Anti-glioma Therapies Research Articles

Microglia and Macrophages in Malignant Gliomas: Recent Discoveries and Implications for Promising Therapies

Malignant gliomas are the most common primary brain tumors. Their deadliest manifestation, glioblastoma multiforme (GBM), accounts for 15% of all primary brain tumors and is associated with a median survival of only 15 months even after multimodal therapy. There is substantial presence of microglia and macrophages within and surrounding brain tumors. These immune cells acquire an alternatively activated phenotype with potent tumor-tropic functions that contribute to glioma growth and invasion. In this review, we briefly summarize recent data that has been reported on the interaction of microglia/macrophages with brain tumors and discuss potential application of these findings to the development of future antiglioma therapies.

Cellular Host Responses to Gliomas

BackgroundGlioblastoma multiforme (GBM) is the most aggressive type of malignant primary brain tumors in adults. Molecular and genetic analysis has advanced our understanding of glioma biology, however mapping the cellular composition of the tumor microenvironment is crucial for understanding the pathology of this dreaded brain cancer. In this study we identified major cell populations attracted by glioma using orthotopic rodent models of human glioma xenografts. Marker-specific, anatomical and morphological analyses revealed a robust influx of host cells into the main tumor bed and tumor satellites.Methodology/Principal FindingsHuman glioma cell lines and glioma spheroid orthotopic implants were used in rodents. In both models, the xenografts recruited large numbers of host nestin-expressing cells, which formed a ‘network’ with glioma. The host nestin-expressing cells appeared to originate in the subventricular zone ipsilateral to the tumor, and were clearly distinguishable from pericytes that expressed smooth muscle actin. These distinct cell populations established close physical contact in a ‘pair-wise’ manner and migrated together to the deeper layers of tumor satellites and gave rise to tumor vasculature. The GBM biopsy xenografts displayed two different phenotypes: (a) low-generation tumors (first in vivo passage in rats) were highly invasive and non-angiogenic, and host nestin-positive cells that infiltrated into these tumors displayed astrocytic or elongated bipolar morphology; (b) high-generation xenografts (fifth passage) had pronounced cellularity, were angiogenic with ‘glomerulus-like’ microvascular proliferations that contained host nestin-positive cells. Stromal cell-derived factor-1 and its receptor CXCR4 were highly expressed in and around glioma xenografts, suggesting their role in glioma progression and invasion.Conclusions/SignificanceOur data demonstrate a robust migration of nestin-expressing host cells to glioma, which together with pericytes give rise to tumor vasculature. Mapping the cellular composition of glioma microenvironment and deciphering the complex ‘crosstalk’ between tumor and host may ultimately aid the development of novel anti-glioma therapies.

Novel report of expression and function of CD97 in malignant gliomas: correlation with Wilms tumor 1 expression and glioma cell invasiveness

The Wilms tumor 1 (WT1) protein-a developmentally regulated transcription factor-is aberrantly expressed in gliomas and promotes their malignant phenotype. However, little is known about the molecular allies that help it mediate its oncogenic functions in glioma cells. The authors used short interfering RNA (siRNA) to suppress WT1 expression in glioblastoma (GBM) cells and evaluated the effect of this on GBM cell invasiveness. Gene expression analysis was then used to identify the candidate genes that were altered as a result of WT1 silencing. One candidate target, CD97, was then selected for further investigation into its role by suppressing its expression using siRNA silencing, followed by proliferation and invasion assays. WT1 levels were reliably and reproducibly suppressed by siRNA application. This resulted in a significant decrease in cellular invasiveness. Microarray analyses identified the gene products that were consistently downregulated (27) and upregulated (11) with WT1 silencing. Of these, CD97 expression was consistently suppressed across the 3 different GBM cell lines studied and was found on further investigation to significantly impact GBM cell invasiveness. Although CD97 expression in gliomas has not been described previously, we conclude that the possible upregulation of CD97 mediated by WT1 promotes cellular invasiveness-one of the most characteristic and challenging aspects of glial tumor cells. Further studies are needed to clarify the nature of this regulation and its impact, as CD97 could represent a novel target for antiglioma therapies.

The Role of Integrins in Glioma Biology and Anti-Glioma Therapies

The tumor environment is critical for tumor maintenance and progression. Integrins are a large family of cell surface receptors mediating the interaction of tumor cells with their microenvironment and play important roles in glioma biology, including migration, invasion, angiogenesis and tumor stem cell anchorage. Here, we review preclinical and clinical data on integrin inhibition in malignant gliomas. Various pharmacological approaches to the modulation of integrin signaling have been explored including antibodies and peptide-based agents. Cilengitide, a cyclic RGD-mimetic peptide of αvβ3 and αvβ5 integrins is in advanced clinical development in glioblastoma. Cilengitide had only limited activity as a single agent in glioblastoma, but, when added to standard radiochemotherapy, appeared to prolong progression-free and overall survival in patients with newly diagnosed glioblastomas and methylation of the promoter of the O⁶ methylguanine methyltransferase (MGMT) gene. MGMT gene promoter methylation in turn predicts benefit from alkylating chemotherapy. A phase III randomized clinical trial in conjunction with standard radiochemotherapy in newly diagnosed glioblastoma patients with MGMT gene promoter methylation has recently completed accrual (EORTC 26071-22072). A companion trial explores a dose-escalated regimen of cilengitide added to radiotherapy plus temozolomide in patients without MGMT gene promoter methylation. Promising results in these trials would probably result in a broader interest in integrins as targets for glioma therapy and hopefully the development of a broader panel of anti-integrin agents.

Mutant EGFR is required for maintenance of glioma growth in vivo, and its ablation leads to escape from receptor dependence

Epidermal growth factor receptor (EGFR) gene amplification is the most common genetic alteration in high-grade glioma, and approximately 50% of EGFR-amplified tumors also harbor a constitutively active mutant form of the receptor, DeltaEGFR. Although DeltaEGFR greatly enhances tumor growth and is thus an attractive target for anti-glioma therapies, recent clinical experiences with EGFR kinase inhibitors have been disappointing, because resistance is common and tumors eventually recur. Interestingly, it has not been established whether DeltaEGFR is required for maintenance of glioma growth in vivo, and, by extension, if it truly represents a rational therapeutic target. Here, we demonstrate that in vivo silencing of regulatable DeltaEGFR with doxycycline attenuates glioma growth and, therefore, that it is crucial for maintenance of enhanced tumorigenicity. Similar to the clinical experience, tumors eventually regained aggressive growth after a period of stasis, but interestingly, without re-expression of DeltaEGFR. To determine how tumors acquired this ability, we found that a unique gene, KLHDC8, herein referred to as SDeltaE (Substitute for DeltaEGFR Expression)-1, is highly expressed in these tumors, which have escaped dependence on DeltaEGFR. SDeltaE-1 is also expressed in human gliomas and knockdown of its expression in DeltaEGFR-independent "escaper" tumors suppressed tumor growth. Taken together, we conclude that DeltaEGFR is required for both glioma establishment and maintenance, and that gliomas undergo selective pressure in vivo to employ alternative compensatory pathways to maintain aggressiveness in the event of EGFR silencing. Such alternative pathways function as substitutes for DeltaEGFR signaling and should therefore be considered as potential targets for additional therapy.

Turning Cancer Stem Cells Inside Out: An Exploration of Glioma Stem Cell Signaling Pathways

Tumors are complex collections of heterogeneous cells with recruited vasculature, inflammatory cells, and stromal elements. Neoplastic cells frequently display a hierarchy in differentiation status. Recent studies suggest that brain tumors have a limited population of neoplastic cells called cancer stem cells with the capacity for sustained self-renewal and tumor propagation. Brain tumor stem cells contribute to therapeutic resistance and tumor angiogenesis. In this minireview, we summarize recent data regarding critical signaling pathways involved in brain tumor stem cell biology and discuss how targeting these molecules may contribute to the development of novel anti-glioma therapies.

Folate Supplementation Limits the Aggressiveness of Glioma via the Remethylation of DNA Repeats Element and Genes Governing Apoptosis and Proliferation

We have investigated whether the folate supplementation could be used to limit the aggressiveness of glioma through the DNA remethylation because (a) the cancer genome is characterized by a low level of DNA methylation (or 5-methylcytosine, 5 mC); and (b) folate is the main generator of S-adenosyl-methionine, the methyl donor molecule in the DNA methylation reaction catalyzed by the DNA methyltranferases. The effects of folate supplementations were analyzed on the global DNA methylation status, the methylation status of DNA repeat element, the sensitivity of temozolomide-induced apoptosis, and the proliferation index of glioma cells. Finally, we analyzed whether the DNA methylation level could be used as a prognostic factor and/or a biomarker in an antiglioma therapy using folate supplementation as an adjuvant. Our data show that gliomagenesis is accompanied by a reduction in 5 mC levels and that this low level of 5 mC is a poor prognostic factor in Glioblastoma Multiforme patients. We also show that folate supplementation enhanced the DNA remethylation through the Sp1/Sp3-mediated transcriptional up-regulation of genes coding for Dnmt3a and Dnmt3b proteins, two de novo methyltranferases. Finally, we show that the folate-induced DNA methylation limits proliferation and increases the sensitivity to temozolomide-induced apoptosis in glioma cells through methylation of the genes implicated in these processes (PDGF-B, MGMT, survivin, and bcl-w). This study suggests that folate supplementation could be a promising adjuvant for the future design of antiglioma therapies in preclinical and/or clinical studies.

An oncolytic adenoviral vector carrying the tyrosinase promoter for glioma gene therapy

Targeting gene expression to cancer cells remains a challenge for the development of gene and viral therapy for gliomas. Recent studies have highlighted transcriptional targeting as one of the possible solutions to overcome this limitation. In this context, melanoma associated antigens (MAAs) are usually over-expressed in brain tumors in comparison to normal brain tissue. For this reason, we investigated the use of the tyrosinase promoter as a transcriptional element to target oncolytic therapy for gliomas. Tyrosinase mRNA expression was evaluated by qRT-PCR in normal human brain tissue as well as in human glioma specimens. We found that this gene was significantly over-expressed in glioma cell lines and in primary glioma samples. Tyrosinase expression correlated with the grade of the tumor (p-value range: 0.05-0.001). Furthermore, transfection of several cell cultures with human and mouse tyrosinase promoters driving a luciferase reporter gene confirmed the activity of this promoter in mouse and human cells. To evaluate whether tyrosinase-activated conditionally replicative adenoviruses (CRAds) could induce toxicity in glioma cells, two vectors (Ad h/m and Ad24TYR) were tested in a mouse glioma model. C57BL/6 mice underwent intracranial injection of tumor cell line GL261. Survival was used to evaluate efficacy of the tested vectors. Mice receiving 1 x 10(9) MOI of Ad h/m and Ad24TYR following intracranial tumor implants had a median survival of 46+/-3 days (p<0.05); in contrast, those treated with medium had a median survival of 31+/-2 days. These results suggest that injection of tyrosinase CRAds leads to prolongation of survival in mice with experimental brain tumors. The tyrosinase promoter stands as a proof of principle of the potential use of MAA over-expression patterns for targeting novel anti-glioma therapies.

Optimization of adenoviral vector-mediated transgene expression in the canine brain in vivo, and in canine glioma cells in vitro

Expression of the immune-stimulatory molecule Fms-like tyrosine kinase 3 ligand (Flt3L) and the conditional cytotoxic enzyme herpes simplex virus type 1 thymidine kinase (HSV1-TK) provides long-term immune-mediated survival of large glioblastoma multiforme (GBM) models in rodents. A limitation for predictive testing of novel antiglioma therapies has been the lack of a glioma model in a large animal. Dogs bearing spontaneous GBM may constitute an attractive large-animal model for GBM, which so far has remained underappreciated. In preparation for a clinical trial in dogs bearing spontaneous GBMs, we tested and optimized adenovirus-mediated transgene expression with negligible toxicity in the dog brain in vivo and in canine J3T glioma cells. Expression of the marker gene beta-galactosidase (beta-Gal) was higher when driven by the murine (m) than the human (h) cytomegalovirus (CMV) promoter in the dog brain in vivo, without enhanced inflammation. In the canine brain, beta-Gal was expressed mostly in astrocytes. beta-Gal activity in J3T cells was also higher with the mCMV than the hCMV promoter driving tetracycline-dependent (TetON) transgene expression within high-capacity adenovirus vectors (HC-Ads). Dog glioma cells were efficiently transduced by HC-Ads expressing mCMV-driven HSV1-TK, which induced 90% reduction in cell viability in the presence of ganciclovir. J3T cells were also effectively transduced with HC-Ads expressing Flt3L under the control of the regulatable TetON promoter system, and as predicted, Flt3L release was stringently inducer dependent. HC-Ads encoding therapeutic transgenes under the control of regulatory sequences driven by the mCMV promoter are excellent vectors for the treatment of spontaneous GBM in dogs, which constitute an ideal preclinical animal model.

Molecular targets of glioma invasion

Glioblastoma multiforme is the most common and lethal primary malignant brain tumor. Although considerable progress has been made in technical proficiencies of surgical and radiation treatment for brain tumor patients, the impact of these advances on clinical outcome has been disappointing, with median survival time not exceeding 15 months. Over the last 30 years, no significant increase in survival of patients suffering from this disease has been achieved. A fundamental source of the management challenge presented in glioma patients is the insidious propensity of tumor invasion into distant brain tissue. Invasive tumor cells escape surgical removal and geographically dodge lethal radiation exposure and chemotherapy. Recent improved understanding of biochemical and molecular determinants of glioma cell invasion provide valuable insight into the underlying biological features of the disease, as well as illuminating possible new therapeutic targets. These findings are moving forward to translational research and clinical trials as novel antiglioma therapies.

O6-methylguanine-DNA methyltransferase is downregulated in transformed astrocyte cells: implications for anti-glioma therapies

BackgroundA novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas. However, expression of O6-methylguanine-DNA methyltransferase (MGMT) renders glioma cells resistant to the treatment, indicating that identification of mechanisms underlying the gene regulation of MGMT is highly required. Although glioma-derived cell lines have been widely employed to understand such mechanisms, those models harbor numerous unidentified genetic lesions specific for individual cell lines, which complicates the study of specific molecules and pathways.ResultsWe established glioma models by transforming normal human astrocyte cells via retroviral-mediated gene transfer of defined genetic elements and found that MGMT was downregulated in the transformed cells. Interestingly, inhibitors of DNA methylation and histone deacetylation failed to increase MGMT protein levels in the transformed astrocyte cells as well as cultured glioblastoma cell lines, whereas the treatment partially restored mRNA levels. These observations suggest that downregulation of MGMT may depend largely on cellular factors other than promoter-hypermethylation of MGMT genes, which is being used in the clinic to nominate patients for temozolomide treatment. Furthermore, we discovered that Valproic acid, one of histone deacetylase inhibitors, suppressed growth of the transformed astrocyte cells without increasing MGMT protein, suggesting that such epigenetic compounds may be used to some types of gliomas in combination with alkylating agents.ConclusionNormal human astrocyte cells allow us to generate experimental models of human gliomas by direct manipulation with defined genetic elements, in contrast to tumor-derived cell lines which harbor numerous unknown genetic abnormalities. Thus, we propose that the study using the transformed astrocyte cells would be useful for identifying the mechanisms underlying MGMT regulation in tumor and for the development of rational drug combination in glioma therapies.

Genetically modified adenoviruses against gliomas: from bench to bedside.

Oncolytic or tumor-selective adenoviruses are constructed as novel antiglioma therapies. After infection, the invading genetic adenoviral material is activated within the host cell. E1A and E1B adenoviral proteins are expressed immediately. E1A protein interacts with cell cycle regulatory proteins, such as retinoblastoma (Rb), driving the cell into the S phase and ensuing viral replication. The action of E1A stimulates the cellular p53 tumor suppressor system, which results in growth arrest or apoptosis, and halts adenovirus replication. However, adenoviral E1B interacts with p53 protein, preventing the DNA replication process from being abrogated by the induction of p53-mediated apoptosis. It was subsequently hypothesized that mutant adenoviruses that were unable to express wild-type E1A or E1B proteins could not replicate in normal cells with functional Rb or p53 pathways but instead would replicate and kill glioma cells that had defects in the regulation of these tumor suppressor pathways. Mutant E1B adenoviruses have already entered the clinical setting as an experimental treatment for patients with malignant gliomas. Mutant E1A adenoviruses are now in preclinical development as antiglioma therapy. In this review, the authors describe the mechanisms underlying the production of oncolytic adenoviruses, preclinical and clinical experiences with specific oncolytic adenoviruses, and the possibilities of combining mutant oncolytic adenoviruses with gene therapy or conventional therapies for managing malignant gliomas.

“...those left behind.” Biology and Oncology of Invasive Glioma Cells

Although significant technical advances in surgical and radiation treatment for brain tumors have emerged in recent years, their impact on clinical outcome for patients has been disappointing. A fundamental source of the management challenge presented by glioma patients is the insidious propensity of the malignant cells to invade into adjacent normal brain. Invasive tumor cells escape surgical removal and geographically dodge lethal radiation exposure. Recent improved understanding of the biochemistry and molecular determinants of glioma cell invasion provide valuable insight to the underlying biological features of the disease, as well as illuminating possible new therapeutic targets. Heightened commitment to migrate and invade is accompanied by a glioma cell's reduced proliferative activity. The microenvironmental manipulations coincident to invasion and migration may also impact the glioma cell's response to cytotoxic treatments. These collateral aspects of the glioma cell invasive phenotype should be further explored and exploited as novel antiglioma therapies.

A syngeneic mouse glioma model for study of glioblastoma therapy.

Animal models of human tumors serve a vital role in the development and testing of new anticancer therapies. Since the immune system is likely to play an essential role in tumor eradication, there is a particular need for modeling human disease in immunocompetent hosts. Few models of glioma have been developed in immunocompetent mice that are commercially available and none of these tumors have histological and antigenic characteristics of human gliomas. We have used a cell line, 4C8, derived from a spontaneous glioma-like tumor that arose in a transgenic mouse to develop a new glioma model. The intracranial injection of 4C8 cells into immunocompetent syngeneic B6D2F1 mice resulted in tumors that were densely cellular, developed a pseudopallisading pattern of necrosis, and expressed GFAP; all important features of human malignant gliomas. The average neurological endpoint was 51 days after intracranial injection. The 4C8 cells also grew rapidly in the flank, retaining histologic features seen in intracranial tumors. Flank tumors reached an average volume of 100 mm3, a volume ideal for therapy testing, by 34 days postinjection. These results suggest that the 4C8 mouse glioma model is an excellent system in which to test new antiglioma therapies for use in humans.

Novel way to increase targeting specificity to a human glioblastoma-associated receptor for interleukin 13.

Human brain cancers (gliomas) overexpress large numbers of a receptor for interleukin 13 (IL13), making this receptor an attractive target for anti-glioma therapies. We have recently proposed that the glioma-associated IL13 receptor is different from the one expressed on some hemopoietic and somatic cells. In an attempt to identify an even more glioma-specific target, we have used an antagonist of a related cytokine, IL4, which neutralizes the physiological effects of both IL13 and IL4 on normal cells. Here we demonstrate that the IL4 antagonist also counteracts the action of cytotoxins targeted to the IL13 receptor on normal human cells. Importantly, the IL4 antagonist does not inhibit IL13-based cytotoxins on glioma cells at all. Thus, the IL13 receptor on glioma cells can be categorized as tumor-specific in the presence of an IL4 antagonist. We conclude that IL13 receptor-directed cytotoxins can be delivered to glioma cells without being cytotoxic to normal cells.

Challenges in the design and conduct of phase III brain tumor therapy trials.

Twenty years ago a phase III trial by the Brain Tumor Study Group demonstrated that surgery and radiation were better treatment for malignant glioma than surgery alone.1 Maximal surgical resection and conventional radiotherapy quickly became the standard of care for patients with high-grade glioma. Since then many other treatments, most often cytotoxic drugs,2 have been added to surgery and radiation, and compared with standard therapy in randomized controlled trials(RCTs). None has unequivocally prolonged tumor control or patient survival.3 While these failures speak largely to the resilient nature of malignant glioma, certain issues surrounding the initiation and conduct of phase III trials of brain tumor treatment have surfaced in recent years and warrant discussion before the next generation of antiglioma therapies are tested. Some RCTs were based on inconclusive phase II data and may have been initiated prematurely, while others had divergent entry criteria, inadequate control for prognostic factors, and different outcome measures. Moreover, few studies evaluated quality of life (QOL), toxicity, or cost in a comprehensive way and none considered patient preferences for treatment. In this report we describe methods of assessing the results of phase II trials as a means of selecting treatments that most warrant phase III evaluation, and discuss challenges in the design and conduct of RCTs of new therapies for malignant glioma. When is an RCT warranted? When are the results of a phase II study sufficiently compelling to warrant a randomized trial? Randomized controlled trials are initiated when new treatments are found to have antiglioma activity in phase II evaluation. Phase II trials are of two types:(1) those that measure tumor response and (2) those that measure tumor control. In studies of response , treatment is given to patients with visible tumor, and the rate and duration of the response is recorded. …