| IntroductionEGFR variantⅢ(EGFRvIII) is the most common variant of the EGFR and is present in many different cancer types especially in glioblastomas but not in normal tissue. It results from the fusion of exon 1 to exon 8 of the EGFR gene, which results in a novel glycine at the junction. It is generated from a deletion of exons 2 to 7 of the EGFR gene, which results in an in-frame deletion of 267 amino acids from the extracellular domain of the receptor. This mutant receptor is constitutively active without the ligand in tumors and can lead directly to cancer phenotypes due to its oncogenic properties. EGFRvⅢis an attractive target antigen for cancer immunotherapy because it is not expressed in normal tissue and because cells producing EGFRvIII have enhanced capacity for dysregulated growth, survival, invasion and angiogenesis. EGFR variantⅢ(EGFRvⅢ) promotes glioblastoma (GBM) cell proliferation and invasion, and EGFRvIII may be associated with transforming growth factor beta-2 (TGF-(32) and interleukin-10 (IL-10).Material and methods1.Tumor SamplesGlioblastoma samples were obtained from patients admitted to the Neurosurgery Department at the China Medical University First Affiliated Hospital for surgical resection. All tumors were histologically diagnosed as grade IV, according to the WHO criteria. Samples were stored at-80℃until further analysis by RT-PCR, real-time RT-PCR, and immunohistochemistry.2.RNA isolation and RT-PCR Total RNA was isolated using TaKaRa RNAiso Reagent, according to the manufacturer's instructions. Only RNA samples with OD260/OD280≥1.8 and with no visible degradation by agarose gel electrophoresis were used for RT-PCR and realtime PCR experiments. RT-PCR using 500 ng total RNA and the TaKaRa RNA PCR Kit was performed following the manufacturer's instructions. EGFRvⅢprimers were designed to span exons 1 and 8 of EGFR. The following EGFRvⅢprimers were used: forward,5'-GCGAGTCGGGCTCTGGA-3'and reverse, 5'-TTCACCAATACCTATTCCGTTACAC-3'. After sample denaturation at 94℃for 2 min, PCR amplification was performed at 94℃for 30 s,55℃for 30 s, and 72℃for 1 min for 30 cycles. The RT-PCR products, including 171 bp (EGFRvⅢ) and/or 972 bp (WT-EGFR), were visualized using 2% agarose gel electrophoresis. The RT-PCR products were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit following the manufacturer's instructions and the ABI PRISM 3730XL DNA Analyzer and ABI PRISM 377XL DNA Sequencer.3.ImmunohistochemistryImmunohistochemistry was performed using formalin-fixed and paraffin-embedded tumor tissues cut into 5-μm section and mounted on silanized slides. The sections were deparaffinized and rehydrated in PBS after which heat-induced antigen retrieval was performed by placing the slides in 50 mM citrate buffer, pH 6.0. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS with 0.05% Tween-20 (PBS-T) for 30 min. Sections were washed in PBS and blocked for 2 h in the appropriate species of serum diluted to 10% in PBS. The sections were incubated with mouse anti-human EGFRvIII monoclonal antibodies (DH8.3),overnight at 4℃. After incubation with a biotinylated secondary antibody, the avidin-biotin-peroxidase complex was detected using 3,3-diaminobenzidine (DAB) as the chromogen after which the tissues were counterstained with hematoxylin.4.Cell cultureU87.MG cells were maintained in DMEM supplemented with 10% fetal bovine serum,100 units/mL penicillin, and 100 mg/mL streptomycin. U87.MG.EGFRvIII cells, which express EGFRvIII, were a gift from W. Cavenee and F. Furnari (Ludwig Institute for Cancer Research, San Diego); they were cultured in DMEM with 10% fetal bovine serum,200μg/mL G418,100 units/mL penicillin, and 100 mg/mL streptomycin. Both U87.MG and U87.MG.EGFRvⅢwere cultured in a 5%CO2 atmosphere at 37℃.5.MTT assayU87.MG and U87.MG.EGFRvIII cells (5000 cells per well) were cultured overnight in 96-well plates in growth media. After seeding, cells were maintained in 10% serum for 1,2,3,5, and 7 days, and fresh medium was applied every two days. The cells were incubated with 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide for 4 h after which dimethyl sulfoxide (DMSO) was added, and the cells were disrupted by shaking for 10 min. Optical densities at 490 nm were assessed using a microplate reader.6.In vitro invasion assaysMatrigel Membrane Matrix invasion assays were performed. After the Matrigel inserts were rehydrated,5000 U87.MG.EGFRvⅢand U87.MG cells were seeded onto the chamber wells. The transwell inserts with 8μm pore size were coated with Matrigel. The upper side of the chamber contained serum-free DMEM medium whereas the lower chamber contained DMEM with 20% FBS. After 48 h, non-invading cells within the upper chambers were gently removed, and adherent cells on the lower side of the membrane were fixed and stained with a toluidine blue solution. Ten random fields were chosen and counted under a microscope. Invasion was quantified by counting stained cells using a microgrid, and the mean count was calculated for statistical analysis.7.Real-time PCRTotal RNA was extracted and isolated as described above. Real-time RT-PCR was performed on an ABI PRISM 7000 Real-Time PCR System, using a SYBR PrimeScript RT-PCR Two-Step Kit according to the manufacturer's instructions. The PCR cycling conditions were as follows:95℃for 10 seconds; 95℃for 5 seconds, and 60℃for 20 seconds for 40 cycles. Gene-specific primers for real-time RT-PCR were designed and synthesized by TaKaRa Inc. Primer sequences for IL-10 were as follows:forward,5'-GAGATGCCTTCAGCAGAGTGAAGA -3' and reverse,5'-AGGCTTGGCA ACCCAGGTAAC-3'. The following TGF-β2 primers were used:forward,5'-GCTT TGGATGCGGCCTATTG-3'and reverse,5'-CCAGCACAGAAGTTGGCATTGTA-3'. A standard melting-curve cycle was used to verify the quality of amplification, such as the absence of primer dimer formation. Samples were normalized by comparison withβ-actin gene expression.8.Western blot analysisThe EGFR antibody, which recognizes both wild-type and mutant forms of the receptor, was used for Western blot analysis. Cells were washed with PBS and lysed in RIPA lysis buffer (50 mM Tris HC1,150 mM NaCl,1% NP-40,0.5% sodium deoxycholate,0.1% SDS) which had been supplemented with a protease inhibitor cocktail containing 1 mmol/L phenylmethyl sulfonylfluoride and dithiothreitol. After centrifugation to remove cell debris, the cell lysate was boiled. The proteins were separated on using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in 5% nonfat milk, and probed with the primary antibody overnight at 4℃. Blots were then incubated with horseradish peroxidase-linked secondary antibodies followed by enhanced chemiluminescence detection. Western blot analysis was performed more than three times and representative blots are shown.β-actin expression was used as a loading control.ResultsUp-regulation of TGF-P2 mRNA expression was observed in EGFRvⅢ-positive patients; however, no differences in IL-10 expression levels were detected between the groups. A statistically significant increase in cell proliferation and invasion as well as TGF-β2 and IL-10 expression was observed in U87.MG.EGFRvIII cells as compared to U87.MG cells.ConclusionAssociations between EGFRvIII expression and up-regulation of the immunosuppressive cytokines, TGF-β2 and IL-10, were observed, and EGFRvⅢexpression was associated with increased cell proliferation and invasion. Understanding the immunobiology of EGFRvⅢpositive GBM patients may assist in the development of novel, targeted treatment strategies.
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