Wang et al. BMC Cancer 2010, 10:415 http://www.biomedcentral.com/1471-2407/10/415 tumor cells. The staining index score > 4 was defined as tumors with high expression, and staining index ≤ 4 as tumors with low expression of TLR9. MTT assay MTT assay was used to assess the effect of CpG ODN on glioma cells proliferation. Briefly, 1 × 104 U87, U251 and C6 cells were seeded into 96-well plates in triplicate in a volume of 100 ul per well, then incubated at 37°C for 24 h for cells adherence. CpG ODN(ODN 2006), ODN 2006 control(10 uM) and same volume of medium (10 uM) were added, cell growth was measured at 24, 48, 72 hours after the treatment. MTT (Sigma) was dis- solved in PBS at 5 mg/ml and filtered to be sterilized, 20 ul MTT solution was added at different time points. Plates were then incubated at 37°C for 4 h, 100 ul dimethylsulfoxide was added to each well and mixed thoroughly to dissolve the blue-violet crystals. Cell viabi- lity data were measured with an ELISA reader (TECAN SUNRISE, Austria) at 490 nm. Transwell assay The invasion assay was done in 24 well cell culture cham- bers using transwell inserts (Corning life sciences, NY, Bioscience, San Jose, CA, USA) with 8 um Pore membrane precotated with matrigel (BD biosciense, San Jose, CA, USA). U87 cells were plated at the density of 1 × 104 per upper well in 200 ul culture medium (DMEM, 1%FBS), the lower chamber was filled with 500 μl medium (DMEM, 20% FBS). ODN 2006 (10 uM), ODN 2006 control (10 uM), same volume of the control medium, or Chloro- quine (10 uM) was added to both upper and lower well. The cells were allowed to invade for 24 hours, after which, the noninvading cells with Matrigel matrix were removed from the upper surface of the membrane by scrubbing with a cotton-tipped swab. Cells on the lower surface of the filter were fixed for 30 min in methanol and glacial acetic acid mixture (3:1), air-dried briefly, and stained with Giemsa. The number of invaded cells was counted from five preselected microscopic fields at 200× magnification, all experiments were performed in triplicate. Statistical analysis SPSS statistical software for Windows 13.0 (SPSS, Inc., Chicago, Illinois, USA) was used for all statistical analy- sis. Differences of tumor cell proliferation and invasion between groups were analyzed by t-test. The Jonc- kheere-Terpstra test was used to correlate cumulative TLR9 expression with glioma grading, and the chi- square test was used for comparisons between groups. Cox regression was used to correlate TLR9 expression with PFS while adjusting for clinicopathological vari- ables. Survival curves were constructed using the Kaplan-Meier method. The log-rank test was used for Page 4 of 11 comparison between groups. P ≤ 0.05 was defined as significance. Results TLR9 is expressed in glioma cell lines and tumor samples The expression of TLR9 mRNAs in glioma cell lines and tumor tissues were examined using RT-PCR method. As shown in Fig. 1A, TLR9 is expressed both in human and rat glioma cell lines, TLR9 expression in glioma tissues was also shown in Fig. 1A, all the 34 glioma samples tested in our experiment express TLR9. Housekeeping gene GAPDH was used as positive control. Immunofluorescence was used to determine TLR9 protein expressed in human glioma cell lines. A cyto- plasmic localization of TLR9 in U87 and U251 was con- firmed by confocal microscopy (Fig. 1B). This finding is in agreement with previous studies on the distribution of TLR9 in RAW264.7 cells [19]. Correlation between expression of TLR9 and malignancy of gliomas To determine whether the expression level of TLR9 pro- tein is associated with the histological characteristics of gliomas, glioma tissue microarray was constructed, sec- tions were examined by immunohistochemical staining with an antibody against human TLR9. The anti-TLR9 antibody(Img-305A, clone 26C593.2, Imgenex, San Diego, USA) was widely used by other researchers. In our research, during immunohistochemistry staining, primary antibody control was replaced by normal mouse serum, which was widely used as negative control in immunoas- say. Most importantly, we demonstrated that there is no TLR9 staining in normal brain tissues, which indicated no false positive staining exist in our study. It was demon- strated that kidney tubule but not the glomerus have the expression of TLR9 [8], so we used kidney tubules as posi- tive control for TLR9 staining in this study. As shown in Fig. 2A and Table 2, TLR9, mainly as cytoplasmic staining was found to be expressed in gliomas, no strong immu- noreactivity (score <4) was detected in non-neoplastic brain tissues. TLR9 expression was detected in all high grade glioma cases and was highly expressed(score >4) in 43.78% (88/201) cases (Table 2). We next analyzed the correlation between the expression of TLR9 protein and the histological staging of gliomas. As summary in table 2, expression was significantly higher in high grade gliomas (grade III and grade IV) compared to low-grade gliomas (grade II) (P < 0.01), which supported that the TLR9 expression is associated with the progression of glioma. Association between TLR9 expression and GBM patient prognosis To determine the prognostic value of TLR9 protein for GBM patients, we assessed 69 GBM cases(Recurrent or