Wang et al. BMC Cancer 2010, 10:415 http://www.biomedcentral.com/1471-2407/10/415 glioma [13,14]. In contrast, Ginzkey et al [15] found increase in tumor size following intratumoral injection of immunostimulatory CpG ODN in a rat glioma model, this was consistent with our previous finding that the intratumoral injection of CpG ODN do not increase the survival time in GL261 glioma animal model [16], and these results indicated that directly local injection of CpG may not yield beneficial effects in glioma patients. TLR9 expression has been examined in GBMs but not found to be of prognostic significance [17], however that investigation is at mRNA level, a protein level study is thus needed. The aim of this study was to evaluate the expression of TLR9 in a large series of glioma samples with tissue array and to examine the association between TLR9 expression, clinicopathological variables, and patient outcome. To further characterize the possi- ble role of TLR9 expression on tumor cells, we examine the direct effects of CpG ODN upon the proliferation and invasion of glioma cells in vitro. Methods Cells and Cell culture The human glioma cell lines U87-MG, U251-MG and rat glioma C6 cell lines were first obtained from ATCC and maintained in the Central Lab of First Hospital of China Medical University. Glioma cells were grown in Dulbecco’s modified essential medium (DMEM, Gibcol, Gaithersburg, MD, USA) supplemented with 10% FBS (Hyclone Laboratories, Logan, USA), 100 U/ml penicil- lin, and 100 μg/ml streptomycin at 37°C with 5% CO2 in humidified air. CpG ODN and Cell challenge Nuclease-resistant phosphorothioate-modified oligonu- cleotides (ODNs) were custom synthesized and purified by Takara Biotechnology (Dalian, China), The following sequences were used (the bold letters indicate the CpG motifs or the GpC motifs of the ODN): ODN 2006, TCG TCG TTT TGT CGT TTT GTC GTT; ODN 2006 Control, TGC TGC TTT TGT GCT TTT GTG CTT. The ODNs were dissolved in endotoxin free ster- ile water, aliquot and stored at -80°C until use. Immunofluorescence U87-MG and U251-MG cells were grown on coverslips, after washing with serum free-DMEM, the cells were fixed for 10 min with 4% paraformaldehyde in PBS, per- meabilized with 0.5% Triton X-100 in PBS and blocked 1 h in PBS supplemented with 5% FBS. Cells were incu- bated overnight at 4°C with TLR9 mAb (Img-305A, clone 26C593.2, Imgenex, San Diego, USA, 1:400 dilu- tion), followed by Cy3-labelled anti-mouse secondary antibody (Sigma, USA, 1:400 dilution) for 2 h. The Page 2 of 11 nuclei were counterstained with Hoechst (1:1000 dilu- tions). In the control samples, glioma cells were stained with secondary antibody and Hoechst, but without anti- TLR9 primary antibody. Results were observed and photographed under Leica confocal microscope. Tissue Samples and Patients Tumor specimens were obtained from patients admitted for diagnosis and treatment to First Hospital of China Medical University and Beijing Tiantan Hospital. The diagnosis was made according to WHO criteria. The study was approved by the Ethics Committee of each institution and was based on the criteria of the Helsinki convention. Informed consent was obtained from each patient. Fresh surgical samples from glioma patients and non-neoplastic brain tissues(temporal lobectomy from epilepsy surgery) were immediately snap-frozen in liquid nitrogen upon surgical removal. One part of each sam- ple was fixed with formalin, embedded with paraffin wax and kept at RT. The including criteria for the patients in this study are: 1) Patients do not have other cancers or diseases such as acute infection, diabetes and did not receive previous radiotherapy, chemotherapy, or corticosteroid therapy; 2). Patients had taken routine contrast-enhanced MRI examination before and after surgical operation; 3) Patients gave informed consent; 4). Patients who died from non-glioma-related cause were excluded. The extent of tumor resection was defined as follows: (0) residual tumor >30% or biopsy; (1) partial removal with residual tumor ≤ 30%; (2) gross total resection. Clinicopathological variables were retro- spectively collected from medical records until July 2009. Progress-free survival (PFS) was defined from the date of surgery to the first MRI-confirmed recurrence or death, whichever occurred first. For the survival analysis, to avoid the bias caused by different treatment strategy, in addition to the above screening criteria, patients must have received similar treatments: surgical resection performed by neurosur- geons who used similar operational techniques and prin- ciples. Every patient in the study received the same radiotherapy regimen. All Patients received chemother- apy after radiotherapy. The criteria ensured that the patient group was uniform, which strengthens the analy- sis. We stopped to collect data at July 2009, the observa- tion for each patient will end once the patient is dead or have recurrent. 69 GBM patients fulfill these criteria and were used for the survival analysis. There are no patients survived more than 24 months. RNA Extraction and RT-PCR assay for detection of Toll-Like Receptor 9 Total RNA was extracted from 3 glioma cell lines and 34 clinical glioma samples using Trizol reagent (Invitrogen,