Rat C6 Glioma Model Research Articles

Photodynamic therapy of C6-implanted glioma cells in the rat brain employing second-generation photosensitizer talaporfin sodium

The usefulness of photodynamic therapy (PDT) as a local therapy for malignant glioma was evaluated by investigating histological changes in a rat C6 glioma model treated with a combination of talaporfin sodium, a water-soluble photosensitizer derived from chlorophyll and exposure to a diode laser. Glioma cells (C6) at the confluence stage were transplanted stereotactically into the right frontal lobe of SD rats. Five days later, the rats underwent right frontal craniotomy and intravenous administration of talaporfin sodium. One hour after talaporfin sodium administration, each rat was irradiated by a 664 nm diode laser beam. The brain was removed 1, 3 or 6h after laser irradiation for histological examination of tumor-affected brain tissue and surrounding normal brain tissue. In addition to the tumor mass, tumor cells invading surrounding edematous brain tissue were seen in untreated rats, ranging from the brain surface to a depth of 2mm. One hour after PDT, coagulation necrosis as well as disappearance of indication of cell viability such as disappearance of tumor cell processes and foamy changes of cytoplasm were noted in the tumor tissue at a depth of 0.5mm, accompanied by reduction of cytoplasmic glial fibrillary acidic protein (GFAP) expression and appearance of granular M30 cytodeath positivity. Three hours later, the cytoplasm of the residual tumor cells showed disappearance of GFAP expression and increased expression of M30 cytodeath. Six hours later, the foamy cytoplasm of swollen tumor cells demonstrated strong positivity for M30 cytodeath. PDT using talaporfin sodium induced coagulation necrosis and apoptosis in rats with C6 glioma.

Tissue-type plasminogen activator has antiangiogenic properties without effect on tumor growth in a rat C6 glioma model

Tissue-type plasminogen activator (tPA) plays a major role in the fibrinolytic system. According to several reports, tPA may also have antiangiogenic properties, especially in combination with a free sulfhydryl donor (FSD). In the rat C6 glioma model, in vitro and in vivo tPA synthesis by glioma cells is enhanced by differentiation therapy. To address the antiangiogenic potential of tPA in this model, tPA was overexpressed in glioma tumors by ex vivo transduction of C6 cells with a lentiviral vector encoding tPA. The transduced cells were subcutaneously implanted into nude mice. Gene transfer allowed for efficient synthesis of tPA by the C6 tumors. Although the treatment of tPA+ tumor-bearing animals with the FSD captopril generated angiostatin in situ and reduced endothelial vascularization of the tumors, it had no effect on tumor growth. Alternative mechanisms could account for this lack of effect and consequently have important implications for vascular the treatment of glioblastoma.

The effects of systemic and intratumoral interleukin-12 treatment in C6 rat glioma model

Objective: Cytokine based immunotherapy has long been an exciting field for many investigators aiming to provide an effective alternative treatment modality for glioma management. Among these cytokines, interleukin-12 (IL-12) plays a crucial role in mediating inflammatory and antitumoral activity on the host defence. We have investigated the therapeutic role of systemic and local delivery of IL-12 in C6 rat glioma model and compared these two modalities.Methods: The donor C6 glioma cells were injected stereotactically to 32 Wistar rats and right frontal tumor formation was established in all subjects. The rats were evenly divided into four groups as intratumoral (i.t.) control group (Group IA), intraperitoneal (i.p.) control group (Group IB), i.t. treatment group (Group II) and i.p. treatment group (Group III). Magnetic resonance imaging were performed to 12 rats (three from each group) on the seventh post-inoculation day. Recombinant mouse IL-12 (rmIL-12) was administered via i.t. (0.1 μg 5 μl/day/rat) and i.p. (0.1 μg 20 μl/day/rat) routes to treatment groups between days 9 and 11 following tumor inoculation, for 3 consecutive days. The rats which were unresponsive to the external stimuli, unable to feed themselves or having severe neurological impairment were decapitated and the specimens were histopathologically examined.Results: The subjects of Group III (i.p.) showed a statistically significant prolongation in survival time (mean=39 days) when compared to the control group (mean=31.7 days) (p=0.035) and Group II (i.t.) (mean=24.5 days) (p=0.005). Histopathologic examination of Group III revealed markedly increased intratumoral and peritumoral lymphocyte infiltration compared with the other groups.Conclusion: This study demonstrated that systemic administration of IL-12 in C6 glioma model in rats prolongs the survival, probably by stimulating the cellular immunity leading to lymphocytic infiltration.

FDG-PET imaging for the evaluation of antiglioma agents in a rat model

The increasing development of novel anticancer agents demands parallel advances in the methods used to rapidly assess their therapeutic efficacy (TE) in the preclinical phase. We evaluated the ability of small-animal PET, using the (18)F-fluoro-deoxy-D-glucose (FDG) radiotracer, to predict the TE of a number of anticancer agents in the rat C6 glioma model following 3 days of treatment. Semi-quantitative measurements of changes in FDG uptake during the course of treatment (standardized uptake value response [SUV(r)]) were found to be significantly lower in tumors treated with the hypoxia-inducible factor-1alpha inhibitor YC-1 (15 mg/kg) than in tumors in the control group. No significant SUV(r) change was observed following a similar 3-day regimen with the proapoptotic agent NS1619 (20 microg/kg), the combination of YC-1 and NS1619, or the alkylating agent temozolomide (7.5 mg/kg). Quantitative immunohistochemical studies demonstrated significantly lower levels of glucose transporter-1 (GLUT-1) expression in the YC-1-treated tumors, thereby correlating with the low SUV(r) observed in this group. The ability of SUV(r) to predict gold-standard outcomes of TE was further validated as YC-1-treated tumors had decreased volumes compared to control tumors. As such, we successfully demonstrated the ability of FDG-PET to rapidly determine the TE of novel agents for the treatment of glioma in the preclinical phase of evaluation.

Use of trans sodium crocetinate for sensitizing glioblastoma multiforme to radiation

Adjuvant treatment with radiation (radiation therapy or radiosurgery) is a mainstay of treatment for patients harboring glioblastomas multiforme (GBM). Hypoxic regions within the tumor make cells less sensitive to radiation therapy. Trans sodium crocetinate (TSC) has been shown to increase oxygen diffusion in the brain and elevate the partial brain oxygen level. The goal of this study was to evaluate the radiosensitizing effects of TSC on GBM tumors. A rat C6 glioma model was used, in which C6 glioma cells were stereotactically injected into the rat brain to create a tumor. Following creation of a right frontal tumor, animals were randomized into 1 of 4 groups: 1) TSC alone (animal treated with moderate-dose TSC only); 2) radiation (animals receiving 8 Gy of cranial radiation); 3) radiation and low-dose TSC (animals receiving 8 Gy of radiation and 50 microg/kg of TSC); or 4) radiation and moderate-dose TSC (animals receiving 8 Gy of radiation and 100 microg/kg of TSC). Animals were observed clinically for 60 days or until death. Magnetic resonance (MR) imaging was performed at 2-week intervals on each animal and quantitatively evaluated for tumor response. Immunohistochemical analysis was performed on all brain tumors. Survival differences were also evaluated using the Kaplan-Meier method. On MR imaging, a statistically significant reduction in tumor size was seen in the group receiving moderate-dose TSC and radiation treatment compared with the group receiving radiation treatment alone. The rate of tumor growth was significantly less for the combination of TSC and radiation treatment compared with either modality alone. Median survival times for the TSC-only and the radiation therapy-only groups were 15 and 30 days, respectively. The 60-day median survival times for the groups receiving a combination of either low- or moderate-dose TSC with radiation therapy were statistically improved compared with those for the other treatment groups. Use of TSC improves the extent of GBM tumor regression following radiation therapy and enhances survival. Radiosensitization of hypoxic tumors through increased oxygen diffusion may have clinical utility in patients with GBM tumors but must be explored in a clinical trial.

In vivo detection of c‐Met expression in a rat C6 glioma model

The tyrosine kinase receptor, c-Met, and its substrate, the hepatocyte growth factor (HGF), are implicated in the malignant progression of glioblastomas. In vivo detection of c-Met expression may be helpful in the diagnosis of malignant tumours. The C6 rat glioma model is a widely used intracranial brain tumour model used to study gliomas experimentally. We used a magnetic resonance imaging (MRI) molecular targeting agent to specifically tag the cell surface receptor, c-Met, with an anti-c-Met antibody (Ab) linked to biotinylated Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-albumin in rat gliomas to detect overexpression of this antigen in vivo. The anti-c-Met probe (anti-c-Met-Gd-DTPA-albumin) was administered intravenously, and as determined by an increase in MRI signal intensity and a corresponding decrease in regional T1 relaxation values, this probe was found to detect increased expression of c-Met protein levels in C6 gliomas. In addition, specificity for the binding of the anti-c-Met contrast agent was determined by using fluorescence microscopic imaging of the biotinylated portion of the targeting agent within neoplastic and ‘normal’brain tissues following in vivo administration of the anti-c-Met probe. Controls with no Ab or with a normal rat IgG attached to the contrast agent component indicated no non-specific binding to glioma tissue. This is the first successful visualization of in vivo overexpression of c-Met in gliomas.

Intratumoral vs Systemic Administration of Meta-Tetrahydroxyphenylchlorin for Photodynamic Therapy of Malignant Gliomas: Assessment of Uptake and Spatial Distribution in C6 Rat Glioma Model

Malignant gliomas, with an incidence of 5 cases per 100,000 population per year, represent the most common primary brain tumour. They have an overall survival length of less than 2 years. Many different adjuvant therapies have been developed. Among them, Photodynamic Therapy (PDT), that is based on photochemical reactions between light and tumoral tissue selectively labelled with exogenous photosensitizing agents. Among photosensitizers, m-THPC (Temoporfin), seems to be the most promising one for the treatment of brain tumors, but, unfortunately, it causes problems of high skin photosensitivity. To by-pass this problem, we devised an intratumoral route of administration of this photosensitizer. The aim of this study is to investigate and compare the uptake of m-THPC in brain tumor and normal tissue after systemic and intratumoral administration of the drug. 30 female Wistar rats received m-THPC 12 days after C6 tumor implantation. Temoporfin was administered intratumorally in 24 rats at two different concentrations. 6 rats constituted the control group and received m-THPC by means of an intraperitoneal injection. The brains were extracted at 4 h, 24 h and 96 h after Temoporfin injection. The samples were examined with a confocal laser scanning microscope. All samples showed high fluorescence emission exclusively in the tumour area, without appreciable differences between the samples taken at the different times of sacrifice and the two routes of administration. No fluorescence whatsoever was detected among normal brain tissue surrounding the tumour. The intratumoral route appears to give comparable results to the systemic one, regarding intracellular uptake efficiency and tumour--normal tissue ratio, with the advantage of a much shorter time needed to reach optimal intratumoural concentration--that is just four hours from m-THPC injection.

Phenyl-tert-butylnitrone induces tumor regression and decreases angiogenesis in a C6 rat glioma model

The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T1/T2-weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.

Convection-enhanced delivery in the treatment of malignant glioma

Despite advancements in glioma therapy, median survival remains low because of rapid post-resection recurrence. A regional method of drug delivery to address local invasion may improve clinical outcomes. Convection-enhanced delivery (CED) is a novel therapy that allows distribution of substances throughout the interstitium via positive-pressure infusion. Studies using various agents have investigated the parameters that affect CED including infusion rate, cannula size, infusion volume, extracellular space, particle characteristics and tumor tissue structure. We review models of small animal glioma that have been successfully treated using different substances administered through CED, particularly our favorable results using topotecan in a C6 rat glioma model. We also review Phase I/II trials utilizing CED which have shown promising response rates and acceptable safety profiles. Future studies should include prospective clinical trials and investigation of novel antitumor agents that are ineffective with systemic delivery. Development of a large animal glioma model would enhance pre-clinical investigation of CED. Clinically, methods to monitor distribution of therapeutic agents and real-time patient response should likewise be explored.

L‐type amino acid transporter 1 as a potential molecular target in human astrocytic tumors

L-type amino acid transporter 1 (LAT1) is a Na+-independent neutral amino acid transport agency and essential for the transport of large neutral amino acids. LAT1 has been identified as a light chain of the CD98 heterodimer from C6 glioma cells. LAT1 also corresponds to TA1, an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells. We have investigated for the first time, the expression of the transporter in the human primary astrocytic tumor tissue from 60 patients. LAT1 is unique because it requires an additional single membrane spanning protein, the heavy chain of 4F2 cell surface antigen (4F2hc), for its functional expression. 4F2hc expression was also determined by immunohistochemistry. Kaplan-Meier analyses demonstrated that high LAT1 expression correlated with poor survival for the study group as a whole (p<0.0001) and for those with glioblastoma multiforme in particular (p=0.0001). Cox regression analyses demonstrated that LAT1 expression was one of significant predictors of outcome, independent of all other variables. On the basis of these findings, we also investigated the effect of the specific inhibitor to LAT1, 2-aminobicyclo-2 (2,2,1)-heptane-2-carboxylic acid (BCH), on the survival of C6 glioma cells in vitro and in vivo using a rat C6 glioma model. BCH inhibited the growth of C6 glioma cells in vitro and in vivo in a dose-dependent manner. Kaplan-Meier survival data of rats treated with BCH were significant. These findings suggest that LAT1 could be one of the molecular targets in glioma therapy.

Patupilone (epothilone B, EPO906) and imatinib (STI571, Glivec) in combination display enhanced antitumour activity in vivo against experimental rat C6 glioma

The microtubule-stabilizing agent patupilone (epothilone B, EPO906) and the tyrosine kinase inhibitor imatinib (STI571, Glivec) which primarily inhibits Bcr-Abl, PDGF and c-Kit tyrosine kinase receptors, were combined in vivo to determine if any interaction would occur with respect to antitumour effect and tolerability using rat C6 glioma xenografted into nude mice. Patupilone and imatinib were administered alone or in combination at suboptimal doses. Imatinib treatment (orally once daily) was initiated 4 days after s.c. injection of rat C6 glioma cells into athymic nude mice and patupilone administration (i.v. once per week) was started 3 or 4 days after imatinib treatment. As a single agent, imatinib was inactive in the regimens selected (100 mg/kg: T/C 86% and 116%; 200 mg/kg: T/C 68% and 84%; two independent experiments), but well tolerated (gain in body weight and no mortalities). Patupilone weekly monotherapy demonstrated dose-dependent antitumour effects (1 mg/kg: T/C 67% and 70%; 2 mg/kg: T/C 32% and 63%; 4 mg/kg: T/C 3% and 46%). As expected, dose-dependent body weight losses occurred (final body weight changes at 1 mg/kg were -7% and -3%; at 2 mg/kg were -23% and -13%; and at 4 mg/kg were -33% and -15%). Combining 2 mg/kg patupilone and 200 mg/kg per day imatinib in one experiment produced a non-statistically significant trend for an improved antitumour effect over patupilone alone (combination, T/C 9%), while in the second experiment, enhancement was seen with the combination and reached statistical significance versus patupilone alone (combination, T/C 22%; P=0.008). Reduction of the imatinib dose to 100 mg/kg per day resulted in no enhancement of antitumour activity in combination with 2 mg/kg patupilone. Reduction of the patupilone dose to 1 mg/kg resulted in a reduced antitumour effect, and only a trend for synergy with either imatinib dose (combination, T/C 46% and 40%). Pooling the data from the two experiments confirmed a significant synergy for the combination of 2 mg/kg patupilone and 200 mg/kg per day imatinib (P=0.032), and a trend for synergy at the 1 mg/kg patupilone dose. Reduction in the imatinib dose to 100 mg/kg per day resulted only in additivity with either dose of patupilone. Body weight losses were dominated by the effect of patupilone, since no greater body weight loss was observed in the combination groups. Combining patupilone with high-dose imatinib produced an increased antitumour effect without affecting the tolerability of treatment in a relatively chemoresistant rat C6 glioma model. Such results indicate that further evaluation is warranted, in particular to elucidate possible mechanisms of combined action.

Enhancement of blood-tumor barrier permeability by Sar-[D-Phe8]des-Arg9BK, a metabolically resistant bradykinin B1 agonist, in a rat C6 glioma model.

BackgroundWhile it is well known that bradykinin B2 agonists increase plasma protein extravasation (PPE) in brain tumors, the bradykinin B1 agonists tested thus far are unable to produce this effect. Here we examine the effect of the selective B1 agonist bradykinin (BK) Sar-[D-Phe8]des-Arg9BK (SAR), a compound resistant to enzymatic degradation with prolonged activity on PPE in the blood circulation in the C6 rat glioma model.ResultsSAR administration significantly enhanced PPE in C6 rat brain glioma compared to saline or BK (p < 0.01). Pre-administration of the bradykinin B1 antagonist [Leu8]-des-Arg (100 nmol/Kg) blocked the SAR-induced PPE in the tumor area.ConclusionsOur data suggest that the B1 receptor modulates PPE in the blood tumor barrier of C6 glioma. A possible role for the use of SAR in the chemotherapy of gliomas deserves further study.

Expression of P2X 4 receptor in rat C6 glioma by tumor-associated macrophages and activated microglia

Here we report an immunohistochemical analysis of P2X 4 receptor (P2X 4R), an ATP-gated ion channel, expression in rat C6 glioma model. Striking P2X 4R expression was detected in compact and infiltrative tumor growth areas. Expression of P2X 4R by perivascular cells was observed in normal control brain and in brain areas not affected by tumor growth. Double-immunolabeling revealed that P2X 4R was co-expressed by ED1 +, AIF-1 +, and EMAP II + tumor-infiltrating microglia/macrophages; whereas on GFAP + and nestin + astrocytes P2X 4R was hardly seen. Our results indicate that P2X 4R expression defines a distinct subset of tumor-associated macrophages and activated microglia in glioma.

Feasibility and limitations of the rat model by C6 gliomas implanted at the subcutaneous region

Although rat models implanted with C6 glioma cells have been widely utilized for the assessment of new therapeutic modalities, a convenient in vivo model is still required. We implanted C6 glioma cells into the brains and the abdominal subcutaneous regions of Wistar rats, and evaluated the volumes of the growing tumors. The cultures of 5 × 106 cells had successfully formed tumors at 100% (52/52) of sites in the subcutaneous regions on day 5 after implantation. It was easy to measure the visible tumors with a sliding caliper. The tumor volume at the subcutaneous region reached a maximum volume (950 mm3 ± 167 SE) on day 15, after which it diminished. In contrast, tumors implanted in the intracerebral region showed a maximum volume on day 20 after the implantation of 1 × 105 cultured cells. Pathological examinations of the subcutaneous and intracranial tumors on day 15 showed similar findings exhibiting high nuclear cell ratio, mitosis and pseudopalisading with small populations of GFAP positive cells. The results suggested that rat C6 glioma models with implantation into the subcutaneous abdomen are available from day 5 to day 15 as a convenient model for the assessment of anti-proliferative drugs. The usefulness and the limitations of the rat C6 glioma model are discussed in this manuscript. [Neurol Res 2002; 24: 485-490]

Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma

The highly unsaturated fatty acids (HUFA) of the n-6 and n-3 series are involved in cell signalling in normal and transformed cells and have recently been associated with pathways leading to tumour cell death. The antitumour activity of three HUFA (arachidonic acid, gamma linolenic acid and eicosapentaenoic acid) were studied in glioma cells and tissue. Using five glioma models, including primary cell suspensions prepared from 46 human glioma samples and an in vivo rat C6 glioma model, we obtained evidence that, following exposure to HUFA, either administered into the medium surrounding human glioma cells or in 16 preparations of multicellular spheroids derived from human and rodent glioma cell lines (C6, MOG, U87, U373) or administered intra-tumourally by infusion using osmotic mini-pumps in 48 rats, glioma regression and apoptosis were detected. Additionally, synergy between gamma irradiation and HUFA administration was observed in 13 experiments analyzing C6 glioma cell apoptosis in vitro. These pro-apoptotic and antiproliferative activities were observed using both C18 and C20 fatty acids of the n-6 and n-3 series, but not when saturated and monounsaturated C18 and C20 fatty acid preparations were used. In the glioma infusion model, in addition to the apoptosis detected in glioma tissue infused with HUFA for 3–7 days, preservation of normal neural tissue and vasculature in adjacent brain was observed. Also, there was little evidence of acute inflammatory infiltration in regressing tumours. Our findings suggest that intraparenchymal infusion of HUFA may be effective in stimulating glioma regression.

Antiangiogenic activity of endostatin inhibits C6 glioma growth.

Angiogenesis is a vital component of the development and progression of many human solid tumors. Glioblastoma multiforme is one of the most highly vascularised class of solid tumors. Thus, we have investigated the potential antitumourigenic activity of endostatin, an angiogenic inhibitor, in the rat C6 glioma model. We have engineered C6 cells that endogenously express mouse endostatin in order to assess the growth of C6 tumors in vivo when endostatin is constitutively expressed. Endostatin secreted by stably transfected C6 cells is biologically active as shown by its inhibition (26%) of bFGF-stimulated proliferation of BAECs in culture. The subcutaneous implantation of endostatin-C6 cells in athymic (nu/nu) mice resulted in a reduced tumor growth rate (90% inhibition) compared to control cell lines throughout the duration of our experiments. Tumor inhibition was associated with a 50% reduction in the number of vessels, which were also smaller in morphology. However, endostatin-C6 tumors were no more necrotic than control tumors. The implantation of endostatin-C6 cells into immunocompetent Wistar rat brains also resulted in reduced tumor volumes (71% inhibition) compared to controls. Tumor cells were sparsely localised along the injection tract but had not formed discrete tumors. Despite the inhibitory response mediated by endostatin on C6 growth, complete tumor inhibition or dormancy was not observed in either the athymic or immunocompetent tumor models. These findings demonstrate that the endogenous expression of endostatin by C6 glioma cells results in a reduced tumor growth rate in vivo that is associated with an inhibition of tumor angiogenesis. Our data suggest that endostatin should be developed as an adjuvant gene therapy for the effective treatment of gliomas.

Therapeutic effects of sodium butyrate on glioma cells in vitro and in the rat C6 glioma model.

Preliminary in vitro studies have indicated that sodium butyrate inhibits the proliferation of cultured glioma cells and induces cellular differentiation, making it potentially useful as a therapeutic agent for patients with glioblastoma multiforme. The purpose of this study was to expand on the preliminary research by investigating the effects of sodium butyrate on multiple cell lines, explanted cells from glioblastoma tumor specimens, and in vivo in the rat C6 glioma brain tumor model. Four malignant glioma cell lines (A-172, T98G, U118MG, and C6) and two primary cell cultures derived from human glioblastoma tumor specimens were treated with 2 mmol/L sodium butyrate for up to 72 hours. Sodium butyrate-induced effects on cell morphology, proliferation, cell cycle distribution, migration, glial fibrillary acidic protein staining, and S100beta protein content were determined. For in vivo studies, a total of 64 male Wistar-Furth rats underwent operations to implant C6 glioma cells stereotactically or were used as controls. The rats were treated with escalating doses of sodium butyrate by microinfusion with Alzet minipumps (Durect Corp., Cupertino, CA). Sodium butyrate treatment in vitro produced changes in morphology and glial fibrillary acidic protein expression indicative of cellular differentiation. In cell lines and explanted cells, sodium butyrate consistently inhibited glioblastoma cell proliferation (to 51 +/- 6% that of controls) and migration (to 46 +/- 17%). Intratumoral infusion of 40 mmol/L sodium butyrate prolonged the survival of Wistar-Furth rats with intracerebral C6 tumors (P = 0.013) without detectable toxicity. These data support further consideration of direct interstitial infusion of sodium butyrate in a Phase I clinical study for patients with recurrent glioblastoma multiforme.

Combined Antitumor Effects of an Adenoviral Cytosine Deaminase/Thymidine Kinase Fusion Gene in Rat C6 Glioma

In this study, we investigated the feasibility of a double-suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type 1 thymidine kinase (TK) gene and the Escherichia coli cytosine deaminase (CD) gene, via a recombinant adenoviral vector, and ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) treatment, in a rat C6 glioma model. Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the beta-galactosidase gene and then staining cells with X-5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene (ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-deltaE1 (as a control). After the addition of a variety of concentrations of GCV and 5-FC, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. For in vivo antitumor experiments, 1x10(5) cells were stereotactically injected into the right caudate-putamen of female Wistar rat brains. At 3 days after implantation, 1x10(8) plaque-forming units of ad-CD/TK or ad-deltaE1 (as a control) were stereotactically injected into the tumors and GCV (25 mg/kg) and 5-FC (250 mg/kg), alone or in combination, were intraperitoneally administered. Animals were then killed, and tumor volumes were measured by determining the tumor area in every fifth section, using a light microscope. C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity (>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30 microg/ml or GCV at concentrations greater than 0.3 microg/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after single-prodrug treatments, indicating additive effects of the prodrug treatments. In in vivo experiments, the tumor volumes of the rats treated with GCV or 5-FC alone after ad-CD/TK injection (59.1+/-4.6 and 57.4+/-7.1 mm3, respectively) were significantly smaller than that of the control rats (157+/-8.9 mm3, P<0.05). Furthermore, the tumor volume of the rats treated with GCV and 5-FC in combination was 14.7+/-1.8 mm3. These results demonstrated the efficient transduction of C6 glioma cells with a recombinant adenovirus and the additive effects of CD/TK fusion gene/GCV/5-FC treatment, compared with single-gene therapy with the TK or CD gene. Therefore, our data suggest that the direct administration of a double-suicide gene/prodrug therapy has great potential in the treatment of brain tumors.

Limitations of the C6/Wistar Rat Intracerebral Glioma Model: Implications for Evaluating Immunotherapy

Intracranial rat glioma models are a useful method for evaluating the efficacy and toxicity of novel therapies for malignant glioma. The C6/Wistar model has been used extensively as a reproducible in vivo model for studying primary brain tumors including anti-glioma immune responses. The objective of the present study is to provide in vivo evidence that the C6 rat glioma model is allogeneic within Wistar rats and is therefore inappropriate for evaluating immune responses. Growth patterns and immune responses of C6 cells implanted into the brain and flank of Wistar rats were analyzed and compared to an immunogenic syngeneic model (9L/Fischer). Wistar rats with C6 tumors developed a potent humoral and cellular immune response to the tumor. Wistar rats given simultaneous flank and intracerebral tumors had a survival rate of 100% compared to an 11% survival rate in control animals receiving only intracranial C6 cells. The C6 rat glioma induces a vigorous immune reaction that may mimic a specific anti-tumor response in Wistar rats. Efficacy of immunotherapy within this model must be cautiously interpreted.

Beneficial effects of the radioprotectant 21-aminosteroid U-74389G in a radiosurgery rat malignant glioma model

Purpose: To evaluate the radioprotectant effects of the 21-aminosteroid U-74389G on the rat C6 glioma model after stereotactic radiosurgery. Because radiosurgery causes both tumor cytotoxicity, as well as regional brain edema, we hypothesized that this drug might exhibit advantageous or deleterious effects on healthy and neoplastic tissue. Methods: Rats were implanted with 10 6 C6 glioma cells into the right frontal brain and randomized to a Control Group ( n = 18), radiosurgery on Day 14 (50% isodose = 35 Gy) ( n = 15), or radiosurgery preceded by a single 15 mg/kg intravenous dose of 21-aminosteroid ( n = 27). All animals were killed by 90 days and evaluated for survival, tumor size, the presence or absence of regional parenchymal edema, or radiation-induced vasculopathy. Results: After tumor implantation, median survival in the Control Group was 23 days. Significant improvements in median survival were noted after RS alone (median, 31 days; p = 0.02), and RS plus 21-aminosteroid (median, 59 days; p < 0.0001). In the Control Group, mean tumor diameter was 5.4 mm. After RS alone, the mean diameter was 3.2 mm ( p = 0.002), and after RS plus 21-aminosteroid, 2.9 mm ( p = 0.0002). In the Control Group, the tumor grew as a hypercellular, compact mass. Only 3 of 18 animals had peritumoral edema. In contrast, 7 of 15 animals in the RS group had evidence of edema ( p = 0.006), but rats that received 21-aminosteroid showed no increase compared to controls ( p = 0.38). Similarly, 6 of 15 animals that had radiosurgery alone showed evidence of vasculopathy ( p = 0.005) compared to no animals in the control group and only 2 of 27 aminosteroid-treated animals. Conclusions: The 21-aminosteroid U-74389G exhibits a radioprotectant effect on normal brain tissue, but does not appear to protect the tumor in an in vivo rat radiosurgery model. We believe that the observed beneficial effects on healthy brain led to significant prolongation of animal survival; perhaps, by limiting the adverse effects of high-dose radiosurgery. This radioprotectant should now be evaluated in randomized clinical trials in patients with malignant brain tumors.