Inhibition of glioma angiogenesis and growth in vivo by systemic treatment with a monoclonal antibody against vascular endothelial growth factor receptor-2.

Using an orthotopic intracerebral model, we investigated whether systemic treatment with DC101, a monoclonal antibody against vascular endothelial growth factor receptor (VEGFR)-2, could inhibit angiogenesis and the growth of human glioblastoma cells in severe combined immunodeficient mice. Intraperitoneal treatment with DC101, control IgG, or PBS was initiated either on day 0 or, in another series, on day 6 after tumor cell implantation, and animals were killed approximately 2 weeks after tumor cell injection. Tumor volumes in animals treated with DC101 were reduced by 59 and 81% compared with IgG and PBS controls, respectively (P < 0.001), when treatment was initiated immediately, and similar results were obtained when treatment started on day 6. Microvessel density in tumors of DC101-treated animals was reduced by at least 40% compared with animals treated with control IgG or PBS (P < 0.01). We observed a reduction in tumor cell proliferation and an increase in apoptosis in DC101-treated animals (P < 0.001). However, in mice treated with DC101, we also noticed a striking increase in the number and total area of small satellite tumors clustered around, but distinct from, the primary. These satellites usually contained central vessel cores, and tumor cells often had migrated over long distances along the host vasculature to eventually reach the surface and spread leptomeningeally. We conclude that systemic antagonization of VEGFR-2 can inhibit glioblastoma neovascularization and growth but can lead to increased cooption of preexistent cerebral blood vessels. Therefore, a combination of different treatment modalities which also include anti-invasive therapy may be needed for an effective therapy against glioblastoma, and the use of an antibody against VEGFR-2 may be one effective component.

Regulation of BRCA1 and BRCA2 expression in human breast cancer cells by DNA-damaging agents.

Germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 have been linked to the development of breast cancer, ovarian cancer, and other malignancies. Recent studies suggest that the BRCA1 and BRCA2 gene products may function in the sensing and/or repair of DNA damage. To investigate this possibility, we determined the effects of various DNA-damaging agents and other cytotoxic agents on the mRNA levels of BRCA1 and BRCA2 in the MCF-7 and other human breast cancer cell lines. We found that several agents, including adriamycin (a DNA intercalator and inhibitor of topoisomerase II), camptothecin (a topoisomerase I inhibitor), and ultraviolet radiation induced significant decreases in BRCA1 and BRCA2 mRNA levels. Decreased levels of BRCA1 and BRCA2 mRNAs were observed within 6-12 h after treatment with adriamycin and persisted for at least 72 h. Adriamycin also induced decreases in BRCA1 protein levels; but these decreases required several days. U.V. radiation induced dose-dependent down-regulation of BRCA1 and BRCA2 mRNAs, with significant decreases in both mRNAs at doses as low as 2.5 J/m2, a dose that yielded very little cytotoxicity. Adriamycin-induced down-regulation of BRCA1 and BRCA2 mRNAs was first observed at doses that yielded relatively little cytotoxicity and little or no apoptotic DNA fragmentation. Adriamycin and U.V. radiation induced distinct dose- and time-dependent alterations in the cell cycle distribution; but these alterations did not correlate well with corresponding changes in BRCA1 and BRCA2 mRNA levels. However, the adriamycin-induced reduction in BRCA1 and BRCA2 mRNA levels was correlated with p53 functional status. MCF-7 cells transfected with a dominant negative mutant p53 (143 val-->ala) required at least tenfold higher doses of adriamycin to down-regulate BRCA1 and BRCA2 mRNAs than did parental MCF-7 cells or control-transfected MCF-7 clones. These results suggest that BRCA1 and BRCA2 may play roles in the cellular response to DNA-damaging agents and that there may be a p53-sensitive component to the regulation of BRCA1 and BRCA2 mRNA expression.

Allelic losses in neurofibromatosis 2-associated meningiomas.

More than 50% of patients with neurofibromatosis 2 (NF2) develop meningiomas. Recently, a higher proliferative activity, more mitotic figures, and greater nuclear pleomorphism have been described for NF2-associated meningiomas compared with sporadic ones. To analyze whether such histological differences could reflect underlying genetic differences, we examined 30 meningiomas from 22 NF2 patients for allelic losses on those chromosome arms that are frequently affected by deletions in sporadic meningiomas. In addition, we assessed the proliferative activity of the tumors and studied NF2 germline mutations. Twenty-three meningiomas corresponded to WHO grade I (10 fibrous, 6 psammomatous, 4 transitional, 3 meningothelial) and 7 to WHO grade II. The average MIB-1 index was 1.60 +/- 0.85 (WHO grade I: 1.41 +/- 0.80, WHO grade II: 2.13 +/- 0.82). When compared with several published studies of sporadic meningiomas, the MIB-1 index in NF2-associated meningiomas was not higher. Loss of heterozygosity (LOH) flanking or within the NF2 locus at 22q12 was detected in 100% of the tumors. LOH on 1p was the second most frequent abnormality (40%), followed by losses on 10q (27%), 6q and 14q (24%), 18q (23%), and 9p (17%). LOH on 19q and 17p, which is not commonly seen in sporadic meningiomas, was also only rarely detected in NF2-associated meningiomas. NF2 gene mutations were detected in 8 of 15 patients analyzed and were located in exons 2, 5, 6, 7, and 8. We conclude that sporadic and NF2-associated meningiomas share a common spectrum and frequency of allelic deletions as well as, in contrast to previous observations, a similar proliferative activity.

Expression and localization of scatter factor/hepatocyte growth factor in human astrocytomas.

Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotropic cytokine that has been implicated in glioma invasion and angiogenesis. The SF/HGF receptor, MET, has been found to be expressed in neoplastic astrocytes as well as in endothelial cells of the tumor vasculature. Both SF/HGF and MET expression have also been described to correlate with the malignancy grade of human gliomas. However, most glioblastoma cell lines lack SF/HGF expression, raising the question of the cellular origin of SF/HGF in vivo. Using in situ hybridization, we analyzed glioblastomas, anaplastic astrocytomas, diffuse astrocytomas, pilocytic astrocytomas, and normal brain for the expression of SF/HGF mRNA. We detected strong SF/HGF expression by the majority of the tumor cells and by vascular endothelial cells in all glioblastoma specimens analyzed. Combined use of in situ hybridization with fluorescence immunohistochemistry confirmed the astrocytic origin of the SF/HGF-expressiong cells. In contrast, CD68-immunoreactive microglia/macrophages, as well as vascular smooth muscle cells reactive to alpha-smooth muscle actin, lacked SF/HGF expression. In anaplastic, diffuse, and pilocytic astrocytomas, SF/HGF expression was confined to a subset of tumor cells, and signals were less intense than in glioblastomas. In addition, we detected SF/HGF mRNA in cortical neurons. SF/HGF expression was not up regulated around necroses or at tumor margins. MET immunoreactivity was observed in GFAP-expressing astrocytic tumor cells and endothelial cells as well as in a subset of microglia/macrophages. We conclude that in vivo, both autocrine and paracrine stimulation of tumor cells and endothelium through the SF/HGF-MET system are likely to contribute to tumor invasion and angiogenesis. Lack of SF/HGF expression by most cultured glioblastoma cells is not representative of the in vivo situation and most likely represents a culture artifact.

Isolated angiitis of the central nervous system: lack of inflammation after long-term treatment.

A case of isolated angiitis of the CNS was observed for 5 years. Initial response to cyclophosphamide was followed by relapse on therapy interruption. After renewed treatment, clinical stabilization was achieved despite progressive stenoses shown by angiography. The patient died of cyclophosphamide-induced myelodysplastic syndrome. Autopsy revealed lack of inflammation, vascular scarring, and amyloid angiopathy. We conclude that cure from isolated angiitis of the CNS is possible and that the risk of overtreatment should be minimized.

Allelic losses at 1p, 9q, 10q, 14q, and 22q in the progression of aggressive meningiomas and undifferentiated meningeal sarcomas.

Meningiomas are usually benign tumors; however, they can recur after surgical resection and occasionally show histological progression to a higher malignancy grade. Five such rare cases of aggressively recurring meningiomas were present in our departmental cohort of 923 primary meningeal neoplasms operated over a 17-year period. Four other aggressively recurring meningeal tumors with a very similar clinical and histomorphological appearance (three undifferentiated meningeal sarcomas, one hemangiopericytoma) was also included in this study. We investigated whether disease progression can be traced by genetic alterations and whether a pattern of genetic alterations is specific for meningiomas. A total of 40 specimens from primary tumors and multiple recurrences of the nine patients were analyzed with 26 polymorphic allelic markers for deletions on 1p, 1q, 9q, 10q, 14q, and 22q. Loss of heterozygosity (LOH) at 22q was observed in all meningiomas cases at the earliest time point analyzed. Allelic loss at 1p was seen in the original tumor in two cases and upon meningioma recurrence in two others. Deletion on 10q occurred during tumor progression in two cases, and on 9q and 14q in one case. While allelic loss at 22q appears to be an early event in aggressive meningioma disease, there is a clear correlation of further deletions on chromosome arms 1p, 9q, 10q, and 14q with histopathological and clinical progression, as shown in these intraindividual trackings. None of these genetic findings were present in the non-meningiomatous meningeal tumors, indicating that meningothelial cells have their own lineage-specific genetic pathways towards clinical malignancy.

Scatter factor/hepatocyte growth factor (SF/HGF) content and function in human gliomas.

Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotrophic cytokine that stimulates motility and invasion of several cancer cell types and induces angiogenesis. Its receptor MET is a transmembrane tyrosine kinase encoded by the C-MET proto-oncogene. To assess the potential relevance of SF/HGF in gliomas we performed functional studies in vivo and in vitro, expression analyses and correlative studies. We showed that both SF/HGF and MET are expressed in gliomas in vivo and are upregulated during transition from low grade to malignant glioma. When SF/HGF cDNA was transfected into glioma cells that expressed the MET receptor the cells formed considerably larger and more vascularized intracranial tumors in vivo than SF/HGF negative control clones. In other glioma cells, which constitutively expressed both SF/HGF and MET, we abolished SF/HGF expression by antisense ribozyme-targeting, which led to a significant decrease in tumorigenicity and tumor growth. In vitro SF/HGF strongly stimulated glioma cell motility and to a lesser degree proliferation. SF/HGF also strongly increased endothelial cell motility in vitro and extracts of tumors derived from SF/HGF-transfected glioma cells were more mitogenic for endothelial cells and more angiogenic in the rat cornea angiogenesis assay than extracts from control tumors. In a three-dimensional in vitro angiogenesis assay basic fibroblast growth factor (bFGF) was found to synergize with either SF/HGF or vascular endothelial growth factor (VEGF) in inducing endothelial capillary-like tubes, whereas neither SF/HGF nor VEGF alone or in combination were effective. Interestingly, while both VEGF and SF/HGF levels appeared to be increased in malignant gliomas compared with low grade ones, this was not the case for bFGF of which biologically relevant levels were already present in low grade gliomas. It thus seems that bFGF alone is insufficient to induce angiogenesis in gliomas but may act synergistically with either VEGF and/or SF/HGF when these become upregulated during malignant progression. In conclusion, we showed that SF/HGF may contribute to glioma progression by stimulating tumor invasiveness, proliferation and neovascularization.

Multiple vascular abnormalities and a paradoxical combination of vitamin B12 deficiency and thrombocytosis in a case with POEMS syndrome.

POEMS/Crow-Fukase syndrome is a rare multisystem disorder associated with elevated vascular endothelial growth factor (VEGF), which clinically presents with polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes. We report a case of POEMS syndrome due to a gammopathy of undetermined significance with thrombocytosis, vitamin B(12) deficiency, highly elevated VEGF and in addition to glomeruloid angiomas two previously undescribed proliferative vascular lesions: a spinal arteriovenous fistula and a plexogenic pulmonary arteriopathy, which ultimately resulted in lethal pulmonary hypertension. We assume that the high VEGF levels caused the vascular abnormalities observed in our patient.

Vascular endothelial growth factor, hepatocyte growth factor/scatter factor, basic fibroblast growth factor, and placenta growth factor in human meningiomas and their relation to angiogenesis and malignancy.

OBJECTIVE: Angiogenesis is mediated by a number of different growth factors and appears vital for tumor growth. The understanding of angiogenic mechanisms could offer new therapeutic perspectives; in this context, the role of four potentially angiogenic growth factors was analyzed in a large series of meningiomas of different grades. METHODS: Vascular endothelial growth factor (VEGF), placenta growth factor, hepatocyte growth factor/scatter factor, and basic fibroblast growth factor were quantified in 69 tumors by enzyme-linked immunosorbent assay. Microvessel density and proliferative activity were determined on paraffin sections, and clinical tumor invasiveness was rated. Induction of endothelial chemotaxis and capillary-like tube formation were studied in vitro using modified Boyden chamber assays and three-dimensional collagen gel assays, respectively. RESULTS: Tumors included 40 benign (World Health Organization [WHO] Grade I), 21 atypical (WHO Grade II), and 8 anaplastic/malignant (WHO Grade III) meningiomas. We found a correlation between meningioma grade and VEGF content (r = 0.37, P = 0.002), which was 2-fold higher in atypical than in benign meningiomas (P = 0.022) and 10-fold higher in malignant than in benign meningiomas (P = 0.025). Among different subtypes of Grade I meningiomas, VEGF levels were 10-fold higher in meningothelial than in fibrous meningiomas (P = 0.015). None of the other three factors investigated showed any association with tumor grade, microvessel density, or invasiveness, and VEGF also did not correlate with vascularity or invasiveness. Moreover, vascularity did not increase with malignancy grade. Endothelial chemotaxis and capillary-like tube formation in vitro were induced by meningioma extracts and were most effectively blocked by co-addition of antibodies against basic fibroblast growth factor, followed by anti-VEGF, whereas anti-hepatocyte growth factor/scatter factor was not effective. The chemotactic activity of meningioma extracts on endothelial cells correlated with their VEGF content (r = 0.6, P = 0.003). CONCLUSION: Meningiomas do not show an angiogenic switch involving VEGF and/or hepatocyte growth factor/scatter factor, as has previously been found in gliomas. Nevertheless, the biological activity of VEGF and basic fibroblast growth factor in meningiomas suggests that both are potential targets for antiangiogenic therapy in meningiomas of all WHO grades.

Intracavitary chemotherapy for glioblastoma: present status and future directions.

Considerable efforts have been invested to improve local control of the glioma disease although its infiltrative nature leading to whole brain involvement is a fundamental characteristic and antagonistic to this endeavour. The typically local recurrence of glioblastoma in about 80% of the cases has prompted intracavitary treatments of which presently only a biodegradable wafer containing carmustine has shown statistically significant benefit regarding survival in three phase III trials. Based on that proof of principle, many new developments are attempting to improve on this concept, introducing different agents with otherwise high systemic toxicity and poor penetration. New pharmacological formulations offer longer sustained release, better adaptation to the geometry of the resection cavity, and allow repeated administration. Should local recurrence become effectively controlled, significant progress can be made to increase survival with very limited local and virtually no systemic side effects. Since all agents so far show only limited activity against solid tumor, complete resection seems to be the prerequisite for effective local therapies.

Invasion as limitation to anti-angiogenic glioma therapy.

The inhibition of tumor angiogenesis could be an efficient therapeutic strategy for the treatment of malignant gliomas. Prominent neovascularization is induced by these tumors, and microvascular proliferation is a malignancy grading criterion. However, glioma cells can also invade the brain diffusely over long distances without necessarily requiring angiogenesis. Experimentally, it was shown that especially during early stages of growth in rodent brain, glioma cells can coopt the preexistent host vasculature to recruit their blood supply in the absence of neovascularization. This phenomenon was only observed in orthotopic models in which the tumor cells were implanted into the brain which is a densely vascularized environment, but not in subcutaneous models in which tumor cells are implanted into a virtual space. Using an orthotopic mouse model, we analyzed whether systemic anti-angiogenic therapy with an antibody against the vascular endothelial growth factor receptor-2 (VEGFR-2) could inhibit intracerebral growth of xenografted human glioblastoma cells and what effect this treatment had on tumor morphology and invasiveness. We found that anti-angiogenic therapy inhibited tumor growth by 80% compared to buffer-treated controls. The intratumoral microvessel density was reduced by at least 40% in treated animals compared to controls. However, in mice treated with the anti-VEGFR-2 antibody, we noticed a striking increase in the number and total area of small satellite tumors clustered around the primary mass. These satellites usually contained central vessel cores, and tumor cells often had migrated along blood vessels over long distances to eventually reach the surface and spread in the subarachnoid space. Systemic anti-angiogenic therapy can thus apparently increase the invasiveness of gliomas in the orthotopic model. Tumor cell invasion was tightly associated with preexistent blood vessels, suggesting that increased cooption of the host vasculature could represent a compensatory mechanism that is selected for by inhibiting adequate tumor vascularization.

Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria.

Tumor cells with stem cell-like properties can be cultured from human glioblastomas by using conditions that select for the expansion of neural stem cells. We generated cell lines from glioblastoma specimens with the goal to obtain model systems for glioma stem cell biology. Unsupervised analysis of the expression profiles of nine cell lines established under neural stem cell conditions yielded two distinct clusters. Four cell lines were characterized by the expression of neurodevelopmental genes. They showed a multipotent differentiation profile along neuronal, astroglial and oligodendroglial lineages, grew spherically in vitro, expressed CD133 and formed highly invasive tumors in vivo. The other five cell lines shared expression signatures enriched for extracellular matrix-related genes, had a more restricted differentiation capacity, contained no or fewer CD133+ cells, grew semiadherent or adherent in vitro and displayed reduced tumorigenicity and invasion in vivo. Our findings show that stable, multipotent glioblastoma cell lines with a full stem-like phenotype express neurodevelopmental genes as a distinctive feature, which may offer therapeutic targeting opportunities. The generation of another distinct cluster of cell lines showing similarly homogeneous profiling but restricted stem cell properties suggests that different phenotypes exist, each of which may lead to the typical appearance of glioblastoma.

[Social support of radiotherapy patients in emotional stress and crisis situations]. / Ansprechpartner von Strahlentherapiepatienten bei seelischen Belastungen und in Krisensituationen.

PURPOSE: This study assesses the psychosocial strains imposed upon radio-oncological patients throughout their treatment. We investigated patients' resources of social support and their relationship towards doctors, nursing staff and technical staff. PATIENTS AND METHODS: One hundred and fifty radio-oncological follow-up patients were asked retrospectively to complete a standardised written questionnaire. This was preceded by 30 in-depth interviews led with radio-oncological in-patients who currently received treatment. RESULTS: Radiotherapy patients reported a spectrum of major stresses, which showed a considerable individual variability. Many patients had experienced emotional crises during hospitalization for their radiation treatment. Most frequently the main social support was provided by relatives. Still, medical staff also played an important supportive role in alleviating emotional distress. Almost half of all patients appeared to have a generally positive attitude towards psychological counseling. CONCLUSION: This study is part of a psychosocial intervention project established in a radiotherapy department. Support is provided for patients directly, e.g. through crisis intervention, supportive therapy, creative therapy, etc. In addition, indirect intervention is aimed at enhancing psychosocial competences of the clinical staff, through lectures, case conferences and seminars.

Somatostatin and somatostatin receptors in the diagnosis and treatment of gliomas.

Somatostatin analogues are in clinical use for the diagnosis and treatment of several oncological indications, namely pituitary adenomas and endocrine gastrointestinal tumors. In addition for a variety of malignancies their potential value is being studied. It has been speculated that somatostatin plays a role in the homeostasis of gliomas, and that gliomas could be susceptible to antiproliferative effects of somatostatin analogues. These assumptions were tested in 20 human cell lines derived from malignant gliomas and 4 glioblastoma tissue specimens, which were analyzed for their expression of the five known somatostatin receptor genes (SSTR1-5) and for the receptor function. Using semiquantitative PCR techniques, SSTR2 transcripts were found in all 20 cell lines and 4 glioblastomas, SSTR1 transcripts were detected in 9 cell lines and 4 glioblastomas, and SSTR3 transcripts were noted in 7 cell lines and 1 glioblastoma. SSTR4 and SSTR5 transcripts were only rarely detected. Gene expression profiles in glioblastoma tissue specimens resembled those of the cell lines in quality as well as quantity, with average transcript levels being highest for the SSTR2, followed by SSTR1 and SSTR3. However, when compared to GH3 anterior pituitary tumor cells, the relative amounts of PCR amplified DNA fragments were found to be at least 120 fold lower in glioblastoma cell lines and tumor specimens. Binding studies indicated that glioblastoma derived cells contained only minute amounts of SSTRs. No inhibition of proliferation was observed when 10 selected cell lines were incubated with somatostatin-14 (SST-14) or octreotide (SMS 201-995) at concentrations ranging from 10(-9) M to 10(-6) M, however, the proliferation of two cell lines was weakly stimulated after 6 days of incubation with 10(-6) M octreotide. The activity of adenylate cyclase, stimulated by forskolin, was inhibited by maximally 25% at 10(-6) M SST-14 or octreotide in one of 5 selected glioblastoma cell lines. Somatostatin peptides do not seem to exert anti-proliferative effects on glioblastoma cells and therefore appear to be of no obvious value for glioblastoma therapy. Most likely the amount of cell surface SSTRs is not sufficient to mediate antiproliferative effects. Since it has been described that SSTRs are detectable on most differentiated gliomas as well as astrocytes, it may be speculated that SSTRs may be relevant only in the context of well differentiated cellular programs but lose their significance with progressive dedifferentiation.

Diffuse arachnoidal enhancement of a well differentiated choroid plexus papilloma.

INTRODUCTION: The case of a 38-year old man with a histologically benign choroid plexus papilloma arising within the fourth ventricle with en plaque growth around the brain stem and medulla is described in detail. Up to this point this particular growth pattern has not been published and is a rare presentation for this tumour. CLINICAL PRESENTATION: The patient presented with a 1.5 year history of headache, nausea, and vomiting in the morning. Additional symptoms like blurred vision and gait ataxia lead to hospital admission. MRI demonstrated a homogeneously contrast-enhancing tumour completely filling the fourth ventricle and subsequent obstructive hydrocephalus. In addition Gd enhancement encasing the brain stem, the lower aspect of the medulla and the conus medullaris was seen suggesting a disseminated ependymoma or medulloblastoma. INTERVENTION: An extensive resection of the tumour in the fourth ventricle and CP angle was performed. Infiltrative growth into the structures of the left CP angle and into the rhomboid fossa hampered complete removal. Surprisingly histological examination revealed a well-differentiated papillary choroid plexus papilloma without signs of anaplasia. On follow up imaging the Gd enhancement encasing the pons vanished completely. A growing cyst adjacent to a small tumour residuum left behind on the floor of the fourth ventricle led to re-operation after 8 months with complete removal. DISCUSSION: This case presents several biological, neuroradiological and surgical aspects which make it noteworthy and we hope that the informations provided add to the understanding of these tumours, expand the differential diagnostic thinking of lesions which present with diffuse arachnoid Gd enhancement upon first presentation.

[How do patients experience radiotherapy?]. / Wie erleben Patienten die Strahlentherapie?

PURPOSE: In the course of a radiotherapy treatment oncological patients--in addition to some physical symptoms--often experience severe psychological distress. This study assesses and analyses the psychological strains emerging throughout a radio-oncological treatment. Specific intervention measures are being suggested to alleviate patient's psychosocial distress. This study is part of a psychosocial intervention project performed in a radio-oncological department. PATIENTS AND METHODS: Using a semi-standardised interviewing technique 30 radio-oncological in-patients were questioned in depth about their psychosocial situation. Subsequently a fully standardised written questionnaire was designed, enabling a further 150 patients to be investigated retrospectively, when presenting themselves for routine radio-oncological follow-up examinations. RESULTS: In particular, the irradiation session itself, the waiting time beforehand, and the relationship to fellow patients were found to be potentially stressful. More than half of all patients reported anxiety during their first irradiation, which usually decreased during subsequent sessions, and in no case increased. One third of the 30 inpatients wished to have more contact and conversations with fellow-patients, e.g. while waiting for their treatment. The 150 retrospectively assessed patients stated a substantially lower wish for further contact. Almost one third of the in-patients apparently gained comfort from a downward comparison to patients who they perceived as being worse off than themselves. CONCLUSION: Psychosocial intervention can help radiotherapy patients to cope with fears and distress, and thus enhance their quality of life. Support should be provided by psychological specialists, as well as by clinical staff in various ways. Anxiety concerning radiotherapy can often be alleviated through a sensitive and empathetic manner while explaining the radiotherapy process. Patient groups and seminars offer patients the opportunity to exchange experiences and to gain further information about their disease and treatment. In situations of extreme distress psychologists can accompany patients, and if necessary perform crisis intervention. Psychological specialists can also provide further education and support for the clinical staff.

Improved method for the intracerebral engraftment of tumour cells and intratumoural treatment using a guide screw system in mice.

BACKGROUND: Orthotopic in vivo models for the experimental treatment of malignant gliomas have become indispensable to evaluate the efficacy of novel therapeutic regimens. Recently, a guide screw system was introduced which is implanted into the cranium of small rodents where it facilitates fast, repeated and exactly reproducible intracerebral injections, avoiding time consuming stereotactic procedures. Here we report our experience with this system, and describe several modifications which we introduced to improve its reliability and to simplify its application. FINDINGS: The most important modification made was the optimization of the guide screw implantation site, which needs to be adapted to the age of the mice. Other improvements were the fixation of the guide screw to the cranium of the mice using a two component adhesive. This avoids the tendency, when daily intratumoural injections are performed over several weeks, for the screw to loosen, making precise injections impossible. The injection procedure, initially described as a free-hand approach, was improved by mounting the syringe to a Greenberg retractor. This provided additional stability and speeded up the process. Applying this modified technique, we were able to achieve reproducible results with regard to engraftment rate, tumour growth, and intratumoural treatment in immunocompromized mice. INTERPRETATION: We introduced several major and some minor improvements to make working with the guide screw system more reliable, faster and more comfortable. Daily injections over a period of three weeks using this system are feasible and well tolerated by mice.

Isolation and culture of human neuromicrovascular endothelial cells for the study of angiogenesis in vitro.

Neovascularization in the adult central nervous system occurs as a response to several pathophysiological conditions such as ischemia, wound repair, or neoplasia. Endothelial cells from different blood vessel types, different organs, and different species are heterogeneous; therefore, the appropriate cell type should be used to study specific aspects of vascular pathology. We have developed a method to isolate human cerebral microvascular endothelial cells (CMECs) from small, freshly obtained specimens of normal brain adherent to human arteriovenous malformations (AVMs). The isolation procedure involves enzymatic digestions and gradient centrifugations, yielding over 95% pure primary cultures. Alternative isolation methods using magnetic beads, panning, or cloning were not superior with regard to cell purity or yield. CMECs were identified by their immunoreactivity for vWF, CD34, EN4, binding of Ulex europeus lectin, and uptake of DiI-Ac-LDL. They displayed ultrastructural features characteristic of blood-brain barrier endothelial cells and expressed GLUT-1. CMECs were subcultured; however, prolonged culture led to reduced culture purity. Vascular endothelial growth factor, basic fibroblast growth factor and hepatocyte growth factor/scatter factor stimulated the directional motility of CMECs, with dose-response profiles similar to human umbilical vein endothelial cells (HUVECs). In contrast, to stimulate proliferation, lower concentrations of growth factors tended to be necessary for CMECs than for the large vessel endothelial cells. CMECs formed capillary tube-like structures in an in vitro angiogenesis assay using matrigel. This study expands the spectrum of available tissue sources for the isolation of human neuromicrovascular endothelial cells, which are essential for the in vitro study of blood-brain barrier function and cerebral angiogenesis.