H.J.J.A. Bernsen

Spatial relationship between hypoxia and the (perfused) vascular network in a human glioma xenograft: a quantitative multi-parameter analysis.

PURPOSE To quantitatively study the spatial distribution of tumor hypoxia in relation to the perfused vasculature. METHODS AND MATERIALS Using a human glioma xenograft model, nude mice were administered two different hypoxia markers (NITP or pimonidazole) and the perfusion marker Hoechst 33342. Frozen tumor sections were sequentially scanned for perfusion, hypoxia, and vasculature, respectively, to quantitate perfusion, vasculature, and hypoxia parameters in the same section. RESULTS All tumors showed incomplete perfusion. Both NITP and pimonidazole stained the same hypoxic tumor areas. No statistically significant differences between the two markers were observed. The density of the perfused vessels was inversely related to the hypoxic fraction. At critical distances from perfused vessels, hypoxia occurred. These data suggest that predominantly diffusion-limited hypoxia was detected, based on the spatial distribution of nearby vessels. Also, the proportion of hypoxia distributed over arbitrary zones of 50 microm around perfused vessels was calculated. The largest proportion of hypoxia was found at distances beyond 100 microm from perfused vessels. CONCLUSION With the multiple staining and functional microscopic imaging technique described here, the spatial relationship between perfused vessels and hypoxia was quantified in whole tumor cross-sections. The usefulness of this histologically-based method to quantitate morphological and physiological aspects of the tumor microenvironment was evaluated.

Vascularity and perfusion of human gliomas xenografted in the athymic nude mouse

The vascularisation and perfusion of seven subcutaneously xenografted human glioma lines established from surgical specimens has been analysed using an anti-collagen type IV antibody to visualise the vascular walls in combination with a perfusion marker (Hoechst 33342). A computer-based digital image processing system was employed for quantitative analysis of the parameters. The vascular architecture of individual tumours belonging to the same tumour line showed a consistent similarity, while substantial differences occurred between the various tumour lines derived from different patients. Despite the presence of a large inter-tumour variation in vascular area as a proportion of the tumour area, this vascular parameter clearly showed tumour line-specific characteristics. The perfused fraction of the tumour vessels also showed a large inter-tumour variation for all tumour lines ranging from 20% to 85%, but the majority of tumours of all lines had perfusion fractions of more than 55%. Despite large variation, the perfused vascular area as a proportion of the tumour cross-sectional area exhibited clear tumour line-specific tendencies. These observations suggest that consistent differences in vascular parameters are present between glioma xenograft lines, although the tumour lines all originated from histologically similar human high-grade gliomas. These differences may have important consequences for treatment and clinical behaviour of this type of tumour.

Suramin treatment of human glioma xenografts; effects on tumor vasculature and oxygenation status

In this study the effect of suramin on tumor growth, vascularity and oxygenation of a human glioma xenografted in the nude mouse was examined. Vascular parameters and oxygenation status of the xenografts were determined immunohistochemically in frozen sections of the tumors, using the hypoxia marker pimonidazole-hydrochloride to detect hypoxic areas. Tumor vessels in these sections were stained by an endothelial cell marker and perfusion of vessels was visualized by administration of the perfusion marker Hoechst 333342 before harvesting the tumors. The vascular parameters were quantified with an image analysis system. The results show that tumor growth was reduced considerably after suramin treatment. This growth suppression was accompanied by marked changes in vascular architecture. Although the total vascular area and perfused fraction of tumor vessels remained unchanged after suramin treatment, vascular density increased, indicating that more but smaller vessel structures had developed during therapy. These vessel structures were also more homogeneously spread over the tumor area. Control tumors showed extensive areas of hypoxia while in treated tumors hypoxic areas had mostly disappeared. This effect was probably due to the higher density of homogeneously distributed perfused vessel structures in the treated tumors, contributing to an increased oxygenation of the tumor. These observations suggest that suramin therapy can result in marked changes not only in tumor vascularity but also in tumor oxygenation status which may have important consequences for sensitivity of these tumors to other therapies such as radiation treatment.

Antiangiogenic Therapy in Brain Tumor Models

The prognosis of patients with malignant brain tumors remains poor despite new developments in neurosurgery, chemotherapy and radiotherapy. Malignant gliomas are highly vascularized, and there is ample evidence that their growth is angiogenesis-dependent. Therefore, new therapeutic approaches often include the inhibition of angiogenesis. In this review, experimental studies of antiangiogenic agents in brain tumor models are summarized. The results of these experiments as well as potential pitfalls in extrapolation to the clinic are discussed.

The effect of the anti-angiogenic agent TNP-470 on tumor growth and vascularity in low passaged xenografts of human gliomas in nude mice

The effect of the anti-angiogenic agent TNP-470 on tumor growth, vascular area, vascular density and tumor perfusion of two different subcutaneously implanted human glioma xenografts (E98 and E106) in nude mice was evaluated. Vascular parameters were investigated with an image analysis system. For both tumor lines a small but significant tumor growth suppression was observed. However, no differences in vascular parameters between TNP-470 treated tumors and controls could be found after 6 weeks of treatment. It is concluded that although TNP-470 is a promising anti-angiogenic agent in many tumor types, at least 2 glioma lines seem to be partly resistant to its anti-angiogenic effects. Further evaluation of the effects of combination of TNP-470 and cytostatic agents or radiotherapy in human glioma xenografts are required to determine the place of anti-angiogenic therapy in general and treatment with the anti-angiogenic agent TNP-470 more specifically in the treatment of human gliomas.

Delayed Vascular Changes after Antiangiogenic Therapy with Antivascular Endothelial Growth Factor Antibodies in Human Glioma Xenografts in Nude Mice

OBJECTIVE The purpose of this study was to examine the delayed effects of antivascular endothelial growth factor treatment on tumor growth and vascularity in a subcutaneous mouse tumor model of human glioblastoma. METHODS Antivascular endothelial growth factor antibody treatment was administered for a period of 6 weeks, to suppress tumor growth. To detect late vascular effects, tumor vascular parameters for treated tumors and control tumors were analyzed 4 weeks thereafter. By that time, tumors had grown to adequate sizes (diameter, 8-10 mm) for comparison with untreated control tumors. Vascular parameters were quantified by using an image-analysis system. RESULTS Vascular density was significantly lower in antivascular endothelial growth factor antibody-treated tumors, compared with control tumors of similar size. The vascular architecture of treated tumors was also distinctly different, compared with control tumors, showing larger but sparser vessel structures. CONCLUSION These findings suggest that antiangiogenic therapy may have a prolonged effect on the vascular architecture of certain tumors, resulting in enduring changes in the tumor vessels. Because tumor vasculature plays an important role in the sensitivity to various treatment modalities, these changes are likely to influence the responses of these tumors to further therapy.

In vivo 31P magnetic resonance spectroscopy and morphometric analysis of the perfused vascular architecture of human glioma xenografts in nude mice.

The relationship between the bioenergetic status of human glioma xenografts in nude mice and morphometric parameters of the perfused vascular architecture was studied using (31)P magnetic resonance spectroscopy (MRS), fluorescence microscopy and two-dimensional digital image analysis. Two tumour lines with a different vascular architecture were used for this study. Intervascular distances and non-perfused area fractions varied greatly between tumours of the same line and tumours of different lines. The inorganic phosphate-nucleoside triphosphate (P(i)/NTP) ratio increased rapidly as mean intervascular distances increased from 100 microm to 300 microm. Two morphometric parameters - the percentage of intervascular distances larger than 200 microm (ivd200) and the non-perfused area fraction at a distance larger than 100 microm from a nearest perfused vessel (area100), - were deduced from these experiments and related to the P(i)/NTP ratio of the whole tumour. It is assumed that an aerobic to anaerobic transition influences the bioenergetic status, i.e. the P(i)/NTP ratio increased linearly with the percentage of ivd200 and the area100.

Effect of carbogen breathing on the radiation response of a human glioblastoma xenograft : Analysis of hypoxia and vascular parameters of regrowing tumors

Background and Purpose:The aim of these experiments was to study the relationship between the previously demonstrated efficacy of carbogen breathing on tumor oxygenation status and the response to radiation assessed by a growth delay assay. This study was also developed to investigate the microenvironmental changes caused by combined treatment compared to irradiation only in regrowing tumors.Material and Methods:A human glioblastoma xenograft tumor line was implanted in nude mice. Irradiations consisted of 10 Gy or 20 Gy with and without carbogen breathing. Several microenvironmental parameters (tumor cell hypoxia, tumor blood perfusion, vascular volume, and microvascular density) were analyzed after immunohistochemical staining. Tumor growth delay was monitored for up to 120 days after treatment.Results:In general, there was no benefit of combined treatment. However, a small subgroup with good response to combined radiation and carbogen treatment was identified showing little hypoxia and mainly necrosis in the regrowing tumors. These microenvironmental characteristics were not seen in tumors of the other treatment groups.Conclusion:The observations suggest that a subgroup of patients, who could potentially benefit from the combined carbogen and radiation treatment, might be identified. However, the heterogeneous response to treatment illustrates the need for selection of patients before start of treatment.Hintergrund und Ziel:In der Vergangenheit wurde die Effektivität von Carbogenbeatmung auf den Oxygenationsstatus von Tumoren nachgewiesen. Ziel dieser Experimente war es, den Zusammenhang zwischen o. g. Carbogenbeatmung und der Reaktion auf Bestrahlung mit Hilfe der Wachstumsverzögerung zu analysieren. Des Weiteren wurde diese Studie entwickelt, um durch die Kombinationsbehandlung mit Bestrahlung und Carbogen verursachte Veränderungen des Mikromilieus im Vergleich zu alleiniger Strahlenbehandlung zu untersuchen.Material und Methodik:Eine humane Glioblastom-Xenotransplantat-Tumorlinie wurde in Nacktmäuse implantiert. Die Bestrahlungsdosis betrug 10 Gy oder 20 Gy mit und ohne Carbogenbeatmung. Verschiedene Parameter des Mikromilieus (Tumorzellhypoxie, Tumordurchblutung, vaskuläres Volumen und mikrovaskuläre Dichte) wurden nach immunhistochemischer Färbung analysiert. Die Tumorwachstumsverzögerung wurde bis zu 120 Tage nach Behandlung beobachtet.Ergebnisse:Im Allgemeinen hatte die kombinierte Behandlung keinen Vorteil. Eine kleine Untergruppe wies allerdings eine gute Reaktion auf Kombinationsbehandlung mit Bestrahlung und Carbogenbeatmung auf; diese erneut wachsenden Tumoren zeigten nur sehr wenig Hypoxie und waren größtenteils nekrotisch. Diese Eigenschaften im Mikromilieu wurden nicht in den Tumoren gesehen, die schnell gewachsen waren.Schlussfolgerung:Die Beobachtungen suggerieren, dass eine Untergruppe von Patienten, die möglicherweise von der Kombinationsbehandlung profitieren, identifiziert werden könnte. Die unterschiedliche Reaktion auf Behandlung veranschaulicht allerdings die Notwendigkeit einer Patientenauswahl vor Behandlungsbeginn.

Effect of breathing a hyperoxic hypercapnic gas mixture on the oxygenation of meningiomas; preliminary results

For meningiomas in which complete resection is impossible stereotactic radiosurgery and radiotherapy are increasingly important therapeutical options. The radiosensitivity of meningiomas may be improved by increasing tumor oxygen levels. Hyperoxygenating agents, like breathing a hyperoxic hypercapnic gas mixture, have already been applied successfully in the treatment of other tumors. The aim of this study was to explore the effect of breathing a hyperoxic hypercapnic gas mixture on tumor blood oxygenation of meningiomas using magnetic resonance imaging (MRI) methods. Three patients with convexity meningiomas were each measured twice; with and without breathing the hyperoxic hypercapnic gas mixture. Tumor blood oxygenation changes were measured using blood oxygen level dependent MR imaging. Dynamic contrast enhanced MRI was used to assess functional changes of tumor vasculature. A significant increase in tumor blood oxygenation was observed under hypercapnic hyperoxic conditions in all patients, exceeding the increase in normal brain tissue. It was concluded that the oxygenation status of meningiomas can be improved by breathing a hyperoxic hypercapnic gas mixture.

Effect of Carbogen Breathing on the Radiation Response of a Human Glioblastoma Xenograft

Background and Purpose:The aim of these experiments was to study the relationship between the previously demonstrated efficacy of carbogen breathing on tumor oxygenation status and the response to radiation assessed by a growth delay assay. This study was also developed to investigate the microenvironmental changes caused by combined treatment compared to irradiation only in regrowing tumors.Material and Methods:A human glioblastoma xenograft tumor line was implanted in nude mice. Irradiations consisted of 10 Gy or 20 Gy with and without carbogen breathing. Several microenvironmental parameters (tumor cell hypoxia, tumor blood perfusion, vascular volume, and microvascular density) were analyzed after immunohistochemical staining. Tumor growth delay was monitored for up to 120 days after treatment.Results:In general, there was no benefit of combined treatment. However, a small subgroup with good response to combined radiation and carbogen treatment was identified showing little hypoxia and mainly necrosis in the regrowing tumors. These microenvironmental characteristics were not seen in tumors of the other treatment groups.Conclusion:The observations suggest that a subgroup of patients, who could potentially benefit from the combined carbogen and radiation treatment, might be identified. However, the heterogeneous response to treatment illustrates the need for selection of patients before start of treatment.Hintergrund und Ziel:In der Vergangenheit wurde die Effektivität von Carbogenbeatmung auf den Oxygenationsstatus von Tumoren nachgewiesen. Ziel dieser Experimente war es, den Zusammenhang zwischen o. g. Carbogenbeatmung und der Reaktion auf Bestrahlung mit Hilfe der Wachstumsverzögerung zu analysieren. Des Weiteren wurde diese Studie entwickelt, um durch die Kombinationsbehandlung mit Bestrahlung und Carbogen verursachte Veränderungen des Mikromilieus im Vergleich zu alleiniger Strahlenbehandlung zu untersuchen.Material und Methodik:Eine humane Glioblastom-Xenotransplantat-Tumorlinie wurde in Nacktmäuse implantiert. Die Bestrahlungsdosis betrug 10 Gy oder 20 Gy mit und ohne Carbogenbeatmung. Verschiedene Parameter des Mikromilieus (Tumorzellhypoxie, Tumordurchblutung, vaskuläres Volumen und mikrovaskuläre Dichte) wurden nach immunhistochemischer Färbung analysiert. Die Tumorwachstumsverzögerung wurde bis zu 120 Tage nach Behandlung beobachtet.Ergebnisse:Im Allgemeinen hatte die kombinierte Behandlung keinen Vorteil. Eine kleine Untergruppe wies allerdings eine gute Reaktion auf Kombinationsbehandlung mit Bestrahlung und Carbogenbeatmung auf; diese erneut wachsenden Tumoren zeigten nur sehr wenig Hypoxie und waren größtenteils nekrotisch. Diese Eigenschaften im Mikromilieu wurden nicht in den Tumoren gesehen, die schnell gewachsen waren.Schlussfolgerung:Die Beobachtungen suggerieren, dass eine Untergruppe von Patienten, die möglicherweise von der Kombinationsbehandlung profitieren, identifiziert werden könnte. Die unterschiedliche Reaktion auf Behandlung veranschaulicht allerdings die Notwendigkeit einer Patientenauswahl vor Behandlungsbeginn.