Wang et al. BMC Cancer 2010, 10:415 http://www.biomedcentral.com/1471-2407/10/415 Page 8 of 11 Figure 4 Invasion of GBM cell line U87 in response to CpG ODN(ODN 2006). U87 cells were plated in 24 well cell culture chambers using transwell inserts precotated with matrigel. ODN 2006 (10 uM), ODN 2006 control (10 uM), same volume of the control medium, or TLR signaling inhibitor chloroquine (10 uM) were added and incubated for 24 hours, the number of invaded cells was stained and counted, all analysis were performed in triplicate. The invasion ability was represented as numbers of invaded cells. Bars refer to the standard error of the mean value. Comparison between groups was made by using student’s t test, P ≤ 0.05 was defined as significant. et al [14] further confirmed their RT-PCR result with FCM. We preferred Grauer’s data because their result is consistent with the function of TLR9 signal pathway. As a receptor for CpG ODN, TLR9 play a key role in tissue repair and cancer progression, it was demonstrated that CpGs stimulated the invasion of TLR9+ cancer cells, but not TLR9- cancer cells [11]. Grauer et al [13] showed that GL261 is TLR-, which indicated that CpG ODN may not affect the invasion of GL261 cells, this was supported by widely accepted results which indi- cated that CpG ODN induced apoptosis of GL261 in vitro and cured GL261 glioma animal model in vivo. Meng et al [17] and Andaloussi et al [14] found that U87 and U251 express high level of TLR9 mRNA, how- ever no data about the expression of TLR9 protein in these two cell lines was reported. To avoid the contro- versy data like GL261, we investigated the expression of TLR9 in U87 and U251 cells at both the mRNA level (RT-PCR) and protein level(immunofluoresence), our result for the first time showed that TLR9 staining located intracellularly and could be seen throughout the cytoplasm in U87 and U251 glioma cells. The relationship between the expression level of TLR9 and tumor grade of glioma is unknown, to investigate this, large amount of clinical samples are required. Meng et al [17]checked the expression of TLR9 in 37 frozen glioma samples, and they found no relationship between the TLR9 expression and survival, however, there are several factors may affect their results in their study: 1. some of the their samples were frozen for a very long time since 1990. 2. their samples were frozen under -180°C, the frozen and thawing process may damage the cells and thus affect the result of immuno- histochemistry analysis. 3. for the QPCR experiment, they set a QPCR threshold(0.025) to divide the patients into low TLR9 group and high TLR9 group, however, this threshold is not a widely accepted criteria and may affect their result. 4. Only quantitative RT-PCR data was used and there is no quantitative protein level data, the non-neoplastic brain tissues and necrotic tissue, which have low expression of TLR9, may be included in the tissues for the extraction of mRNA from the specimen, and thus may interfere with the result. Because of these reasons, their results require confirmation with larger specimen numbers. We screened the expression of TLR9 in a large collec- tion of human glioma samples in tissue array with immunochemistry. With the mutivariant analysis (Table 3), we found that TLR9 expression is an indepen- dent prognostic factor to predict patient PFS in GBM (P <0.05), which indicated that the expression levels of TLR9 correlate significantly with the clinical outcome of patients with GBM. Other biological factors such as age, gender, KPS before surgery, the extent of resection were not found associated with prognosis. Our research include same group of patients that was used in one previous published study [30], which showed that the resection extent and KPS were not related to the survi- val, and these data were further supported by other researcher’s results. Based on the published literature, it remains unclear whether the extent of surgical resection correlates with survival [31-33]. Even for KPS, there are many studies found that it is not associated with survi- val of GBM patients [34-36]. In addition to this, our study for the first time demonstrated that TLR9 expres- sion increased significantly from low grade (grade II) glioma to high grade (grade III and grade IV) glioma (Table 2), this result is similar to results obtained by other researchers with clinical breast specimen [37] and cervical neoplasia [38]. Present data indicated that TLR9 expression increases according to the histopathological grade of glioma, and the TLR9 expression level is related to the PFS of GBM patients. The potential relationship between TLR9 pathway and cell proliferation was explored, we demonstrated that TLR9 activator CpG ODN did not affect the prolifera- tion of glioma cells in vitro. Merrell et al [11] showed that treatment of TLR9 expressing breast and brain