Tailoring vascular phenotype through AAV therapy promotes anti-tumor immunity in glioma.

Glioblastomas are aggressive brain tumors that are largely immunotherapy resistant. This is associated with immunosuppression and a dysfunctional tumor vasculature, which hinder T cell infiltration. LIGHT/TNFSF14 can induce high endothelial venules (HEVs) and tertiary lymphoid structures (TLS), suggesting that its therapeutic expression could promote T cell recruitment. Here, we use a brain endothelial cell-targeted adeno-associated viral (AAV) vector to express LIGHT in the glioma vasculature (AAV-LIGHT). We found that systemic AAV-LIGHT treatment induces tumor-associated HEVs and T cell-rich TLS, prolonging survival in αPD-1-resistant murine glioma. AAV-LIGHT treatment reduces T cell exhaustion and promotes TCF1+CD8+ stem-like T cells, which reside in TLS and intratumoral antigen-presenting niches. Tumor regression upon AAV-LIGHT therapy correlates with tumor-specific cytotoxic/memory T cell responses. Our work reveals that altering vascular phenotype through vessel-targeted expression of LIGHT promotes efficient anti-tumor T cell responses and prolongs survival in glioma. These findings have broader implications for treatment of other immunotherapy-resistant cancers.

The effect of thermal therapy on the blood-brain barrier and blood-tumor barrier.

The blood-brain and blood-tumor barriers represent highly specialized structures responsible for tight regulation of molecular transit into the central nervous system. Under normal circumstances, the relative impermeability of the blood-brain barrier (BBB) protects the brain from circulating toxins and contributes to a brain microenvironment necessary for optimal neuronal function. However, in the context of tumors and other diseases of central nervous system, the BBB and the more recently appreciated blood-tumor barrier (BTB) represent barriers that prevent effective drug delivery. Overcoming both barriers to optimize treatment of central nervous system diseases remains the subject of intense scientific investigation. Although many newer technologies have been developed to overcome these barriers, thermal therapy, which dates back to the 1890 s, has been known to disrupt the BBB since at least the early 1980s. Recently, as a result of several technological advances, laser interstitial thermal therapy (LITT), a method of delivering targeted thermal therapy, has gained widespread use as a surgical technique to ablate brain tumors. In addition, accumulating evidence indicates that laser ablation may also increase local BBB/BTB permeability after treatment. We herein review the structure and function of the BBB and BTB and the impact of thermal injury, including LITT, on barrier function.

The CXCR2/CXCL2 signalling pathway - An alternative therapeutic approach in high-grade glioma.

OBJECTIVE: Besides VEGF, alternative signalling via CXCR2 and its ligands CXCL2/CXCL8 is a crucial part of angiogenesis in glioblastoma. Our aim was to understand the role of CXCR2 for glioma biology and elucidate the therapeutic potential of its specific inhibition. METHODS: GL261 glioma cells were implanted intracranially in syngeneic mice. The 14 or 7 days of local or systemic treatment with CXCR2-antagonist (SB225002) was initiated early on the day of tumour cell implantation or delayed after 14 days of tumour growth. Glioma volume was verified using MRI before and after treatment. Immunofluorescence staining was used to investigate tumour progression, angiogenesis and microglial behaviour. Furthermore, in vitro assays and gene expression analyses of glioma and endothelial cells were performed to validate inhibitor activity. RESULTS: CXCR2-blocking led to significantly reduced glioma volumes of around 50% after early and delayed local treatments. The treated tumours were comparable with controls regarding invasiveness, proliferation and apoptotic cell activity. Furthermore, no differences in CXCR2/CXCL2 expression were observed. However, immunostaining revealed reduction in vessel density and accumulation of microglia/macrophages, whereas interaction of these myeloid cells with tumour vessels was enhanced. In vitro analyses of the CXCR2-antagonist showed its direct impact on proliferation of glioma and endothelial cells if used at higher concentrations. In addition, expression of CXCR2/CXCL2 signalling genes was increased in both cell types by SB225002, but VEGF-relevant genes were unaffected. CONCLUSION: The CXCR2-antagonist inhibited glioma growth during tumour initiation and progression, whereas treatment was well-tolerated by the recipients. Thus, the CXCR2/CXCL2 signalling represents a promising therapeutic target in glioma.

Cannabidiol Increases Proliferation, Migration, Tubulogenesis, and Integrity of Human Brain Endothelial Cells through TRPV2 Activation.

The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.

Targetting αvß3 and α5ß1 integrins with Ecballium elaterium (L.) A. Rich. seed oil.

In the present study, the effect of Ecbalium elaterium seed oil on adhesion, migration and proliferation of human brain cancer cell line (U87) was determined. Treatment of U87 cell line with the seed oil resulted in strong inhibition of their adhesion to fibrinogen (Fg), fibronectin (Fn). It also reduced their migration and proliferation in a dose-dependent manner without being cytotoxic. Concomitantly, by using Matrigel™ assays, the oil significantly inhibited angiogenesis. The anti- tumor effect of the oil is specifically mediated by αvß3 and α5ß1 integrins. The presence of integrin antagonists in seed oil from E. elaterium could be used for the development of anticancer drugs with targeted "multi-modal" therapies combining anti-adhesif, antiproliferative, antimetastasic and anti-angiogenic, approaches.

MRI venous architecture of the thalamus.

PURPOSE: Our purpose is to describe the thalamic veins using a novel approach named venous gliography in cases with primary or secondary gliomas of the thalamus. Venous gliography is defined by authors as a method to visualize veins on MRI Brain T1-weighted post contrast scans containing gliomas which have induced regional venous congestion. METHODS: Routine clinical MR Imaging studies were reviewed to assess the presence of thalamic veins in 29 glioma cases. In addition, confocal reconstruction techniques (Anatom-e and Osirix) were used in cases that had thin sections (1.0-1.5mm) post contrast T1 weighted sequences. Multiplanar MIP and confocal volume rendered images were generated to evaluate the thalamic veins in those cases. RESULTS: Using venous gliography and confocal reconstruction techniques, two patterns in the venous architecture of the thalamus were documented. First, the branching pattern created by the tributaries of the internal cerebral vein, namely the superior thalamic vein and the anterior thalamic vein, which together formed the superior group of thalamic veins. Second, the pattern created by the un-branched vertically oriented veins, namely the inferior thalamic veins and the posterior thalamic veins, which joined the basal vein of Rosenthal and constituted the inferior group of thalamic veins. CONCLUSIONS: Venous gliography combined with the use of confocal reconstruction techniques provided a novel approach to display the thalamic veins that are usually not seen. The understanding of the venous architecture is mandated by the recent research where veins have taken on an important role in the perivenular spread of gliomas.

Compliant neurovascular balloon catheters may not be compatible with liquid embolic materials: intraprocedural rupture of the protecting balloon during tumor embolization using n-butyl cyanoacrylate and lipiodol mixture.

BACKGROUND: Simultaneous use of balloon catheters with embolic materials can cause unwanted rupture of the balloon occlusion catheters, which might cause a serious problem. Therefore, knowledge of the compatibility of occlusion balloon catheters with liquid embolic materials is important in various interventional procedures. OBJECTIVE: To determine the compatibility of occlusion balloon catheters with commonly used embolic materials in vitro. METHODS: We used three types of occlusion balloon catheters (Scepter C, Microvention, Tustin, California, USA; Hyperform, Covidien, Irvine, California, USA; and Ascent, Codman Neurovascular, Raynham, Massachusetts, USA) to test their tolerances to Lipiodol, n-butyl cyanoacrylate (NBCA; Histoacryl; B Braun, Melsungen, Germany), and dimethyl sulfoxide (DMSO) with Onyx. The balloon was inflated just as it is in an endovascular procedure, then put on a Petri dish to observe its morphological change after one drop of liquid embolic material was added using a 1 mL syringe. The presence of rupture and the time to rupture were evaluated by constant video monitoring. Additionally, we observed morphological changes of the balloon catheter surface after contact with embolic materials with a scanning electron microscope. RESULTS: Lipiodol or a 33% NBCA-Lipiodol mixture dropping onto the three types of balloon catheter resulted in ruptures of all three. All three types of balloon catheter were tolerant to NBCA and to DMSO followed by Onyx. CONCLUSIONS: Glue embolization should not be performed with all three kinds of balloon catheter on the market, but DMSO and Onyx are compatible with those balloon catheters.

Ultrasensitive detection of 3D cerebral microvascular network dynamics in vivo.

Despite widespread applications of multiphoton microscopy in microcirculation, its small field of view and inability to instantaneously quantify cerebral blood flow velocity (CBFv) in vascular networks limit its utility in investigating the heterogeneous responses to brain stimulations. Optical Doppler tomography (ODT) provides 3D images of CBFv networks, but it suffers poor sensitivity for measuring capillary flows. Here we report on a new method, contrast-enhanced ODT with Intralipid that significantly improves quantitative CBFv imaging of capillary networks by obviating the errors from long latency between flowing red blood cells (low hematocrit ~20% in capillaries). This enhanced sensitivity allowed us to measure the ultraslow microcirculation surrounding a brain tumor and the abnormal ingrowth of capillary flows in the tumor as well as in ischemia triggered by chronic cocaine in the mouse brain that could not be detected by regular ODT. It also enabled significantly enhanced sensitivity for quantifying the heterogeneous CBFv responses of vascular networks to acute cocaine exposure. Inasmuch as lipid emulsions are widely used for parenteral nutrition the Intralipid contrast method has translational potential for clinical applications.

Effects of all­trans retinoic acid on VEGF and HIF­1α expression in glioma cells under normoxia and hypoxia and its anti­angiogenic effect in an intracerebral glioma model.

All­trans retinoic acid (ATRA) is one of the most potent inducers of differentiation and is capable of inducing differentiation and apoptosis in glioma cells. However, the effect of ATRA on glioma angiogenesis is yet to be elucidated. The present study investigated the effects of ATRA on the expression of vascular endothelial growth factor (VEGF) and hypoxia­inducible factor­1α (HIF­1α) in various glioma cell lines under normoxia and hypoxia. The effect of ATRA on angiogenesis in a rat intracerebral glioma model was also investigated, with the aim of revealing the effect of ATRA on glioma angiogenesis. In the present study, U­87 MG and SHG44 glioma cells were treated with ATRA at various concentrations (0, 5, 10, 20 and 40 µmol/l) under normoxia or hypoxia. Quantitative polymerase chain reaction and western blot analysis were used to investigate VEGF and HIF­1α mRNA and protein expression, respectively. An intracerebral glioma model was generated using intracerebral implantation of C6 glioma cells into rats. Tumor­bearing rats were treated with ATRA at different doses (0, 5 and 10 mg/kg/day) for two weeks, and immunohistochemical assays were performed to detect the cluster of differentiation 34­positive cells in order to evaluate the microvessel density (MVD) in each group. Following ATRA treatment, the expression of VEGF and HIF­1α was found to vary among the different concentration groups. In the glioma cells in the lower concentration groups (5 and 10 µmol/l ATRA), a significant increase in VEGF and HIF­1α expression was observed. Conversely, a significant decrease in VEGF and HIF­1α expression was found in the glioma cells in the high ATRA concentration group (40 µmol/l), compared with that in the cells in the control group. Furthermore, in the rat intracerebral glioma model, ATRA decreased glioma MVD, particularly in the high­dose group (10 mg/kg/day), compared with the control group. These results suggest that ATRA may exhibit a dose­dependent effect on glioma angiogenesis and may inhibit glioma angiogenesis in vivo.

In vivo imaging of vessel diameter, size, and density: a comparative study between MRI and histology.

The aim of this study was to compare magnetic resonance imaging (MRI) and histological estimates of the mean vessel diameter (mVD), the vessel density (Density), and the vessel size index (VSI) obtained in the same tumor-bearing animals. Twenty-seven rats bearing intracranial glioma (C6 or RG2) were imaged by MRI. Changes in transverse relaxations (ΔR 2* and R(2)) were induced by the injection of an iron-based contrast agent and were mapped using a multi gradient-echo spin-echo sequence. Then, brain vascular network was studied ex vivo by histology. Three regions of interest were drawn in apparently normal tissue (neocortex and striatum) and in the tumor. In vivo mVD(MRI), Density(MRI), and VSI(MRI) were measured; ex vivo, mVD(histo), Density(histo), and VSI(histo) were quantified on the same animals. MRI and histology measurements differed by -15 to 26%. A positive correlation was found between MRI and histology for mVD, Density, and VSI counterparts (R(2) = 0.62, 0.50, 0.73, respectively; P < 0.001 in all cases). This study indicates that MRI and histology yields well correlated the estimates of mVD, Density, and VSI. VSI is the closest MRI estimate to histology. As Density and mVD or VSI provide complementary information, it is worth computing them to characterize angiogenesis beyond blood volume fraction.

A phase I study of the combination of sorafenib with temozolomide and radiation therapy for the treatment of primary and recurrent high-grade gliomas.

PURPOSE: Despite recent advances in the management of high-grade and recurrent gliomas, survival remains poor. Antiangiogenic therapy has been shown to be efficacious in the treatment of high-grade gliomas both in preclinical models and in clinical trials. We sought to determine the safety and maximum tolerated dose of sorafenib when combined with both radiation and temozolomide in the primary setting or radiation alone in the recurrent setting. METHODS AND MATERIALS: This was a preclinical study and an open-label phase I dose escalation trial. Multiple glioma cell lines were analyzed for viability after treatment with radiation, temozolomide, or sorafenib or combinations of them. For patients with primary disease, sorafenib was given concurrently with temozolomide (75 mg/m(2)) and 60 Gy radiation, for 30 days after completion of radiation. For patients with recurrent disease, sorafenib was combined with a hypofractionated course of radiation (35 Gy in 10 fractions). RESULTS: Cell viability was significantly reduced with the combination of radiation, temozolomide, and sorafenib or radiation and sorafenib. Eighteen patients (11 in the primary cohort, 7 in the recurrent cohort) were enrolled onto this trial approved by the institutional review board. All patients completed the planned course of radiation therapy. The most common toxicities were hematologic, fatigue, and rash. There were 18 grade 3 or higher toxicities. The median overall survival was 18 months for the entire population. CONCLUSIONS: Sorafenib can be safely combined with radiation and temozolomide in patients with high-grade glioma and with radiation alone in patients with recurrent glioma. The recommended phase II dose of sorafenib is 200 mg twice daily when combined with temozolomide and radiation and 400 mg with radiation alone. To our knowledge, this is the first publication of concurrent sorafenib with radiation monotherapy or combined with radiation and temozolomide.

Vascular-targeted photothermal therapy of an orthotopic murine glioma model.

AIM: To develop nanoshells for vascular-targeted photothermal therapy of glioma. MATERIALS & METHODS: The ability of nanoshells conjugated to VEGF and/or poly(ethylene glycol) (PEG) to thermally ablate VEGF receptor-2-positive endothelial cells upon near-infrared laser irradiation was evaluated in vitro. Subsequent in vivo studies evaluated therapy in mice bearing intracerebral glioma tumors by exposing tumors to near-infrared light after systemically delivering saline, PEG-coated nanoshells, or VEGF-coated nanoshells. The treatment effect was monitored with intravital microscopy and histology. RESULTS: VEGF-coated but not PEG-coated nanoshells bound VEGF receptor-2-positive cells in vitro to enable targeted photothermal ablation. In vivo, VEGF targeting doubled the proportion of nanoshells bound to tumor vessels and vasculature was disrupted following laser exposure. Vessels were not disrupted in mice that received saline. The normal brain was unharmed in all treatment and control mice. CONCLUSION: Nanoshell therapy can induce vascular disruption in glioma.

Valproic acid inhibits angiogenesis in vitro and glioma angiogenesis in vivo in the brain.

Antiangiogenic strategy is promising for malignant glioma. Histone deacetylase inhibitors (HDACIs) are unique anticancer agents that exhibit antiangiogenic effects. The in vitro and in vivo antiangiogenic effects of HDACIs, valproic acid (VPA), were investigated in malignant glioma in the brain. In vitro, VPA preferentially inhibited endothelial cell proliferation compared to glioma cell proliferation at the optimum concentration in a dose-dependent manner. VPA reduced vascular endothelial growth factor (VEGF) secretion of glioma cells in a dose-dependent manner under both normoxic and hypoxic conditions. VPA was also found to inhibit tube formation in the angiogenesis assay. In vivo, treatment with VPA combined with irinotecan reduced the number of vessels expressing factor VIII in the brain tumor model. VPA inhibits glioma angiogenesis by direct (inhibition of endothelial cell proliferation and tube formation) and indirect (decreased secretion of VEGF by glioma cells) mechanisms. These data suggest a potential role for VPA as an adjuvant therapy for patients with malignant glioma.

Preclinical development of novel anti-glioma drugs targeting the endoplasmic reticulum stress response.

The endoplasmic reticulum (ER) stress response represents a cellular "yin-yang" process, where low to moderate activity is cell protective and supports chemoresistance (yang), but where more severe conditions will aggravate these mechanisms to the point where they abandon their protective efforts and instead turn on a cell death program (yin). Because tumor cells frequently experience chronic stress conditions (due to hypoxia, hypoglycemia, acidification, etc.), the protective yang components of their ER stress response are continuously engaged and thus less able to neutralize additional insults taxing the ER stress response. This tumor-specific situation may provide therapeutic opportunities for pharmacologic intervention, where further aggravation of ER stress would lead to the activation of pro-apoptotic yin components and result in tumor cell death. This review will describe the yin-yang principle of ER stress, and will present pharmacologic agents and combination strategies aimed at exploiting the ER stress response for improved therapeutic outcomes, particularly in the setting of difficult to treat tumor types such as glioblastoma.

Angioarchitectural morphometrics of brain tumors: are there any potential histopathological biomarkers?

Normal brain parenchyma has a highly complex microvascular structure that undergoes re-modelling in the presence of a tumor. A detailed understanding of the clinical and pathological aspects of neo-angiogenesis in malignant human brain tumors is essential in order to ensure the successful clinical application of anti-angiogenic therapy. As the quantification of various aspects of tumor vasculature may provide an indication of angiogenic activity, it is reasonable to assume that identifying specific new biomarkers will help investigators to design more appropriate and less toxic anti-angiogenic treatment strategies. Research into the quantitative analysis of microangioarchitectures can be divided into two main branches: morphometrics and biomarker research. Morphometrics adds quantitative elements to the qualitative description of tissues by means of Euclidean and fractal geometrical parameters, whereas biomarker research seeks predictors that can be used in clinical applications. All of the many morphometric parameters used to analyze microvascular networks in brain tumors have their pros and cons, but none has yet been validated as a reliable biomarker. This review describes the helpfulness and limitations of the morphometric parameters that have so far been proposed, and the current "state-of-the-art" in planning future research into biomarkers and morphometry of the microangioarchitecture of brain tumors. It concludes by discussing the need to make a multiparametric analysis of histological specimens that can be integrated with qualitative and quantitative analyses based on neuroradiological and nuclear medicine techniques in order to provide a valid holistic representation of brain tumors. An empirical algorithm for assessing the microangioarchitecture of brain tumors is also provided.

Effect of cervical spinal cord stimulation on cerebral glucose metabolism.

OBJECTIVE: Syndromes resulting from decreased cerebral blood flow and metabolic activity have significant clinical and social repercussion. However, treatment options are limited. Cervical spinal cord stimulation has shown clinical benefit in the management of several ischemic syndromes. The aim of this report was to assess the effect of cervical spinal cord stimulation on cerebral glucose metabolism. MATERIALS AND METHODS: Between April 2000 and December 2005, 16 patients with brain tumors were assessed. Before and during spinal cord stimulation, they had cerebral glucose metabolism evaluated using 18fluoro-2-deoxyglucose positron emission tomography (18FDG-PET) in the healthy cerebral hemisphere contralateral to the lesion area. RESULTS: Following cervical spinal cord stimulation, there was a significant (p<0.001) increase in glucose metabolism in healthy cerebral hemisphere. The measured increase was 37.7%, with an estimated potential maximal contribution of the first 18fluoro-2-deoxyglucose injection to the quantification of the second positron emission tomography study (carry-over effect)

Pilot study of anti-angiogenic vaccine using fixed whole endothelium in patients with progressive malignancy after failure of conventional therapy.

Vaccines targeting tumour angiogenesis were recently shown to inhibit tumour growth in animal models. However, there is still a lack of information about the clinical utility of anti-angiogenic vaccination. Therefore, here, we aimed to test the clinical effects of a vaccine using glutaraldehyde-fixed human umbilical vein endothelial cells (HUVECs). Six patients with recurrent malignant brain tumours and three patients with metastatic colorectal cancer received intradermal injections of 5x10(7) HUVECs/dose (in total 230 vaccinations). ELISA and flow cytometry revealed immunoglobulin response against HUVECs' membrane antigens. ELISPOT and chromium-release cytotoxicity assay revealed a specific cellular immune response against HUVECs, which were lysed in an effectors:targets ratio-dependent manner. Gadolinium-contrasted MRI showed partial or complete tumour responses in three malignant brain tumour patients. Except for a DTH-like skin reaction at the injection site, no adverse effect of vaccination could be observed. Our results suggest that the endothelial vaccine can overcome peripheral tolerance of self-angiogenic antigens in clinical settings, and therefore should be useful for adjuvant immunotherapy of cancer.

The role of spinal cord stimulation in the management of patients with brain tumors.

High grade gliomas (HGG) have decreased blood flow resulting in hypoxia, limited access by chemotherapeutic agents, and reduced radiation-sensitivity. Spinal cord stimulation (SCS) has been used successfully in the treatment of pain and ischemic syndromes. The present article summarizes our investigations into the effects of SCS in patients with HGG. Before their scheduled radio-chemotherapy, 23 patients with HGG were assessed pre- and post-SCS using several evaluation techniques: (1) transcranial Doppler (TCD) for middle cerebral artery blood flow; (2) color Doppler for common carotid artery blood flow; (3) single photon emission computed tomography (SPECT) for tumor blood flow; (4) polarographic probe technique for tumor pO2 measurement; (5) positron emission tomography (PET) for tumor glucose metabolism. Pre-SCS, the tumors were more ischemic and more hypoxic than healthy tissues. Post-SCS, there was significant: (1) increase in blood flow measured by TCD (> or =18%), color Doppler (> or =61%) and SPECT (15%), (2) increase in oxygenation and decrease (> or =45%) in percentage of hypoxic values <10 mmHg and <5 mmHg, and (3) increase (43%) in glucose metabolism. Our studies show that SCS can modify loco-regional blood flow and oxygen supply, as well as glucose-metabolism in HGG. This suggests that SCS could prove useful as an adjuvant treatment to radio-chemotherapy. These data merit further confirmatory studies.

Hyperoxia retards growth and induces apoptosis, changes in vascular density and gene expression in transplanted gliomas in nude rats.

This study describes the biological effects of hyperoxic treatment on BT4C rat glioma xenografts in vivo with special reference to tumor growth, angiogenesis, apoptosis, general morphology and gene expression parameters. One group of tumor bearing animals was exposed to normobaric hyperoxia (1 bar, pO(2) = 1.0) and another group was exposed to hyperbaric hyperoxia (2 bar, pO(2) = 2.0), whereas animals housed under normal atmosphere (1 bar, pO(2) = 0.2) served as controls. All treatments were performed at day 1, 4 and 7 for 90 min. Treatment effects were determined by assessment of tumor growth, vascular morphology (immunostaining for von Willebrand factor), apoptosis by TUNEL staining and cell proliferation by Ki67 staining. Moreover, gene expression profiles were obtained and verified by real time quantitative PCR. Hyperoxic treatment caused a approximately 60% reduction in tumor growth compared to the control group after 9 days (p < 0.01). Light microscopy showed that the tumors exposed to hyperoxia contained large "empty spaces" within the tumor mass. Moreover, hyperoxia induced a significant increase in the fraction of apoptotic cells ( approximately 21%), with no significant change in cell proliferation. After 2 bar treatment, the mean vascular density was reduced in the central parts of the tumors compared to the control and 1 bar group. The vessel diameters were significantly reduced (11-24%) in both parts of the tumor tissue. Evidence of induced cell death and reduced angiogenesis was reflected by gene expression analyses.Increased pO(2)-levels in experimental gliomas, using normobaric and moderate hyperbaric oxygen therapy, caused a significant reduction in tumor growth. This process is characterized by enhanced cell death, reduced vascular density and changes in gene expression corresponding to these effects.