells using fluorescence activated cell sorting on a Sony SH800.
Single-guide RNA (sgRNA) protospacer sequences were individually cloned into the pCRISPRia-v2 vector (Addgene plasmid #84832), between the BstXI and BlpI sites, by ligation. Each vector was verified by Sanger sequencing of the protospacer. One sgRNA expression vector with the following protospacer was cloned for non-targeting control sgRNAs (ncRNA) (5′-GCTGCATGGGGCGCGAATCA-3′), PTCH1 sgRNAs (sgPTCH1) (5′-AAATGTACGAGCACTTCAAG-3′) and SMO sgRNAs (sgSMO) (5′-CAAGAACTACCGATACCGTG-3′). Lentivirus was generated as described above for each sgRNA expression vector, and M10GdCas9-KRAB cells were independently transduced with lentivirus from each sgRNA expression vector, then selected to purity using 20 μg/mL puromycin over 7 days.
Meningiomas
Meningioma tissue microarrays were comprised of cases treated with surgery at the University of California San Francisco (UCSF) between 2003 to 2012. Demographic and clinical data were obtained from the medical record. For all cases, diagnostic imaging was re-reviewed to confirm the extent of resection and determine the occurrence and timing of local recurrence, which was defined as local recurrence of any size after gross-total resection, or growth of ≥20% along any dimension after subtotal resection. Mortality data and cause of death were extracted from the electronic medical record, institutional cancer registry, Surveillance, Epidemiology, and End Results (SEER), Department of Motor Vehicles (DMV), Social Security, and nationwide hospital databases, and publicly available obituaries. Overall survival was defined as the length of time from resection of meningioma to death. This study was approved by the Institutional Review Board, Human Research Protection Program Committee on Human Research, protocol 18–24,633.
Meningioma and meningioma tissue microarray ciliary immunofluorescence
Meningioma tissue microarrays were created as previously described [14]. In brief, two 2 mm cores were taken from each meningioma block, along with control tissue from normal adult brain, normal adult meninges, 2 lung adenocarcinomas, and placenta. All meningioma diagnoses and grades were re-reviewed according to current diagnostic criteria by a board-certified neuropathologist (MP) [13]. Only patients with demographic, histopathologic, radiologic, and comprehensive clinical follow-up data who consented to tumor sampling for research were included. Ciliary immunofluorescence was performed as previously described [11]. In brief, sections were deparaffinized in xylene, rehydrated through graded ethanol dilutions and subjected to antigen retrieval using CC1 TRIS buffer (Ventana Medical Systems); labeled with primary antibodies including Pericentrin (PA5–54109, Thermo Fisher Scientific) and γTubulin (T5192, Sigma) to mark centrosomes, and acetylated tubulin to mark cilia (T6793, Sigma). Alexa Fluor secondary antibodies and DAPI (62,248, Thermo Fisher Scientific) were used, and sections were mounted in mounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific).
Microscopy
Fluorescence microscopy was performed on a Zeiss LSM 800 confocal laser scanning microscope with a PlanApo 20X air objective. Images were processed and quantified from a representative region of each tumor using ImageJ. Cilia prevalence was quantified as the ratio of cilia to nuclei. Cilia length was quantified using a region of interest calculation.
NanoString transcriptional profiling
NanoString transcriptional profiling from formal-fixed paraffin-embedded meningiomas was performed as previously described [21]. In brief, total RNA was extracted from tumor cores containing 75% or more tumor cells, as determined by hematoxylin and eosin (H&E) staining. The GX Human Cancer Reference NanoString panel codeset, containing 30 additional meningioma related genes, were synthesized by NanoString Technologies. Of note, SMO was the only Hedgehog pathway gene included in this panel. RNA (200 ng per meningioma) was analyzed by the NanoString nCounter Analysis System at NanoString Technologies, according to the manufacturer’s protocol. Data were preprocessed, normalized against a panel of housekeeping genes, and log2-transformed count data were centered and scaled within-meningiomas using a Z-score transformation.
Primary meningioma cell immunofluorescence
Immunofluorescence for acetylated tubulin (AcTub) and Ki67 from meningioma cells was performed on glass coverslips. Cells were fixed in 4% paraformaldehyde, blocked in 2.5% BSA (Sigma) and 0.1% Triton X-100 (Sigma) in Phosphate Buffered Saline (PBS) for 30 min at room temperature (Thermo Fisher Scientific), and labeled with anti-Ki67 (ab15580, Abcam), anti-Smoothened (20787–1-AP, ProteinTech), anti-Centriolin (sc-365,521, Santa Cruz), anti-Arl13b (17711–1-AP, ProteinTech) or anti-acetylated tubulin (T6793, Sigma) primary antibodies at room temperature for 3 h. Cells were labeled with Alexa Fluor secondary antibodies and DAPI to mark DNA (Life Technologies, H3570) for 1 h at room temperature, and were mounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific).
Quantitative reverse transcriptase polymerase chain reaction
RNA was isolated from cells using the RNEasy Mini Kit and a QiaCube (QIAGEN), and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad) and a ProFlex thermocycler (Thermo Fisher Scientific). Target genes were amplified using PowerUp SYBR Green Master Mix and a QuantStudio 6 thermocycler (Thermo Fisher Scientific). Gene expression was calculated using the ΔΔCt method, with normalization to human GAPDH (sense: 5′-CTTCACCACCATGGAGAAGGC-3′, antisense: 5′-GGCATGGACTGTGGTCATGAG-3′) or mouse Gapdh (sense: 5′-TGCCCCCATGTTTGTGATG-3′, antisense: 5′- TGTGGTCATGAGCCCTTCC-3′). Target human gene primers were PTCH1 (sense: 5′-GAAGAAGGTGCTAATGTCCTGAC − 3′, antisense: 5′-GTCCCAGACTGTAATTTCGCC -3′), SMO (5′-GAAGTGCCCTTGGTTCGGA − 3′, antisense: 5′-GCAGGGTAGCGATTCGAGTT − 3′) and GLI1 (sense 5′-AGCCTTCAGCAATGCCAGTGAC-3′, antisense: 5′- GTCAGGACCATGCACTGTCTTG-3′). Target mouse gene primers were Gli1 (sense 5′- GGTGCTGCCTATAGCCAGTGTCCTC-3′, antisense: 5′-GTGCCAATCCGGTGGAGTCAGACCC − 3′).
Statistics
All experiments were performed with at least 3 biologic replicates and at least 3 technical replicates. Lines represent mean and error bars represent standard error of the mean. Local recurrence free and overall survival were estimated using the Kaplan-Meier method and compared by log-rank tests. ANOVA and two-tailed Student’s unpaired t-test were used to compare groups, and in all cases, statistical significance, as denoted by (*), was defined as p ≤ 0.05.