Wang et al. BMC Cancer 2010, 10:415 http://www.biomedcentral.com/1471-2407/10/415 cancer cells(U373 and D54MG) with CpG ODN stimu- lates their invasion via MMP 13 activation. In our research, a different glioma cell line(U87 cells) were used to ensure whether or not TLR9 is responsible for the invasive of glioma cells induced by CpG ODN. We believe that, just like the TLR9 signal pathways in other cancer cells, the activation of TLR9 may lead to enhanced invasiveness of U87 glioma cells, this was sup- ported by our research, we found that the TLR9 agonist CpG ODN(ODN 2006) can significantly increased the invasiveness of U87 glioma cells(Fig. 4). Most impor- tantly, we found that the CpG induced invasion could be blocked with chloroquine, which is an inhibitor of TLR9 signaling pathway. All the results suggested that the expression of TLR9 in glioma cells is functional and related to the invasiveness of glioma cells. In this research, we found that non-CpG ODN(ODN2006 con- trol) can also increase the invasion of glioma cells through TLR9 signaling pathway, which indicated that non-CpG ODNs (ODNs from in vivo cells such as dying tumor cells) may also be able to increase the invasion of glioma through TLR9 pathway. In addition to this, for the first time, we showed that chloroquine was able to inhibit the TLR9 induced invasiveness of glioma cells (Fig. 4), and thus may be used as an adjuvant for the GBM therapy, this was confirmed by Briceno et al [39,40], who showed that chloroquine may improve mid-term survival when given in addition to conven- tional therapy for GBM. However, the exact mechanism for the TLR9 regulation of glioma invasion need further investigation and is out of the focus of this research. Our finding has several important implications: First, they may give novel clues for the investigation of the mechanism of glioma initiation and development. Sec- ondly, our findings warrant caution in the directly injec- tion of TLR9 agonist CpG ODN into glioma tissues for the glioma immunotherapy., this conclusion may be contradictory to some researcher’s work [13,41,42]. Although there is contradictory results for the expres- sion of TLR9 in the GL261 cells between Grauer’s result [13] and Meng’s result [17], both of them confirmed that the prolonged survival seen in their study was not due to the expression of TLR9 expression in glioma cells, but rather due to increased apoptosis of GL261 cells in vivo and enhanced modulation of the local CNS immune response. TLR9 expression in non-tumoral cells or microglia activation may play an important role in the CpG induced anti-cancer immunotherapy, how- ever, the inflammatory response observed following CpG treatment do not always prevent growth of solid tumor masses, in one CpG treated glioma animal model [43], intratumoral application of CpG proved ineffective. In addition to this, Deng et al [44] showed that intracer- ebral injection of ODN containing CpG motif induce Page 9 of 11 meningitis in mice and rat. In a phase II trial evaluating the efficacy of CpG in 34 recurrent GBM patients [45], patients usually experienced transient neurological wor- sening or fatigure after intratumoral application of CpG. In 3 patients, seizure occurred just after administration of CpG. Our research indicated that locally administered CpG ODN may increase the invasion of glioma cells. Based on these data, carefully safety evaluation in animal model and in large amount of glioma patients should be performed before CpG can be routinely intratumoral administered in clinic. For the glioma immunotherapy strategy with intratumoral injection of CpG ODN, the most successful animal model is GL261 mouse model [13,14]. However, we think GL261 was not a perfect glioma model, since CpG ODN can inhibit GL261 cell proliferation in a cell type specific manner [13], but for human cell lines such as U87 and U251, or rat glioma cell line such as C6, we did not find such effect in this study(Fig. 3). So, it is difficult to translate the data from GL261 model into other cell lines or other models. We did not deny the effectiveness of CpG ODN on tumor immunotherapy, our previous experiment demonstrated that the CpG ODN can activate immune system sys- temically and cure the brain tumor when it was deliv- ered subcutaneously [16]. We think CpG ODN may activate TLR9 and promote tumor progression if it was delivered locally into the tumor within the brain. CpG ODN may be a magic antitumor weapon, but how to use it and where to use it is important question, which may lead to very different result. We must emphasis that we did not think the TLR9 only express in glioma cells, the infiltrated lymphocyte, the microglial cells may also express high level of TLR9, Meng et al [17] indicated that TLR9 was mainly expressed in infiltrated lymphocyte or microglial cells in GBM tissues. However, in our study, we found the glioma cells do express TLR9(Fig. 1). We consider the glioma tissue as an unseperatabe whole, which was con- sist of glioma cells, infiltrated lymphocytes and micro- glial cells et al. The expression of TLR9 in different kind of cells within the glioma tissue, but not only in glioma cells, will affect the prognosis of patients with glioma., we think the TLR9 expression may reflect inflammatory microenviroment within glioma tissues. There may be several reasons for the high expression of TLR9 in GBM tissues: 1. by stander phenomenon, the promoter of TLR9 was activated because of other activated pathways in GBM. 2. The upregulation of TLR9 could be beneficial to the tumor, promoting survi- val and the invasiveness of glioma cells, the selective pressure of the local microenvironment might result in the ultimate higher expression of TLR9. 3. The presence of HCMV in gliomas [24] may responsible for the high TLR9 expression, however, the relationship between the