Inhibition of tumor-induced migration of bovine capillary endothelial cells by mouse and rabbit tumor necrosis factor.

The effect of tumor necrosis factor (TNF) on the tumor-induced endothelial migration was evaluated with the use of a phagokinetic track assay. TNF from both ddy mice and Japanese albino rabbits (sp act, 3-5 X 10(5) U/mg, respectively) was found to inhibit the migration of bovine capillary endothelial (BCE) cells stimulated by factors from tumor cells, such as the medium conditioned with mouse sarcoma 180 cells or human meningioma extract. These TNF preparations, however, did not affect the spontaneous migration of the BCE cells. When mouse TNF was further fractionated by polyacrylamide gel electrophoresis, only TNF-positive fractions showed an inhibitory activity on the tumor-induced endothelial motility. Moreover, monoclonal antibody against rabbit TNF completely neutralized its migration-inhibitory activity. These findings indicate that the observed inhibitory effect of TNF preparations on the endothelial motility evoked by tumor is exclusively ascribed to the function of TNF. This activity presumably is involved in the suppression of tumor angiogenesis in vivo.

Characterization of the epidermal growth factor receptor in human meningioma.

We have characterized the epidermal growth factor (EGF) receptor in human meningioma (biopsy) microsomes, cellularly derived microsomes, and intact meningioma cells in culture. Scatchard analysis of competition studies reveals both high and low affinity EGF binding sites in the meningiomas tested [dissociation constant (Kd) = 0.9 nM, maximum number of binding sites (Bmax) = 280 fmol/mg protein; Kd = 5.0 nM, Bmax = 660 fmol/mg protein, respectively]. The binding of 125I-EGF is specific since it is abolished by excess unlabeled EGF but not by excess unlabeled platelet-derived growth factor or insulin. Meningioma cultures preincubated with platelet-derived growth factor (10 ng/ml) at 37 degrees C shifted the 125I-EGF competition curve to the right but did not affect receptor number (100,000 sites/cell) when compared to cultures preincubated at 4 degrees C. Cross-linking studies performed with ethyleneglycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal a major band of specifically bound EGF (Mr approximately 150,000), although the normal (Mr approximately 170,000) and another putative proteolytic form (Mr approximately 125,000) can also be seen. These results indicate that human meningiomas contain a mixed population of EGF binding sites and exhibit properties of previously described EGF receptors.

Search for estrogen receptors in human meningioma tissue sections with a monoclonal antibody against the human estrogen receptor.

Meningiomas are rich in progestin receptors, whereas estrogen receptors (ER) are virtually undetectable. The present experiments were performed to evaluate whether the absence of ER from the majority of human meningioma cytosols can be attributed to: occurrence of only a small number of ER-positive cells in an otherwise ER-negative tissue; resistance of nuclear receptors to extraction; or impairment of steroid binding. Twenty-one specimens were selected from our total series of 67 meningiomas. Based on cytosol assays, five of these meningiomas were considered to be ER positive (10-42 fmol/mg protein) and five had marginal ER concentrations (4-9 fmol/mg protein), whereas the remaining tissues were ER negative. For comparison, human breast cancer tissues were used. Tissues were sectioned at 6 micron and stained immunocytochemically using a monoclonal antibody against the human ER. The breast cancer specimens showed specific nuclear staining in part of the tumor cells. The sensitivity of the immunocytochemical assay was found to be sufficient to detect staining in breast cancer tissues containing as little as 17 fmol ER/mg cytosol protein. No specific staining was observed in meningioma tissues. It is concluded that the majority of meningiomas are truly devoid of ER and that the estrogen binding agent detected in low concentration in some meningioma cytosols is immunologically different from the human breast cancer ER. The presence of progestin receptors in meningioma apparently does not require the continuous presence of ER.

Distribution and biochemical characterization of somatostatin receptors in tumors of the human central nervous system.

Fifty-two brain tumors, consisting of 17 astrocytomas, 4 oligodendrogliomas, 20 glioblastomas, 3 neurinomas, 2 ependymomas, 1 neurofibroma, 1 ganglioneuroblastoma, 1 medulloblastoma, 1 plexus papilloma, 1 teratoma, and 1 germinoma, were tested for their content of specific somatostatin receptors using autoradiographic techniques or in vitro binding assays with membrane homogenates. Somatostatin receptors were found in most of the differentiated glia-derived tumors such as astrocytomas and oligodendrogliomas whereas the poorly differentiated glioblastomas were usually free of receptors. Tumors originating from neuroblasts, i.e., ganglioneuroblastoma and medulloblastoma, contained a high density of somatostatin receptors, whereas neurinomas and neurofibromas as well as the ependymomas, one teratoma, and one plexus papilloma were lacking such receptors. In one germinoma, low amounts of somatostatin receptors were observed over the lymphocytic elements. Receptor-positive tumors had saturable and high affinity receptors with pharmacological specificity for somatostatin and somatostatin analogues resembling that of normal human central nervous system tissue. In most instances, they could be labeled with two different iodinated radioligands, a somatostatin octapeptide derivative (204-090) or a somatostatin-28 analogue. This is the first time that somatostatin receptors have been shown to exist not only on neuronal structures of the central nervous system but also on glial elements. The precise function of such somatostatin receptors on glial cells, which may be different from neurotransmission, remains to be determined.

Intermediate filaments in meningiomas.

The presence of intermediate filaments (IF) (diameter 10 nm) is a characteristic electron microscopic finding in the cytoplasm of meningioma cells. To identify these IF, immunohistochemical staining for cytokeratins and vimentin and two-dimensional (2-D) gel electrophoresis followed by immunoblot analysis were applied to a group of 16 meningiomas. Thirteen meningiomas were obtained directly from surgery and three came from an autopsy in which they were found in close proximity as discrete tumor masses. Except for the angioblastic type, all major histological variants were represented (nine transitional, four syncytial, and three fibroblastic). None of the meningiomas stained for epithelial or internal organ cytokeratins. With monoclonal antibodies, each of the meningiomas stained positively for vimentin. Two-D gels revealed vimentin and vimentin breakdown products as the only IF present; these findings were verified by immunoblots. The study concludes that vimentin is the IF present in fibroblastic, syncytial, and transitional meningiomas.

An ultrastructural and immunocytochemical analysis of leptomeningeal and meningioma cultures.

Using immunocytochemical methods, we localized several glycoproteins of the extracellular matrix to leptomeningeal cells and meningiomas in vitro. Three cell lines derived from normal human leptomeninges and seven from meningiomas were studied by indirect immunofluorescence to evaluate the cellular production of fibronectin, laminin, collagen type IV, and procollagen type III. All leptomeningeal cell lines stained intensely and uniformly for all matrix proteins; all meningioma cell cultures stained uniformly, but the intensity of staining varied considerably. After removal of the cells in culture adherent to glass with 25 mM ammonium hydroxide, indirect immunofluorescence demonstrated an exuberant residual extracellular residue enriched with fibronectin, laminin, collagen type IV, and procollagen type III. Electron microscopic examination of all leptomeningeal and meningioma cultures revealed desmosomes and dense tonofilament formation; in addition, granular, filamentous basement membrane-like material was abundant in the extracellular spaces of all cultures. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the cell layer of two leptomeningeal and four meningioma cultures showed production of interstitial collagen types I and III; diethylaminoethyl (DEAE)-cellulose chromatography of the medium demonstrated preferential production of procollagen type I. Our findings show conclusively that normal arachnoid cells in vitro synthesize several of the collagen subtypes and may be responsible for the "fibrous response" of the leptomeninges to trauma, infection, or infiltration by tumor. The similarities between leptomeningeal cells and meningiomas demonstrated by electron microscopy and by indirect immunofluorescence support the notion that meningiomas are derived from arachnoid cells. The localization of various mesenchymal glycoproteins within the intra- and extracellular spaces and the ubiquity of specialized intercellular junctions suggest that leptomeningeal cells in culture have the potential to behave like both stromal and epithelial cells.

Brain glycoprotein in tumours of the nervous system.

We studied various tumours of the nervous system by the immunofluorescence technique using an anti-brain specific alpha 2 glycoprotein antiserum (anti-NSA3 antiserum). We found the antigen in 24/27 astrocytomas and 4/4 oligodendrogliomas but in none of the 8 meningiomas tested. There was an identity between the astrocytoma/oligodendroglioma antigen and that of normal brain as shown by the immunoprecipitation technique. By the immunofluorescence technique using inhibition of the antiserum we demonstrated that the tumour antigen is devoid of some specific nervous system determinants present in normal brain.

Immunolocalization of laminin in neoplasms of the central and peripheral nervous systems.

Laminin is a basement membrane glycoprotein that is expressed in vitro by immature and neoplastic astrocytes. The expression of laminin in vivo was examined immunohistochemically in normal adult brain and 90 neoplasms of the central and peripheral nervous systems. In normal adult brain, laminin was detected in the vasculature, arachnoid, pial-glial membrane, and choroid plexus. The vasculature in all 90 tumors demonstrated intense laminin immunoreactivity. Deposits of laminin were observed at the glioma-mesenchymal junction in several neoplasms, but never between or within neuroepithelial cells. The glial basement membrane often remained intact although surrounded on both sides by invasive glioma or medulloblastoma. However, there was always fragmentation and disruption of the glial membrane in adjacent fields. Laminin expression by tumor cells was observed in 10/10 schwannomas, 9/10 fibroblastic meningiomas, 3/19 nonfibroblastic meningiomas, and 3/6 mixed glioma-sarcomas. Laminin expression in the normal nervous system and in neuroepithelial neoplasms corresponds to regions of recognized basal lamina formation, including the junction between glial and mesenchymal elements. Although invasive gliomas are able to break down the pial-glial basement membrane and gain access to the perivascular or subarachnoid space, this membrane often remains intact late in the invasive process and may represent a partial barrier to tumor invasion. Laminin may be a useful marker for schwannomas, fibroblastic meningiomas, and vascular neoplasms of the nervous system.

Intracellular mucoid changes in tumor cells of meningiomas: a manifestation of polyvinylpyrrolidone (PVP) effect on tissues with mesenchymal characteristics.

In two cases of meningiomas in Taiwanese patients, extensive intracellular mucoid changes were found within tumor cells, together with deposits of polyvinylpyrrolidone (PVP) granules. Both patients had in the past received intravenous PVP as a plasma expander. Recipients of PVP have previously been reported to develop a form of PVP thesaurismosis with concomitant mucoid changes in the cells storing this substance. Such changes, as a rule, were limited to cells of mesenchymal origin. By storing PVP granules and undergoing mucoid changes as a result, meningioma tumor cells behave as cells with mesenchymal characteristics.

Simian virus 40-related DNA sequences in a human brain tumor.

Papovaviruses can induce experimental brain neoplasms in animals, and some papovaviruses have been implicated in the formation of various human tumors. We examined a series of seven human brain tumors removed at craniotomy for the presence of viral DNA sequences by the technique of DNA-DNA hybridization. Simian virus 40 (SV40) DNA was labeled in vitro and used as a "probe" for detecting related DNA sequences in cellular DNA extracted from brain tumors. SV40-related DNA sequences were found in DNA extracted from one tumor, a glioblastoma multiforme. It was calculated that approximately 1.2 viral genome equivalents per diploid cell were present in the tumor. Since the rate of reassociation of the probe deviated from ideal second-order kinetics, it is surmised that either only a portion of the SV40 genome was present in tumor cells or, alternatively, that the probe detected a related human papovavirus.

Meningioma, meningeal hemangiopericytoma (angioblastic meningioma), peripheral hemangiopericytoma, and acoustic schwannoma. A comparative immunohistochemical study.

The relationship between meningeal hemangiopericytoma (angioblastic meningioma), meningiomas of meningothelial derivation, and peripheral hemangiopericytoma is controversial; and immunohistochemical studies have yielded conflicting results. Likewise, immunohistochemistry has been touted as a reliable means of differentiating fibrous meningioma from acoustic schwannoma. By the immunoperoxidase method, we studied 40 meningiomas (11 meningotheliomatous, four transitional, 11 fibrous, three secretory, four metaplastic, one xanthomatous, one papillary, four atypical, one malignant), five arachnoid granulations, 13 angioblastic meningiomas, nine peripheral hemangiopericytomas, and seven acoustic schwannomas. Antisera to vimentin, epithelial membrane antigen (EMA), keratin, S-100 protein, carcinoembryonic antigen (CEA), desmin, factor VIII, Ulex europeaus, and glial fibrillary acidic protein (GFAP) were utilized. All meningiomas and arachnoid granulations stained for vimentin and EMA; 15% and 12% of meningiomas were S-100 and keratin positive, respectively. The latter was noted primarily in areas of secretory (pseudopsammomatous) differentiation. In contrast, all angioblastic meningiomas stained for only vimentin. This profile of immunoreactivity was also seen in the peripheral hemangiopericytomas, with the exception of single cases that stained focally for EMA and S-100 protein, respectively. Acoustic schwannomas all stained positively for S-100 protein, vimentin, and were variably reactive for EMA, a pattern not distinct from meningioma. We conclude that (a) meningiomas express both epithelial and mesenchymal markers as do arachnoid granulations, (b) that angioblastic meningiomas demonstrate only mesenchymal markers, (c) that angioblastic meningiomas express identical markers to peripheral hemangiopericytoma and should thus be considered a variant thereof, (d) among meningiomas, CEA and keratin appear to be relatively specific markers for the "secretory" variant, and (e) because of overlap in S-100 and EMA reactivity, these markers are unreliable in differentiating meningioma from acoustic schwannoma.

Secretory meningioma. A distinct subtype of meningioma.

Six meningiomas with abundant hyaline inclusions (pseudopsammoma bodies) were studied. As seen by light and electron microscopy, hyaline inclusions are composed of material of varying structures located in intracellular lumina lined by microvilli. A remarkable pericytic proliferation within the vessel walls was found in five cases. In all six cases, immunohistochemical examination for multiple antigens showed positive staining for carcinoembryonic antigen and epithelial membrane antigen in inclusions and surrounding cells. Weak positivity was found for keratin and secretory component in five cases and for alpha-1-antitrypsin and IgM in four cases. It is concluded that secretory meningioma is a distinct type of meningioma, usually meningothelial in type. It shows characteristic light-microscopic, ultrastructural, and immunohistochemical features of epithelial and secretory differentiation with accumulation of secretory material in the form of hyaline inclusions; marked vascular pericytic proliferation is also frequently present.

Subcellular localization of sialic acid in human brain tumors.

The subcellular localization of sialic acid has been investigated in various types of human brain tumors, such as astrocytoma, meningioma and primary malignant lymphoma. High levels of sialic acid were observed in the microsomal fraction of all types of tumors. However, there was no significant difference between values obtained in meningioma, primary malignant lymphoma, and normal tissue, in the fractions examining nucleus, mitochondria and supernatant.

The expression of S-100 protein and neuron-specific enolase in meningiomas.

The distribution of S-100 protein and neuron-specific enolase (NSE) was examined in 19 meningiomas using the peroxidase-antiperoxidase (PAP) immunohistochemical technique. Positive cytoplasmic staining for NSE was observed in 16 tumors, and comparable staining for S-100 protein was observed in eight tumors. The finding of S-100 protein and NSE immunoreactivity in fibroblastic, meningiotheliomatous, and transitional meningiomas raises the possibility that these morphologically distinct neoplasms derive from a common pluripotential cell capable of differentiation along diverse paths.

Epithelial differentiation in meningiomas. An immunohistochemical, histochemical, and ultrastructural study--with review of the literature.

Meningiomas possess features indicative of epithelial differentiation. The present study further documents this finding by a multimodality approach on serial sections from 25 meningiomas of all histologic and clinical categories. Standard light microscopic examination, immunohistochemical staining with a variety of epithelial and nonepithelial markers, and histochemical staining for mucin were performed on each case. Directed electron microscopic examination was performed on selected examples of epithelial feature-positive and -negative cases. All cases were immunoreactive for vimentin and epithelial membrane antigen. Thirty-two percent were reactive for cytokeratin and 39% for S-100. Twenty percent showed mucin positivity by histochemistry. Electron microscopic examination of cytokeratin-positive cases showed either intracellular lumina (secretory areas) or cytoplasmic tonofibrils, whereas cytokeratin-negative tumors lacked lumina and tonofibrils. Previous studies showing epithelial features in meningiomas are reviewed with emphasis on the studies using immunohistochemistry. These histochemical, immunohistochemical, and ultrastructural characteristics are of utility in the examination of tumors of suspected meningeal origin, particularly when dealing with atypical presentations or histomorphologic features.

Cryostat section assay of oestrogen and progesterone receptors in meningiomas: a clinicopathological study.

Oestrogen receptors and progesterone receptors were measured in the cytosols from cryostat sections of 45 meningiomas from 40 patients (12 men, 28 women) using an isoelectric focusing technique. Near fascimile adjacent sections from the same tissue blocks were stained and examined to determine the histological subtype of the neoplasms. Appreciable levels of progesterone receptor (greater than 10 fmol/mg cytosol protein) were present in 24 (53.3%) of of the neoplasms, but no clinically important oestrogen receptor was detected in any of the tumours. Competitive binding studies on control tissue confirmed the specificity of the assay procedures. No correlation was found between progesterone receptor state and the age, sex, or menopausal state of the patients, or the histological subtype and site of the neoplasms. Four of the patients studied had multiple intracranial neoplasms, which in two were of differing progesterone receptor state. The presence of specific progesterone receptor in meningioma cytosols raises the possibility of hormonal manipulation in the treatment of this group of neoplasms.

Epithelial membrane antigen and cytokeratin expression by meningiomas: an immunohistological study.

Thirteen meningiomas of varying light microscopic features were studied immunohistologically using a panel of monoclonal antibodies directed against epithelial, mesenchymal, and neural components. All 13 meningiomas showed expression of epithelial membrane antigen, vimentin, and S100 protein, as did normal meninges. Five of the 13 meningiomas also showed focal expression of cytokeratins, with double labelling showing expression of cytokeratins and vimentin by different cells. The cytokeratin expression was especially noticeable in cells surrounding the hyaline bodies of meningiomas. These results provide further evidence that meningiomas have features of both mesenchymal and epithelial tissues.

Immunoreactive thymopoietin in the mouse central nervous system.

A thymopoietin-immunoreactive substance (TP-IRS) has been detected in homogenates of mouse spinal cord and brain using a radioimmunoassay; levels were maximal at birth. TP-IRS was also detected in supernatants of mouse neuroblastoma (NIE-115) and primary spinal cord cultures but not human astrocytic and meningeal tumors or mouse primary astrocyte cultures. With affinity purified rabbit anti-TP globulin, immunofluorescent staining was seen in mouse spinal cord cultures in association with nuclear membranes of neurons and, to a lesser degree, flat background cells. From supernatants of NIE-115 cells grown in tritiated leucine and lysine, proteins of approximately 8000 and 4500 Da were isolated by TP affinity chromatography (compared with 5562 Da for thymic thymopoietin). When injected into mice, these neural proteins partially blocked neuromuscular transmission in a manner similar to thymic thymopoietin.

Expression of muscarinic binding sites in primary human brain tumors.

The expression of muscarinic binding sites was examined in a collection of primary brain tumors of different cellular origins and various degrees of dedifferentiation, as compared to control specimens. Eleven gliogenous tumors were examined, all of which contained substantial amounts of muscarinic binding sites. Most of the other tumor types examined did not display detectable binding of [3H]N-methyl-4-piperidyl benzilate ([3H]4NMPB). Scatchard analysis indicated the existence of homogeneous antagonist sites in both normal forebrain and glioblastoma multiforme, with Kd values of 1.2 nM and 0.9 nM, respectively. The density of muscarinic binding sites varied between tumors from different patients, and also between specimens prelevated from different areas of the same tumor. This variability, as well as the average density of binding sites, appeared to be larger in highly malignant tumors than in less malignant ones. In contrast, the density of muscarinic receptors from control specimens was invariably high, but within the same order of magnitude. To test whether the muscarinic binding activity in the brain tumors is correlated to other cholinoceptive properties, cholinesterase activity was also examined. Individual data for density of [3H]4NMPB binding sites were then plotted against corresponding values of cholinesterase activity. The pattern of distribution of these values was clearly different in tumor specimens, when compared to that observed in samples derived from non-malignant brain. Our observations indicate that human brain cells of gliogenous origin are capable of expressing muscarinic binding sites, and that, if a correlation exists between muscarinic receptors and cholinesterase levels in gliogenous tumors, it differs from that of non-malignant brain tissue.

Gonadal steroid receptors in meningiomas.

Oestrogen receptor (ER) analysis was performed in 70 meningiomas with an enzyme immunoassay, using monoclonal antibodies against human oestrogen receptor protein (oestrophilin) and with a sensitive radioligand binding assay, using 125I-oestradiol as radioligand. Low levels of ER immunoreactivity were found in tumours from 51% of patients, whereas ER binding activity was demonstrated in 40% of the meningiomas examined. In 8 (11%) tissue samples multiple binding sites for oestradiol were observed. The immunoreactive binding sites corresponded to the classical, high-affinity ER. In ligand binding studies, however, measurement of classical ER was considerably influenced by a second low-affinity, high-capacity oestrogen binding component even at low ligand concentrations. 3H-methylpromegestone and 3H-methyltrienolone, a synthetic gestagen and androgen, were used for concurrent determination of the progesterone receptor (PR) and androgen receptor (AR) binding activity. High concentrations of PR were detected in 53 (76%), whereas moderate levels of AR binding sites were demonstrated in 33 (47%) tumours. A positive correlation between ER immunoreactivity and AR binding activity is indicative for an oestrogen regulation of AR via the ER system. The presence of gonadal steroid receptors in a large proportion of meningiomas and the tendency for a dependence of receptor concentrations on the histological subtype could have implications for tumour therapy.