Experimental Glioma Model Research Articles

Effect of Human Skin-Derived Stem Cells on Vessel Architecture, Tumor Growth, and Tumor Invasion in Brain Tumor Animal Models

Glioblastomas represent an important cause of cancer-related mortality with poor survival. Despite many advances, the mean survival time has not significantly improved in the last decades. New experimental approaches have shown tumor regression after the grafting of neural stem cells and human mesenchymal stem cells into experimental intracranial gliomas of adult rodents. However, the cell source seems to be an important limitation for autologous transplantation in glioblastoma. In the present study, we evaluated the tumor targeting and antitumor activity of human skin-derived stem cells (hSDSCs) in human brain tumor models. The hSDSCs exhibit tumor targeting characteristics in vivo when injected into the controlateral hemisphere or into the tail vein of mice. When implanted directly into glioblastomas, hSDSCs distributed themselves extensively throughout the tumor mass, reduced tumor vessel density, and decreased angiogenic sprouts. In addition, transplanted hSDSCs differentiate into pericyte cell and release high amounts of human transforming growth factor-beta1 with low expression of vascular endothelial growth factor, which may contribute to the decreased tumor cell invasion and number of tumor vessels. In long-term experiments, the hSDSCs were also able to significantly inhibit tumor growth and to prolong animal survival. Similar behavior was seen when hSDSCs were implanted into two different tumor models, the chicken embryo experimental glioma model and the transgenic Tyrp1-Tag mice. Taken together, these data validate the use of hSDSCs for targeting human brain tumors. They may represent therapeutically effective cells for the treatment of intracranial tumors after autologous transplantation.

Establishment of experimental glioma models at the intrinsic brainstem region of the rats

Objectives: As the treatment of human intrinsic brainstem gliomas remains challenging, experimental glioma models are needed.Methods: We developed a rat model of intrinsic brain stem glioma that uses a stereotactic frame to fix the head for the delivery of C6 glioma cells to target sites via a permanently implanted cannula. We inoculated the rat midbrain, pons or cerebral cortex with 5 × 104 cells suspended in 1 μl culture medium over the course of 2 minutes.Results: Three days post-implantation, tumor formation was visible in the periaqueductal gray matter in the midbrain and the tegmentum of the pons. On the tenth day, the tumor diameter exceeded over 2 mm; there was no tumor cell seeding into the cerebrospinal fluid space. The tumor manifested the histological features typical of glioblastoma; Ki-67 labeling index was 32%.Discussion: Because in our model the cannula is permanently implanted, additional inocula can be delivered. Here we detail our rat brainstem glioma model and discuss its usefulness for the investigation of these tumor in humans.

Cellules dendritiques et gliomes : un espoir en immunothérapie ?

Immunotherapy has been explored for several decades to try to improve the prognosis of gliomas, but until recently no therapeutic benefit has been achieved. The discovery of dendritic cells, the most potent professional antigen presenting cells to initiate specific immune response, and the possibility of producing them ex vivo gave rise to new protocols of active immunotherapy. In oncology, promising experimental and clinical therapeutic results were obtained using these dendritic cells loaded with tumor antigen. Patients bearing gliomas have deficit antigen presentation making this approach rational. In several experimental glioma models, independent research teams have showed specific antitumor responses using these dendritic cells. Phase I/II clinical trials have demonstrated the feasibility and the tolerance of this immunotherapeutic approach. In neuro-oncology, the efficiency of such an approach remains to be established, similarly in oncology where positive phase III studies are missing. Nevertheless, dendritic cells comprise a complex network which is only partially understood and capable of generating either immunotolerance or immune response. Numerous parameters remain to be explored before any definitive conclusion about their utility as an anticancer weapon can be drawn. It seems however logical that immunotherapy with dendritic cells could prevent or delay tumor recurrence in patients with minor active disease. A review on glioma and dendritic cells is presented.

Identification of mobile cholesterol compounds in experimental gliomas by 1H MRS in vivo: Effects of ganciclovir-induced apoptosis on lipids

This letter presents a novel identification and analysis of mobile cholesterol compounds in an experimental glioma model by 1H MRS in vivo. The cholesterol compounds turned out to comprise as much as 17 mol% of MRS visible total lipids. The results also imply partly associated accumulation of 1H MRS detectable cholesterol compounds and unsaturated lipids during gene therapy-induced apoptosis, and indicate that the contribution of cholesterol compounds cannot be bypassed in spectral lipid analysis. The introduced 1H MRS approach facilitates a non-invasive follow-up of mobile cholesterol compounds, paving way for studies of tumour cholesterol metabolism in vivo.

‘Pseudopalisading’ Necrosis in Glioblastoma: A Familiar Morphologic Feature That Links Vascular Pathology, Hypoxia, and Angiogenesis

Glioblastoma (GBM) is a highly malignant, rapidly progressive astrocytoma that is distinguished pathologically from lower grade tumors by necrosis and microvascular hyperplasia. Necrotic foci are typically surrounded by "pseudopalisading" cells-a configuration that is relatively unique to malignant gliomas and has long been recognized as an ominous prognostic feature. Precise mechanisms that relate morphology to biologic behavior have not been described. Recent investigations have demonstrated that pseudopalisades are severely hypoxic, overexpress hypoxia-inducible factor (HIF-1), and secrete proangiogenic factors such as VEGF and IL-8. Thus, the microvascular hyperplasia in GBM that provides a new vasculature and promotes peripheral tumor expansion is tightly linked with the emergence of pseudopalisades. Both pathologic observations and experimental evidence have indicated that the development of hypoxia and necrosis within astrocytomas could arise secondary to vaso-occlusion and intravascular thrombosis. This emerging model suggests that pseudopalisades represent a wave of tumor cells actively migrating away from central hypoxia that arises after a vascular insult. Experimental glioma models have shown that endothelial apoptosis, perhaps resulting from angiopoetin-2, initiates vascular pathology, whereas observations in human tumors have clearly demonstrated that intravascular thrombosis develops with high frequency in the transition to GBM. Tissue factor, the main cellular initiator of thrombosis, is dramatically upregulated in response to PTEN loss and hypoxia in human GBM and could promote a prothrombotic environment that precipitates these events. A prothrombotic environment also activates the family of protease activated receptors (PARs) on tumor cells, which are G-protein-coupled and enhance invasive and proangiogenic properties. Vaso-occlusive and prothrombotic mechanisms in GBM could readily explain the presence of pseudopalisading necrosis in tissue sections, the rapid peripheral expansion on neuroimaging, and the dramatic shift to an accelerated rate of clinical progression resulting from hypoxia-induced angiogenesis.

Multiphoton Excitation of Autofluorescence for Microscopy of Glioma Tissue

Intraoperative detection of residual tumor tissue in glioma surgery remains an important challenge because the extent of tumor removal is related to the prognosis of the disease. Multiphoton excited fluorescence tomography of living tissues provides high-resolution structural and photochemical imaging at a subcellular level. In this conceptual study, we have used multiphoton microscopy and fluorescence lifetime imaging (4D microscopy) to image cultured glioma cell lines, solid tumor, and invasive tumor cells in an experimental mouse glioma model and human glioma biopsy specimens. A laser imaging system containing a mode-locked 80 MHz titanium:sapphire laser with a tuning range of 710 to 920 nm, a scan unit, and a time correlated single photon counting board was used to generate autofluorescence intensity images and fluorescence lifetime images of cultured cell lines, experimental intracranial gliomas in mouse brain, and biopsies of human gliomas. Multiphoton microscopy of native tumor bearing brain provided structural images of the normal brain anatomy at a subcellular resolution. Solid tumor, the tumor-brain interface, and single invasive tumor cells could be visualized. Fluorescence lifetime imaging demonstrated significantly different decay of the fluorescent signal in tumor versus normal brain, allowing a clear definition of the tumor-brain interface based on this parameter. Distinct fluorescence lifetimes of endogenous fluorophores were found in different cellular compartments in cultured glioma cells. The analysis of the relationship between the laser excitation wavelength and the lifetime of excitable fluorophores demonstrated distinct profiles for cells of different histotypes. Multiphoton excited fluorescence of endogenous fluorophores allows structural imaging of tumor and central nervous system histo-architecture at a subcellular level. The analysis of the decay of the fluorescent signal within specific excitation volumes by fluorescent lifetime imaging discriminates glioma cells and normal brain, and the excitation/lifetime profiles may further allow differentiation of cellular histotypes. This technology provides a noninvasive optical tissue analysis that may potentially be applied to an intraoperative analysis of resection plains in tumor surgery.

302. Anti-Angiogenic Treatment Enhances Anti- Tumor Effects of an Oncolytic Virus in an Experimental Rat Glioma Model

Oncolytic viruses (OVs) are viruses with selective ability to replicate & lyse in tumors. Recent results from clinical trials have established their safety yet efficacy remains elusive. We are examining stromal responses elicited upon treatment of intracerebral brain tumor by hrR3 (type 1 Herpes simplex virus (HSV-1) derived OV). Effect of OV treatment on host tumor vasculature. 7 days post intracerebral tumor implantation (D74HveC rat glioma cells), rats were treated with intratumoral injection of hrR3 or PBS.

Selective accumulation and strong photodynamic effects of a new photosensitizer, ATX-S10·Na (II), in experimental malignant glioma

We investigated the feasibility of a novel photosensitizer, ATX-S10.Na (II), in photodynamic therapy (PDT) for glioma. First, PDT was performed in various brain tumor cell lines in vitro. Cytotoxicity depended upon both drug concentration and laser energy and the 50% inhibitory concentration ranged from 3.5 to 20 microg/ml. Next, PDT was performed in the subcutaneous and intracranial 9L tumor models in Fischer rats using ATX-S10.Na (II) and light from a 670-nm diode laser delivered by intratumoral insertion of an optical fiber. The effect of PDT on brain tumors was evaluated using magnetic resonance imaging. Sequential changes of the ATX-S10.Na (II) concentrations were also measured quantitatively by fluorospectrometry up to 12 h after intravenous administration in rats with intracranial and subcutaneous tumors. The concentration of ATX-S10.Na (II) in the brain tumor reached a maximum at 2 h after administration and the tumor/normal brain concentration ratio was as high as 131 at 8 h. Intratumoral PDT for intracranial tumors irradiated at this timing showed an obvious anti-tumor effect without severe side effects. The present study demonstrated the highly selective accumulation of ATX-S10.Na (II) in tumor tissue and its potent photodynamic effect in an experimental malignant glioma model.

The Characterization of Tumor Apoptosis after Experimental Radiosurgery

We sought to evaluate whether radiosurgery induces apoptosis in an experimental glioma model and to elucidate the time course of this radiobiologic phenomenon. Fischer 344 rats harboring established intracranial 9L gliosarcomas underwent radiosurgery (n = 42) or no radiosurgery (n = 45). Animals were sacrificed at 3, 6, 12, 24, 48, 72 h, and 1 or 2 weeks after treatment and in situ tumor apoptosis was assessed by specific staining. Tumor apoptosis was noted to be statistically higher in radiosurgery-treated animals relative to controls at the 6-, 24-, and 48-hour time points following radiosurgery. Radiosurgery induces apoptosis in the rat intracranial 9L gliosarcoma in a time-dependent fashion. The time course of this radiobiologic phenomenon begins at approximately 6 h following radiosurgery, continues up to 48 h, and begins to decline by 72 h.

Imaging in neurooncology

Imaging in patients with brain tumors aims toward the determination of the localization, extend, type, and malignancy of the tumor. Imaging is being used for primary diagnosis, planning of treatment including placement of stereotaxic biopsy, resection, radiation, guided application of experimental therapeutics, and delineation of tumor from functionally important neuronal tissue. After treatment, imaging is being used to quantify the treatment response and the extent of residual tumor. At follow-up, imaging helps to determine tumor progression and to differentiate recurrent tumor growth from treatment-induced tissue changes, such as radiation necrosis. A variety of complementary imaging methods are currently being used to obtain all the information necessary to achieve the abovementioned goals. Computed tomography and magnetic resonance imaging (MRI) reveal mostly anatomical information on the tumor, whereas magnetic resonance spectroscopy and positron emission tomography (PET) give important information on the metabolic state and molecular events within the tumor. Functional MRI and functional PET, in combination with electrophysiological methods like transcranial magnetic stimulation, are being used to delineate functionally important neuronal tissue, which has to be preserved from treatment-induced damage, as well as to gather information on tumor-induced brain plasticity. In addition, optical imaging devices have been implemented in the past few years for the development of new therapeutics, especially in experimental glioma models. In summary, imaging in patients with brain tumors plays a central role in the management of the disease and in the development of improved imaging-guided therapies.

Imaging in Neurooncology

Imaging in patients with brain tumors aims toward the determination of the localization, extend, type, and malignancy of the tumor. Imaging is being used for primary diagnosis, planning of treatment including placement of stereotaxic biopsy, resection, radiation, guided application of experimental therapeutics, and delineation of tumor from functionally important neuronal tissue. After treatment, imaging is being used to quantify the treatment response and the extent of residual tumor. At follow-up, imaging helps to determine tumor progression and to differentiate recurrent tumor growth from treatment-induced tissue changes, such as radiation necrosis. A variety of complementary imaging methods are currently being used to obtain all the information necessary to achieve the above mentioned goals. Computed tomography and magnetic resonance imaging (MRI) reveal mostly anatomical information on the tumor, whereas magnetic resonance spectroscopy and positron emission tomography (PET) give important information on the metabolic state and molecular events within the tumor. Functional MRI and functional PET, in combination with electrophysiological methods like transcranial magnetic stimulation, are being used to delineate functionally important neuronal tissue, which has to be preserved from treatment-induced damage, as well as to gather information on tumor-induced brain plasticity. In addition, optical imaging devices have been implemented in the past few years for the development of new therapeutics, especially in experimental glioma models. In summary, imaging in patients with brain tumors plays a central role in the management of the disease and in the development of improved imaging-guided therapies.

Characterization of Gastrin-Induced Proangiogenic Effects In vivo in Orthotopic U373 Experimental Human Glioblastomas and In vitro in Human Umbilical Vein Endothelial Cells

This study aims to investigate the role of gastrin-17 (G17) on angiogenesis features in gliomas both in vitro and in vivo. The influences of G17 and G17 receptor antagonists were characterized in vitro in terms of angiogenesis on human umbilical vein endothelial cell (HUVEC) tubulogenesis processes on Matrigel and in vivo with respect to U373 orthotopic glioma xenografts. The influence of phosphatidylinositol 3'-kinase, protein kinase C, and nuclear factor-kappaB inhibitors was characterized in vitro on G17-mediated HUVEC tubulogenesis. G17-mediated release of interleukin (IL)-8 from HUVECs and G17-induced modifications in nuclear factor-kappaB DNA binding activity were characterized by means of specific enzyme-linked immunosorbent assays. The influence of G17 on E- and P-selectin expression was determined by means of computer-assisted microscopy, whereas the influence of E- and P-selectin on HUVEC migration was approached by means of antisense oligonucleotides. The chemotactic influence of G17 and IL-8 on HUVEC migration was characterized by means of computer-assisted videomicroscopy with Dunn chambers. Messenger RNAs for cholecystokinin (CCK)A, CCKB, and CCKC receptors were present in HUVECs and microvessels dissected from a human glioblastoma. Whereas G17 significantly increased the levels of angiogenesis in vivo in the U373 experimental glioma model and in vitro in the HUVECs, the CCKB receptor antagonist L365,260 significantly counteracted the G17-mediated proangiogenic effects. G17 chemoattracted HUVECs, whereas IL-8 failed to do so. IL-8 receptor alpha (CXCR1) and IL-8 receptor beta (CXCR2) mRNAs were not detected in these endothelial cells. Gastrin significantly (but only transiently) decreased the level of expression of E-selectin, but not P-selectin, whereas IL-8 increased the expression of E-selectin. Specific antisense oligonucleotides against E- and P-selectin significantly decreased HUVEC tubulogenesis processes in vitro on Matrigel. The present study shows that gastrin has marked proangiogenic effects in vivo on experimental gliomas and in vitro on HUVECs. This effect depends in part on the level of E-selectin activation, but not on IL-8 expression/release by HUVECs.

Therapeutic Efficacy of DTI-015 using Diffusion Magnetic Resonance Imaging as an Early Surrogate Marker

To investigate diffusion weighted magnetic resonance imaging as a quantitative surrogate marker for evaluating the therapy-induced cellular changes in an orthotopic experimental glioma model, tumors were treated with direct intratumoral administration of DTI-015, a solution of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in 100% EtOH. Intracerebral 9L tumors were induced in Fischer 344 rats, and three treatment groups were established: DTI-015, EtOH, and sham. Two groups of rats received intratumoral injection of either 67 mg/mL BCNU in EtOH or EtOH alone at 50% of the tumor volume up to a maximum of 30 mul under stereotactic guidance. Diffusion magnetic resonance images were acquired before treatment and after treatment at 1, 24, 48, and 72 hours and then 3 times per week thereafter. Tumor cell viability was examined using multislice diffusion weighted magnetic resonance imaging with diffusion weighted transverse magnetic resonance images and histogram plots of each tumor quantified over time. Control animals (EtOH- or sham-treated animals) showed mean apparent diffusion coefficients (ADCs) that remained essentially unchanged over the experimental time course. In contrast, rats treated with DTI-015 showed a significant increase in ADC relative to the pretreatment within 24 hours, which further increased over time, followed by a significant therapeutic response as evidenced by subsequent tumor volume shrinkage, development of a cystic region, and enhanced animal survival. Finally, not only were ADC measurements predictive of differences between treatment groups, but they also yielded spatial and temporal data regarding the efficacy of treatment within individual treated animals that could be used to guide subsequent therapy.

T11TS/S-LFA3 induces apoptosis of the brain tumor cells: a new approach to characterise the apoptosis associated genetic changes by arbitrarily primed-PCR

Genetic alterations in ethyl nitrosourea (ENU) induced brain tumor model were analysed by simple PCR based technique with arbitrary primers. T11TS/SLFA3 was established previously as a potent immune stimulator with antineoplastic property in experimental glioma model. The goal of this study was to reveal whether T11TS induces apoptosis of the neural neoplastic cell and to decipher the DNA polymorphism level of the cells undergoing apoptosis. The results clearly establish the apoptogenic role of T11TS/S-LFA3 and along with the detection of cancer associated genomic instability, AP-PCR analysis is useful for the detection of DNA level fragmentation, a unique feature of apoptosis.

Evaluation of Anti-tumor property of Specific and Non-specific BRMs in Experimental Glioma by Assessing the Microglial Cell Functional and Phenotypic Modulations

Background: Microglial cells are considered to be the chief immunomodulatory cells of the brain. These cells play a crucial role against various neurodegenerative diseases. When modulated microglia have been shown to exert a potential anti-tumor immune response against brain neoplasms. Although several specific BRMs like IL-2, IFN-g have been shown to modulate the microglia to get an effective anti-tumor immune response, associated toxicities and detrimental side effects have posed severe limitation in there use particularly for therapeutic purposes.Objectives: In the present study, attempts have been made to elicit the modulations of microglia cell function and phenotypic expression following specific (IL-2, IFN-g and a novel nonspecific BRM (corpuscular antigen) in order to determine their anti-tumor property in experimental glioma model. Materials And Methods: Brain was experimentally induced in young Druckray rats of both sexes with N-N-ethyl nitroso urea (ENU). These ENU treated animals were administered with both specific and nonspecific BRMs like IL-2, IFN-a and SRBC either singly or in combination 5 months after ENU administration and after ascertaining its degree of malignancy. Results :Microglial cells separated from different experimental groups were found to be positive for CD11b, MHC II, CD4 and negative for GFAP.FACS analysis demonstrated that different subtypes of microglial cell populations (CD25+, MHC II+ and CD25+MHC II+) in the brain tissue based on phenotypic expression, which were downregulated in tumor bearing animals, and subsequently restored with increased expression, particularly with SRBC administration. The Scanning Electron Microscopic (SEM) study also depicted modulation of cellular morphology of microglial cell predominantly with SRBC administration. Studies conducted ton evaluate immunological functions at the cellular level showed increased antigen presenting caopacity of microglial cell with SRBC administration, which was significantly greater then IL-2, IFN- and combined doses (E2_S). However phagocytic functions of microglial cells were found not to be significantly modulated with BRM treatment.Conclusion: The results suggest that the nonspecific BRM SRBC can exert greater modulatory effect on microglila cell function and phenotypic expression than the other BRMs used, and in doing so culminate in a potent anti tumor immune response in experimental glioma with compatible tolerance profile.

Cationic liposomes conjugation to recombinant adenoviral vectors containing herpes simplex virus thymidine kinase gene followed by ganciclovir treatment reduces viral antigenicity and maintains antitumor activity in mouse experimental glioma models.

Gene therapy using adenoviral (Ad) vector containing herpes simplex virus thymidine kinase (AxCAHSV-tk) followed by the administration of ganciclovir (GCV) has been a promising therapy for cancer including malignant gliomas. However, there remain numerous problems to overcome, such as the high immunogenicity and toxicity of Ad vector. To optimize the therapy, we investigated whether a conjugation of our original cationic liposomes and Ad vectors reduces viral antigenicity and maintains the antitumor activity in mouse experimental (subcutaneous and intracranial) glioma models. Our original liposomes consist of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride, dilauroyl phosphatidylcholine, and dioleoyl phosphatidyl-ethanolamine in a molar ratio of 1:2:2. AxCAHSV-tk and GCV showed a remarkable inhibition of experimental glioma growth. The growth-inhibitory effect decreased in mice previously immunized with another Ad vector (AxCALacZ). In contrast, the conjugation of AxCAHSV-tk and liposomes did not diminish the growth-inhibitory effect. Furthermore, the conjugation reduced antigenicity for Ad vector in vivo. These findings suggest that suicide gene therapy, using a conjugation of AxCAHSV-tk and our liposomes, is a feasible approach for human cancer gene therapy, especially malignant gliomas.

Brain tumor invasion model system using organotypic brain-slice culture as an alternative to in vivo model.

The primary cause of local recurrence and therapeutic failure in the treatment of malignant gliomas is the invasion of tumor cells into the surrounding normal brain. While it is known that malignant gliomas infiltrate diffusely into regions of normal brain, it is frequently very difficult to unequivocally identify the solitary invading glioma cell in histopathological preparations, or in experimental glioma models. We have developed an experimental invasion assay system, which allows us to track the solitary invasive glioma cell, using human brain tissue obtained from routine craniotomies for seizures or trauma. This tissue is cut into 1-mm thick slices and cultured in the upper chamber of Transwell culture dishes on top of a 0.4- micro m pore size polyester membrane, which is fed on medium provided in the lower chamber. Glioma cells are stably transfected with vectors containing a green fluorescent protein (GFP) cDNA. Stable, high-level expression GFP transfectants were selected by direct visualization under fluorescence microscope. In addition, various tumor spheroids are stained with vital dye, DiI, to track the invading cells. GFP-expressing glioma cells or stained spheroids were then implanted on the center of the brain slice, and the degree of brain tumor invasion into the brain tissue was evaluated at different time points by optical sectioning using a confocal microscope. We observed that GFP-expressing glioma cells or stained spheroids could be readily tracked and followed with this model system. Individual tumor cells that exhibited green or red fluorescence could be identified and their migration path through the brain slices unequivocally followed. This experimental invasion system may be of considerable utility in studying the process of brain tumor invasion and in evaluating its invasiveness in individual brain tumor because it not only provides a better representation of extracellular matrix molecules normally encountered by invading glioma cells, but also provides the fluorescent tag applied to the tumor cells.

Antitumor Effect of an Adeno‐associated Virus Vector Containing the Human Interferon‐β Gene on Experimental Intracranial Human Glioma

We constructed an adeno‐associated virus (AAV) vector containing the human interferon‐β(HuIFN‐β) gene (AAV‐IFN‐β) and investigated its antitumor effect against human glioma cells (U251‐SP) inoculated into the brain of nude mice. Prior to this, we examined human glioma cells transduced with AAV‐IFN‐β using video‐enhanced contrast differential interference contrast (VEC‐DIC) microscopy. Infection of AAV‐IFN‐β induced apoptosis and secondary necrosis in human glioma cells. In in vivo experiments, we confirmed production of HuIFN‐β and induction of heat‐shock protein (HSP) in glioma cells transduced with AAV‐IFN‐β. Growth of the experimental gliomas was completely inhibited by six injections of AAV‐IFN‐β, starting 7 days after transplantation of glioma cells. In addition, the survival of mice treated with AAV‐IFN‐β was remarkably prolonged. These results indicate that AAV‐IFN‐β induces apoptosis of glioma cells and has a strong antitumor effect in this experimental glioma model.

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.

Dexamethasone-induced abolition of the inflammatory response in an experimental glioma model: a flow cytometry study.

Commonly used for management of cerebral edema in patients with brain tumors, steroid medications also have immunosuppressive functions. To characterize the effects of steroids on the central nervous system's response to tumors more clearly, flow cytometry was used to quantify the extent of inflammatory cell infiltration in an immunogenic rat glioma model. Freshly prepared 11-day-old intracranial C6 tumors that had been excised from dexamethasone-treated and untreated rats were labeled ex vivo with monoclonal antibodies against CD 11b/c, CD45, and CD8a antigens. The extent of microglia (CD11b/c-highly positive, CD45-slightly positive cell), macrophage (CD11b/c-highly positive, CD45-highly positive cell), lymphocyte (CD11b/c-negative, CD45-highly positive cell), and cytotoxic T-cell (CD8a-positive cell) infiltration into each rat's tumor, tumor periphery, and contralateral tumor-free hemisphere was analyzed using flow cytometry. Microglia and lymphocytes constituted a significant component of infiltrating cells in this model, comprising 23 +/- 3% and 33 +/- 5% of viable cells, respectively. Macrophages, on the other hand, accounted for only 9 +/- 1% of infiltrating cells. Treatment of rats with a 7-day course of low-dose dexamethasone (0.1 mg/kg/day) resulted in a greater than 50% inhibition of microglia (p = 0.03) and lymphocyte (p = 0.001) infiltration into tumors. Increasing the dexamethasone dose to 1 mg/kg/day further abolished lymphocyte infiltration (89% inhibition, p = 0.001) but had no additional inhibitory effect on microglia invasion. Macrophage infiltration of tumors was not inhibited at the dexamethasone doses used in this study (p = 0.42). Flow cytometry is a valuable technique for characterizing tumor-associated inflammatory cells in gliomas. Even at low doses, dexamethasone was found to inhibit significantly the infiltration of brain tumors by lymphocytes and microglia. These findings should be considered when experimental immunotherapeutic strategies are evaluated for clinical application.