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TLR9 expression is associated with prognosis in patients with glioblastoma multiforme
Journal of Clinical Neuroscience 19 (2012) 75–80
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
TLR9 expression is associated with prognosis in patients with glioblastoma multiforme Lige Leng a,b, Tao Jiang b,c, Yazhuo Zhang a,b,⇑ a
Beijing Neurosurgical Institute, 6 Tiantan Xili Street, Beijing 100050, China Capital Medical University, Beijing, China c Beijing Tiantan Hospital, Beijing, China b
a r t i c l e
i n f o
Article history: Received 15 February 2011 Accepted 29 March 2011
Keywords: Glioblastoma MMP2 MMP9 Prognosis TLR9
a b s t r a c t The aim of this study was to determine if there was an association between expression of toll-like receptor 9 (TLR9) in glioblastoma tissue and patient outcome in glioblastoma multiforme. Further, we characterized the direct in vitro effects of the TLR9 agonist, CpG oligodeoxynucleotide (ODN), commonly used as a vaccine adjuvant in cancer immunotherapy, on glioma cells. TLR9 expression was assessed using immunohistochemical techniques, and enzyme-linked immunosorbent assays were used to investigate the expression of other proteins in glioma cells relevant to immunotherapy. There was a highly significant difference in both progression-free survival and overall survival between TLR9+ and TLR9 patients, with poorer outcome in TLR9+ patients. In in vitro glioma cells, there was a positive correlation between the protein levels of TLR9 and both matrix metalloproteinase (MMP)-2 and MMP-9 (p < 0.05), but no relationship between TLR9 levels and levels of interleukin-6, transforming growth factor-b2 or signal transducer and activator of transcription (STAT)-3 (p > 0.05). Our data indicate that expression of TLR9 correlates with shorter progression-free survival and overall survival in patients with glioblastoma multiforme. Our findings also indicate that caution is warranted when directly injecting the TLR9 agonist CpG ODN into glioma tissues as part of glioma immunotherapy. Because the CpG ODN is a TLR9 agonist, we recommend caution when using CpG ODN in immunotherapy. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Glioblastoma multiforme (GBM), the most malignant form of glioma, is the most common primary malignant brain tumor in adults worldwide.1,2 Patients with this condition still face a poor prognosis, despite multimodal intensive treatment regimens comprising maximum surgical resection, radiation and chemotherapy. The median survival time of patients with GBM is only 12–15 months from the initial diagnosis.3 Immunotherapy, in which activated lymphocytes specifically target and destroy tumor cells, is a promising treatment strategy for gliomas.4 In recent years, CpG-based immunotherapy has been intensively investigated as a treatment option. CpG is a synthetic oligodeoxynucleotide (ODN) with unmethylated CG dinucleotides within particular sequence contexts.5 CpG ODN have been used in clinical trials as vaccine adjuvants in cancer immunotherapy regimens, given that they can mimic microbial DNA and activate the immune system through the binding of toll-like receptor 9 (TLR9). TLR9, a member of the TLR family, is an evolutionarily well-conserved transmembrane protein that recognizes microbe-derived molecular patterns.6 TLR9 resides in the endoplasmic reticulum ⇑ Corresponding author. Tel.: +86 135 2213 0701. E-mail address: [email protected] (Y. Zhang). 0967-5868/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2011.03.037
and its activation results in increased production of inflammatory mediators.7,8 In addition to being expressed in plasmacytoid DC and B cells, TLR9 is also expressed in breast,9 gastric,10 lung11 and prostate12 cancer cells. Accumulating data indicate that CpG ODN, which are TLR9 agonists, can promote tumor development and metastasis.9,12–14 We hypothesized that the TLR9 signal pathway may also be important in glioma growth and progression. TLR9 expression has been previously examined in a study of 37 GBM but found to not have prognostic significance.15 The aim of the present study was to evaluate the expression of TLR9 in a large series of glioblastoma samples, and to investigate a possible association between TLR9 expression, clinicopathological variables, and patient outcome. We also aimed to investigate the direct effects of the TLR9 agonist CpG ODN on glioma cells and to characterize the microenvironment of TLR9 in vitro. 2. Methods 2.1. Cells and cell cultures U87-MG and U251-MG glioma cells were obtained from the American Type Culture Collection and maintained in the Central Laboratory of the First Hospital of China Medical University. Cells
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Nuclease-resistant phosphorothioate-modified ODN 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 ODN were dissolved in endotoxin-free sterile water and stored at 80 °C until use. The cell challenge was conducted in 6-well cell culture chambers. Cells were plated at 1 106 per well (in DMEM and 10% FBS). ODN 2006 (10 lM), or ODN 2006 control (10 lM), or chloroquine (10 lM) was added the well. The cells were cultured for 24 hours.
(HRP)-labeled secondary antibody (KIT-5930, MaxVision HRP-polymer antimouse/rabbit IHC kit; Maixin-Bio, Fuzhou, China) was applied, and the sections were incubated first for 30 minutes at room temperature, then for 5 minutes at room temperature with diaminobenzidine. The sections were finally counterstained with hematoxylin and mounted with Permount (BIOS, Beijing, China). Sections were visualized and photographed using a light microscope (Olympus BX-51; Olympus Optical, Tokyo, Japan). The degree of immunostaining was scored separately by two independent investigators. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. Scores from the two investigators were averaged for further comparative evaluation of TLR9 and MMP9 expression. The proportion of positively stained tumor cells was assessed as follows: 0, <30% positive cells; 1, >30% positive cells. The intensity of staining was assessed using the following scale: 0, no staining or weak staining (light yellow); 1, moderate staining (yellowish brown) or strong staining (brown). The stain index was calculated as staining intensity proportion of positively stained cells (Supplementary Fig. 1).
2.3. Patients and treatment
2.5. Enzyme-linked immunosorbent assay
Tumor specimens were obtained from 125 patients admitted to Beijing Tiantan Hospital for diagnosis and treatment. A diagnosis of GBM was made according to World Health Organization criteria. The study was approved by the ethics committees of all institutions involved and was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all patients. GBM tissues, and non-neoplastic brain tissues obtained during temporal lobectomy for the treatment of epilepsy, were immediately snap-frozen in liquid nitrogen upon surgical removal. A portion of each sample was fixed with formalin, embedded in paraffin wax and kept at room temperature. Patients were eligible for inclusion if they had no other cancer or disease (e.g. acute infection or diabetes); if they had not previously undergone radiotherapy, chemotherapy, or corticosteroid therapy; and if they had undergone routine contrast-enhanced MRI examinations before and after surgery. Patients who died from a non-glioma-related cause were excluded. To minimize the confounding effects of different surgical procedures, all patients studied underwent surgical resection performed by neurosurgeons using similar operational techniques and principles, and every patient in the study received the same radiotherapy regimen. The extent of tumor resection was defined as follows: 0, partial removal with 630% residual tumor; 1, gross total resection. Clinicopathological data were retrospectively collected from patients’ medical records up to July 2010. Observations for each patient ended once the patient died or was found to have recurrent disease. Progression-free survival (PFS) was defined as being the time from the day of surgery to the first MRI-confirmed recurrence or death, whichever occurred first. Overall survival (OS) was defined as being the time from the day of surgery to death.
TLR9 protein was extracted using the Mem-PER eukaryotic membrane protein extraction reagent kit (Pierce, Rockford, IL, USA). Other enzyme-linked immunosorbent assay (ELISA) kits used were as follows: human prolactin (PRL; Uscn Life Sciences, Wuhan, China); human signal transducer and activator of transcription-3 (STAT3; Cell Signaling, Beverley, MA, USA); human interleukin (IL)-6 and transforming growth factor (TGF)-b2 (Quantikine; R&D Systems, Minneapolis, MN, USA); and human MMP2 and MMP9 (RayBiotech, Norcross, GA, USA).
were grown in Dulbecco’s modified essential medium (DMEM; Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, IL, USA), 100 U/mL penicillin and 100 lg/mL streptomycin at 37 °C in an atmosphere of humidified air with 5% carbon dioxide CO2. 2.2. CpG oligodeoxynucleotide and cell challenge
2.4. Immunohistochemistry Paraffin-embedded specimens were cut into 4-lm sections and baked at 65 °C for 30 min. The sections were deparaffinized with xylene, rehydrated, submerged in ethylenediamine tetraacetic acid (pH 8.0), autoclaved for antigen retrieval, treated with 3% hydrogen peroxide, then incubated with 1% fetal bovine serum. Anti-TLR9 and anti-matrix metalloproteinase-9 (MMP9; Imgenex, San Diego, USA; 1:100 dilution) antibodies were added and sections were incubated overnight at 4 °C. For negative controls, the primary antibody was replaced with normal mouse serum. Normal human kidney samples were used as positive controls. Horseradish peroxidase
2.6. Statistical analysis The Statistical Program for the Social Sciences statistical software for Windows 13.0 (SPSS, Chicago, IL, USA) was used for all statistical analyses. Cell growth curve plotting and ELISA tests were performed in quadruplicate three times. Overall and progressionfree survival curves were constructed using the Kaplan–Meier technique and compared using a 2-sided log-rank test. The Cox proportional-hazards model was fitted to assess the prognostic and predictive values of TLR9 expression. Differences in in vitro protein expression between groups were analyzed using a t-test. Crosstab was used to analyze the correlation between TLR9 and MMP9 activity. A p 6 0.05 was defined as significant. 3. Results Clinical data for the 125 patients are presented in Table 1. The difference in PFS and OS between TLR9+ patients (77 patients, of whom 57 died) and TLR9 patients (48 patients, of whom 41 died) was highly significant (p < 0.01 according to the log-rank test in both cases), indicating that TLR9 expression status has prognostic value. For PFS among the TLR9 patients, there was a risk reduction of 43% (hazard ratio [HR] for death, 0.57; 95% confidence interval [CI], 0.41–0.71) as compared with the TLR9+ group (Fig. 1). For OS, similarly, the TLR9 patient group had a risk reduction of 66% (HR for death, 0.34; 95% CI, 0.23–0.51) as compared with the TLR9+ group. The median OS among TLR9 patients was 559 days (95% CI, 464–654), as compared with 437 days (95% CI, 346–528) among the TLR9+ patients (Fig. 2). Crosstab was used to analyze the correlation between TLR9 and MMP9 expression status in glioblastoma tissue. The median expression rates of TLR9 and MMP9 in tumor cells were 61.6%
L. Leng et al. / Journal of Clinical Neuroscience 19 (2012) 75–80 Table 1 Clinical and demographic data for 125 patients with glioblastoma multiforme Mean age (years; ±standard deviation) Sex (male/female)
46.29 ± 18.05 76/49
KPS score 0–80 81–100
48 77
Underwent radiation therapy (n) Underwent chemotherapy (n)
80 80
Extent of tumor resection (n) Gross total resection Subtotal resection
65 60
KPS = Karnofsky performance scale. As estimated by postoperative CT scan or MRI; gross total resection was considered to have taken place when there was no residual tumor evident, and >70% tumor removal was considered subtotal resection.
and 76.8%, respectively. TLR9 expression status and MMP9 expression status were significantly correlated in glioblastoma tissue (p < 0.05; Table 2). To further analyze the influence of TLR9 expression, we performed multivariate analysis using a Cox proportional-hazards model, stratified according to treatment and including known clinical prognostic factors (Table 3). Expression status of TLR9 (p = 0.021) emerged as a significant independent prognostic factor. Irrespective of whether U87 and U251 glioma cells were cultured with medium, CpG ODN or chloroquine, there was no significant difference in growth at any time point (p > 0.05; Fig. 3). Protein expression of TLR9, MMP9, IL-6, TFG-b2, MMP2 and STAT3 in the U87 and U251 cells when cultured with medium, CpG ODN or chloroquine is shown in Fig. 4. There were positive relationships between the protein levels of TLR9 and both MMP2 and MMP9 (p < 0.05). There was no relationship between the protein level of TLR9 and IL-6, TFG-b2 or STAT3 (p > 0.05).
4. Discussion Despite recent advances, the prognosis for patients with GBM has altered little over the past 10 years, with a median survival
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of only about 1 year,16 but immunotherapy may be a promising treatment strategy.17 TLR9 belongs to the TLR family of pattern recognition receptors, which recognize structures that are highly conserved among microorganisms.18 TLR9 itself recognizes ODNs with a CpG motif. After binding with the ligand, the TLR9 signal pathway subsequently activates the mitogen-activated protein kinase signaling pathway,19 which may responsible for the proinflammatory or progrowth microenvironment of tumors. Meng et al.15 and Andaloussi et al.20 found that U87 and U251 cells express high levels of TLR9 mRNA; however, to date there have been no data available about the expression of TLR9 protein in these two cell lines. In the present study, we used immunohistochemical techniques to screen a large collection of human glioblastoma samples for expression of TLR9. Multivariate analysis revealed that TLR9 expression is an independent prognostic factor for OS of patients with GBM (p < 0.05), indicating that expression of TLR9 correlates significantly with clinical outcome among patients with GBM. Other biological factors, including age, sex, Karnofsky performance scale (KPS) score before surgery, and extent of resection, were not associated with prognosis. The present study group included the same group of patients included in a previously published study,21 in which it was also determined that KPS score was not associated with prognosis. Based on information in the literature, it remains unclear whether the extent of surgical resection correlates with survival.22–24 Even KPS score is not indisputably associated with survival in GBM patients.25–27 The TLR9 signaling pathway seems to be important in gliomas; 28 however, other independent retrospective and prospective studies will be necessary before direct clinical application of the current findings is possible. The exact pathophysiological role of TLR9 in glioma is not known, but it may represent a useful prognostic biomarker. The exact mechanism by which TLR9 exerts its effects in gliomas is poorly understood; therefore, in the present study we further investigated the microenvironment of TLR9 in vitro. STAT3 affects proliferation, growth and apoptosis, and aberrant activation of STAT3 has been identified not only in glioblastoma but also in a number of other human cancers, including breast, lung, ovarian, pancreatic, skin and prostate cancers, and Hodgkin lymphoma, myeloma and acute myeloid leukemia.29 Like all STAT proteins, STAT3 is activated by tyrosine phosphorylation in response to stimulation by cytokines and growth factors. Specifically, STAT3 activation is downstream of receptor engagement by
Fig. 1. Kaplan–Meier curves showing progression-free survival (PFS) for 125 patients with glioblastoma multiforme, according to toll-like receptor-9 (TLR9) expression status. There was a highly significant difference between TLR9+ and TLR9 patients (p < 0.01, log-rank test). Among TLR9 patients, there was a risk reduction of 43% (hazard ratio for death, 0.57; 95% confidence interval, 0.41–0.71) as compared with TLR9+ patients.
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Fig. 2. Kaplan–Meier curves showing overall survival (OS) for 125 patients with glioblastoma multiforme, according to toll-like receptor-9 (TLR9) expression status. There was a highly significant difference between TLR9+ and TLR9 patients (p < 0.01, log-rank test). Among TLR9 patients, there was a risk reduction of 66% (hazard ratio for death, 0.34; 95% confidence interval, 0.23–0.51) as compared with TLR9+ patients.
Table 2 Expression of toll-like receptor-9 (TLR9) and matrix metalloproteinase-9 (MMP9) in tumor specimens from 125 patients with glioblastoma multiforme TLR9+
TLR9
MMP9+ MMP9
61 16
35 13
Total 96 29
Total
77
48
125
Crosstabs, p = 0.011.
Table 3 Multivariate analysis of prognostic parameters for overall survival among 125 patients with glioblastoma multiforme Variable
Hazard ratio (95% CI)
p
Age Sex KPS score Extent of resection Radiation therapy Chemotherapy TLR9 expression
0.992–1.028 0.655–1.539 0.447–0.948 0.635–1.472 0.466–1.200 0.465–1.178 1.183–2.602
0.294 0.974 0.071 0.875 0.228 0.276 0.021
CI = confidence interval, KPS = Karnofsky performance scale, TLR9 = toll-like receptor-9.
members of the IL-6 cytokine family, including IL-6, oncostatin M (OSM), and leukemia inhibitory factor, and by growth factors such as platelet-derived growth factor, fibroblast growth factor and epidermal growth factor.30 Substantial data gathered from cell lines, mouse models, and patient samples support a role for IL-6 proteins in glioblastoma-associated STAT3 activation. In several studies, constitutive expression of IL-6 and OSM has been observed in human glioblastoma cells, both in vivo and in vitro.31–33 Elevated expression of IL-6 cytokines is thought to be responsible, at least in part, for both constitutive and induced activation of STAT3 in glioblastoma. In some glioblastoma cell lines, autocrine IL-6 expression resulted in constitutive activation of STAT3, and antibodies neutralizing IL-6 reduced STAT3 activation, inhibited cell proliferation and induced apoptosis.34 In a number of other human brain tumor cell lines, stimulation with IL-6, leukemia inhibitory factor, ciliary neurotrophic factor, or OSM induced STAT3 activation,35 and treatment of human astroglioma cells with OSM increased the STAT3-dependent expression and activation of MMP9 and vascular endothelial-derived growth factor.36,37 Our study demonstrated that TLR9 activator CpG ODN does not affect the expression of IL-6, STAT3 or TGF-b2 in glioma cells in vitro. Wang et al.28 also found that TLR9 activator CpG ODN did not affect the
Fig. 3. Progression-free survival) (PFS) of U87 glioblastoma (left) and U251 glioma cells (right) when grown in medium alone, or with CpG oligodeoxynucleotide or chloroquine showing that there was no significant difference between treatment groups at any time point.
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Fig. 4. Protein expression of (A) toll-like receptor-9 (TLR9), (B) matrix metalloproteinase-2 (MMP2), (C) MMP9, (D) interleukin (IL)-6, (E) transforming growth factor (TGF)-b2, and (F) signal transducer and activator of transcription (STAT3) by U87 glioblastoma and U251 glioma cells when grown in medium alone, or with CpG oligodeoxynucleotide (ODN) or chloroquine. There was a statistically significant association between TLR9 levels and MMP2 and MMP9 levels (Student’s t-test was used to compare groups; p 6 0.05), but not between TLR9 and any other protein (p > 0.05). The TLR9 activator CpG ODN increased the expression of TLR9, MMP-2, and MMP-9, but not IL-6, STAT3 or TGF-b2 in glioma cells in vitro.
proliferation of glioma cells in vitro. These findings indicate that the TLR9 signaling pathway does not affect glioma proliferation, growth or apoptosis. Glioblastoma invasion is facilitated by the expression MMP, a family of zinc-dependent endopeptidases. They mediate the degradation of protein components of the extracellular matrix. Twentythree family members are known, and elevated levels of several of them have been reported in GBM. In our study we found that TLR9 activator CpG ODN affected the expression of MMP2 and MMP9 in in vitro glioma cells. In a previous study, a glioma cell line (U251) and a glioblastoma cell line (U87) were used to determine whether TLR9 is responsible for the CpG ODN-induced invasiveness of glioma cells.28 It was found that TLR9 agonist CpG ODN (ODN 2006) can significantly increase the invasiveness of U87 and U251 glioma cells. Most importantly, CpG-induced invasion could be blocked with chloroquine, which is an inhibitor of the TLR9 signaling pathway. Thus, chloroquine may be used as an adjuvant in GBM therapy, as confirmed by Briceno et al.38,39 who showed that chloroquine may improve mid-term survival when given in addition to conventional therapy. A better understanding of the function and regulation of TLR signaling pathways in glioma may shed new light on understanding the mechanisms of glioma formation and progression, as well as provide new targets for more effective treatment regimens. 5. Conclusions Our data indicate that TLR9 expression correlates with PFS and OS in patients with GBM. The immune microenvironment of TLR9 can be simulated in vitro. Because the CpG ODN is a TLR9 agonist, we recommend caution when injecting the TLR9 agonist CpG ODN directly into glioma tissues as part of glioma immunotherapy. Acknowledgements We thank Yuling Yang and Na Liu for their help with tissue sample collection and clinical data retrieval.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jocn.2011.03.037. References 1. Frosch MP, Anthony DC, Girolami UD. The central nervous system. In: Kumar V, Abbas AK, Fausto N, editors. Robbins and Cotran pathologic basis of disease. 7th ed. Elsevier Saunders; 2005. p. 1401–10. 2. Rosell R, de Las Peñas R, Balaña C. Translational research in glioblastoma multiforme: molecular criteria for patient selection. Future Oncol 2008;4:219–28. 3. Laperriere N, Zuraw L, Cairncross G. Radiotherapy for newly diagnosed malignant glioma in adults: a systematic review. Radiother Oncol 2002;64:259–73. 4. Walker PR, Calzascia T, de Tribolet N, et al. T-cell immune responses in the brain and their relevance for cerebral malignancies. Brain Res Brain Res Rev 2003;42:97–122. 5. Vollmer J, Krieg AM. Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists. Adv Drug Deliv Rev 2009;61:195–204. 6. Kadowaki N, Ho S, Antonenko S, et al. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med 2001;194:863–9. 7. Boonstra A, Rajsbaum R, Holman M, et al. Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88and TRIF-dependent TLR signals, and TLR independent signals. J Immunol 2006;177:7551–8. 8. Klinman DM, Yi AK, Beaucage SL, et al. CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma. Proc Nat Acad Sci USA 1996;93:2879–83. 9. Ilvesaro JM, Merrell MA, Li L, et al. Toll-like receptor 9 mediates CpG oligonucleotide induced cellular invasion. Mol Cancer Res 2008;6:1534–43. 10. Schmausser B, Andrulis M, Endrich S, et al. Toll-like receptors TLR4, TLR5 and TLR9 on gastric carcinoma cells: an implication for interaction with Helicobacter pylori. Int J Med Microbiol 2005;295:179–85. 11. Droemann D, Albrecht D, Gerdes J, et al. Human lung cancer cells express functionally active Toll-like receptor 9. Respir Res 2005;6:121–6. 12. Ilvesaro JM, Merrell MA, Swain TM, et al. Toll like receptor-9 agonists stimulate prostate cancer invasion in vitro. Prostate 2007;67:774–81. 13. Merrell MA, Ilvesaro JM, Lehtonen N, et al. Toll-like receptor 9 agonists promote cellular invasion by increasing matrix metalloproteinase activity. Mol Cancer Res 2006;4:437–47. 14. Ren T, Wen ZK, Liu ZM, et al. Functional expression of TLR9 is associated to the metastatic potential of human lung cancer cell: functional active role of TLR9 on tumor metastasis. Cancer Biol Ther 2007;6:1704–9. 15. Meng Y, Kujas M, Marie Y, et al. Expression of TLR9 within human glioblastoma. J Neurooncol 2008;88:19–25.
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16. Bondy ML, Scheurer ME, Malmer B, et al. Brain tumor epidemiology: consensus from the Brain Tumor Epidemiology Consortium. Cancer 2008;113:1953–68. 17. Walker PR, Calzascia T, de Tribolet N, et al. T-cell immune responses in the brain and their relevance for cerebral malignancies. Brain Res Brain Res Rev 2003;42:97–122. 18. Wagner H. The immunobiology of the TLR9 subfamily. Trends Immunol 2004;25:381–6. 19. Takeshita F, Gursel I, Ishii KJ, et al. Signal transduction pathways mediated by the interaction of CpG DNA with Toll-like receptor 9. Semin Immunol 2004;16:17–22. 20. Andaloussi A, Sonabend AM, Han Y, et al. Stimulation of TLR9 with CpG ODN enhances apoptosis of glioma and prolongs the survival of mice with experimental brain tumors. Glia 2006;54:526–35. 21. Lin Y, Jiang T, Zhou K, et al. Plasma IGFBP-2 levels predict clinical outcomes of patients with high grade gliomas. Neuro Oncol 2009;11:468–76. 22. Sanai N, Berger MS. Operative techniques for gliomas and the value of extent of resection. Neurotherapeutics 2009;6:478–86. 23. Hess KR. Extent of resection as a prognostic variable in the treatment of gliomas. J Neurooncol 1999;42:227–31. 24. Pang BC, Wan WH, Lee CK, et al. The role of surgery in high grade glioma – is surgical resection justified? A review of the current knowledge. Ann Acad Med Singapore 2007;36:358–63. 25. Chinnaiyan P, Wang M, Rojiani AM, et al. The prognostic value of nestin expression in newly diagnosed glioblastoma: report from the Radiation Therapy Oncology Group. Radiat Oncol 2008;3:32–6. 26. Reithmeier T, Graf E, Piroth T, et al. BCNU for recurrent glioblastoma multiforme: efficacy, toxicity and prognostic factors. BMC Cancer 2008;10:30–4. 27. Batchelor TT, Betensky RA, Esposito JM, et al. Age-dependent prognostic effects of genetic alterations in glioblastoma. Clin Cancer Res 2004;10:228–33.
28. Wang C, Cao S, Yan Y, et al. TLR9 expression in glioma tissues correlated to glioma progression and the prognosis of GBM patients. BMC Cancer 2010;10:415–9. 29. Bromberg J. Stat proteins and oncogenesis. J Clin Invest 2002;109:1139–42. 30. Heinrich PC, Behrmann I, Haan S, et al. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 2003;374:1–20. 31. Van NJ, Benveniste EN. Interleukin-6 expression and regulation in astrocytes. J Neuroimmunol 1999;100:124–39. 32. Van ME, Sawamura Y, Diserens AC, et al. Human glioblastoma cells release interleukin 6 in vivo and in vitro. Cancer Res 1990;50:6683–8. 33. Van WNJ, Oh JW, Repovic P, et al. Interleukin-6 (IL-6) production by astrocytes: autocrine regulation by IL-6 and the soluble IL-6 receptor. J Neurosci 1999;19:5236–44. 34. Rahaman SO, Harbor PC, Chernova O, et al. Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells. Oncogene 2002;21:8404–13. 35. Schaefer LK, Menter DG, Schaefer TS. Activation of stat3 and stat1 DNA binding and transcriptional activity in human brain tumour cell lines by gp130 cytokines. Cell Signal 2000;12:143–51. 36. Repovic P, Fears CY, Gladson CL, et al. Oncostatin-M induction of vascular endothelial growth factor expression in astroglioma cells. Oncogene 2003;22:8117–24. 37. Chen SH, Gillespie GY, Benveniste EN. Divergent effects of oncostatin M on astroglioma cells: influence on cell proliferation, invasion, and expression of matrix metalloproteinases. Glia 2006;53:191–200. 38. Briceno E, Calderon A, Sotelo J. Institutional experience with chloroquine as an adjuvant to the therapy for glioblastoma multiforme. Surg Neurol 2007;67:388–91. 39. Briceno E, Reyes S, Sotelo J. Therapy of glioblastoma multiforme improved by the antimutagenic chloroquine. Neurosurg Focus 2003;14:E3.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
TLR9 expression is associated with prognosis in patients with glioblastoma multiforme Lige Leng a,b, Tao Jiang b,c, Yazhuo Zhang a,b,⇑ a
Beijing Neurosurgical Institute, 6 Tiantan Xili Street, Beijing 100050, China Capital Medical University, Beijing, China c Beijing Tiantan Hospital, Beijing, China b
a r t i c l e
i n f o
Article history: Received 15 February 2011 Accepted 29 March 2011
Keywords: Glioblastoma MMP2 MMP9 Prognosis TLR9
a b s t r a c t The aim of this study was to determine if there was an association between expression of toll-like receptor 9 (TLR9) in glioblastoma tissue and patient outcome in glioblastoma multiforme. Further, we characterized the direct in vitro effects of the TLR9 agonist, CpG oligodeoxynucleotide (ODN), commonly used as a vaccine adjuvant in cancer immunotherapy, on glioma cells. TLR9 expression was assessed using immunohistochemical techniques, and enzyme-linked immunosorbent assays were used to investigate the expression of other proteins in glioma cells relevant to immunotherapy. There was a highly significant difference in both progression-free survival and overall survival between TLR9+ and TLR9 patients, with poorer outcome in TLR9+ patients. In in vitro glioma cells, there was a positive correlation between the protein levels of TLR9 and both matrix metalloproteinase (MMP)-2 and MMP-9 (p < 0.05), but no relationship between TLR9 levels and levels of interleukin-6, transforming growth factor-b2 or signal transducer and activator of transcription (STAT)-3 (p > 0.05). Our data indicate that expression of TLR9 correlates with shorter progression-free survival and overall survival in patients with glioblastoma multiforme. Our findings also indicate that caution is warranted when directly injecting the TLR9 agonist CpG ODN into glioma tissues as part of glioma immunotherapy. Because the CpG ODN is a TLR9 agonist, we recommend caution when using CpG ODN in immunotherapy. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Glioblastoma multiforme (GBM), the most malignant form of glioma, is the most common primary malignant brain tumor in adults worldwide.1,2 Patients with this condition still face a poor prognosis, despite multimodal intensive treatment regimens comprising maximum surgical resection, radiation and chemotherapy. The median survival time of patients with GBM is only 12–15 months from the initial diagnosis.3 Immunotherapy, in which activated lymphocytes specifically target and destroy tumor cells, is a promising treatment strategy for gliomas.4 In recent years, CpG-based immunotherapy has been intensively investigated as a treatment option. CpG is a synthetic oligodeoxynucleotide (ODN) with unmethylated CG dinucleotides within particular sequence contexts.5 CpG ODN have been used in clinical trials as vaccine adjuvants in cancer immunotherapy regimens, given that they can mimic microbial DNA and activate the immune system through the binding of toll-like receptor 9 (TLR9). TLR9, a member of the TLR family, is an evolutionarily well-conserved transmembrane protein that recognizes microbe-derived molecular patterns.6 TLR9 resides in the endoplasmic reticulum ⇑ Corresponding author. Tel.: +86 135 2213 0701. E-mail address: [email protected] (Y. Zhang). 0967-5868/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2011.03.037
and its activation results in increased production of inflammatory mediators.7,8 In addition to being expressed in plasmacytoid DC and B cells, TLR9 is also expressed in breast,9 gastric,10 lung11 and prostate12 cancer cells. Accumulating data indicate that CpG ODN, which are TLR9 agonists, can promote tumor development and metastasis.9,12–14 We hypothesized that the TLR9 signal pathway may also be important in glioma growth and progression. TLR9 expression has been previously examined in a study of 37 GBM but found to not have prognostic significance.15 The aim of the present study was to evaluate the expression of TLR9 in a large series of glioblastoma samples, and to investigate a possible association between TLR9 expression, clinicopathological variables, and patient outcome. We also aimed to investigate the direct effects of the TLR9 agonist CpG ODN on glioma cells and to characterize the microenvironment of TLR9 in vitro. 2. Methods 2.1. Cells and cell cultures U87-MG and U251-MG glioma cells were obtained from the American Type Culture Collection and maintained in the Central Laboratory of the First Hospital of China Medical University. Cells
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Nuclease-resistant phosphorothioate-modified ODN 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 ODN were dissolved in endotoxin-free sterile water and stored at 80 °C until use. The cell challenge was conducted in 6-well cell culture chambers. Cells were plated at 1 106 per well (in DMEM and 10% FBS). ODN 2006 (10 lM), or ODN 2006 control (10 lM), or chloroquine (10 lM) was added the well. The cells were cultured for 24 hours.
(HRP)-labeled secondary antibody (KIT-5930, MaxVision HRP-polymer antimouse/rabbit IHC kit; Maixin-Bio, Fuzhou, China) was applied, and the sections were incubated first for 30 minutes at room temperature, then for 5 minutes at room temperature with diaminobenzidine. The sections were finally counterstained with hematoxylin and mounted with Permount (BIOS, Beijing, China). Sections were visualized and photographed using a light microscope (Olympus BX-51; Olympus Optical, Tokyo, Japan). The degree of immunostaining was scored separately by two independent investigators. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. Scores from the two investigators were averaged for further comparative evaluation of TLR9 and MMP9 expression. The proportion of positively stained tumor cells was assessed as follows: 0, <30% positive cells; 1, >30% positive cells. The intensity of staining was assessed using the following scale: 0, no staining or weak staining (light yellow); 1, moderate staining (yellowish brown) or strong staining (brown). The stain index was calculated as staining intensity proportion of positively stained cells (Supplementary Fig. 1).
2.3. Patients and treatment
2.5. Enzyme-linked immunosorbent assay
Tumor specimens were obtained from 125 patients admitted to Beijing Tiantan Hospital for diagnosis and treatment. A diagnosis of GBM was made according to World Health Organization criteria. The study was approved by the ethics committees of all institutions involved and was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all patients. GBM tissues, and non-neoplastic brain tissues obtained during temporal lobectomy for the treatment of epilepsy, were immediately snap-frozen in liquid nitrogen upon surgical removal. A portion of each sample was fixed with formalin, embedded in paraffin wax and kept at room temperature. Patients were eligible for inclusion if they had no other cancer or disease (e.g. acute infection or diabetes); if they had not previously undergone radiotherapy, chemotherapy, or corticosteroid therapy; and if they had undergone routine contrast-enhanced MRI examinations before and after surgery. Patients who died from a non-glioma-related cause were excluded. To minimize the confounding effects of different surgical procedures, all patients studied underwent surgical resection performed by neurosurgeons using similar operational techniques and principles, and every patient in the study received the same radiotherapy regimen. The extent of tumor resection was defined as follows: 0, partial removal with 630% residual tumor; 1, gross total resection. Clinicopathological data were retrospectively collected from patients’ medical records up to July 2010. Observations for each patient ended once the patient died or was found to have recurrent disease. Progression-free survival (PFS) was defined as being the time from the day of surgery to the first MRI-confirmed recurrence or death, whichever occurred first. Overall survival (OS) was defined as being the time from the day of surgery to death.
TLR9 protein was extracted using the Mem-PER eukaryotic membrane protein extraction reagent kit (Pierce, Rockford, IL, USA). Other enzyme-linked immunosorbent assay (ELISA) kits used were as follows: human prolactin (PRL; Uscn Life Sciences, Wuhan, China); human signal transducer and activator of transcription-3 (STAT3; Cell Signaling, Beverley, MA, USA); human interleukin (IL)-6 and transforming growth factor (TGF)-b2 (Quantikine; R&D Systems, Minneapolis, MN, USA); and human MMP2 and MMP9 (RayBiotech, Norcross, GA, USA).
were grown in Dulbecco’s modified essential medium (DMEM; Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, IL, USA), 100 U/mL penicillin and 100 lg/mL streptomycin at 37 °C in an atmosphere of humidified air with 5% carbon dioxide CO2. 2.2. CpG oligodeoxynucleotide and cell challenge
2.4. Immunohistochemistry Paraffin-embedded specimens were cut into 4-lm sections and baked at 65 °C for 30 min. The sections were deparaffinized with xylene, rehydrated, submerged in ethylenediamine tetraacetic acid (pH 8.0), autoclaved for antigen retrieval, treated with 3% hydrogen peroxide, then incubated with 1% fetal bovine serum. Anti-TLR9 and anti-matrix metalloproteinase-9 (MMP9; Imgenex, San Diego, USA; 1:100 dilution) antibodies were added and sections were incubated overnight at 4 °C. For negative controls, the primary antibody was replaced with normal mouse serum. Normal human kidney samples were used as positive controls. Horseradish peroxidase
2.6. Statistical analysis The Statistical Program for the Social Sciences statistical software for Windows 13.0 (SPSS, Chicago, IL, USA) was used for all statistical analyses. Cell growth curve plotting and ELISA tests were performed in quadruplicate three times. Overall and progressionfree survival curves were constructed using the Kaplan–Meier technique and compared using a 2-sided log-rank test. The Cox proportional-hazards model was fitted to assess the prognostic and predictive values of TLR9 expression. Differences in in vitro protein expression between groups were analyzed using a t-test. Crosstab was used to analyze the correlation between TLR9 and MMP9 activity. A p 6 0.05 was defined as significant. 3. Results Clinical data for the 125 patients are presented in Table 1. The difference in PFS and OS between TLR9+ patients (77 patients, of whom 57 died) and TLR9 patients (48 patients, of whom 41 died) was highly significant (p < 0.01 according to the log-rank test in both cases), indicating that TLR9 expression status has prognostic value. For PFS among the TLR9 patients, there was a risk reduction of 43% (hazard ratio [HR] for death, 0.57; 95% confidence interval [CI], 0.41–0.71) as compared with the TLR9+ group (Fig. 1). For OS, similarly, the TLR9 patient group had a risk reduction of 66% (HR for death, 0.34; 95% CI, 0.23–0.51) as compared with the TLR9+ group. The median OS among TLR9 patients was 559 days (95% CI, 464–654), as compared with 437 days (95% CI, 346–528) among the TLR9+ patients (Fig. 2). Crosstab was used to analyze the correlation between TLR9 and MMP9 expression status in glioblastoma tissue. The median expression rates of TLR9 and MMP9 in tumor cells were 61.6%
L. Leng et al. / Journal of Clinical Neuroscience 19 (2012) 75–80 Table 1 Clinical and demographic data for 125 patients with glioblastoma multiforme Mean age (years; ±standard deviation) Sex (male/female)
46.29 ± 18.05 76/49
KPS score 0–80 81–100
48 77
Underwent radiation therapy (n) Underwent chemotherapy (n)
80 80
Extent of tumor resection (n) Gross total resection Subtotal resection
65 60
KPS = Karnofsky performance scale. As estimated by postoperative CT scan or MRI; gross total resection was considered to have taken place when there was no residual tumor evident, and >70% tumor removal was considered subtotal resection.
and 76.8%, respectively. TLR9 expression status and MMP9 expression status were significantly correlated in glioblastoma tissue (p < 0.05; Table 2). To further analyze the influence of TLR9 expression, we performed multivariate analysis using a Cox proportional-hazards model, stratified according to treatment and including known clinical prognostic factors (Table 3). Expression status of TLR9 (p = 0.021) emerged as a significant independent prognostic factor. Irrespective of whether U87 and U251 glioma cells were cultured with medium, CpG ODN or chloroquine, there was no significant difference in growth at any time point (p > 0.05; Fig. 3). Protein expression of TLR9, MMP9, IL-6, TFG-b2, MMP2 and STAT3 in the U87 and U251 cells when cultured with medium, CpG ODN or chloroquine is shown in Fig. 4. There were positive relationships between the protein levels of TLR9 and both MMP2 and MMP9 (p < 0.05). There was no relationship between the protein level of TLR9 and IL-6, TFG-b2 or STAT3 (p > 0.05).
4. Discussion Despite recent advances, the prognosis for patients with GBM has altered little over the past 10 years, with a median survival
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of only about 1 year,16 but immunotherapy may be a promising treatment strategy.17 TLR9 belongs to the TLR family of pattern recognition receptors, which recognize structures that are highly conserved among microorganisms.18 TLR9 itself recognizes ODNs with a CpG motif. After binding with the ligand, the TLR9 signal pathway subsequently activates the mitogen-activated protein kinase signaling pathway,19 which may responsible for the proinflammatory or progrowth microenvironment of tumors. Meng et al.15 and Andaloussi et al.20 found that U87 and U251 cells express high levels of TLR9 mRNA; however, to date there have been no data available about the expression of TLR9 protein in these two cell lines. In the present study, we used immunohistochemical techniques to screen a large collection of human glioblastoma samples for expression of TLR9. Multivariate analysis revealed that TLR9 expression is an independent prognostic factor for OS of patients with GBM (p < 0.05), indicating that expression of TLR9 correlates significantly with clinical outcome among patients with GBM. Other biological factors, including age, sex, Karnofsky performance scale (KPS) score before surgery, and extent of resection, were not associated with prognosis. The present study group included the same group of patients included in a previously published study,21 in which it was also determined that KPS score was not associated with prognosis. Based on information in the literature, it remains unclear whether the extent of surgical resection correlates with survival.22–24 Even KPS score is not indisputably associated with survival in GBM patients.25–27 The TLR9 signaling pathway seems to be important in gliomas; 28 however, other independent retrospective and prospective studies will be necessary before direct clinical application of the current findings is possible. The exact pathophysiological role of TLR9 in glioma is not known, but it may represent a useful prognostic biomarker. The exact mechanism by which TLR9 exerts its effects in gliomas is poorly understood; therefore, in the present study we further investigated the microenvironment of TLR9 in vitro. STAT3 affects proliferation, growth and apoptosis, and aberrant activation of STAT3 has been identified not only in glioblastoma but also in a number of other human cancers, including breast, lung, ovarian, pancreatic, skin and prostate cancers, and Hodgkin lymphoma, myeloma and acute myeloid leukemia.29 Like all STAT proteins, STAT3 is activated by tyrosine phosphorylation in response to stimulation by cytokines and growth factors. Specifically, STAT3 activation is downstream of receptor engagement by
Fig. 1. Kaplan–Meier curves showing progression-free survival (PFS) for 125 patients with glioblastoma multiforme, according to toll-like receptor-9 (TLR9) expression status. There was a highly significant difference between TLR9+ and TLR9 patients (p < 0.01, log-rank test). Among TLR9 patients, there was a risk reduction of 43% (hazard ratio for death, 0.57; 95% confidence interval, 0.41–0.71) as compared with TLR9+ patients.
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Fig. 2. Kaplan–Meier curves showing overall survival (OS) for 125 patients with glioblastoma multiforme, according to toll-like receptor-9 (TLR9) expression status. There was a highly significant difference between TLR9+ and TLR9 patients (p < 0.01, log-rank test). Among TLR9 patients, there was a risk reduction of 66% (hazard ratio for death, 0.34; 95% confidence interval, 0.23–0.51) as compared with TLR9+ patients.
Table 2 Expression of toll-like receptor-9 (TLR9) and matrix metalloproteinase-9 (MMP9) in tumor specimens from 125 patients with glioblastoma multiforme TLR9+
TLR9
MMP9+ MMP9
61 16
35 13
Total 96 29
Total
77
48
125
Crosstabs, p = 0.011.
Table 3 Multivariate analysis of prognostic parameters for overall survival among 125 patients with glioblastoma multiforme Variable
Hazard ratio (95% CI)
p
Age Sex KPS score Extent of resection Radiation therapy Chemotherapy TLR9 expression
0.992–1.028 0.655–1.539 0.447–0.948 0.635–1.472 0.466–1.200 0.465–1.178 1.183–2.602
0.294 0.974 0.071 0.875 0.228 0.276 0.021
CI = confidence interval, KPS = Karnofsky performance scale, TLR9 = toll-like receptor-9.
members of the IL-6 cytokine family, including IL-6, oncostatin M (OSM), and leukemia inhibitory factor, and by growth factors such as platelet-derived growth factor, fibroblast growth factor and epidermal growth factor.30 Substantial data gathered from cell lines, mouse models, and patient samples support a role for IL-6 proteins in glioblastoma-associated STAT3 activation. In several studies, constitutive expression of IL-6 and OSM has been observed in human glioblastoma cells, both in vivo and in vitro.31–33 Elevated expression of IL-6 cytokines is thought to be responsible, at least in part, for both constitutive and induced activation of STAT3 in glioblastoma. In some glioblastoma cell lines, autocrine IL-6 expression resulted in constitutive activation of STAT3, and antibodies neutralizing IL-6 reduced STAT3 activation, inhibited cell proliferation and induced apoptosis.34 In a number of other human brain tumor cell lines, stimulation with IL-6, leukemia inhibitory factor, ciliary neurotrophic factor, or OSM induced STAT3 activation,35 and treatment of human astroglioma cells with OSM increased the STAT3-dependent expression and activation of MMP9 and vascular endothelial-derived growth factor.36,37 Our study demonstrated that TLR9 activator CpG ODN does not affect the expression of IL-6, STAT3 or TGF-b2 in glioma cells in vitro. Wang et al.28 also found that TLR9 activator CpG ODN did not affect the
Fig. 3. Progression-free survival) (PFS) of U87 glioblastoma (left) and U251 glioma cells (right) when grown in medium alone, or with CpG oligodeoxynucleotide or chloroquine showing that there was no significant difference between treatment groups at any time point.
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Fig. 4. Protein expression of (A) toll-like receptor-9 (TLR9), (B) matrix metalloproteinase-2 (MMP2), (C) MMP9, (D) interleukin (IL)-6, (E) transforming growth factor (TGF)-b2, and (F) signal transducer and activator of transcription (STAT3) by U87 glioblastoma and U251 glioma cells when grown in medium alone, or with CpG oligodeoxynucleotide (ODN) or chloroquine. There was a statistically significant association between TLR9 levels and MMP2 and MMP9 levels (Student’s t-test was used to compare groups; p 6 0.05), but not between TLR9 and any other protein (p > 0.05). The TLR9 activator CpG ODN increased the expression of TLR9, MMP-2, and MMP-9, but not IL-6, STAT3 or TGF-b2 in glioma cells in vitro.
proliferation of glioma cells in vitro. These findings indicate that the TLR9 signaling pathway does not affect glioma proliferation, growth or apoptosis. Glioblastoma invasion is facilitated by the expression MMP, a family of zinc-dependent endopeptidases. They mediate the degradation of protein components of the extracellular matrix. Twentythree family members are known, and elevated levels of several of them have been reported in GBM. In our study we found that TLR9 activator CpG ODN affected the expression of MMP2 and MMP9 in in vitro glioma cells. In a previous study, a glioma cell line (U251) and a glioblastoma cell line (U87) were used to determine whether TLR9 is responsible for the CpG ODN-induced invasiveness of glioma cells.28 It was found that TLR9 agonist CpG ODN (ODN 2006) can significantly increase the invasiveness of U87 and U251 glioma cells. Most importantly, CpG-induced invasion could be blocked with chloroquine, which is an inhibitor of the TLR9 signaling pathway. Thus, chloroquine may be used as an adjuvant in GBM therapy, as confirmed by Briceno et al.38,39 who showed that chloroquine may improve mid-term survival when given in addition to conventional therapy. A better understanding of the function and regulation of TLR signaling pathways in glioma may shed new light on understanding the mechanisms of glioma formation and progression, as well as provide new targets for more effective treatment regimens. 5. Conclusions Our data indicate that TLR9 expression correlates with PFS and OS in patients with GBM. The immune microenvironment of TLR9 can be simulated in vitro. Because the CpG ODN is a TLR9 agonist, we recommend caution when injecting the TLR9 agonist CpG ODN directly into glioma tissues as part of glioma immunotherapy. Acknowledgements We thank Yuling Yang and Na Liu for their help with tissue sample collection and clinical data retrieval.
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