Wang et al. BMC Cancer 2010, 10:415 http://www.biomedcentral.com/1471-2407/10/415 Page 6 of 11 Figure 2 TLR9 expression in glioma tissues correlated to glioma progression and the prognosis of GBM patients. A. Representative immunohistochemistry staining photos of the glioma tissue arrays. Normal human kidney tissues provided by Department of Surgery were used as positive control. For negative controls, the primary antibody was replaced by normal mouse serum. Cell nuclei were counterstained with haematoxylin. B. Kaplan-Meier analysis showing the PFS(progress free survival) of glioblatoma (GBM) patients in low TLR9 expression group (bold line) and high TLR9 expression group (dotted line). The difference of PFS between the two groups is significant(p = 0.001). CpG enhance glioma invasion in vitro To study the effects of TLR9 stimulation on the invasion behavior of glioma cells, we performed invasion assays using the well characterized TLR9 agonist, ODN2006. As shown in Fig. 4, the invasion of glioma U87 cells was sig- nificantly elevated by CpG ODN(ODN2006), compared with that of glioma cells treated with medium. Similar to other researchers’ results [11], the non-CpG ODN (ODN2006 control) can also enhance the invasion of U87 cells. The increased cell numbers seen in the invasion assays were not due to effect of proliferation, because the Table 2 TLR9 expression is correlated with glioma progression Degree of TLR9 immunoreactivity (%) Low High Characteristics Normal brain Low Gradea High Gradeb IIIb1 IVb2 a, bP = 0.000; b1, b2P = 0.078. 13/13(100.00) 80/91 (87.91) 113/201(56.22) 47/73(64.38) 66/128 (51.56) 0/13(0. 00) 11/91(12.09) 88/201(43.78) 26/73(35.62) 62/128(48.44) ODN had no effects on cell viability(Fig. 3). In order to ensure that TLR9 is responsible for the invasion effects induced by CpG ODN, glioma cells were treated with CpG ODN in the presence of chloroquine, which is an inhibitor of endosomal acidification resulting in inhibi- tion of TLR9 signaling [21]. As shown in Fig. 4, chloro- quine significantly inhibited CpG ODN induced invasion in glioma cells, which suggest that TLR9 signaling is responsible for the enhanced invasion of U87 cells induced by CpG ODN. Discussion Glioma is the most frequent tumor originating from glial cells in the nervous system, various glioma subtypes has been divided by grade or by the cell type. Each sub- type constitutes different genetic mutations [22] and likely differ in prognosis and response to therapy. We proposed that there may exist some specific biological markers related to the clinical behaviors of gliomas, these markers may also involved in signal pathways that driving the initiation and development of gliomas. Recently, researchers showed that virus may be responsible for the initiation and development of glioma