The Impact Of RAGE Ablation In Tumor Microenvironment

Receptor for advanced glycation end-products (RAGE) is a multiligandreceptor of the immunoglobulin superfamily that has been implicated in avariety of human disease processes. Under the conditions of tumor growth，upregulation of RAGE and its ligands play an important role in immuneresponse, inflammation and angiogenesis of tumor. Therefore, by blockingthe binding of RAGE and its ligands can be an effective means to preventdevelopment of tumor, bringing hope to the treatment of cancer patients.Purpose:Evaluate the effect of gene knockout of RAGE in tumormicroenvironment on growth of gliomas. In two different syngeneicintracranial glioma models, animal survival was significantly longer inRage-/-mice. Improved animal survival in Rage-/-mice, however, was notdue to changes in tumor growth rate, but a reduction in tumor-associatedinflammation. Furthermore, the knockout of RAGE affected angiogenesisin GL261non invasive tumors by resulting in abnormal leaky dilatedvessels, but did not seen in K-Luc invasive tumors. These results indicatethat glioma heterogeneity directly impacts response to anti-RAGEtherapies.Materials and Methods：1. Application of quantitative PCR, real-time quantitative PCR,immune-ofluorescence staining to evaluate the expression of RAGE inRAGE-/-mice.2. Construct K-Luc and GL261glioma orthotopic mice model.Comparing cell survival time of RAGE-/-and wild type mice, differences in tumor volume, analysis the affect of RAGE-/-to the survival of gliomamice.3. Application of real-time quantitative PCR to detect theInflammatory cytokines, such as IL-6, IL-1β, TNF-α, et al. Analysis therole of RAGE knockout in the tumor inflammation. Applying westernblotting to measure the expression of RAGE and its various ligands（S100B,S100A9, HMGB1）in K-Luc and GL261glioma tumor model.4. Take14-day brain tumor tissue of two kinds of glioma model,applying immunofluorescence staining to measure CD31and calculate thetotal area of CD31, Application of real-time quantitative PCR to measurethe expression of angiogenesis-related factors, such as VEGFR1，VEGF-a，MMP2，MMP9. And Evaluate the affect of RAGE knockout on gliomaangiogenesis.5. Create the chimeric mice through cross bone marrowtransplantation, apply real-time quantitative PCR and immunofluorescencestaining to evaluate the affect of MP and MG in TAMs on RAGE-mediatedtumor angiogenesis.Result：1. RAGE-/-mice was evaluated and successfully established RAGE-/-mice glioma model.2.Survival of glioma cell lines of GL261and K-Luc in RAGE-/-micehave significantly prolonged.3. GL261tumors had a much lower expression levels of Gliomainflammatory cytokines in Rage-/-mice. Continued higher inflammatoryresponse in the K-Luc tumor model may caused by other ProinflammatoryRAGE ligands(S100A9and HMGB1). 4. GL261Rage-/-tumors had larger dilated vessels, various angiogenicfactors in both gliomas were significantly decreased.5. Construct chimeric mice through cross bone marrow transplantationbetween Rage-/-mice and wide-type mice, then established GL261tumormodel. Only in the chimeric mice，which Rage-/-mice was used as donor,and wide-type mice was used as acceptor, that is only mice that lackedRAGE expression in both MG and MP possessed large dilated vessels.Conclusion：1. Animal survival was significantly prolong in glioma mice whenRAGE was knockout.2. RAGE play the role of proinflammatory receptor in gliomas, whichis combined with a variety of ligands play a role in the inflammatoryresponse in the tumor.3. The role of RAGE knockout on glioma growth appears to be in partfrom the decrease of tumor inflammation and angiogenesis injury when thetumor is big enough.4. TAMs play a role in tumor angiogenesis through the expression ofRAGE.