Glioma Cell Populations Research Articles

Endogenous growth inhibition of angiogenesis in brain tumors

The growth inhibition of remote metastases by a primary tumor is known as endogenous growth inhibition leading to tumor dormancy. Such a phenotype has not been described in primary malignant gliomas. However, although glioma cells have frequently spread to other parts of the brain at the time of diagnosis, formation of solid secondary tumors is uncommon. We hypothesize that a dormant population of distant glioma cells exist. The purpose of this study was to investigate whether primary gliomas could inhibit secondary tumor formation. Subcutaneous tumors from human gliomas were grown as xenografts in Swiss nude mice. At a tumor size of at least one cm(3), the same amount of cells was injected into the contralateral flank or into the right cerebral hemisphere. Control mice without a primary tumor were injected with tumor cells either into the right flank, the right hemisphere, or bilaterally subcutaneously. Only one of 18 human gliomas demonstrated inhibition at the subcutaneous and intracerebral secondary implantation sites. Growth inhibition of the secondary tumors was accompanied by a significant reduction in microvessel density, upregulation of vascular endothelial growth factor mRNA and downregulation of basic fibroblast growth factor mRNA. Therefore, endogenous inhibition of secondary tumors may represent a rare phenotype in malignant glioma.

B-FABP-Expressing Radial Glial Cells: The Malignant Glioma Cell of Origin?

Brain fatty acid-binding protein (B-FABP) is normally expressed in radial glial cells, where it plays a role in the establishment of the radial glial fiber network required for neuronal migration. B-FABP is also expressed in astrocytoma tumors and in some malignant glioma cell lines. To address the role of B-FABP in malignant glioma, we have studied the growth properties of clonal populations of malignant glioma cells modified for B-FABP expression. Here, we demonstrate that expression of B-FABP in B-FABP-negative malignant glioma cells is accompanied by the appearance of radial glial-like properties, such as increased migration and extended bipolar cell processes, as well as reduced transformation. Conversely, B-FABP depletion in B-FABP-positive malignant glioma cells results in decreased migration, reduction in cell processes, a more transformed phenotype. Moreover, expression of B-FABP in astrocytomas is associated with regions of tumor infiltration and recurrence. Rather than being a direct manifestation of the tumorigenic process, we propose that the ability of high-grade astrocytoma cells to migrate long distances from the primary tumor reflects properties associated with their cell of origin. Thus, targeting B-FABP-expressing cells may make a significant impact on the treatment of these tumors.

Cellular and Functional Characterization of Immunoresistant Human Glioma Cell Clones Selected With Alloreactive Cytotoxic T Lymphocytes Reveals Their Up-regulated Synthesis of Biologically Active TGF-β

Two immunoresistant (IR) glioma cell variants, 13-06-IR29 and 13-06-IR30, were cloned from 13-06-MG glioma cell populations after receiving continuous immunoselective pressure from multiple alloreactive cytotoxic T lymphocyte (aCTL) preparations. Reapplication of aCTL immunoselective pressure to the IR clones, displaying a partial regain in sensitivity to aCTL after removal of the selective pressure, restored the resistance. The IR variants exhibited cross-resistance to non-human leukocyte antigen (HLA)-restricted effector cells and gamma-irradiation, but not to carmustine. The IR clones were characterized for factors that might contribute to the immunoresistance. The aCTL adhesion to extracellular matrix extracts derived from either the IR clones or the parental cells was similar and not impaired. Furthermore, aCTL binding to parental cells and IR clones was equal. Down-regulation of the cell recognition molecules, class I HLA or intercellular adhesion molecule-1 (ICAM-1), that would inhibit their recognition by aCTL was not observed on the IR clones. The down-regulation of Fas by the IR clones correlated with their resistance to FasL-induced apoptosis. HLA-G or FasL that might provide an immunotolerant environment or provide a means of counterattack to aCTL, respectively, were not associated with the IR phenotype. The aCTL, coincubated with the IR clones and parental cells, displayed up-regulation of multiple secreted cytokines. A significant up-regulation of bioactive transforming growth factor (TGF)-beta was observed in the IR clones compared with the parental cells. These data suggest that increased secretion of bioactive TGF-beta may inhibit aCTL lysis of the IR clones. Disruption of the TGF-beta signaling pathway may circumvent the resistance.

Polyamine depletion and cell cycle manipulation in combination with HSV thymidine kinase/ganciclovir cancer gene therapy

We have shown earlier that polyamine biosynthesis inhibition is accompanied by cell cycle alterations that can be utilized to enhance the efficacy of herpes simplex virus thymidine kinase - ganciclovir (HSV-TK/GCV) cancer gene therapy. In the present study, we asked 1) can the activated polyamine catabolism instead of biosynthesis inhibition be utilized to enhance the efficacy of HSV-TK/GCV gene therapy, and 2) can other known cell cycle inhibitors be used to make tumor cells more sensitive to this form of gene therapy? We show, using rat (9L) and human (U251-MG) glioma cell populations with 15% of HSV-TK-positive cells that DENSPM-induced activation of polyamine catabolism caused a profound polyamine deprivation in U251-MG cells, but there were no associated cell cycle effects in these cells. Consequently, we did not see any enhancement of the HSV-TK/GCV system. Aphidicolin, hydroxyurea, mimosine and resveratrol, but not lovastatin induced an apparent cell cycle arrest, followed by an intense but transient increase of the S phase cells after removal of the drug. This effect was shown to potentiate the HSV-TK/GCV cytotoxicity to some extent, especially in 9L cells and when the GCV treatment was started 0-24 h before the drug treatment. However, the enhancement was weaker than observed earlier with DFMO-induced cell cycle arrest and a considerable degree of the effect appeared to result from the growth-inhibitory actions of the drugs. In summary, we demonstrate that polyamine deprivation via DENSPM action is not associated with cell cycle effects and is not sufficient to cause enhancement of the HSV-TK/GCV system. Also, drugs with a rapid effect to the cell cycle are weak boosters of the HSVTK/GCV gene therapy, thus being less useful than DFMO for enhancement of this gene therapy form in animal studies and clinical trials.

CXCR4 expression mediates glioma cell invasiveness

Glioblastoma multiforme is a highly invasive tumor bearing a dismal prognosis. Experimental strategies that focus on the specific biological cues governing the invasive capacity of these tumors may hold significant therapeutic promise. In this context, we describe the in vitro and in vivo association of the cell surface chemokine receptor, CXCR4, with the development of an invasive phenotype in malignant glioblastoma. We demonstrate that invasive populations of glioma cells overexpress CXCR4 at the message and protein levels, and that this expression ranges from 25- to 89-fold higher than that found in noninvasive tumor cells. Furthermore, neutralization of CXCR4 significantly impairs the in vitro invasive capacity of malignant glial cells. In addition, glioma cells secrete CXCL12 and demonstrate robust invasive capacity toward a CXCL12 gradient in vitro. These findings underscore the importance of CXCR4 as a potential therapeutic target for the treatment of invasive glioblastoma.

Targeted cytoplasmic irradiation induces bystander responses.

The observation of radiation-induced bystander responses, in which cells respond to their neighbors being irradiated, has important implications for understanding mechanisms of radiation action particularly after low-dose exposure. Much of this questions the current dogma of direct DNA damage driving response in irradiated systems. In this study, we have used a charged-particle microbeam to target individual helium ions ((3)He(2+)) to individual cells within a population of radioresistant glioma cells cultured alone or in coculture with primary human fibroblasts. We found that even when a single cell within the glioma population was precisely traversed through its cytoplasm with one (3)He(2+) ion, bystander responses were induced in the neighboring nonirradiated glioma or fibroblasts so that the yield of micronuclei was increased by 36% for the glioma population and 78% for the bystander fibroblast population. Importantly, the yield of bystander-induced micronuclei was independent of whether the cytoplasm or nucleus of a cell was targeted. The bystander responses were fully eliminated when the populations were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide or filipin, which scavenge nitric oxide (NO) and disrupt membrane rafts, respectively. By using the probe 4-amino-5-methylamino-2',7'-difluorofluorescein, it was found that the NO level in the glioma population was increased by 15% after 1 or 10 cytoplasmic traversals, and this NO production was inhibited by filipin. This finding shows that direct DNA damage is not required for switching on of important cell-signaling mechanisms after low-dose irradiation and that, under these conditions, the whole cell should be considered a sensor of radiation exposure.

Intracellular oxidation by human glioma cell populations: effect of arachidonic acid

Arachidonic acid (AA) and Gamma linolenic acid have been shown to limit glioma cell growth, stimulate apoptosis and lipid peroxidation. However, brain tumours are characterised by cellular heterogeneity and responding cell populations have not been identified. Brain tumour samples from patients were disaggregated. In cell preparations from 7 gliomas, reactive oxygen species (ROS), morphology and plasma membrane integrity were monitored ±18–36 μM AA for 15–120 min using flow cytometry. Basal oxidative activity related to cell size/morphology, small granular cells showed lower activity. AA stimulation of ROS formation depended on cell size/morphology. Large, less granular cells showed greater AA stimulation. In 17 gliomas, GFAP immunofluorescence was demonstrated in larger cell populations. The large GFAP positive cell population with low side scatter was the highest responding cell population, suggesting selective tumour cell sensitivity to AA induced ROS formation. ROS may have a role in AA induced cell death and anti-tumour activity of AA in glioma.

Improved herpes simplex virus type 1 amplicon vectors for proportional coexpression of positron emission tomography marker and therapeutic genes.

For the development of efficient and safe gene therapy protocols for clinical application it is desirable to determine the tissue dose of vector-mediated therapeutic gene expression noninvasively in vivo. The herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET). Using bicistronic or multicistronic gene-expressing cassettes with tk as the PET marker gene, the quantitative analysis of tk gene expression may indirectly indicate the distribution and the level of expression of linked and proportionally coexpressed genes. Here, we describe the construction and functional evaluation of HSV-1 amplicon vectors mediating proportional coexpression of HSV-1-tk as PET marker gene and the enhanced green fluorescent protein gene (gfp) as proof of principle and cell culture marker gene and the Escherichia coli cytosine deaminase (cd) as therapeutic gene. Several double-/triple-gene constructs expressing HSV-1-tk, gfp, and E. coli cd were engineered based on gene fusion or the use of an internal ribosome entry site (IRES). Functional analysis in cell culture (green fluorescent protein [GFP] fluorescence and sensitivity to the prodrugs ganciclovir [GCV] and 5-fluorocytosine [5-FC]) and Western blots were carried out after infection of proliferating rat 9L gliosarcoma and human Gli36 glioma cells with helper virus-free packaged HSV-1 amplicon vectors. To study the ability of PET to differentiate various levels of tk expression noninvasively in vivo, retrovirally transduced and selected populations of rat F98 and human Gli36dEGFR glioma cells with defined levels of proportionally coexpressed tk and gfp genes were grown as subcutaneous tumors in nude rats and nude mice, and tk imaging by PET was performed. To study HSV-1 amplicon vector-mediated gene coexpression in vivo, HSV-1 amplicon vectors bearing coexpression constructs were injected (4 x 10(7) to 1 x 10(8) transducing units) into subcutaneously growing Gli36dEGFR gliomas in nude animals, and tk imaging was performed 24 hr later. All vector constructs mediated GFP expression and sensitized 9L and Gli36 cells toward GCV- and 5-FC-mediated cell killing in a drug dose-dependent manner, respectively. The levels of gene expression varied depending on the location of the genes within the constructs indicating the influence of the IRES on the level of expression of the second gene. Moreover, functional proportional coexpression of the PET marker gene HSV-1-tk and the linked therapeutic E. coli cd gene was observed. In selected tumor cell populations, subtle IRES-dependent differences of tk gene expression could be noninvasively distinguished by PET with good correlation between quantitative assays for IRES-dependent attenuated GFP and TK expression in culture and in vivo. After infection of subcutaneously growing gliomas with HSV-1 amplicon vectors, various levels of TK expression were found ranging from 0.011-0.062 percentage injected dose per gram (%ID/g). These values were 4.0- to 5.7-fold lower than positive control tumor cells. TK expression could be imaged by PET in vivo even with the tk gene located at the weak position downstream from the IRES. In conclusion, these HSV-1 amplicon vectors carrying HSV-1-tk as PET marker gene and any linked therapeutic gene will serve an indirect noninvasive assessment of the distribution of therapeutic gene expression by PET. Monitoring the correlation between primary transduction and therapeutic efficiency of a given vector is highly desirable for the development of safe and efficient gene therapy and vector application protocols in clinical applications.

Alginate-encapsulated producer cells: a potential new approach for the treatment of malignant brain tumors.

In recent years gene therapy has evolved as a new treatment for brain tumors, where genetically engineered cells can be used to deliver specific substances to target cells. However, clinical success has been limited due to insufficient gene transfer, lack of prolonged gene expression, and immunorejection of producer cells. These obstacles may be overcome by encapsulating producer cells into immunoisolating substances such as alginate. This may provide a stable in situ delivery system of specific proteins, which can interfere with tumor growth and differentiation. This article represents a fundamental study describing the in vitro and the in vivo behavior of alginate-encapsulated producer cells. The viability and cell cycle distribution of encapsulated NIH 3T3 cells was studied by confocal laser scanning microscopy (CLSM) and by flow cytometry. The CLSM study showed a high viability of the encapsulated NIH 3T3 cells during 9 weeks in culture. The flow cytometric analysis revealed a change in cellular ploidy after 1 week in culture, with normalization in ploidy after 3 and 9 weeks. The production of the bacterial E. coli beta-galactosidase in alginate-encapsulated BT4CnVlacZ cells was studied by x-gal staining, and the cells expressed prolonged beta-galactosidase activity. H528 hybridoma cells producing monoclonal antibodies (mAbs) against the human epidermal growth factor receptor (EGFR) were encapsulated in alginate, and the mAb release was determined. The release of mAbs stabilized around 400 ng/ml/h after 12 days in vitro. To actually demonstrate that alginate-encapsulated H528 cells potentially inhibit a heterogeneous glioma cell population, cell migration from human GaMg glioma spheroids was studied during stimulation with EGF in the presence of encapsulated H528 cells. The migration in vitro was totally inhibited in the presence of H528 encapsulated cells. Alginate beads with H528 cells were also implanted into rat brains, and after 9 weeks the distribution of mAbs within the brain was studied by immunohistochemistry. It is shown that the alginate entrapped H528 cells produce mAbs inside the brain for prolonged periods and that the mAbs are distributed within all CSF compartments. Encapsulated producer cells represent a potential delivery system for specific proteins to brain tumors. Different producer cells may be encapsulated in alginate to target phenotypic features and microenvironmental factors, which may influence the progressive growth of brain tumors.

Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes.

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is upregulated in response to a hypoxic or hypoglycemic stress. Here we show that the increase in steady-state levels of VEGF mRNA is partly due to transcriptional activation but mostly due to increase in mRNA stability. Both oxygen and glucose deficiencies result in extension of the VEGF mRNA half-life in a protein synthesis-dependent manner. Viewing VEGF as a stress-induced gene, we compared its mode of regulation with that of other stress-induced genes. Results showed that under nonstressed conditions, VEGF shares with the glucose transporter GLUT-1 a relatively short half-life (0.64 and 0.52 h, respectively), which is extended fourfold and more than eightfold, respectively, when cells are deprived of either oxygen or glucose. In contrast, the mRNAs of another hypoxia-inducible and hypoglycemia-inducible gene, grp78, as well as that of HSP70, were not stabilized by these metabolic insults. To show that VEGF and GLUT-1 are coinduced in differentially stressed microenvironments, multicell spheroids representing a clonal population of glioma cells in which each cell layer is differentially stressed were analyzed by in situ hybridization. Cellular microenvironments conducive to induction of VEGF and GLUT-1 were completely coincidental. These findings show that two different consequences of tissue ischemia, namely, hypoxia and glucose deprivation, induce VEGF and GLUT-1 expression by similar mechanisms. These proteins function, in turn, to satisfy the tissue needs through expanding its vasculature and improving its glucose utilization, respectively.

Glioma-stimulated chemoattraction of endothelial cells and fibroblasts in vitro: a model for the study of glioma-induced angiogenesis.

Induction of angiogenesis is essential for the continued growth of solid tumors, and one critical component of tumor-induced angiogenesis involves the stimulation of microvascular cells to migrate into the growing mass. We have developed a convenient model system utilizing dual co-culture chambers to study cellular chemotaxis induced by glioma cells in vitro. In this system, rat C6 glioma cells induced migration of fibroblasts and brain capillary endothelial cells. The migratory response was directly related to the number of C6 cells serving as stimulus in the lower chamber. Similar migratory responses were induced by C6 cell conditioned medium in a concentration dependent fashion. Medium conditioned by cultured human anaplastic astrocytoma cells was also found to contain potent chemotactic factor(s). This system may ultimately be employed in the identification of particular glioma cell population(s) and secreted factor(s) responsible for the chemoattraction of microvascular cells.

Transient heat shock in mid-G1-phase of the C6 glioma cell cycle impairs entry into S-phase

Synchronous populations of rat C6 glioma cells have been obtained by a mitotic selection procedure and used to establish the effect of transient temperature elevation on their progression through the cell cycle. A transient 3-h exposure to a 42°C heat shock within defined area in the mid-Gl-phase of the cell cycle has been demonstrated to impair progression to S-phase. This anti-proliferative heat shock effect was not apparent in asynchronous cell populations. A small, but significant, decrease in 3H-thymidine incorporation was observed (approximately 5%) following an 8-h exposure to heat shock.

Experimental Therapy of Human Glioma by Means of a Genetically Engineered Virus Mutant

Malignant gliomas are the most common malignant brain tumors and are almost always fatal. A thymidine kinase-negative mutant of herpes simplex virus-1 (dlsptk) that is attenuated for neurovirulence was tested as a possible treatment for gliomas. In cell culture, dlsptk killed two long-term human glioma lines and three short-term human glioma cell populations. In nude mice with implanted subcutaneous and subrenal U87 human gliomas, intraneoplastic inoculation of dlsptk caused growth inhibition. In nude mice with intracranial U87 gliomas, intraneoplastic inoculation of dlsptk prolonged survival. Genetically engineered viruses such as dlsptk merit further evaluation as novel antineoplastic agents.

Monoclonal antibodies against human astrocytomas and their reactivity pattern

The establishment of hybridomas after fusion of X63-Ag8.653 mouse myeloma cells and splenocytes from BALB c mice hyperimmunized against human astrocytomas is presented. The animals were primed with 5 × 10 6 chemically modified uncultured or cultured glioma cells. Six weeks after the last immunization step an intrasplenal booster injection was administrated and 3 days later the spleen cells were prepared for fusion experiments. According to the specificity analysis of the generated antibodies 7 hybridoma products (MUC 7-22, MUC 8-22, MUC 10-22, MUC 11-22, MUC 14-22, MUC 15-22 and MUC 2-63) react with gliomas, neuroblastomas and melanomas as well as with embryonic and fetal cells but do not recognize non-neurogenic tumors. The selected monoclonal antibodies (McAbs) of IgG 1 and IgG 2a isotypes are not extensively characterized but these antibodies have been demonstrated to be reactive with a panel of glioma cell lines with varying patterns of antigen distribution. Using the McAbs described above and a series of cryosections of glioma biopsies and paraffin sections of the same material as well as glioma cultures established from these, variable antigenic profiles among glioma cell populations could be demonstrated. From these results it is evident that there is not only a distinct degree of antigenic heterogeneity among and within brain tumors, but also that the pattern of antigenic expression can change continuously. Some of the glioma associated antigens recognized by the selected antibodies persist after fixation with methanol/acetone and Karnovsky's fixative and probably are oncoembryonic/oncofetal antigen(s). The data suggest that the use of McAbs recognizing tumor associated oncofetal antigens in immunohistochemistry facilitates objective typing of intracranial malignancies and precise analysis of fine needle brain/tumor biopsies in a sensitive and reproducible manner.

The failure of human leukocyte interferon to influence the growth of human glioma cell populations: in vitro and in vivo studies.

Five high-grade (3 grade III and 2 grade IV) astrocytoma tumour cell populations were treated with a preparation of Human Leukocyte Interferon either in monolayer cell culture or as multicellular spheroids in vitro or as xenografts growing in immune-deprived mice in vivo. A moderate and transient sensitivity was seen in one grade III tumour when tested in both of the in vitro assays, but no inhibition of growth was seen in vivo. Two tumours which were apparently resistant to Interferon treatment responded to orthodox chemotherapy. When used in conjunction with BCNU, Interferon was not effective in prolonging delay in tumour growth. It is concluded that Interferon is unlikely to be an effective agent in the treatment of malignant brain tumours.