40 human glioma samples (20 GBM; 5 anaplastic astrocytoma; 6 anaplastic oligodendroglioma; 9 oligodendroglioma) were acquired from University of Texas MD Anderson Cancer Center’s Brain Tumor Center tissue bank. 51 human glioma samples (28 GBM; 23 low-grade glioma) were acquired from the Tumor Tissue Bank of the Tianjin Medical University Cancer Institute and Hospital. Tissues were obtained from surgery and snap frozen.
RNA extraction for transcriptome sequencing.
While frozen, each tissue (weighing up to 50 mg) was transferred to a liquid nitrogen–cooled mortar and pestle, crushed into powder, and dissolved in 1 ml TRIzol reagent (Invitrogen). Chloroform (200 μl) was added to the sample, which was then vortexed at high speed for 15 seconds and centrifuged at 12,000 g for 15 minutes at 4°C. The aqueous phase was transferred to a fresh 1.5-ml Eppendorf tube, and an equal volume of 70% ethanol was added and the contents mixed by tube inversion. A Qiagen RNeasy mini column (Qiagen) was used to further purify the sample. The column was washed twice with 500 μl RPE buffer (Qiagen), and RNA was eluted with 50 μl nuclease-free water. The RNA was quantified using a spectrophotometer. RNA integrity was verified using an Agilent 2100 BioAnalyzer. Poly-A selection was not performed on any of the sample pools.
Samples were pooled for SOLiD sequencing according to tumor type. GBM samples were divided into 4 pools of 5 tumor samples. Anaplastic astrocytoma and anaplastic oligodendroglioma samples were pooled into 2 separate pools, and the oligodendroglioma samples were further split into 2 pools. 2 pools of commercial normal brain RNA (Ambion) were also acquired: 1 pool contained RNA from adult brain, the other from fetal brain.
Library preparation for whole transcriptome sequencing.
Libraries for both whole transcriptome and small RNA sequencing were prepared using the small RNA expression kit from Applied Biosystems Inc. (PN 4397682; Life Technologies Corp.), based on the SOLiD System whole transcriptome and small RNA sequencing protocols provided by Applied Biosystems. rRNA was depleted from total RNA using the Invitrogen Ribominus Eukaryotic Kit (PN A1083708; Life Technologies Corp.), and 0.5–1.0 μg rRNA-depleted total RNA was fragmented using RNase III. The fragmented rRNA-depleted total RNA was hybridized and ligated with truncated adaptor mix A from the SOLiD small RNA expression kit. Next, reverse transcription was performed to generate cDNA templates. cDNA was size selected from Novex 6% TBE-urea gel (Invitrogen). The excised gel piece containing DNA 100–200 bp in length was split vertically into 4 pieces using a razor blade. The size-selected cDNA was further amplified using the supplied primer set containing a 6-base-long sequence-specific barcode in approximately 12–15 cycles of PCR. The purified PCR products with barcodes served as a library. Libraries ranged in size from 150 to approximately 250 bp and contained 50- to 150-bp cDNA inserts, quantitated and qualitated by Agilent Bioanalyzer 2100.
Library preparation for small RNA sequencing.
The sample containing small RNA was hybridized with truncated adaptor mix A provided in the small RNA expression kit. The adaptor mixes were sets of RNA/DNA oligonucleotides with a single-stranded degenerate sequence at one end and a defined sequence required for SOLiD sequencing at the other. Hybridizing and ligating the sample with adaptor mix A sequentially yielded the template for SOLiD sequencing from the 5′ end of the small RNA. The small RNA population of 18–40 nt ligated with adaptor mix A was reverse transcribed to generate cDNAs. To meet the sample quantity requirement for SOLiD sequencing and to append the required terminal sequences to each molecule, the cDNA libraries were amplified using one of the supplied primer sets containing a 6-base-long sequence-specific barcode in approximately 12–15 cycles of PCR. The individual library PCR products, containing small RNA of 18–40 nt with barcode(s), were purified and size-selected for 108–130 bp by electrophoresis on 6% polyacrylamide gel.
Template bead preparation.
The individual prepared library was quantitated and titrated, before multiplexing, as pooled templates for emulsion PCR; the template molecules were attached to 1 μm beads as described in the Applied Biosystems SOLiD emulsion PCR protocol. After emulsion PCR, the template beads with amplified monoclonal templates were enriched by adaptor P2-affinity binding of polystyrene beads in 60% glycerol gradient by centrifugation. The P2-enriched template beads were further modified by terminal transferase with oligo linker for immobilization to the slide and prepared for deposit on substrate-coated glass slides, as described in the Applied Biosystems protocol.
Sequencing runs were performed on SOLiD version 3.5 for both whole transcriptome RNA sequencing and small RNA sequencing. The library template beads were titrated by workflow analysis to determine the percentage of P2-positive beads in the total template before they were deposited onto slides for sequencing. The number of P2-positive template beads deposited in full chambers of slides with multiplexed 5 barcoded whole transcriptome libraries and performed in 50 nt of whole transcriptome sequencing, and of P2-positive beads of 10 pooled barcoded libraries on full slide for small RNA sequencing in 35 nt small RNA sequencing, was determined. The average depth of colorspace reads per sample pool was 6.7 × 107.
Fusion gene discovery.
To identify fusion gene candidates, RNA-seq reads from each sample pool were aligned against transcripts from NCBI RefSeq 38. Reads that aligned to known transcripts were assumed to result from normal transcription and were discarded from further analysis. The 5′ and 3′ ends of unaligned reads were split into 2 anchors, each 18 colors in length. The paired anchors were aligned against human exon sequences from NCBI RefSeq 38. Anchors with more than 3 alignments against the exome were discarded as noninformative. Since anchors were short, no mismatches against the reference exon sequences were allowed in anchor alignments. For each read with alignments for both anchors, a list of anchor-based exon-exon junctions was generated by taking a Cartesian product of the 2 sets of exons to which the anchors aligned. If an anchor pair aligned to an exon-exon junction between 2 exons from the same gene, all junctions for that anchor pair were discarded, because the read was assumed to originate from some form of unannotated transcription or splicing. We were then left with fusion candidates represented by exon-exon junctions between distinct genes. Once we had the lists of anchor-based junctions for every sample, we combined them and verified the junctions by aligning the full transcriptome-unaligned reads against them.
To reduce the number of false positives, each fusion candidate was required to fulfill the following requirements (Supplemental Figure 1). (a) The fusion candidate must not involve rRNA genes or other highly abundant genes, and must not involve genes located at hypervariable genomic sites. The list of blacklisted gene name patterns included RN18S1, RN28S1, RPPH1, SNORD*, SNORA*, RNY*, RN7SL, RN7SK, RNU*, and HLA-*. (b) Full 50-base reads aligning to the fusion candidate must together cover at least 15 bases on both sides of the fusion junction. (c) The fusion candidate must not be found in Ambion commercial normal brain samples. (d) The fusion candidate’s supporting reads must not contain more than an average of 0.7 nt mismatches per read against the reference transcriptome. (e) The 3′ side of the fusion junction must have no perfect alignments against any sequence within 50 kb of the genomic alignment for the 5′ side of the fusion junction. The 5′ side of the fusion junction must have no perfect alignments against any sequence within 50 kb of the genomic alignment for the 3′ side of the fusion junction.
For each fusion fulfilling these requirements, we counted the number of reads in each of the 8 tumor sample pools and calculated a P value using the Pearson χ2 test to compare goodness-of-fit against a uniform reference distribution. Fusions were then ranked in ascending order by P value, so that fusions whose read distributions deviated most significantly from a uniform distribution were ranked at the top. In total, we identified 17,564 putative fusion junctions supported by 1 or more RNA-seq reads; filtering yielded a ranked list of 52 fusion candidates.
Identification of FGFR3-TACC3 fusions in TCGA samples.
We downloaded TCGA GBM transcriptome sequencing data for 169 patients using the CGHub system. To look for evidence of FGFR3-TACC3–positive cases, we executed our fusion gene discovery algorithm on the samples and identified 2 patients (TCGA-27-1835 and TCGA-76-4925) with hundreds of reads overlapping an FGFR3-TACC3 fusion junction. Patient samples with 1–4 reads overlapping an FGFR3-TACC3 fusion junction were ignored because all such samples exclusively originated from the same sequencing batch as TCGA-27-1835 and exhibited the same fusion variant as TCGA-27-1835. We concluded that these weakly expressing fusion genes resulted from nucleic acid contamination between samples. RPKM gene expression values were calculated for FGFR3 and TACC3 to show their overexpression in fusion-positive samples relative to fusion-negative controls.
Assessment of mutual exclusivity between FGFR3-TACC3 and RTK amplifications.
Custom Agilent CGH microarrays on 4 fusion-positive and 3 fusion-negative GBM samples from the University of Texas MD Anderson Cancer Center and the Tianjin Medical University Cancer Institute and Hospital were analyzed for EGFR, PDGFRA, and MET amplification by calculating the median log ratio of all probes within each gene relative to commercial pooled reference DNA.
Agilent HG CGH 244A microarray data for 444 GBM samples was downloaded from TCGA GBM project. These data included fusion-positive samples TCGA-27-1835 and TCGA-06-6390, reported by Singh et al. (21
, and MET
amplification was assessed by calculating the median log ratio of all probes within each gene relative to commercial pooled reference DNA.
TCGA GBM exome sequencing data was downloaded for the fusion-positive patients TCGA-06-6390, TCGA-19-5958, and TCGA-76-4925 reported by Singh et al. (21
). Paired tumor DNA and normal blood DNA was available for all 3 cases. EGFR
, and MET
amplification was assessed by calculating the number of reads overlapping each gene in tumor versus normal blood samples.
A sample was considered to harbor high-level EGFR, PDGFRA, or MET amplification if data indicated 5 or more extra copies of the gene. Mutual exclusivity of FGFR3-TACC3 fusion and EGFR, PDGFRA, and MET amplification was assessed using Fisher exact test.
Small RNA expression analysis based on sRNA-seq.
Small RNA sequencing reads were trimmed to a length of 18 colors and aligned against mature microRNA sequences from miRBase version 14 using Bowtie (35
). Mature microRNA expression levels were calculated by counting reads that aligned completely within an annotated mature microRNA site in the pre-microRNA sequence. Read distributions of selected microRNAs are shown in Supplemental Figure 3. Trimming of reads to 18 colors prior to alignment was necessary because the sRNA-sequence protocol resulted in reads that included a 3′ adapter sequence. The trimming step also allowed us to effectively calculate the expression level of each microRNA as the sum of its iso-miR expressions. MicroRNA expression levels were normalized by the total number of mappable reads per sequencing experiment (36
Array CGH analysis.
Customized CGH microarrays with high probe density at the fusion region were designed using the Agilent eArray tool. The microarray design was based on the Agilent 105A backbone profile with 105,000 probes and 22-kbp median spacing. Extra probes (22,500 at 200-bp intervals) were designed for cytoband 4p16.3 (containing FGFR3-TACC3
). Customized microarrays were ordered from Agilent. To analyze copy numbers, all microarray probes were remapped against the hg19 genome assembly, and log ratios were calculated between Cy3 and Cy5 channels. The log ratios were then segmented using circular binary segmentation (37
). Microarray files have been deposited in GEO (accession no. GSE42400).
RT-PCR validation of fusion transcripts.
To create a mammalian expression vector, cDNA was synthesized using SuperScript III (catalog no. 18080-051; Invitrogen) and random hexamers followed by PCR amplification using Advantage HD polymerase mix (639241; Clontech) with FGFR3 primer TCGCCAGTCTCCCGAGC and TACC3 primer GACAGCGGCTCCGTGGAGG. The fusion PCR products were TOPO cloned (45-0640; Invitrogen). Finally, the MSC enzymes BamHI and XbaI were used to move the fusion genes into pcDNA3.1+. All final constructs were sequence verified.
Immunoblot validation of fusion proteins.
Total protein was isolated from tumor tissue by subjecting it to lysis buffer (1× RIPA containing 0.1% Halt protease) and phosphatase inhibitor cocktail (Fisher Scientific) and brief sonication (Branson Digital Sonifer Model 450) at 10% amplitude, followed by rotation at 40°C for 30 minutes. Total protein was isolated by centrifugation at 1,214 g for 15 minutes, and lysates were stored at –80°C.
Detection of fusion breakpoint at the DNA level.
Genomic DNA was isolated from fusion-positive patient samples and used as a template for PCR with Extensor Long Range PCR Polymerase (Thermo Scientific). Primers were designed against exon 18 (5′-CCCTCCCAGAGGCCCACCTT-3′) of FGFR3 and exon 11 (5′-CCTGCTCCTCAGCTCCCGGT-3′) or the intronic region after exon 4 (5′-GCAGACCCACGGCCAAGACC-3′) of TACC3. PCR products were gel purified and cloned with TOPO TA Cloning Kit for Sequencing with chemically One Shot TOP10 Chemically Competent E. coli (Invitrogen). At least 2 fusion-positive clones from each patient were subjected to capillary sequencing with plasmid-specific primers.
cDNA (random primed, superscript III) was made from GBM-13, the patient sample with the highest detected fusion level. The complete 3.0-kb fusion transcript was amplified using the forward primer 5′-TCGCCAGTCTCCCGAGC-3′ (upstream of the FGFR3 start codon) and the reverse primer 5′-GACAGCGGCTCCGTGGAGG-3′ (downstream of the TACC3 stop codon) with the Clontech Advantage — LA polymerase kit (catalog no. 639152). The PCR product was cloned into pCR2.1 (catalog no. K4500; Invitrogen). An error-free subclone was created in pcDNA3.1 (by way of a pBluescript II intermediate to pick up required HindIII-Xba cloning sites). WT FGFR3 and TACC3 constructs were purchased (Origene) and subcloned into the pcDNA3.1 expression vector.
Cell line generation and immunoblotting.
All tissue cultures were maintained in DMEM/F12 medium supplemented with 10% FBS in a 37°C humidified incubator containing 5% CO2
. Control cell lines containing pcDNA3.1 expression vector only were obtained as previously described (38
). The FGFR3-TACC3
construct sequenced from GBM-13 was inserted into the pcDNA3.1 expression plasmid (Invitrogen) under control of the cytomegalovirus promoter. 6 × 105
SNB19 and U251 cells were transfected with 10 mg FGFR3-TACC3
, WT FGFR3
, or WT TACC3
cDNA with Lipofectamine (Invitrogen) per the manufacturer’s instructions. Stably transfected cells were selected for with 0.4 mg/ml G418 (Invitrogen) for 2 weeks, after which SNB19 clones were selected and amplified. Relative expression of either WT FGFR3
or the FGFR3-TACC3
fusion between SNB19 cell clones and the mixed population was measured by immunoblot analysis using a mouse monoclonal antibody probing for FGFR3
amino acids 25–124 (sc-13121; Santa Cruz Biotechnology Inc.). Downstream analysis after FGFR3-TACC3
fusion overexpression was performed, probing for β-tubulin (9F3), phosphorylated STAT3 (Tyr705; 3E2), total STAT3 (79D7), phosphorylated p44/42 MAPK (ERK1/2; Thr202/Tyr204), total p44/42 MAPK (ERK1/2), and total TACC3
(Santa Cruz Biotechnology Inc.)
Cell viability, proliferation, apoptosis, and soft-agar assays.
An MTT assay was performed to measure cell viability. Cells were seeded at 650 cells/well in a 96-well plate in quadruplicate and allowed to attach overnight. Cell viability was measured by incubating cells with 0.5 mg/ml MTT reagent in PBS (Sigma-Aldrich) for 2 hours. MTT reagent was then aspirated, and cells were subjected to lysis with 100% DMSO. Plates were read at 590 nm using the Tecan SpectraFluor Microplate Reader and Magellan 6 software (Tecan Group Ltd.) at 48, 72, or 96 hours after cell plating. Cell proliferation was measured via BrdU incorporation assay at 7, 11, and 13 hours in triplicate. Briefly, cells were synchronized at G1 by starvation for 3 days. Fresh medium was then added, and the cells were incubated for 12 hours, followed by pulse labeling with 20 mm BrdU for 1 hour at the indicated time points. The cells were stained with FITC-conjugated anti-BrdU antibody, incubated with 7-aminoactinomycin-D (7-AAD), and quantified via flow cytometry. To determine whether FGFR3-TACC3 promotes colony formation in soft agar, empty vector, FGFR3-TACC3, WT FGFR3, and WT TACC3 SNB19 or U251 stable cells were cultured in DMEM/F12 medium with 10% FBS in the log phase. The soft agar was prepared by mixing 1.5% sterile low melting point agarose in PBS with fresh medium at a 1:2 dilution. Soft agar (1 ml of 0.5%) was added to each well of a 6-well plate and kept at room temperature for 15 minutes. Cells were then harvested and suspended in 0.375% soft agar at 1,000 cells/ml. This cell suspension (1 ml) was added on top of the prepared base agar layer. The plates were incubated for 2 weeks and fed 2 times per week. Colonies were counted under a microscope, and relative colony size was measured using AxioVision 3.1 software.
Gene expression microarray analysis on fusion clones.
fusion– or WT FGFR3
–transfected SNB19 mixtures or clones were hybridized onto dual-channel Agilent Whole Human Genome 4 × 44K v1 microarrays. RNA from parental SNB19 cells was hybridized onto the reference channel. Microarray slides were imaged, and background adjusted probe intensities were calculated using Agilent Feature Extraction Software version 18.104.22.168. Probe sequences were aligned against RefSeq 38 transcript sequences, and the probes were arranged into probesets based on the genes they aligned against. Background-adjusted probe intensities were quantile normalized and summarized using the robust multiarray analysis (RMA) algorithm (39
). Differential gene expression was calculated by comparing against the reference channel. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA; version 11904312). Microarray files have been deposited in GEO (accession no. GSE42401).
miR-99a reporter gene assay.
The 3′-UTR of FGFR3 in the pMirTarget reporter vector was purchased from Origene Technologies Inc. The miR-99a binding site was mutated using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies), creating a deleted mutant from 5′-AAUACGGGUA-3′ to 5′-AA––––––UA-3′. To test the ability of miR-99a to target the 3′-UTR of FGFR3, WT and mutant constructs were transfected into SNB19 cells concurrently with TK Renilla luciferase reporter vector (Promega) and either control (scrambled) microRNA or miR-99a mimic (Thermo Fisher Scientific). At 48 hours after transfection, cells were assayed for relative luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). Transfections were replicated in 9 independent experiments. In each experiment, relative luciferase activity following miR-99a overexpression was normalized to scrambled controls and converted to a log ratio. An unpaired 1-tailed t test was used to determine whether the log ratios were different between WT FGFR3 UTR and deletion mutant cells.
qRT-PCR validation of successful miR-99a transfection in SNB19 cells.
To confirm successful transfection, miR-99a mimic (300516-03), anti–miR-99a (IH-300516-05), and scrambled (control) microRNA (Thermo Fisher Scientific) were transfected into SNB19 parental cells with 9 biological replicates (transfections) per group. For each of the biological replicates, miR-99a (assay ID 000435; Applied Biosystems) levels were quantified using TaqMan qRT-PCR (microRNA protocol 4364031 revision B; Applied Biosystems) with 3 technical replicates that were averaged and normalized to endogenous U6 (assay ID 001093; Applied Biosystems). A 2-tailed Mann-Whitney U test was used to assess whether miR-99a/U6 log ratios differed between groups.
qRT-PCR validation of FGFR3 mRNA level regulation by miR-99a, miR-21, and miR-125b.
miR-99a mimic (300516-03), anti–miR-99a (IH-300516-05), miR-21 mimic (C-301023-01), miR-125b mimic (C-300595-03), or scrambled (control) microRNA (Thermo Fisher Scientific) were transfected into SNB19 parental cells with 9 biological replicates (transfections) per group. 0.5 μg total RNA from each transfection was reverse transcribed in 20-μl reactions. A ratio of 1 μg total RNA to 0.4 μg random hexamers was maintained, and the mixtures were heated at 70°C for 10 minutes. The tubes were then incubated at room temperature for 10 minutes, and the following components were added: 1× Superscript II RT Buffer (Invitrogen), 10 mM dithiothreitol (Invitrogen), 0.5 mM dNTPs (ISC Bioexpress), 20 U RNase Inhibitor (Ambion), and 200 U Superscript II Reverse Transcriptase (Invitrogen). The reaction was again incubated for 10 minutes at room temperature and then held at 37°C for 1 hour. The reaction was incubated at 42°C for 1.5 hours and then at 50°C for 30 minutes. Real-time PCR was performed on the Applied Biosystems Prism 7900 using an FGFR3 assay (Hs00179829_m1; Applied Biosystems) and human cyclophilin A (4326317e) Vic-labeled Pre-Developed Assay Reagent (Applied Biosystems); the 15-μl final reaction volume contained 1× TaqMan Universal PCR Master Mix (Applied Biosystems) and 1× Assay-on-Demand. cDNA (25 ng/well) was amplified with the following cycling conditions: 10 minutes at 95°C, followed by 50 cycles at 95°C for 15 seconds and 60°C for 1 minute. Each qRT-PCR measurement was performed in 3 technical replicates that were averaged and normalized to cyclophilin A. A 2-tailed Mann-Whitney U test was used to assess whether FGFR3/cyclophilin A log ratios differed between groups.
Measurement of FGFR3 protein level regulation by miR-99a, miR-21, and miR-125b.
To determine whether overexpression of miR-99a mimic or anti-miR, miR-21 mimic, and miR-125b mimic affected FGFR3 protein expression, each respective microRNA was transfected into SNB19 parental cells, and relative FGFR3 expression was measured 48 hours later via immunoblot analysis (Santa Cruz Biotechnology Inc.). Band intensity was quantified and normalized to β-tubulin expression using Image J software (NIH). The experiment was replicated 3 times.
Generation of FGFR3 WT and fusion constructs containing the 3′-UTR of FGFR3.
To generate WT FGFR3 and FGFR3-TACC3 fusion constructs containing the 3′-UTR of FGFR3, the 3′-UTR of FGFR3 was amplified by PCR from a FGFR3 3′-UTR plasmid (SC215711; Origene Technologies) to introduce the NheI cloning sites at both sides (underlined below), using the forward primer 5′-GCTAGCGGGCTCGCGGACGTGAAG-3′ and the reverse primer 5′-GCTAGCGGTTAGCAACCAGGTGTC-3′. The PCR product was cloned into pCR2.1 and verified by DNA sequencing. FGFR3 3′-UTR was then digested with NheI from pCR2.1–
FGFR3 3′-UTR and subcloned into the XbaI sites downstream of pcDNA3.1+ WT FGFR3 and pcDNA3.1+
FGFR3-TACC3 vectors. All constructs were verified by DNA sequencing. To determine the ability of miR-99a to regulate expression of FGFR3 3′-UTR–containing constructs, miR-99a or scrambled control was transfected and assayed via immunoblot as described previously.
To determine whether the FGFR3-TACC3 fusion exhibits ligand dependence or independent firing, EV, FGFR3-TACC3 fusion, and WT FGFR3 cells were seeded in 6-well plates and incubated with 50 ng/ml bFGF ligand for 30 minutes at 37°C. Cells were then harvested and subjected to immunoblot, probing for FGFR3, phosphorylated ERK, total ERK, and actin (Santa Cruz Biotechnology Inc.).
Intracranial xenograft implantation and tissue preparation.
Male athymic mice (nu/nu
) were implanted in the brain with EV, FGFR3-TACC3
fusion, or WT FGFR3
cells. Briefly, mice were anesthetized with 0.25 ml of a cocktail of 10 mg/ml ketamine and 1 mg/ml xylazine, and cells were implanted using cranial guide screws as previously described (40
). A Hamilton syringe and microinfusion syringe pump (0.5 ml/min; Harvard Apparatus) were used to implant 1 × 106
cells into the brain of 10 mice simultaneously, as described previously (41
). Upon detection of an external tumor or obvious declining health, mice were sacrificed by intracardiac perfusion of PBS and 4% paraformaldehyde. Brains were extracted and fixed in 10% formalin for 24 hours, embedded in paraffin, and sectioned into 5-mm slices.
For immunohistochemical staining, Dako Envision+System–horseradish peroxidase and diaminobenzidine (DAB) were used (Dako). Briefly, after antigen retrieval for 10 minutes in 0.1 M citrate buffer (pH 6.0) or 1 mM EDTA (pH 8.0) and subsequent incubation in peroxidase block solution for 5 minutes at room temperature, the sections were incubated overnight at 4°C with rabbit anti-human FGFR3 (1:600; Abcam), pSTAT3 (1:200, Tyr705 D3A7; Cell Signaling Technology), and pERK (1:200, T202/Y204; Cell Signaling Technology). The sections were incubated in peroxidase-labeled polymer for 30 minutes at room temperature, and the signals were revealed with DAB+ substrate–chromogen solution. For negative controls, primary antibodies were replaced with PBS.
Drug treatment studies.
For studies measuring cell viability after U0126 drug treatment, EV, WT FGFR3, and FGFR3-TACC3 fusion cells were plated at 100,000 cells/well in a 96-well plate. 12 hours after plating time, “0” was read to ensure cells were plated in equal numbers. At this time, drugs were added in increasing concentrations, with DMSO only as control. Plates were assayed 48 hours later, and values were normalized to DMSO controls. Cells were also seeded in 6-well plates and incubated with each inhibitor for 1 hour. These cells were assayed via immunoblot to determine the potency of each drug concentration.
Unless otherwise indicated, data are represented as mean ± SEM. Statistical analyses were performed using Fisher exact test, Mann-Whitney U test, 2-way ANOVA, 1- or 2-tailed Student’s t test, and log-rank test, as indicated. Activation z scores were calculated as defined in the Ingenuity white paper “Ingenuity Downstream Effects Analysis in IPA.” Mice that died of causes other than cancer were considered censored. A P value of 0.05 or less was considered significant.
Glioma tissue samples from the Brain Tumor Center tissue bank of the University of Texas MD Anderson Cancer Center were collected under an institutional review board–approved protocol. Glioma tissue samples from the Tumor Tissue Bank of the Tianjin Medical University Cancer Institute and Hospital were collected with approval from the institutional review board. All subjects provided informed consent of their participation in the study. Implantation of cells into the brains of male athymic mice were performed according to institution-approved protocols.