Invasion of human glioma biopsy specimens in cultures of rodent brain slices: a quantitative analysis

2002 ◽  
Vol 97 (1) ◽  
pp. 169-176 ◽  
Author(s):  
Sophie de Boüard ◽  
Christo Christov ◽  
Jean-Sébastien Guillamo ◽  
Lina Kassar-Duchossoy ◽  
Stéphane Palfi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Brain Slices ◽  
Lower Grade ◽  
Human Glioma ◽  
Newborn Mice ◽  
Content Type ◽  
Biopsy Specimens ◽  
Rodent Brain ◽  
Fine Print

Object. The reliable assessment of the invasiveness of gliomas in vitro has proved elusive, because most invasion assays inadequately model in vivo invasion in its complexity. Recently, organotypical brain cultures were successfully used in short-term invasion studies on glioma cell lines. In this paper the authors report that the invasiveness of human glioma biopsy specimens directly implanted into rodent brain slices by using the intraslice implantation system (ISIS) can be quantified with precision. The model was first validated by the demonstration that, in long-term studies, established glioma cells survive in the ISIS and follow pathways of invasion similar to those in vivo. Methods. Brain slices (400 µm thick) from newborn mice were maintained on millicell membranes for 15 days. Cells from two human and one rodent glioblastoma multiforme (GBM) cell lines injected into the ISIS were detected by immunohistochemistry or after transfection with green fluorescent protein—containing vectors. Preferential migration along blood vessels was identified using confocal and fluorescent microscopy. Freshly isolated (≤ 24 hours after removal) 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate—prelabeled human glioma biopsy specimens were successfully implanted in 19 (83%) of 23 cases, including 12 GBMs and seven lower grade gliomas (LGGs). Morphometric quantification of distance and density of tumor cell invasion showed that the GBMs were two to four times more invasive than the LGGs. Heterogeneity of invasion was also observed among GBMs and LGGs. Directly implanted glioma fragments were more invasive than spheroids derived from the same biopsy specimen. Conclusions. The ISIS combines a high success rate, technical simplicity, and detailed quantitative measurements and may, therefore, be used to study the invasiveness of biopsy specimens of gliomas of different grades.

Differential effects of vincristine and phenytoin on the proliferation, migration, and invasion of human glioma cell lines

1995 ◽  
Vol 82 (6) ◽  
pp. 1035-1043 ◽  
Author(s):  
Jörg-Christian Tonn ◽  
Hans Kristian Haugland ◽  
Jaakko Saraste ◽  
Klaus Roosen ◽  
Ole Didrik Laerum
Keyword(s):  
Cell Lines ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Migration And Invasion ◽  
Human Glioma ◽  
Human Glioma Cells ◽  
Content Type ◽  
Glioma Cell Lines ◽  
Dose Dependent ◽  
Fine Print

✓ The aim of this study was to investigate the antimigratory and antiinvasive potential of vincristine sulfate (VCR) on human glioma cells and to analyze whether phenytoin (5,5-diphenylhydantoin; DPH) might act synergistically with VCR. Vincristine affects the cytoplasmic microtubules; DPH has been reported to enhance VCR cytotoxicity in murine cells. In two human glioma cell lines, GaMG and D-37MG, we found VCR to reduce monolayer growth and colony formation in a dose-dependent fashion at concentrations of 10 ng/ml and above. Phenytoin increased the cytotoxic and cystostatic effects of VCR in monolayer cells but not in spheroids. Multicellular spheroids were used to investigate directional migration. A coculture system of GaMG and D-37MG spheroids with fetal rat brain aggregates was used to analyze and quantify tumor cell invasion. A dose-dependent inhibition of migration and invasion by VCR was observed in both cell lines without further enhancement by DPH. Immunofluorescence microscopy with antibodies against α-tubulin revealed dose-dependent morphological alterations in the microtubules when the cells were exposed to VCR but not after incubation with DPH. Based on the combination of standardized in vitro model systems currently in use and the present data, the authors strongly suggest that VCR inhibits migration and invasion of human glioma cells. This is not altered by DPH, which inhibits cell proliferation in combination with VCR.

Use of verapamil to enhance the antiproliferative activity of BCNU in human glioma cells: an in vitro and in vivo study

1990 ◽  
Vol 73 (2) ◽  
pp. 248-253 ◽  
Author(s):  
Alfred P. Bowles ◽  
Cooley G. Pantazis ◽  
William Wansley
Keyword(s):  
Tumor Growth ◽  
Cell Lines ◽  
Antiproliferative Activity ◽  
Human Glioma ◽  
Content Type ◽  
Brain Tumor Cells ◽  
Inhibition Of Tumor Growth ◽  
Dose Dependent

✓ The authors have evaluated the antiproliferative activity of verapamil, alone or in combination with 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in brain-tumor cells. These effects were studied in vitro using four human glioma cell lines and in vivo using glioblastoma multiforme cells transplanted to athymic nude mice. The results showed that verapamil when used alone produced inhibition of tumor growth; however, when verapamil was used in combination with BCNU (in vitro), significant dose-dependent suppression of proliferation occurred in all four cell lines. The in vivo results were far more dramatic. Mice treated with BCNU (25 mg/kg) plus verapamil (50 mg/kg) achieved a 200-fold decrease in tumor growth with a greater than 80% regression in tumor size. Complete cures were achieved in 80% of the mice observed for at least 50 days following the completion of therapy. These findings support the use of verapamil in overcoming drug resistance in malignant brain tumors.

Evidence against leukotrienes as mediators of brain edema

1991 ◽  
Vol 74 (5) ◽  
pp. 773-780 ◽  
Author(s):  
Andreas Unterberg ◽  
Walter Schmidt ◽  
Michael Wahl ◽  
Earl F. Ellis ◽  
Anthony Marmarou ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Brain Edema ◽  
Parietal Cortex ◽  
Brain Slices ◽  
Control Group ◽  
Tissue Water ◽  
Content Type ◽  
Cranial Window ◽  
Fine Print

✓ Leukotrienes are powerful metabolites of arachidonic acid which are known to increase the permeability of peripheral blood vessels. These substances are found in brain tissue in association with cerebral ischemia, and in brain tumors. Therefore, it has been proposed that leukotrienes have a mediator function in brain edema. This hypothesis was subjected to further experimental analysis in this study, in which the authors investigated whether: 1) superfusion of the exposed brain surface with leukotrienes increases the permeability of extraparenchymal blood vessels in vivo; 2) intraparenchymal infusion of leukotrienes induces brain edema; and 3) pharmacological inhibition of leukotriene formation by BW755C, an inhibitor of leukotriene synthesis, reduces formation of brain edema from a standardized traumatic insult. The pial vessels of the parietal cortex of cats were examined by fluorescence microscopy during cerebral superfusion with the leukotrienes C4 (LTC4), D4 (LTD4), or E4 (LTE4) by using an open cranial window preparation. Intravenous Na+-fluorescein served as an in vivo blood-brain barrier (BBB) indicator. Superfusion of the pia with leukotrienes (up to 2 µM) did not open the barrier to fluorescein, but was associated with a significant constriction (up to 25%) of arterial and venous vessels. In experiments with slow infusion of leukotriene B4 (LTB4) or LTC4 into the white matter of feline brain, the tissue water content was subsequently determined in serial brain slices using the specific gravity method. Tissue water profiles obtained after a 15- µM infusion of either LTB4 or LTC4 were virtually identical with those of control animals infused with mock cerebrospinal fluid. Thus, neither LTB4 nor LTC4 led to an augmentation of infusion-induced brain edema. In a final series, a cold lesion of the left parietal cortex was induced in rabbits. Twenty-four hours later, swelling of the exposed hemisphere was quantified by gravimetrical comparison of its weight with that of the contralateral nontraumatized hemisphere. Eight animals received BW755C intravenously prior to and after trauma to inhibit formation of leukotrienes. Seven rabbits were infused with an equivalent volume of saline as a control study. The resulting hemispheric swelling was 7.7% ± 0.6% (mean ± standard error of the mean) 24 hours later in animals receiving BW755C and 7.8% ± 1.2% in the control group, indicating that inhibition of leukotrienes was ineffective in preventing formation of vasogenic brain edema. The findings demonstrate that leukotrienes administered to the brain in concentrations occurring under pathological conditions do not open the BBB nor do they induce brain edema. Moreover, formation of brain edema from a standard insult was not therapeutically influenced by inhibition of leukotriene synthesis. Thus, the current findings taken together do not support a role of leukotrienes as mediators in brain edema.

Characterization of neuroectodermal antigen by a monoclonal antibody and its application in CSF diagnosis of human glioma

1988 ◽  
Vol 68 (3) ◽  
pp. 449-455 ◽  
Author(s):  
Toshihiko Wakabayashi ◽  
Jun Yoshida ◽  
Hisao Seo ◽  
Kyoto Kazo ◽  
Yoshiharu Murata ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Brain Tumors ◽  
Cell Lines ◽  
Glioma Cell ◽  
Glioma Cell Line ◽  
Human Glioma ◽  
Content Type ◽  
Sds Page ◽  
Fine Print

✓ Monoclonal antibodies were produced by immunization of the human glioma cell line SK-MG-4. One of the antibodies, designated G-22, reacted with 18 of 20 glioma cell lines, two melanoma cell lines, and three lung cancer cell lines, but not with 39 cell lines derived from sarcoma, carcinoma, or hematopoietic tumors. The antigen was expressed in the brain of human fetuses in early gestation (9 weeks) but not in late gestation (8 months) or in normal adult brain, suggesting that the antibody recognizes neural differentiation antigens expressed by neuroectodermal origin. A high incidence of positive antigens has been observed in gliomas but not in the other neural tumors, such as ependymomas, meningiomas, and neuroblastomas. Thus, the antigen defined by the G-22 monoclonal antibody could be defined as glioma-associated antigen. Pulse-labeling with tritiated leucine and subsequent immunoprecipitation of the solubilized cell membrane revealed that the antigen recognized by this antibody had a molecular weight of 67 kD on sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). It was shown by dot-blot enzyme-linked immunospecific assay (ELISA) that the antigen could be detected in the cerebrospinal fluid (CSF) from patients with gliomas. From analysis of affinity chromatography and SDS-PAGE, the antigen present in the CSF had a molecular weight similar to that of a 1% Nonidet P-40 (NP-40) extract from a glioma cell line. When the antigen in CSF was quantitatively assayed by ELISA, the mean antigen level (expressed as optical density at 450 nm) in the CSF of seven patients was 0.8 ± 0.28 (mean ± standard deviation), which was significantly higher than the 0.38 ± 0.14 level observed in the CSF of 15 patients with nonglioma brain tumors and the 0.23 ± 0.09 level in the CSF of four patients without brain tumors. These results indicate that the monoclonal antibody G-22 is useful for the diagnosis of glioma.

Decreased expression of heparanase in glioblastoma multiforme

2005 ◽  
Vol 102 (3) ◽  
pp. 513-521 ◽  
Author(s):  
Yushi Ueno ◽  
Masaaki Yamamoto ◽  
Israel Vlodavsky ◽  
Iris Pecker ◽  
Kohichi Ohshima ◽  
...  
Keyword(s):  
Glioblastoma Multiforme ◽  
Brain Tumors ◽  
Brain Tissue ◽  
Cell Lines ◽  
Immunohistochemical Analysis ◽  
Metastatic Brain Tumors ◽  
Content Type ◽  
Fine Print

Object. The authors investigated the presence of endoglycosidase heparanase in human glioblastoma multiforme (GBM) and metastatic brain tumors as well as in healthy brain tissue to explore the relationship between the biological characteristics of GBM and the role of heparanase. Methods. Heparanase messenger (m)RNA was almost undetectable in GBMs in vivo, whereas it was frequently seen in metastatic brain tumors according to results of reverse transcription—polymerase chain reaction (RT-PCR). Immunohistochemical analysis of paraffin-embedded tissue sections showed that neoplastic cells in metastatic brain tumors, especially in cells that invaded blood vessels, exhibit intense heparanase immunoreactivity. Heparanase was present in two highly invasive glioma cell lines, U87MG and U251MG, in vitro. These cell lines did not have metastatic capability, which was tested in an experimental pulmonary metastases model in mice. The activity of heparanase in these cell lines was almost the same as that in the highly metastatic melanoma cell line B16-F1. After nude mice were inoculated with U87MG cells, however, heparanase was no longer detected in subcutaneous or intracerebral experimental glioma in vivo based on results of immunohistochemical analysis. According to results of real-time quantitative PCR, there was a 10-fold increase in heparanase mRNA in U87MG glioma cells in vitro compared with that in experimental U87MG glioma tissue in vivo in nude mice. Conclusions. These results indicate that the expression of heparanase was downregulated in GBM in vivo, which rarely metastasizes to distant organs outside the central nervous system. Heparanase is not implicated in the invasiveness of GBM to surrounding healthy brain tissue in vivo.

Expression of P-glycoprotein in human glioma cell lines and surgical glioma specimens

1991 ◽  
Vol 74 (3) ◽  
pp. 460-466 ◽  
Author(s):  
Tsuyoshi Matsumoto ◽  
Eiichi Tani ◽  
Keizo Kaba ◽  
Hideki Shindo ◽  
Katsuya Miyaji
Keyword(s):  
Multidrug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Western Blotting ◽  
Glioma Cell ◽  
Human Glioma ◽  
Content Type ◽  
P Glycoprotein ◽  
Glioma Cell Lines ◽  
Fine Print

✓ The expression of P-glycoprotein, a product of multidrug resistance gene 1, was studied by Western blotting and immunohistochemistry in five human glioma cell lines. One glioma cell line was resistant to vincristine, Adriamycin (doxorubicin), and etoposide, and the other four glioma cell lines were sensitive to each drug. The multidrug-resistant cell line showed a high expression of P-glycoprotein in Western blot analysis and a positive immunostaining for P-glycoprotein mainly along the cell membrane, whereas all multidrug-sensitive glioma cell lines demonstrated no expression of P-glycoprotein in Western blotting and no immunostaining for P-glycoprotein, thus showing a good correlation between the expression level of P-glycoprotein and the extent of multidrug resistance. In 18 human surgical glioma specimens, there was no evidence of complete absence of immunostaining for P-glycoprotein. With a definition of more than 20% of P-glycoprotein-positive cells as positive, from 10% to 20% as intermediate, and less than 10% as negative, immunostaining for P-glycoprotein was positive in one specimen and intermediate in six of 15 specimens taken from virgin gliomas, and positive in two specimens and intermediate in one of three recurrent gliomas treated previously with irradiation, ACNU (1-(4-amino-2-methyl-pyrimidine-5-yl)-methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride), cisplatin, vincristine, and/or procarbazine.

Overexpression of bax in human glioma cell lines

1999 ◽  
Vol 91 (3) ◽  
pp. 483-489 ◽  
Author(s):  
Michael A. Vogelbaum ◽  
Jianxin X. Tong ◽  
Rajashri Perugu ◽  
David H. Gutmann ◽  
Keith M. Rich
Keyword(s):  
Cell Lines ◽  
Glioma Cell ◽  
Human Glioma ◽  
Wild Type ◽  
Content Type ◽  
Human Glioma Cell ◽  
Glioma Cell Lines ◽  
Constitutive Overexpression ◽  
Increased Sensitivity ◽  
Fine Print

Object. Cells that lose their ability to undergo apoptosis may promote the development of neoplasms and result in resistance to clinical treatment with DNA-damaging modalities such as radio- and chemotherapy. Four established human glioma cell lines that are resistant to apoptosis were transfected with the proapoptotic gene bax and assessed for their sensitivity to a proapoptotic stimulus.Methods. Two cell lines had a wild-type p53 genotype (U87 and D247MG) and two had mutant p53 genotypes (U138 and U373). Constitutive overexpression of murine bax was achieved in U138 and U373 only, which resulted in an increased sensitivity of these lines to the apoptosis-inducing effect of cytosine arabinoside (ara-C). Multiple attempts to produce constitutive overexpression of bax in U87 and D247MG cells resulted in spontaneous, near-complete cell loss. Vector-only control transfections were successful in all four cell lines. Inducible overexpression of bax was achieved in the U87 cells and elevated levels of BAX were observed as early as 6 hours after gene induction. This overexpression of BAX resulted in the spontaneous induction of apoptosis in these cells.Conclusions. Overexpression of BAX in four human glioma cell lines resulted in increased sensitivity to apoptosis. In the two lines that had a wild-type p53 genotype, overexpression of BAX produced spontaneous apoptosis. In contrast, the lines that had mutant, nonfunctional P53 did not undergo spontaneous apoptosis, but they were rendered more sensitive to the apoptosis-inducing effect of ara-C. Modulation of BAX expression may be a useful therapeutic modality for gliomas, regardless of p53 genotype.

Cell cycle arrest and astrocytic differentiation resulting from PTEN expression in glioma cells

1999 ◽  
Vol 91 (5) ◽  
pp. 822-830 ◽  
Author(s):  
Jun-ichi Adachi ◽  
Katsumi Ohbayashi ◽  
Tomonari Suzuki ◽  
Tomio Sasaki
Keyword(s):  
Expression System ◽  
Biological Significance ◽  
Glioma Cells ◽  
Genetic Alterations ◽  
Pten Expression ◽  
Human Glioma ◽  
Wild Type ◽  
Content Type ◽  
Astrocytic Differentiation ◽  
Fine Print

Object. Genetic alterations of the PTEN gene (also known as MMAC1 or TEP1) have frequently been identified in high-grade gliomas, indicating that inactivation of PTEN plays a crucial role in human glioma progression. The aim of this study was to assess the biological significance of PTEN inactivation in the development of glioma.Methods. The authors introduced wild-type PTEN complementary DNA into four human glioma cell lines (T98G, U-251MG, U-87MG, and A172) containing endogenous aberrant PTEN alleles. The number of colonies transfected with the wild-type PTEN was reduced to 15 to 32% of those found after transfection of a control vector, suggesting growth suppression by the exogenous PTEN. To analyze phenotypic alterations produced by PTEN expression, T98G-derived clones with inducible PTEN expression were further established using a tetracycline-regulated inducible gene expression system. Induction of PTEN expression suppressed the in vitro growth of T98G cells with accumulation of G1 phase cells. Furthermore, when cells were cultured in the presence of the extracellular matrix (ECM), PTEN expression caused distinct morphological changes, with multiple and elongated cytoplasmic processes similar to those of normal astrocytes. The level of glial fibrillary acidic protein, an intermediate protein specifically expressed in differentiated astrocytes, was upregulated concomitantly.Conclusions. These findings strongly indicate that exogenous PTEN expression inhibits the proliferation of glioma cells by inducing G1 arrest and elicits astrocytic differentiation in the presence of the ECM. Inactivation of PTEN would play an important role in the enhancement of unregulated growth of undifferentiated glioma cells.

Administration of interleukin-12 and -18 enhancing the antitumor immunity of genetically modified dendritic cells that had been pulsed with Semliki Forest virus—mediated tumor complementary DNA

2002 ◽  
Vol 97 (5) ◽  
pp. 1184-1190 ◽  
Author(s):  
Ryuya Yamanaka ◽  
Naoki Yajima ◽  
Naoto Tsuchiya ◽  
Junpei Honma ◽  
Ryuichi Tanaka ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Antitumor Immunity ◽  
Semliki Forest Virus ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
Antitumor Effects ◽  
Content Type ◽  
Immunogene Therapy ◽  
Fine Print

Object. Immunogene therapy for malignant gliomas was further investigated in this study to improve its therapeutic efficacy. Methods. Dendritic cells (DCs) were isolated from bone marrow and pulsed with phosphate-buffered saline or Semliki Forest virus (SFV)—mediated 203 glioma complementary (c)DNA with or without systemic administration of interleukin (IL)-12 and IL-18 to treat mice bearing the 203 glioma. To study the immune mechanisms involved in tumor regression, the authors investigated tumor growth of an implanted 203 glioma model in T cell subset—depleted mice and in interferon (IFN) γ—neutralized mice. To examine the protective immunity produced by tumor inoculation, a repeated challenge of 203 glioma cells was given by injecting the cells into the left thighs of surviving mice and the growth of these cells was monitored. The authors demonstrated that the combined administration of SFV-cDNA, IL-12, and IL-18 produced significant antitumor effects against the growth of murine glioma cells in vivo and also can induce specific antitumor immunity. The synergic effects of the combination of SFV-cDNA, IL-12, and IL-18 in vivo were also observed to coincide with markedly augmented IFNγ production. The antitumor effects of this combined therapy are mediated by CD4+ and CD8+ T cells and by NK cells. These results indicate that the use of IL-18 and IL-12 in DC-based immunotherapy for malignant glioma is beneficial. Conclusions. Immunogene therapy combined with DC therapy, IL-12, and IL-18 may be an excellent candidate in the development of a new treatment protocol. The self-replicating SFV system may therefore provide a novel approach for the treatment of malignant gliomas.

Antitumor effects of antiprogesterones on human meningioma cells in vitro and in vivo

1994 ◽  
Vol 80 (3) ◽  
pp. 527-534 ◽  
Author(s):  
Yasuhiro Matsuda ◽  
Keiichi Kawamoto ◽  
Katsuzo Kiya ◽  
Kaoru Kurisu ◽  
Kazuhiko Sugiyama ◽  
...  
Keyword(s):  
Marked Reduction ◽  
Tumor Volume ◽  
Collagen Gel ◽  
Antitumor Effects ◽  
Histological Changes ◽  
Content Type ◽  
The Relationship ◽  
Fine Print

✓ The presence of the progesterone receptor (PR) in meningioma tissue has been confirmed by previous investigations. Studies have shown that the antiprogesterone drug, mifepristone, is a potent agent that inhibits the growth of cultured meningioma cells and reduces the size of meningiomas in experimental animal models and humans. However, these studies have not fully examined the relationship between the antitumor effects of an antiprogesterone agent and the expression of the PR. The present study examined the antitumor effects of mifepristone and a new potent antiprogesterone agent, onapristone; a correlation between the antitumor effects of these antiprogesterones and the presence of PR's in meningiomas in vitro and in vivo was also investigated. Meningioma tissue surgically removed from 13 patients was used in this study. In the in vitro arm of the study, mifepristone and onapristone exhibited cytostatic and cytocidal effects against cultured meningioma cells, regardless of the presence or absence of PR's; however, three PR-negative meningiomas showed no response to any dose of mifepristone and/or onapristone. In the in vivo arm, meningioma cells, embedded in a collagen gel, were implanted into the renal capsules of nude mice. Antiprogesterone treatment resulted in a marked reduction of the tumor volume regardless of the presence or absence of PR's. No histological changes in the meningioma cells suggestive of necrosis or apoptosis were detected in any of the mice treated with antiprogesterones. These findings suggest that mifepristone and onapristone have an antitumor effect against meningioma cells via the PR's and/or another receptor, such as the glucocorticoid receptor.

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