Studies On Cytokines Affecting The Prognosis And Immune Response In Glioma

Background and ObjectiveThe incidence of malignant gliomas, which is the most common type of primary brain neoplasm, is about 35.12-61.10%. Rapid tumor growth, invasive nature, and presence of the blood-brain barrier, which limits the penetration of large molecules into the central nervous system (CNS), all contribute to a poor response to conventional therapies. Patients with malignant gliomas always get recurred in 1 year despite comprehensive treatments were given to them. Postoperative radiation therapy and chemotherapy prolong the survival time; however, it yields a median survival time of less than 1 year. And long-time survival is extremely rare. Furthermore, brain has been considered to be an immune-privileged site, in part due to lack of lymphatic system and poor penetration of inflammatory cells across an intact blood-brain barrier. Although, recent work has demonstrated the presence CNS immune surveillance in healthy and inflammatory conditions, glioma microenvironment may provide another barrier that attenuates the host anti-tumor response through secretion of inhibitory factors and attraction of immunosuppressive leukocytes. Despite significant infiltration into tumors, the effector function of macrophages (MPs) and microglia (MG) appears to be suppressed in gliomas. Although STAT3 pathway is thought to play a role in this process, the exact mechanism by which gliomas induce STAT3 activation in MPs and MG is still not clear.Therefore, it is very important to explorer new cytokines which can activate the immune system in gliomas.One of the most interesting tumor markers is the tyrosine kinase receptor c-Met, a transmembrane receptor protein. In humans, the c-Met gene is located on chromosome 7, q21-q31, and spans more than 120 kb in length. c-Met plays a key role in the tumorigenicity, malignant progression and tumor angiogenesis by binding with hepatocyte growth factor (HGF),which is one of its ligands. S100B, a member of a multigenic family of Ca2+ binding protein, has been detected in astrocytes, mature oligodendrocytes and many kinds of tumor cells. In humans, the S100B gene is located on chromosome 21, q22. Once released, S100B can differentially affect neurons, astrocytes, and MG mostly through engagement of RAGE (Receptor for Advanced glycation end products), its primary receptor. The activation of RAGE signaling pathway can activate the STAT3 signaling pathway, which can inhibit the immune system in the brain. However, the exact mechanism by which S100B induce STAT3 activation in MPs and MG is still not clear.This paper was divided into two parts:1. Explore the difference of c-Met expression between primary and recurrent gliomas, and to determine whether the dysregulation of c-Met expression has a role in the malignant progression of gliomas.2. S100B-RAGE interaction may play an important role in STAT3 activation and MG/MP suppression in gliomas. A better understanding of this interaction may be beneficial in optimizing immunotherapy approaches against malignant brain tumors.The first part:c-Met expression is associated with time to recurrence in patients with glioblastomaObjective:Explore the difference of c-Met expression between primary and recurrent gliomas, and to determine whether the dysregulation of c-Met expression has a role in the malignant progression of gliomas.Methods:19 Chinese patients from the Department of Neurosurgery, Provincial Hospital affiliated to Shandong University between September 2000 and October 2007 were included. Paired primary and recurrent glioma specimens from the same patient were evaluated using immunohistochemical analysis. The association between c-Met expression and progression-free survival time (PFST) was analyzed by using the Kaplan-Meier method and confirmed using the logrank test.Results:1. c-Met overexpression was found in significantly more recurrent glioma (15/19) than primary glioma (7/19; p= 0.020). Recurrent glioma specimens had obviously higher expression levels of c-Met. Age and sex had little effect on the statistical comparisons.2. The seven patients who had primary glioma overexpressing c-Met had a median PFST of 6.1 months (95% CI:3.7-8.6 months), while patients with primary glioma with little or no c-Met expression had a median PFST of 11.5 months (95% CI:6.9-16 months; log-rank test: p=0.026).Conclusion:The results demonstrate that recurrent GBM tend to express c-Met at a higher level than the primary tumors, and that c-Met overexpression is significantly associated with PFST. c-Met may be a useful target in the diagnosis and treatment of gliomas.The second part:S100B Attenuates Microglia Activation in Gliomas:Possible Role of STAT3 PathwayObjective:Low levels of S100B inhibited MG function and induced STAT3 expression. Inhibition RAGE (S100B receptor) with blocking antibody (Ab) abolished STAT3 activation in N9 MG in vitro and MG/MPs in vivo. These findings suggest that S100B-RAGE interaction may play an important role in STAT3 activation and MG/MP suppression in gliomas. A better understanding of this interaction may be beneficial in optimizing immunotherapy approaches against malignant brain tumors.Methods:1.1 Detect the expression of S100B in GL261 cell line using Elisa; Explore the expresson of S100B in GL261 tumor bearing mice using FACS and immunohistochemical method (IHC).1.2 Explore the changes of RAGE expression in N9 cell line and Bone Marrow Monocyte which were treated with GL261 Condition Medium and different doses of S100B by RT-PCR and WB.1.3 Explore the expression of RAGE in Tumor Bearing mice using FACS and IHC.2. Explore the changes of STAT3 expression in N9 cell line and Bone Marrow Monocyte which were treated with GL261 Condition Medium and different doses of S100B by RT-PCR, WB and FACS.3. Explore the changes of STAT3 signaling pathway after the blocking of RAGE signaling pathway using RAGE antibodies in vitro and vivo. Results:1. S100B Production by GliomasS100B concentration in GCM was 0.22 ng/mL, while in N9 CM was negligible and similar to culture medium (0.02-0.05 ng/mL). Compared with normal brains where very little S100B staining was detected, S100B appeared both intracellularly and in the ECS of gliomas.2. GCM Modulates MG RAGE Expression3. Similar to GCM, S100B upregulated RAGE protein and mRNA expression at very low concentrations.4. S100B upregulated the RAGE expression in tumor bearing mice.5. GCM and S100B Inhibit MAPK Activation and Suppress MG Cytokine Expression6. Low levels of S100B induce STAT3 in MG.7. Blockage of RAGE inhibits STAT3 activity.Conclusion:In summary, we demonstrated that glioma-mediated activation of STAT3 in MG/MP may partly occur through the RAGE pathway. Furthermore, low levels of S100B, a RAGE ligand that is expressed by gliomas, induced STAT3 and inhibited MG activation. These findings suggest that S100B-RAGE interaction may play an important role in STAT3 activation and MG/MP suppression in gliomas.