Implantation Glioma Research Articles

Glioma-derived extracellular vesicles promote tumor progression by conveying WT1.

Glioma persists as one of the most aggressive primary tumors of the central nervous system. Glioma cells are known to communicate with tumor-associated macrophages/microglia via various cytokines to establish the tumor microenvironment. However, how extracellular vesicles (EVs), emerging regulators of cell-cell communication networks, function in this process is still elusive. We report here that glioma-derived EVs promote tumor progression by affecting microglial gene expression in an intracranial implantation glioma model mouse. The gene expression of thrombospondin-1 (Thbs1), a negative regulator of angiogenesis, was commonly downregulated in microglia after the addition of EVs isolated from different glioma cell lines, which endogenously expressed Wilms tumor-1 (WT1). Conversely, WT1-deficiency in the glioma-derived EVs significantly attenuated the Thbs1 downregulation and suppressed the tumor progression. WT1 was highly expressed in EVs obtained from the cerebrospinal fluid of human patients with malignant glioma. Our findings establish a novel model of tumor progression via EV-mediated WT1-Thbs1 intercellular regulatory pathway, which may be a future diagnostic or therapeutic target.

The radiosensitizing effect of CpG ODN107 on human glioma cells is tightly related to its antiangiogenic activity via suppression of HIF-1α/VEGF pathway

Malignant glioma displays invasive growth and is difficult to be completely excised; surgery combined with subsequent radiotherapy is a standard treatment for patients. CpG oligodeoxynucleotides (CpG ODN) can enhance radiotherapeutic effect in some tumors. Angiogenesis is crucial for tumor progression and metastasis. Anti-angiogenic strategy thus may be effective for tumor treatment. Herein, the antiangiogenic activity and radiosensitizing effect of CpG ODN107 on glioma were investigated. Our results showed that the growth of glioma cell line U87 was significantly inhibited by CpG ODN107 (10μg/ml) in combination with irradiation (5Gy) in vitro. In orthotopic implantation model of nude mice, the survival rate of mice significantly increased after treatment with CpG ODN107 (0.083mg/kg) in combination with radiotherapy (10Gy) as compared with treatment with local radiotherapy alone. CpG ODN107 in combination with radiotherapy significantly decreased microvessel density (MVD), VEGF level and HIF-1α expression in orthotopic implantation glioma. In conclusion, CpG ODN107 significantly increased the radiosensitivity of U87 human glioma cells in vitro and in vivo. The radiosensitizing effect of CpG ODN 107 is tightly related to its anti-angiogenic activity via suppression of HIF-1α/VEGF pathway.

Glioma Stem Cell Proliferation and Tumor Growth Are Promoted by Nitric Oxide Synthase-2

Malignant gliomas are aggressive brain tumors with limited therapeutic options, and improvements in treatment require a deeper molecular understanding of this disease. As in other cancers, recent studies have identified highly tumorigenic subpopulations within malignant gliomas, known generally as cancer stem cells. Here, we demonstrate that glioma stem cells (GSCs) produce nitric oxide via elevated nitric oxide synthase-2 (NOS2) expression. GSCs depend on NOS2 activity for growth and tumorigenicity, distinguishing them from non-GSCs and normal neural progenitors. Gene expression profiling identified many NOS2-regulated genes, including the cell-cycle inhibitor cell division autoantigen-1 (CDA1). Further, high NOS2 expression correlates with decreased survival in human glioma patients, and NOS2 inhibition slows glioma growth in a murine intracranial model. These data provide insight into how GSCs are mechanistically distinct from their less tumorigenic counterparts and suggest that NOS2 inhibition may be an efficacious approach to treating this devastating disease.

Glioma tumourgenicity is decreased by iNOS knockout: experimental studies using the C6 striatal implantation glioma model

Nitric oxide synthase (NOS) has recently been shown to be an important pathophysiological regulator in experimental implantation glioma since manipulation of NOS can significantly alter tumoural blood flow and inhibit tumour growth. In this study we investigated the role of iNOS (inducible NOS) in glioma tumourogenisis using the rodent C6 striatal implantation model. We produced genetically engineered C6 clones that do not express iNOS activity even after stimulation with a mixture of lipopolysaccaride (LPS) and tumour necrosis factor (TNF)-α. These iNOS knockout cells showed a similar growth rate to control cells in vivo at 5 days. We then performed an in vivo implantation glioma study using either the iNOS knockout clone or two genetically engineered control C6 clones. There was a significant reduction (p < 0.01) of tumour mass with the iNOS knockout clone 28 days after the implantation. Immunocytochemistry indicated infiltrates of CD3 positive T cells and macrophages in the controls and the iNOS knockout group. These studies indicate that iNOS expression by tumour parenchymal cells is a critical factor for tumour growth with this model. The mechanisms that cause failure of tumour growth need clarification prior to considering that specific iNOS inhibitors might be candidates for adjuvant treatment of malignant glioma.

Highly unsaturated fatty acid induced tumour regression in glioma pharmacodynamics and bioavailability of gamma linolenic acid in an implantation glioma model: effects on tumour biomass, apoptosis and neuronal tissue histology

Highly unsaturated fatty acids (HUFAs) are naturally occurring anti-tumour agents. HUFAs act as intracellular signalling molecules in cell proliferation and death. In human glioma, HUFAs may stimulate tumour regression and apoptosis. An implantation glioma model, using the C6 glioma cell line, was used to investigate the bioactivity of locally infused n-6 HUFA gamma linolenic acid (GLA). Rat brains (15 normal and 37 C6 tumour bearing) were infused with vehicle or GLA 200 μM–2 mM. The most active local concentration of GLA for anti-tumour activity was 2 mM, infused at 1 μ l/h over 7 days. Tumour regression, increased apoptosis and decreased proliferation were observed in tumours of rats infused with this concentration of GLA. Little effect on normal neuronal tissue was detected. The intraparenchymal route was an effective method of GLA administration in the treatment of glioma. These studies provide further insights into the potential role of HUFAs as anti-glioma agents.

Glioma tumourgenicity is decreased by iNOS knockout: experimental studies using the C6 striatal implantation glioma model

Nitric oxide synthase (NOS) has recently been shown to be an important pathophysiological regulator in experimental implantation glioma since manipulation of NOS can significantly alter tumoural blood flow and inhibit tumour growth. In this study we investigated the role of iNOS (inducible NOS) in glioma tumourogenisis using the rodent C6 striatal implantation model. We produced genetically engineered C6 clones that do not express iNOS activity even after stimulation with a mixture of lipopolysaccaride (LPS) and tumour necrosis factor (TNF)-α. These iNOS knockout cells showed a similar growth rate to control cells in vivo at 5 days. We then performed an in vivo implantation glioma study using either the iNOS knockout clone or two genetically engineered control C6 clones. There was a significant reduction (p < 0.01) of tumour mass with the iNOS knockout clone 28 days after the implantation. Immunocytochemistry indicated infiltrates of CD3 positive T cells and macrophages in the controls and the iNOS knockout group. These studies indicate that iNOS expression by tumour parenchymal cells is a critical factor for tumour growth with this model. The mechanisms that cause failure of tumour growth need clarification prior to considering that specific iNOS inhibitors might be candidates for adjuvant treatment of malignant glioma.

Heme oxygenase (HO) isoforms in experimental C6 glioma: an immunocytochemical study

Because of their potential to regulate tumoural blood flow and interactions with nitric oxide the expression of the type 1 and 2 isoforms of heme oxygenase (HO-1 and HO-2) were evaluated in implanted C6 striatal gliomas. Immunocytochemistry using antibodies specific for HO-1 and HO-2 were used in 20 C6 glioma tumours. The bulk of the tumour parenchyma and endothelium was negative for both HO isoforms. Isolated, but weak staining for HO-1 was seen in most tumours with focally increased expression in perinecrotic regions. Cells morphologically resembling macrophages stained with both HO-1 and HO-2, but were not numerous. These findings suggest that carbon monoxide, unlike nitric oxide, does not have a major role in regulating tumoural blood flow in this experimental glioma model. These findings once again demonstrate the differences between human malignant glioma and experimental implantation glioma models.

Can experimental models of rodent implantation glioma be improved? A study of pure and mixed glioma cell line tumours.

To evaluate the hypothesis that co-implantation of different rodent glioma cell lines might result in experimental brain tumours that more closely resemble human gliomas the neuropathology and immunocytochemical features of implantation gliomas derived from single cell lines (C6, A15A5, F98), two cell lines admixed 50:50 prior to implantation (C6 + F98 and C6 + A15A5) and three cell lines equally admixed (C6 + A15A5 + F98) was studied in the adult Wistar rat. Tumours grew consistently following implantation of the single and the two admixed cell lines, however tumour growth following triple mix implantation was considerably and consistently impaired. The tumours derived from admixed cell lines showed regional heterogeneity with areas characteristic of both the primary cell lines. Foci of lymphocytic infiltrates, tumoural necrosis, often with pseudopallisading, and peritumoural edema were consistent features of all tumours. Limited parenchymal and more extensive perivascular tumoural invasion was seen predominantly in tumours containing the C6 cell line. There were no significant differences in GFAP, vimentin and HSP70 staining between the mixed tumours, although the pure F98 and A15A5 tumours were, unlike the pure C6 gliomas, S-100 negative. Using PCNA expression as a measure of the tumour proliferation all except the tumours derived from the three cell lines mix, which had a staining index of 7-10%, had focal staining indices in viable tumour of between 40-80%. There was focal positive staining in both perilesional brain and in regions of all tumours for the macrophage markers ED-1 and ED-2. None of the three cell lines stained in vitro for either ED1 and ED2 but all were constitutively positive in vitro for OX-6, a proposed marker for antigen presenting cells. The macrophage and lymphocytic response suggest a vigorous but largely ineffective immunological response had been mounted against all tumours. The consistent failure of the triple mix tumours to grow is unexplained. This work has shown the feasibility of producing 'mixed' cell line experimental gliomas by combining two cell lines at the time of innoculation. However, the relative failure to produce (i) mixed tumours that have properties not inherent to either parent cell line and (ii) implantation glioma with three cell lines suggest there are limits to this approach. Admixture of cell lines at the time of implantation therefore does not make experimental glioma models that more closely resemble natural gliomas, and also has some particular disadvantages. This experimental approach is therefore not recommended for use in the study of tumour biology and in evaluating the effectiveness of novel therapies.

Progressive focal neurological dysfunction following experimental implantation glioma

One of the difficulties in understanding peritumoural brain dysfunction is the lack of defined clinical deficits in experimental glioma models. In this study progressive focal neurological dysfunction was measured using the staircase test in rodents subjected to striatal implantation of C6 glioma cells. After 22 days none of the animals, all of which had cortico-striatal tumours ranging in size from 93 to 140 mm3, showed any obvious gross behavioural abnormality. However, contralateral forelimb function was significantly worse than that before surgery by day 7 (p < 0.01) and worse than sham-implanted animals by day 12 (p < 0.01). Using this experimental paradigm the staircase test can be used to measure progressive focal neurological deterioration and evaluate both the mechanisms of, and therapies for peritumoural brain dysfunction.

Glioma cells transduced with selection transgenes may not form gliomas in vivo and can also inhibit glioma formation by admixed wild glioma cell lines.

Following in vitro lipofection transfection of the rat glioma cell line A15A5 with the plasmid transgene CMV/HyTK, which confirms hygromycin resistance and ganciclovir sensitivity, a series of experiments was planned in which the "bystander" phenomenon would be evaluated using the rodent implantation glioma model. However examination of the brain of rodents in which the A15A5HyTK cells were implanted showed no evidence of glioma growth. Furthermore, rodents having intracerebral implantation of (i) wild A15A5 and A15A5HyTK cells in a 50/50 mix, (ii) wild A15A5 and A15A5HyTK cells in a 90/10 mixture and (iii) wild C6 and A15A5HyTK cells in a 50/50 mix all failed to grow macroscopic tumours by 15-17 days irrespective of whether the animals had been administered ganciclovir (GCV) in the week before sacrifice. Neuropathological and immunocytochemical analysis of the implantation sites showed no difference between the GCV and saline treated groups of animals for any implantation cell mix. These observations confirm previous results that suggest transduction of malignant rodent glioma cell lines with a variety of selection, oncogenic and marker genes significantly impairs their in vivo tumorigenic potential compared to the wild type cell lines. This study also demonstrates that even without GCV treatment the transduced cells inhibit, by an unknown mechanism(s), the tumorigenicity of other non transfected malignant cells. The implications of this study for gene therapy of human malignant glioma are discussed.

Nitric oxide synthase is expressed in experimental malignant glioma and influences tumour blood flow.

The distribution and function of nitric oxide synthase (NOS) was studied in the rodent C6 implantation glioma model. Using a histochemical stain for NADPH diaphorase, which colocalises with NOS, morphological studies revealed non homogenous staining of the constituent tumour cells and the neoplastic endothelium. Immunocytochemical staining for macrophages (ED1, ED2) showed dense positivity at the tumour brain interface with more patchy positivity within the tumour mass. This finding suggests that both macrophages, which are known to produce large amounts of NO, and the C6 cells contribute to the NADPH diaphorase positivity. Administration of the NOS inhibitor Ng-nitro-L-argine methyl ester (L-NAME) significantly reduced both tumour (40%) and contralateral local cerebral blood flow (20%) compared to control animals. These findings demonstrate that (i) NOS is present in experimental malignant glioma; (ii) NO mediated mechanisms contribute to tumour blood vessel dilatation and blood flow regulation; and (iii) using this model there is a significant differential sensitivity of the tumour and brain parenchymal vascular beds to a NOS inhibitor. Further investigations are required to determine the potential therapeutic and biological relevance of these findings and the relative contributions of tumour cells, neoplastic endothelium and reactive macrophages to NO mechanism in gliomas.

Investigation of an inhibitor of lipid peroxidation U74006F on tumor growth and protein extravasation in the C6 astrocytoma spheroid implantation glioma model

The role played by lipid peroxidation in cerebral tumor growth and protein extravasation has not been defined. A potent in vitro inhibitor of lipid peroxidation (21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16a- methylpregna-1,4,9(11)-triene-3,20-dione monomethane sulfonate (U74006F), has been used to assess its influence on both tumor growth and protein extravasation in a rat C6 astrocytoma implantation model. Following spheroid implantation, rats were treated with one of these U74006F regimens: 12 mg/kg/day for 13 days post-implantation, 3.6 mg/kg/day for 13 days post-implantation or 12 mg/kg/day commencing seven days post-implantation until 13 days post-implantation. Tumor wet weights, dry weight and protein flux as measured by Evans blue dye extravasation were assessed. Neither wet weights nor dry weights of any of the three treatment groups was significantly lower when compared to control tumor wet weights and dry weights. Protein extravasation was not significantly reduced by any of the treatments. It is concluded that U74006F did not significantly decrease tumor growth or permeability in the C6 astrocytoma spheroid implantation model.

Effect of ibuprofen on tumor growth in the C6 spheroid implantation glioma model

The effects of long-term low- and high-dose ibuprofen on tumor growth and permeability were assessed in a glioma model in rats. The rats were treated with ibuprofen (24 mg/kg/day or 96 mg/kg/day) for 24 hours before implantation of C6 astrocytoma spheroids and then for 13 days following implantation. The wet and dry weight of the tumors and protein extravasation were measured by an Evans blue dye technique. Protein extravasation did not appear to be reduced by the treatments when assessed on the basis of tumor dry weight. The treatment significantly reduced the wet weight of the tumors in rats treated with high-dose and low-dose ibuprofen when compared to tumor wet weights in untreated rats. High-dose ibuprofen treatment significantly decreased the dry weight of the tumors compared to that of tumors in untreated control animals. It is hypothesized that the ibuprofen treatment regimen employed inhibits prostaglandin-associated angiogenesis induced by the C6 tumor cell growth and/or the implantation technique, thereby interfering with the ability of the tumors to grow.

A new glioma model in rat: the C6 spheroid implantation technique permeability and vascular characterization.

The C6 spheroid implantation glioma model is a simple, easily reproduced model for primary gliomas in which C6 astrocytoma cells are grown in vitro as spheroids and subsequently implanted into the brains of Sprague Dawley rat hosts. This report describes the growth, histology, vessel architecture and vascular permeability of the resulting tumors. The appearance of the tumor was investigated by light and electron microscopy, and by using the alkaline phosphatase technique. The leakage of tracer was measured from vessels in the tumor and peritumoral area at various times during tumor development. The spheroid implant produces a fully vascularized, rapidly growing tumor with many of the characteristics of glioblastoma multiforme, from an avascular focus of neoplastic cells. The major advantage of this model is that the tumors grow in a spheroidal fashion and the tumor-brain interface can be easily located. Many of the important events in the process of vascularization take place at the tumor-brain interface. Two distinctive vascular events appear to occur simultaneously: proliferation of blood vessels and their growth into the tumor mass so that they develop into typical, permeable tumor vessels, and migration of tumor cells along normal vessels into the surrounding brain. Tumor vessels were permeable to the tracer Evans Blue (EB) from the earliest days of ingrowth. Leakage of the EB increased as the tumors increased in size, but eventually leakage plateaued as tumors developed necrotic centers. It is well known that the structural and permeability characteristics of vessels associated with the tumor affect tumor growth. This model will be useful for a number of proposed studies including assessment of various clinical therapies on tumor growth and development, and more specifically, quantitative analysis of the vascularization process in tumors.