Giles Hamilton Vince

Effect of synthetic matrix‐metalloproteinase inhibitors on invasive capacity and proliferation of human malignant gliomas In vitro

Glioma invasion into the surrounding brain tissue is still a major obstacle for any therapeutical approach. As in other solid tumors, matrix‐metalloproteases (MMPs) have been suggested as being involved. The aim of this study was to evaluate whether the use of MMP inhibitors to target the protease‐mediated invasion process could be a feasible approach. Two human cell lines (U251 and GaMG) and surgical specimens of 6 patients with malignant gliomas were grown as monolayers and spheroid cultures respectively. MMP‐ and u‐PA‐mRNA expression was investigated by semi‐quantitative RT‐PCR. Invasion was studied in Matrigel‐coated Boyden chamber transwell assays for monolayers and in confrontation cultures of tumor spheroids with fetal rat brain aggregates in the presence of the synthetic MMP inhibitors batimastat (BB‐94) and marimastat (BB‐2516). Cytotoxicity/cytostatic effects of high concentrations of both compounds were assessed by growth curves, MTT assays and flow cytometry in human glioma cell lines. Batimastat and marimastat revealed a cytostatic effect at high concentrations (above 1 μM) without cytotoxicity. Both MMP inhibitors effectively reduced glioma invasion in Boyden‐chamber assays at low concentrations of 0.3 μM. In confrontation cultures, concentrations of 10 μM and above were necessary to reduce invasion. This effect was observable with inter‐individual heterogeneity in the patients tumor material. MMP inhibitors effectively reduce glioma invasion, although high concentrations were required in 3‐dimensional culture systems. At these concentrations, both compounds revealed a cytostatic, but no cytotoxic effect. Thus, high local concentrations of MMP inhibitors could offer a new therapeutic strategy for the treatment of gliomas. Int. J. Cancer 80:764–772, 1999.

Hypofractionated stereotactic re-irradiation: treatment option in recurrent malignant glioma

BackgroundHypofractionated stereotactic radiotherapy (HFSRT) is one salvage treatment option in previously irradiated patients with recurrent malignant glioma. We analyzed the results of HFSRT and prognostic factors in a single-institution series.MethodsBetween 1997 and 2003, 19 patients with recurrent malignant glioma (14 glioblastoma on most recent histology, 5 anaplastic astrocytoma) were treated with HFSRT. The median interval from post-operative radiotherapy to HFSRT was 19 (range 3–116) months, the median daily single dose 5 (4–10) Gy, the median total dose 30 (20–30) Gy and the median planning target volume 15 (4–70) ml.ResultsThe median overall survival (OS) was 9.3 (1.9-77.6+) months from the time of HFSRT, 15.4 months for grade III and 7.9 months for grade IV tumors (p = 0.029, log-rank test). Two patients were alive at 34.6 and 77.6 months. OS was longer after a total dose of 30 Gy (11.1 months) than after total doses of <30 Gy (7.4 months; p = 0.051). Of five (26%) reoperations, none was performed for presumed or histologically predominant radiation necrosis. Median time to tumor progression after HFSRT on imaging was 4.9 months (1.3 to 37.3) months.ConclusionHFSRT with conservative total doses of no more than 30 Gy is safe and leads to similar OS times as more aggressive treatment schemes. In individual patients, HFSRT in combination with other salvage treatment modalities, was associated with long-term survival.

Prophylactic intravenous magnesium sulfate for treatment of aneurysmal subarachnoid hemorrhage: A randomized, placebo-controlled, clinical study

Objective:To examine whether the maintenance of elevated magnesium serum concentrations by intravenous administration of magnesium sulfate can reduce the occurrence of cerebral ischemic events after aneurysmal subarachnoid hemorrhage. Design:Prospective, randomized, placebo-controlled study. Setting:Neurosurgical intensive care unit of a University hospital. Interventions:One hundred ten patients were randomized to receive intravenous magnesium sulfate or to serve as controls. Magnesium treatment was started with a bolus of 16 mmol, followed by continuous infusion of 8 mmol/hr. Serum concentrations were measured every 8 hrs, and infusion rates were adjusted to maintain target levels of 2.0–2.5 mmol/L. Intravenous administration was continued for 10 days or until signs of vasospasm had resolved. Thereafter, magnesium was administered orally and tapered over 12 days. Measurements and Main Results:Delayed ischemic infarction (primary end point) was assessed by analyzing serial computed tomography scans. Transcranial Doppler sonography and digital subtraction angiography were used to detect vasospasm. Delayed ischemic neurologic deficit was determined by continuous detailed neurologic examinations; clinical outcome after 6 months was assessed using the Glasgow outcome scale. Good outcome was defined as Glasgow outcome scale score 4 and 5. The incidence of delayed ischemic infarction was significantly lower in magnesium-treated patients (22% vs. 51%; p = .002); 34 of 54 magnesium patients and 27 of 53 control patients reached good outcome (p = .209). Delayed ischemic neurologic deficit was nonsignificantly reduced (9 of 54 vs. 15 of 53 patients; p = .149) and transcranial Doppler-detected/angiographic vasospasm was significantly reduced in the magnesium group (36 of 54 vs. 45 of 53 patients; p = .028). Fewer patients with signs of vasospasm had delayed cerebral infarction. Conclusion:These data indicate that high-dose intravenous magnesium can reduce cerebral ischemic events after aneurysmal subarachnoid hemorrhage by attenuating vasospasm and increasing the ischemic tolerance during critical hypoperfusion.

Microglial/macrophage expression of interleukin 10 in human glioblastomas

Interleukin 10 (IL‐10) expression has been found to be correlated with the extent of malignancy in gliomas. In vitro, IL‐10 increases proliferation and migratory capacity in human glioma cell lines. In this study, we localized the site of IL‐10 synthesis in gliomas to cells of microglial origin. Biopsy specimens from 11 patients with malignant glioma were processed on native tissues and at early cell culture passages (0–4). IL‐10 mRNA was analyzed by RT‐PCR and in situ hybridization. Protein was quantitatively assessed by ELISA in cell culture supernatants, and cells expressing IL‐10 were determined by a combination of immunohistochemistry for CD68 (specific for microglia/macrophage lineage) and IL‐10 in situ hybridization. IL‐10 mRNA decreased from passage 0 to 4 in all samples and was undetectable beyond passage 5. Such downregulation of mRNA leads to a steep decrease of IL‐10 protein in culture supernatants (below detection level, 0.05 ng/ml, beyond passage 1). The combination of in situ hybridization for IL‐10 and CD68 immunostaining revealed that only cells of the microglia/macrophage lineage produced IL‐10 mRNA. Our results identify microglia/macrophage cells as the major source of IL‐10 expression in gliomas which decreases markedly during early passages of primary cultures of human gliomas due to a progressive reduction of microglia/macrophages present. Int. J. Cancer 82:12–16, 1999.

Extraneural metastases of primary brain tumors.

Extraneural metastasis (ENM) of primary brain tumors is a rare occurence. Based on a critical analysis of the literature the present review focuses on illustrating special common features of these tumors with regard to immunological, cytokinetical and tumorbiological issues. In this respect much can be learned from the specific conditions following organ transplantation which is extensively discussed.

Expression of matrix metalloproteinases MMP-1, MMP-11 and MMP-19 is correlated with the WHO-grading of human malignant gliomas

Glioblastomas (GBM) are the most prevalent type of malignant primary brain tumor in adults. They may manifest de novo or develop from low-grade astrocytomas (LGA) or anaplastic astrocytomas. They are characterized by an aggressive local growth pattern and a marked degree of invasiveness, resulting in poor prognosis. Tumor progression is facilitated by an increased activity of proteolytic enzymes such as matrix metalloproteinases (MMPs). Elevated levels of several MMPs were found in glioblastomas compared to LGA and normal brain (NB). However, data for some MMPs, like MMP-1, are controversially discussed and other MMPs like MMP-11 and MMP-19 have as yet not been analysed in detail. We examined the expression of MMP-1, MMP-9, MMP-11 and MMP-19 in NB, LGA and GBM by semiquantitative RT-PCR, Western blotting and immunohistochemistry and found an enhanced expression of these MMPs in GBM compared to LGA or NB in signal strength and in the percentage of tumors displaying MMP expression. The transition from LGA to GBM was characterized by a shift of pro-MMP-11 to expression of the active enzyme. Therefore, MMP-1, MMP-11 and MMP-19 might be of importance for the development of high-grade astrocytic tumors and may be promising targets for therapy.

Heterogeneous regional expression patterns of matrix metalloproteinases in human malignant gliomas.

The aim of the study was to assess the differential intra‐ and intertumoral heterogeneity and patterns of matrix metalloproteinase expression in human glioblastomas in vivo. 12 glioblastoma samples were analyzed for MMP expression by semi‐quantitative RT‐PCR. A total of 56 samples (8 adjoining regions of 6 glioblastoma tumors) were immunohistochemically examined for the expression and regional distribution of gelatinase‐A (MMP‐2), gelatinase‐B (MMP‐9), matrilysin (MMP‐7) and stromelysin‐1 (MMP‐3). Gelatinase‐A mRNA was detected in all samples, gelatinase‐B was found in numerous samples. Correspondingly, strong expression levels of both gelatinase protein was seen in immunohistochemistry. Gelatinase‐A was expressed by both tumor cells and endothelium while gelatinase‐B was found to be restricted to endothelial cells. Stromelysin‐1 protein was not detected in any of the samples. Matrilysin was found around tumor cells of three samples from one patient only. The strong immunoreactivity seen for gelatinase‐A around tumor cells and blood vessels suggests a role in both tissue degradation and tumor neoangiogenesis which is in accordance with previously published in vitro data. The marked localization of gelatinase‐B to the endothelium and its presence in non‐infiltrative benign lesions, however, makes a direct proteolytic role of gelatinase‐B on ECM components during glioma invasion appear unlikely. Its close association with vascular structures, however, might indicate a link to neoangiogenesis. The significance of matrilysin which was only seen in tumor cells in three samples remains unclear. Stromelysin‐1, though strongly expressed in cell lines, does not appear to play a role in glioblastoma tumors in vivo.

Glioma cell migration is associated with glioma-induced angiogenesis in vivo.

To simultaneously assess glioma cell invasion and glioma angiogenesis in vivo by non‐invasive and quantitative means, DiI‐labeled C6 glioma spheroids were implanted into the dorsal skinfold chamber preparation of nude mice (n=6). Heat‐inactivated spheroids served as controls to distinguish between active and passive cell spread. Using multi‐flourescent intravital videomicroscopy, glioma cell migration was analyzed on days 1–4, 6, and 10 and spheroid vascularization was analyzed on days 3, 6, and 10 after implantation. Additionaly, C6 glioma spheroids were implanted into the chronic cranial window of nude mice as an orthotopic implantation site (n=4). In the dorsal skinfold chamber, spheroids were vascularized within 10 days and revealed a tumor‐specific microvasculature. In parallel, individual glioma cells detached from the spheroid edge and migrated centrifugally demonstrating an affinity to tumor and host vessels. Glioma cells demonstrated a heterogeneous pattern of their regional migratory actvity (0.2–9.6 μm/h) which correlated well with regional glioma angiogenesis (r=0.733). Using the cranial window, glioma cells spread similarily demonstrating an affinity to the perivascular space of pial/subpial vessels with preference to the arteriolar segments. Intravital fluorescence microscopy represents a versatile technique to assess the complex relationship between glioma‐driven angiogenesis and glioma cell invasion.

GAPDH is not regulated in human glioblastoma under hypoxic conditions.

BackgroundGene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.ResultsWe observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.ConclusionGAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.

MRI in isolated sixth nerve palsies

Abstract In previous studies the origin of the majority of isolated sixth nerve palsies was not clear or was ascribed to vascular disease. Our purpose was determine how frequently a causative lesion was demonstrated on MRI in patients with an acute unilateral sixth nerve palsy. We performed a prospective study of 43 patients using a standardised protocol. In 27 patients (63 %) a lesion was identified on the initial MRI relevant to the sixth nerve palsy; 21 (49 %) were found to have a tumour or tumour-like lesion; the frequency of presumed vasculopathy in this group was 15 %. There were 16 patients (37 %) with an initially normal MRI, of whom 10 (62 %) had a history of vasculopathy, a significantly different proportion from the group of patients with a visible causative lesion. MRI after 3–6 months was normal in all patients with a normal initial MRI. We suggest that MRI should routinely be performed in patients presenting with an acute sixth nerve palsy, even those with evidence of a vasculopathy. If the symptoms regress spontaneously and there is a history of vasculopathy, follow-up MRI is not necessary.