DRR drives brain cancer invasion by regulating cytoskeletal-focal adhesion dynamics.

Malignant glioma invasion is a primary cause of brain cancer treatment failure, yet the molecular mechanisms underlying its regulation remain elusive. We developed a novel functional-screening strategy and identified downregulated in renal cell carcinoma (DRR) as a regulator of invasion. We show that DRR drives invasion in vitro and in vivo. We found that while DRR is not expressed in normal glial cells, it is highly expressed in the invasive component of gliomas. Exploring underlying mechanisms, we show that DRR associates with and organizes the actin and microtubular cytoskeletons and that these associations are essential for focal adhesion (FA) disassembly and cell invasion. These findings identify DRR as a new cytoskeletal crosslinker that regulates FA dynamics and cell movement.

Inhibition of medulloblastoma cell invasion by Slit.

Invasion of brain tumor cells has made primary malignant brain neoplasms among the most recalcitrant to therapeutic strategies. We tested whether the secreted protein Slit2, which guides the projection of axons and developing neurons, could modulate brain tumor cell invasion. Slit2 inhibited the invasion of medulloblastoma cells in a variety of in vitro models. The effect of Slit2 was inhibited by the Robo ectodomain. Time-lapse videomicroscopy indicated that Slit2 reduced medulloblastoma invasion rate without affecting cell direction or proliferation. Both medulloblastoma and glioma tumors express Robo1 and Slit2, but only medulloblastoma invasion is inhibited by recombinant Slit2 protein. Downregulation of activated Cdc42 may contribute to this differential response. Our findings reinforce the concept that neurodevelopmental cues such as Slit2 may provide insights into brain tumor invasion.

Soluble factors involved in glioma invasion.

Recent studies using molecular and cellular techniques of the factors regulating the invasion process have revealed a crucial role for a number of growth factors and cytokines. Their function lies on the one hand in the autocrine stimulation of the tumor cells themselves, resulting in the stimulation of protease expression and an enhancement of migratory potential. On the other hand, the growth factors and cytokines seem to play a major role in the paracrine activation of the tumor surrounding stroma. Through stimulation of the strong angiogenic response that is characteristic for gliomas and also of the expression of proteases in the stromal cells, they contribute critically to the generation of a stromal environment that is permissive or even inductive for tumor cell invasion. Understanding of the mechanisms by which soluble factors modulate glioma cell invasion therefore will help to determine targets for the modification of existing therapies and lead to the development of novel therapeutic strategies in the management of gliomas.

Evolution of the brain tumour spheroid model: transcending current model limitations.

Tumour recurrence and the high mortality and morbidity associated with malignant brain tumours may be attributed to the failure of current therapeutic modalities (surgery, radiation and chemotherapy) to control the invasion of malignant brain tumour cells into healthy brain tissue. Several in vitro and in vivo models have been developed and used to study brain tumour invasion and cell motility. Here, we review some of the traditional in vitro models of brain tumour invasion and the latest adaptations to the widely used spheroid model. Several research groups studying the mechanisms mediating brain tumour invasion have made important contributions to the field by improving in vitro models of tumour migration and invasion. Sharing these advances will hopefully accelerate experimental discovery and the development of novel anti-invasion brain tumour therapies.

Expression of multidrug-resistance P-glycoprotein (MDR1) in human brain tumors.

Multidrug resistance (MDR) is associated with the expression of P-glycoprotein (P-gp), an ATP-dependent transporter which expels anti-cancer drugs from cells. In the present study, MDR1 P-gp was immunodetected by Western blot analysis in 60 human brain tumors, including meningiomas, schwannomas, low-grade gliomas (astrocytomas, pilocytic astrocytomas) and high-grade gliomas (anaplastic astrocytomas, glioblastomas and anaplastic oligodendrogliomas). Most samples from primary tumors expressed P-gp at the same levels as normal brain tissue except for schwannomas, in which levels were reduced by 65%, and meningiomas, in which levels were more than 10-fold higher in 7 of 10 samples. P-gp levels were 70% and 95% lower in brain metastases from melanomas and lung adenocarcinomas, respectively, than in normal brain tissue. These results indicate that the majority of primary brain tumors express MDR1 P-gp and that its high expression levels in meningiomas may be a marker for this type of brain tumor.

Origin of the Drake fenestrated aneurysm clip.

The development of the Drake fenestrated aneurysm clip is a study in the history of ideas. This communication outlines the conception and solution of a surgical problem involved with the clipping of large basilar tip aneurysms. Dr. Charles G. Drake's ability to modify old ideas and experiment with new ones was instrumental to the conceptual idea of a fenestrated clip. Dr. Frank H. Mayfield and Mr. George Kees, Jr. played essential roles in bringing the idea to a reality. The development of the fenestrated clip has added substantially to the armamentarium of the aneurysm surgeon in dealing with large and complex aneurysms.

Prevention of subdural fluid collections following transcortical intraventricular and/or paraventricular procedures by using fibrin adhesive.

OBJECT: Subdural fluid collections following transcortical intraventricular and/or paraventricular neurosurgical procedures for tumors are common and can be difficult to treat. The authors prospectively studied the efficacy of a fibrin adhesive (Tisseel) in closing cortical and ependymal defects following intraventricular and/or paraventricular lesion resection and in preventing the development of subdural fluid collections. METHODS: Twenty-five patients who underwent 29 transcortical approaches for the resection of intraventricular and/or paraventricular lesions were studied. No patient developed a symptomatic subdural fluid collection and no new seizure or progression of a preexisting seizure disorder was encountered during a median follow-up time of 29 months (range 1-57 months). The incidence of preoperative hydrocephalus was 72% and four (22%) of these patients required postoperative shunt placement. CONCLUSIONS: The use of a fibrin adhesive to seal cortical and ependymal defects after transcortical procedures appears to prevent the development of subdural fluid collections.

Effects of radiation on a three-dimensional model of malignant glioma invasion.

An experimental model of malignant glioma growth involving implantation of spheroids into a gel matrix of collagen type I has been developed. This model has been used to characterize changes in glioma cell invasion in response to single dose and fractionated radiation treatment. Suspensions of C6 astrocytoma cells were grown in spinner culture flasks to yield spheroids of varying size (300-1000 microm). Implantation of spheroids into a gel matrix of collagen type I was associated with measurable invasion of the surrounding gel by individual tumor cells. Changes in the distance of invasion in response to single dose and fractionated radiation were measured. Changes in apoptosis and proliferative indices in different regions of the spheroids in response to radiation were also assessed. In unirradiated gels, maximum depth of invasion, 1300-1750 microm, was achieved by 5 days after implantation. A radiation dose-dependent inhibition of invasion was noted and was most profound for larger spheroids. Fractionation of the radiation dose was associated with a partial recovery of invasion. Changes in apoptotic and proliferative indices in response to radiation depended on the region of the spheroid examined. Increases in apoptosis were noted for cells at the surface of the spheroid and invading cells while cells at the centre of the spheroid demonstrated virtually no increase in apoptosis. Likewise, a dose-dependent decrease in proliferative indices following radiation was noted among the invading cells and cells at the surface of the spheroid but not at the centre of the spheroid. We have described a model of malignant glioma invasion which possesses many of the qualities of in vivo malignant gliomas. Within this model, invasion appeared to be inhibited by radiation in a dose- and fractionation-dependent fashion. Measurement of apoptotic and cell proliferation indices favour a direct cytotoxic effect on the invading cells as the most likely mechanism for this phenomenon.

Ultrastructure of the blood-brain barrier in the rabbit.

The morphology of the normal blood-brain barrier in the rabbit by thin section and freeze-fracture electron microscopy is reported. Exogenous tracer horseradish peroxidase was injected to visualize the integrity of the blood-brain barrier in New Zealand White rabbits. Freeze-fracture was used to determine the intramembrane architecture of the tight junctions. Thin sections (60-100 nm) of brain capillaries from animals injected with horseradish peroxidase (HRP) possessed few pinocytotic vesicles in the cytoplasm. Junctional profiles between adjoining plasma membranes were present. Thin sections of capillaries containing electron dense HRP reaction product (HRP-RP) in the lumen revealed focal fusions of apposing plasma membranes that occluded reaction product from entering the junctional clefts. Some cytoplasmic vesicles were filled with HRP-RP; however, basal laminae and brain interstitium were free of HRP-RP in all vessel profiles examined. Freeze-fracture electron microscopy revealed tight junctions as an elaborate network of interconnecting strands of intramembrane particles appearing as ridges on the EF face and corresponding grooves on the PF face on platinum replicas. Results of this study demonstrate the architecture of rabbit brain microvessel endothelial junctions (blood-brain barrier) and provide evidence that the tight junctions prevent HRP extravasation. It is concluded that rabbit brain endothelial tight junctions (zonulae occludentes), as in other mammals, form the anatomical basis of the blood-brain barrier. Consequently, the rabbit brain microvasculature can be a useful model for establishing stereotactic radiosurgical procedures to treat brain astrocytomas (tumours).

Growth failure after treatment of pediatric brain tumors.

OBJECTIVES: Primary brain tumors are the most common solid tumors that occur in childhood. With improved management of these tumors, there are more survivors with long-term sequelae of radiation and chemotherapy including growth failure. The aim of this study was to assess growth prospectively in children with nonpituitary-related primary brain tumors. METHODS: Forty-one children 3.1 to 13.8 years of age diagnosed consecutively between 1989 and 1992 with a primary nonpituitary-related brain tumor were studied. RESULTS: Of 34 prepubertal children, 14 (41%) were diagnosed as having growth hormone (GH) deficiency. All 14 children were treated with cranial irradiation. During the first year from completion of brain tumor therapy, the annual height velocity of those children confirmed subsequently as being GH-deficient was 3.06 +/- 1.19 cm compared with 5.29 +/- 2.21 cm for those who were not GH-deficient. During the second year, the annual height velocity was 3.29 +/- 1.14 cm per year for the GH-deficient group compared with 5.48 +/- 1.24 cm per year for the non-GH-deficient group. All children with GH deficiency received cranial irradiation and chemotherapy. Two of 34 children developed precocious puberty. Primary hypothyroidism was diagnosed in 6 of 41 children (12%). CONCLUSION: We conclude that GH deficiency and primary hypothyroidism are common after cranial irradiation and chemotherapy for nonpituitary-related brain tumors. Linear growth appears to reflect GH status accurately in children with brain tumors. Precise auxologic evaluation is simple and noninvasive and may reflect more accurately GH status than provocative GH testing. These findings reflect the need for prospective growth monitoring of children with nonpituitary-related brain tumors treated with cranial irradiation and chemotherapy. Early diagnosis of GH deficiency facilitates early initiation of GH therapy and optimization of final height.

Leonardo da Vinci: the search for the soul.

The human race has always contemplated the question of the anatomical location of the soul. During the Renaissance the controversy crystallized into those individuals who supported the heart ("cardiocentric soul") and others who supported the brain ("cephalocentric soul") as the abode for this elusive entity. Leonardo da Vinci (1452-1519) joined a long list of other explorers in the "search for the soul." The method he used to resolve this anatomical problem involved the accumulation of information from ancient and contemporary sources, careful notetaking, discussions with acknowledged experts, and his own personal search for the truth. Leonardo used a myriad of innovative methods acquired from his knowledge of painting, sculpture, and architecture to define more clearly the site of the "senso comune"--the soul. In this review the author examines the sources of this ancient question, the knowledge base tapped by Leonardo for his personal search for the soul, and the views of key individuals who followed him.

The importance of cell density in the interpretation of growth factor effects on collagenase IV activity release and extracellular matrix production from C6 astrocytoma cells.

We have examined the influence of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) on the release of collagenase type IV activity and the production of extracellular matrix (ECM) molecules using C6 astrocytoma cells in monolayer culture. Collagenase type IV activity was significantly increased in a dose dependent manner in the low cell density group by treatment with FGF-2 and VEGF but significantly decreased in a dose dependent fashion in the high cell density group. These results were corroborated using Western blot technique with an antibody to gelatinase A. Addition of exogenous laminin and fibronectin to the media decreased collagenase type IV activity in a dose dependent fashion with the minimum concentration of 0.1 microgram/ml. Laminin and fibronectin reached a concentration of 0.1 microgram/ml in only the high cell density group after treatment with the growth factors tested. These findings indicate that C6 astrocytoma cells appear to have two regulatory mechanisms for collagenase type IV activity which are dependent on cell density. In a low cell density, C6 astrocytoma cells respond to the dominant effect of FGF-2 and VEGF by increasing the release of collagenase IV activity. In a high cell density collagenase type IV activity is decreased due to it's down regulation by released ECM molecules in response to FGF-2 and VEGF. These regulatory mechanisms may be crucial to the understanding of the coordination of tumor-associated angiogenesis by malignant glial cells.

Quantitative comparison of the morphology of the microvasculature of primary lung lesions and metastatic brain tumours.

Endothelial cells that make up microvessels display a differential expression of structure and function that allows them to meet the requirements of the tissues they vascularize. The growth of tumours requires neovascularization that occurs by angiogenesis, a process by which new capillaries are formed from existing vessels. It has been proposed that the local environment determines the morphological characteristics of the vessels that vascularize it. The present study is a quantitative investigation of the properties of capillaries that may contribute to their permeability and their general morphology. Tissue samples were taken from primary non-small cell lung carcinomas and from the same tumour type that had metastasized to brain. Normal samples were taken from tissue distant from the site of pathology. Using transmission electron microscopy, profiles of capillaries from the tissue samples were examined and photographed. Image-analysis was performed to measure vesicular and mitochondrial density, vessel size and vessel wall thickness. The results showed that the morphology of the capillaries that vascularize the primary lung tumours differs from the morphology of the capillaries that vascularize the metastases of these tumours in the brain. In addition to differences in the quantitative observations, qualitative differences were observed with respect to the presence of fenestrations in the vessel wall, found only in brain tumour vessels, and the presence of 'open' junctions, seen only in lung tumour vessels. These results suggest that although the environment of the tumour changed the morphology of the vessels so they were no longer normal, it did not have the same effect on the vessels that vascularize it at the two different sites. Therefore, the response of microvessels in the host tissue to factor(s) produced by tumour cells may be multifaceted and dependent upon the properties of local vessels.

Implantation of C6 astrocytoma spheroid into collagen type I gels: invasive, proliferative, and enzymatic characterizations.

A three-dimensional model has been developed in which C6 astrocytoma spheroids of defined sizes are embedded into collagen type I gels. The authors have monitored cell invasive behavior; obtained quantitative data on cell invasion, proliferation, and enzymatic activity; assessed cell-cell interactions by altering the spheroid size used; and studied cell-matrix interactions by modifying the matrix components. Their results show that C6 astrocytoma cells detach from the spheroid surface and invade the gel as single cells by means of a system that appears to be dependent on metalloprotease function. These invasive cells have a low proliferative index. Larger spheroids with central hypoxic microregions possess cells that invade the gel at faster rates; this could be correlated with the release of increased collagen type I degrading activity. Extracellular matrix proteins, such as laminin, fibronectin, and collagen type IV have no significant influence on invasive activity, whereas hyaluronic acid decreases and human central nervous system myelin increases invasion. New strategies directed at the treatment of malignant gliomas must take into account the subpopulation of malignant cells located long distances from the major tumor mass. The spheroid invasion model may provide specific insights into the behavior of these invasive cells.

Therapy-related chromosomal changes and cytogenetic heterogeneity in human gliomas.

The karyotypes of 18 primary 'untreated' gliomas, 6 recurrent gliomas treated with radiotherapy and/or chemotherapy and 2 gliomas before and after treatment are described, based on observations using standard cytogenetic techniques. In comparison to the untreated gliomas there were relatively consistent chromosome differences in the treated gliomas, including (1) deletions of the long arm of chromosome 7 with breakpoint at q22, possibly induced by alkylating agents, and (2) numerous single cell abnormalities or unrelated clones of structural abnormalities, presumably induced by radiotherapy.

Release of collagen type IV degrading activity from C6 astrocytoma cells and cell density.

Type IV collagen is a major protein component of the vascular basement membrane and its degradation is crucial to the initiation of tumor-associated angiogenesis. The authors have investigated the influence of cell density on the release of collagen type IV degrading activity by C6 astrocytoma cells in monolayer culture. The release of collagen type IV degrading activity was assessed biochemically, immunocytochemically, and by Western blot analysis. The results demonstrate that increasing plating density and increasing cell density are associated with decreased collagen type IV degrading activity released per tumor cell. These findings indicate the existence of regulatory mechanisms dependent on cell-cell communication, which modulate release of collagen type IV degrading activity. The extrapolation of these results to the in vivo tumor microenvironment would suggest that individual and/or small groups of invading tumor cells, distant from the main tumor mass, would release substantial collagen type IV degrading activity, which may be crucial to their continued invasion and to angiogenesis.

Pleomorphic xanthoastrocytoma: case report and analysis of the literature concerning the efficacy of resection and the significance of necrosis.

The case of a patient with a pleomorphic xanthoastrocytoma (PXA), a low-grade glioma of adolescence, is presented. A literature review of 79 patients with PXAs is described and confirms a favorable prognosis in 80% of patients. The sex ratio in the reported cases was almost equal, and the median age at time of diagnosis was 14 years. Seventy-nine percent of the patients presented with seizures. Nine of the 15 deaths from PXA are associated with histological evidence of necrosis at initial presentation or in a recurrent tumor, confirming the poor prognosis associated with the presence of necrosis in these neoplasms. Survival curves confirm that the optimal treatment for PXAs without necrosis is primary surgical resection with subsequent operation for recurrent tumor. The roles of surgery or radiotherapy in necrotic PXA are not clear from the literature.

Cytogenetic evidence that a tumor suppressor gene in the long arm of chromosome 1 contributes to glioma growth.

In a patient with a rare subtype of glioma, pleomorphic xanthoastrocytoma, cytogenetic studies revealed that both homologues of chromosome 1 were involved in translocations at the same band 1q42 but with different partner chromosomes. In addition, 5 glioblastomas out of 25 gliomas karyotyped in our laboratory had lost at least one copy of band 1q42 through deletions, unbalanced rearrangements, or chromosome losses. Twenty-one gliomas that had lost at least one copy of chromosome band 1q42 were identified in the literature; all were astrocytic tumors and the majority were glioblastomas. It indicates a covert tumor suppressor gene in the region that is involved in astrocytic gliomas.