Gene And Immune Therapy Of Malignant Gliomas And Intracranial Metastases

Background and ObjectiveMalignant glioma is the most common central nervous system (CNS) tumor and carries an extremely high mortality rate. Despite optimal treatment, the median lengths of survival is only12to15months for patients with glioblastomas multiforme (GB), is2to3years for patients with anaplastic astrocytoma (AA, WHO grade III) and5to10years for patients with diffuse astrocytoma (DA, WHO grade II). A small subpopulation of cells with tumor stem cells'(TSCs') properties has been identified in brain tumors that express the neural stem cell (NSC) markers CD133and nestin protein. These cells have many distinct characteristics of stem cells including limitless proliferation, invasion, and were responsible for the genesis, growth and recurrence of tumor. Although there are some different voices about this hypothesis, it still draws more and more interests. The molecular and functional characterization of cancer stem cell may be used in the development of novel cancer therapeutic drugs.With the dramatic progress made in pathogenesis and molecular mechanisms of proliferation, invasion and angiogenesis about malignant gliomas, gene therapy is being developed as a more effective strategy in treatment, but it is very difficult to select proper tumor genes for targeted therapies. The high-mobility group Al (HMGA1) protein is a non-histone architectural nuclear factor and encoded by the gene at chromosomal loci6p21.3. HMGA1contains three basic DNA-binding domains termed "AT-hooks", which mediate binding to the AT-rich regions in the narrow minor groove of DNA. Although HMGA1has no intrinsic transcriptional activity alone, through protein-protein and protein-DNA interactions, it can modulate transcription by altering chromatin architecture. It participates in diverse biological processes, including gene transcription, embryogenesis, cell cycle regulation, apoptosis and even neoplastic transformation. Although widely expressed during embryonic development, its expression level is negligible or absent in fully differentiated adult tissues including normal brain tissues. Recently published studies showed that HMGAl was overexpressed in several human benign and malignant tumors. However, the role of HMGA1in tumor progression is still not clear.Besides glioma, another most common type of brain tumor is brain metastasis. The brain is a common location for tumor metastasis; approximately one third of patients with systemic cancer ultimately develop central nervous system (CNS) involvement. This is due in part to the blood-brain barrier and the brain's "immune-privileged" status that each contribute to prevent chemotherapeutic drugs and inflammatory cells from penetrating into the CNS. Toll-like receptor (TLR)-family members as pattern-recognition receptors are wildly expression in brain microglia (MG) and macrophages (MP). Activation of toll-like receptors (TLRs) has been shown to enhance phagocytosis, promote secretion of Thl cytokines, and mediate leukocyte recruitment to infected tissues. Consequently, agonists such as CpG oligodeoxynucleotides (CpG) that bind TLR9have been evaluated as cancer vaccine adjuvants and have shown some efficacy in inducing adaptive and antigen-specific, cellular anti-tumor immune responses in animal models. However, early-stage clinical trials in patients with melanoma and gliomas have been less promising. Although higher CpG doses may improveclinical efficacy, significant inflammatory response in an already edematous brain may hinder this approach. Recently, we showed that intratumoral delivery of low-dose, immunostimulatory CpG conjugated with carbon nanotubes (CNTs) both eradicated intracranial (i.c.) gliomas, and induced antitumor immunity that protected mice from subsequent i.c. tumor rechallenge.This paper was divided into three parts:1. Extract glioma stem cells (GSCs) from glioblastoma cell line U251, investigate the expression of HMGA1in GSCs and U251, and then to further demonstrate HMGA1was correlated with malignant of tumors from the prospective of TSCs.2. Explore the expression of HMGA1in malignant gliomas with different WHO classifications and to study the correlation of HMGA1expression with tumor proliferation, invasion and angiogenesis.3. To enhance CpG activity, functionalized carbon nanotubes (CNT) were conjugated with CpG (CNT-CpG) and evaluated in tumor-bearing mice. We tested this hypothesis and demonstrated that intracerebral CNT-CpG may even be superior to systemic therapy, have systemic antitumor immunity to abrogate the growth of both intracerbral and primary tumors.The first part:the different HMGA1expression of total population of glioblastoma cell line U251and glioma stem cells isolated from U251Objective:Extract glioma stem cells (GSCs) from glioblastoma cell line U251, investigate the expression of HMGA1in GSCs and U251, and then to further demonstrate HMGA1was correlated with malignant of tumors from the prospective of TSCs.Methods:Glioma stem cells (GSCs) expressing the surface marker CD133from human glioblastoma cell line U251were isolated using MACS column and were analyzed using immunofluorescence and flow cytometry (FCM). The different expression of HMGA1was detected using real-time quantitative PCR and Western-Blot at transcription and translation levels between U251and isolated GSCs.Results:1, GSCs were successfully isolated from U251and cultured in serum-free medium (SMF). The percentage of GSCs in U251was0.32±0.07%.2, HMGA1expression was significantly higher in GSCs than in glioblastoma cells (P<0.05), up6.13±0.25-fold and2.75±0.99-fold at transcription and translation level, respectively.Conclusion:These results indicated HMGA1is overexpressed in GSCs as compared to glioblastoma cell line U251, which points to the expression of HMGA1being closely related to malignant proliferation, invasion and differentiation of tumor from the prospective of tumor stem cells (TSCs). We conclude that HMGA1may be a potential rational therapeutic target for glioma and GSC. The second part:HMGA1expression in human gliomas and its correlation with tumor proliferation, invasion and angiogenesisObjective:Investigate the expression of HMGA1in malignant gliomas with different WHO classifications and to study the correlation of HMGA1expression with tumor proliferation, invasion and angiogenesis.Methods:Sixty-four tissue specimens including4normal brain tissue samples from brain contusion and laceration patients,22diffuse astrocytomas,23anaplastic astrocytomas and15glioblastomas multiforme were collected at the Provincial hospital affiliated to Shandong University. The expression of HMGA1in malignant gliomas with different WHO classifications and its correlation with tumor proliferation, invasion and angiogenesis was analyzed by using immunohistochemistry and Real-time quantitative PCR.Results:1, Immunohistochemistry results showed that nuclear immunostaining of HMGA1protein was not observed in normal brain tissues, but was observed in96.7%(58of60) of malignant gliomas including high (+++) in15(25.0%), moderate (++) in28(46.7%), negligible to low (0-+) in17(28.3%) samples. The expression of HMGA1protein was significantly higher in glioblastoma multiforme than in WHO grade Ⅱ(P=0.002) and WHO grade Ⅲ gliomas (P=0.024). HMGA1protein expression has significant correlation with expression of Ki-67(r=0.53, P=0.00), MMP-9(r=0.508, P=0.000), VEGF-A (r=0.316, P=0.014) and MVD (r=0.321, P=0.012).2, Real-time quantitative PCR results also supported HMGA1overexpression in glioblastoma multiforme compared to WHO grade Ⅱ(P=0.043) and WHO grade Ⅲ gliomas (P=0.031). HMGA1gene expression has significant correlation with the gene expressions of Ki-67(r=0.429, P=0.025), MMP-9(r=0.443, P=0.024), VEGF-A(r=0.409, P=0.034).Conclusion:These results indicated that the expression of HMGA1has significant correlation with malignancy, proliferation, invasion and angiogenesis of gliomas. We conclude that HMGA1could be used as an intelligent biomarker in diagnosis and treatment of human tumors. The third part:Intracerebral CpG immunotherapy with carbon nanotubes abrogates growth of primary and intracerbral metastasis melanomasObjective:To enhance CpG activity, functionalized carbon nanotubes (CNTs) were conjugated with CpG (CNT-CpG) and evaluated in tumor-bearing mice. We tested this hypothesis and demonstrated that intracerebral CNT-CpG may even be superior to systemic therapy, have systemic antitumor immunity to abrogate the growth of both intracerbral and primary tumors.Methods:In vitro, the transcription factors NF-κB secreted by RAW MP cells was used for measure TLR-9activation. In vivo, the biodistribution of CpG and CNT-CpG were measured with a Xenogen IVIS Imaging System. Cytotoxicity Assay was measured using a Cobra Quantum gamma counter. Flow cytometry and FlowJo8.4.7software were used for measure the proportion of each cell type was measured as percent of total inflammatory cellsResults:1, In vitro experiments, CNT-CpG preparations that increased NF-κB activity at least2-fold, compared to an equivalent dose of free CpG at24hours,2, In vivo experiments, both CNT-CpG-Cy5.5and free CpG-Cy5.5appeared to clear slower from i.c. than s.c. melanomas, and even after7days, some Cy5.5signal was still detectable in the brains. We next compared the local distribution of free CpG to CNT-CpG in i.c. melanomas. Within a few days, free CpG appeared to diffuse away from the injection site, but CNT-CpG dispersed around the tumor margin.3, Compare with free CNT and free CpG, low doses of i.c. CNT-CpG can induce not only a local, but also a systemic anti-tumor response. Although mice survived longer, in contrast to gliomas, they were not cured from i.c. melanomas by i.c. CNT-CpG4, Treatment with i.c. CNT-CpG increased the infiltration of MP (CD45hi CD11b+), NK, CD8, and CD4cells into i.e. tumors, but had no effect on inflammatory cell infiltration into the non-treated s.c. tumors in the same animals. Interestingly, splenocytes from i.c. CNT-CpG- injected mice elicited a stronger ex vivo anti-tumor response when compared to s.c. treated tumors.Conclusion:We have verified the efficacy of a nanoparticle delivery system for optimizing CpG immunotherapy in a mouse melanoma model. More significantly, we have demonstrated potential value of intracerebral CNT-CpG for metastatic brain tumor therapy. Efficient uptake of CNT-CpG by tumor-associated MG in the brain, and their retention and wider distribution around tumors may account for a stronger immune anti-tumor response following intracerebral CNT-CpG injections. CNT-CpG can induce not only a local, but also a systemic anti-tumor response.