Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells.

Metalloproteinases have been implicated as important factors mediating the tissue migration of a variety of normal and transformed cells. The conditioned medium (CM) of fetal human astrocytes and five glioma cell lines did not degrade azocoll in suspension, but several proteolytic activities, inhibitable by 1,10-phenanthroline, were detected on sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Both cell types secreted three major proteolytic species (Mr 65,000, 57,000, and 52,000). Two of the glioma lines secreted an additional proteinase (Mr 92,000). After treatment with 12-O-tetradecanoylphorbol-13-acetate, the secretion of the Mr 92,000, 57,000, and 52,000 proteinases was induced or enhanced in all of the cells. The Mr 92,000 and 65,000 proteinases bound specifically to a gelatin affinity column. When purified by preparative gel electrophoresis, the Mr 65,000 proteinase was found to degrade type IV procollagen. The Mr 57,000 and 52,000 species were precipitated by anticollagenase IgG. Tissue inhibitor of metalloproteinases was detected in the CM of all of the cells by substrate gel analysis and immunoprecipitation of [35S]methionine-labeled proteins with anti-tissue inhibitor of metalloproteinases IgG. The glioma lines also secreted various amounts of two smaller inhibitors of metalloproteinases (IMPs), also seen in rabbit brain capillary endothelial cell CM (IMP-1 at Mr 22,000 and IMP-2 at Mr 19,000), and an inhibitor not previously identified (IMP-3 at Mr 16,500). 12-O-Tetradecanoylphorbol-13-acetate stimulated the secretion of tissue inhibitor of metalloproteinases in all of the cells and induced IMPs in some of the glioma lines. When gel filtration chromatography of concentrated CM was used to resolve inhibitors from proteinases, the isolated proteinases had activity against azocoll and the glycoprotein and collagen components of an in vitro model of the extracellular matrix. The secretion of a battery of metalloproteinases by astrocytes may be important in facilitating astrocytic migration during development and in pathological conditions such as inflammation or local invasion of astrocytic neoplasms.

The effects of human recombinant tumor necrosis factor on glioma-derived cell lines: cellular proliferation, cytotoxicity, morphological and radioreceptor studies.

To determine whether tumor necrosis factor is of potential value for the treatment of human malignant gliomas, we studied the effects of human recombinant tumor necrosis factor (rTNF-alpha) on the morphology, incorporation of tritiated thymidine, and proliferation of 5 established cell lines derived from human malignant gliomas and 3 normal human brain cell cultures. A radioreceptor analysis for rTNF-alpha was performed on all cell lines and cultures. Two of the 5 human glioma cell lines (SF-188 and U 343 MG-A) demonstrated a marked decrease (60% or less of untreated controls) in the uptake of tritiated thymidine when treated with rTNF-alpha at a concentration of 40 U/ml; rTNF-alpha at 100 U/ml had antiproliferative and cytotoxic effects on both cell lines. The growth and proliferation of cell lines SF-126 and U 251 MG were not affected by rTNF-alpha even at high concentrations (5,000 U/ml). The growth and proliferation of SF-539 were affected to an intermediate degree. A colony-forming efficiency assay corroborated the results of the proliferation studies: SF-126 was relatively resistant (surviving fraction of 0.9 at 500 U/ml) and SF-188 was relatively sensitive (surviving fraction of 0.08 at 500 U/ml) to the cytotoxic effects of rTNF-alpha. Time-sequence electron microscopy showed that rTNF-alpha at a concentration of 500 U/ml caused ultrastructural changes in SF-188, including increased intracytoplasmic vesiculation, swelling and degeneration of mitochondria, loss of cell:cell junctional complexes, and fragmentation of the plasma membrane. Studies with 125I-rTNF-alpha showed a variable degree of binding in all cell lines and cultures. SF-188, a highly sensitive cell line, demonstrated the strongest binding of 125I-rTNF-alpha (3,400 receptors/cell with high affinity; kd = 0.27 nM), while SF-126, a highly resistant cell line, had the weakest binding (809 receptors/cell; kd = 0.25 nM). We conclude that there is a spectrum of antiproliferative and cytotoxic activity among glioma-derived tumor cell lines exposed to rTNF-alpha. An increased number of rTNF-alpha receptors appears to be a necessary but insufficient condition to explain the antiproliferative effects observed in some glioma-derived cell lines.

Partial characterization of a soluble mitogenic factor from medulloblastoma.

To determine how medulloblastoma cells might influence the proliferation and phenotype of normal stromal cells, normal human leptomeningeal cells were treated in culture with medulloblastoma-conditioned medium; their ability to incorporate tritiated thymidine and synthesize collagen was measured. The treated leptomeningeal cells had a significantly greater uptake of tritiated thymidine and grew faster than control leptomeningeal cells. Immunofluorescence studies demonstrated a greater intensity of staining for procollagen type III in the cell layer of the treated cultures than in control cultures; diethylaminoethyl (DEAE)-cellulose chromatography of the medium showed that the treated cells synthesized predominantly type III collagen, whereas control cells synthesized type I collagen. Analysis of the medulloblastoma-conditioned medium revealed that the soluble factor responsible for these effects in an acid- and heat-stable protein. The increased proliferation and altered collagen synthesis induced in leptomeningeal cell cultures by a soluble factor from a medulloblastoma are examples of how tumor and stromal elements interact, and may be related to the process of desmoplasia often observed in medulloblastomas in vivo.

Comparison of in vitro cloning assays for drug sensitivity testing of human brain tumours.

Three in vitro clonogenic assays were used to determine the sensitivity of an established human glioblastoma cell line (U251-MG) to five chemotherapeutic agents. The colony-forming efficiency of untreated culture was 0.695 +/- 0.170 in a monolayer assay with irradiated feeder cells, 0.018 +/- 0.006 in a low-O2 agar assay, and 0.049 +/- 0.021 in a two-layer agar system with nutrient-enriched medium (p less than 0.001). Comparison of the slope of the regression line for the dose-response curve and the interpolated ID90 for each drug showed that U251-MG was equally sensitive to aziridinylbenzoquinone and dianhydrogalactitol in all three assays. The sensitivity of this cell line to 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), cis-dichlorodiammineplatinum (II) (CDDP) and 9-hydroxy-2-N-methylellipticine (HME), however, varied depending on the assay used. In no instance did U251-MG show greater sensitivity (lower ID90 or steeper slope) in the low-O2 agar assay than in the other assays. BCNU and CDDP were least active in the monolayer assay, whereas HME showed both the lowest ID90 and steepest slope using this technique. We conclude that different in vitro tumour clonogenic assays show different colony-forming efficiencies for the same cell line and may show different responses to certain drugs. Identification of accurate predictive models of drug sensitivity will require correlative in vivo and in vitro studies.

Growth of a medulloblastoma on normal leptomeningeal cells in culture: interaction of tumor cells and normal cells.

Well-characterized medulloblastoma cells growing in suspension were placed on top of a confluent monolayer of leptomeningeal cells. In contrast to cells placed on plastic alone, which did not grow or attach, the medulloblastoma cells attached readily to the leptomeningeal cells and grew to form enlarging spheroids. The growth of these spheroids was supported with minimal essential medium containing 10% fetal calf serum or with human cerebrospinal fluid. Medulloblastoma cells grown on plastic remained viable for 7 to 10 days, whereas those grown on a monolayer of leptomeningeal cells remained viable for 40 days. Electron microscopy demonstrated increased interdigitation of the plasma membrane at the sites of contact between leptomeningeal and medulloblastoma cells, the deposition of fine, basement membrane-like material between the two cell types, and an increased number of cytoskeletal filaments in the leptomeningeal cells. We conclude that medulloblastoma cells can be grown on a leptomeningeal monolayer. This in vitro system may be useful in studying the mechanisms by which medulloblastoma cells attach to leptomeningeal elements and grow after dissemination in cerebrospinal fluid.

Inhibition of growth and induction of differentiation in a malignant human glioma cell line by normal leptomeningeal extracellular matrix proteins.

We devised a model system to study the effects of extracellular matrix proteins on the malignant phenotype of an anaplastic glioma cell line, U 343 MG-A. Well-characterized cultures derived from normal human leptomeninges were grown to confluence and maintained for 2 weeks. The leptomeningeal cells were then removed with base and detergent, leaving behind an extracellular matrix enriched in laminin, fibronectin, type I and IV collagen, and procollagen III. U 343 MG-A tumor cells planted on top of this normal extracellular matrix were profoundly growth inhibited compared with glioma cells grown on plastic alone. Glioma cells grown on the extracellular matrix developed multiple, slender processes and assumed a more differentiated astrocytic phenotype; immunostains for glial fibrillary acidic protein revealed a more extensive intracytoplasmic network of intensely staining filaments than in control glioma cells. When glioma cells grown on the extracellular matrix were analyzed by an enzyme-linked immunosorbent assay for glial fibrillary acidic protein, the amount of this intermediate filament per cell was increased 20-fold compared with glioma cells growing on plastic. The growth and differentiation of U 343 MG-A glioma cells in flasks coated with purified fibronectin or laminin was not significantly perturbed; however, glioma cell cultures grown in flasks coated with purified type I or IV collagen showed decreased cellular proliferation, stellate cell formation, and increased levels of glial fibrillary acidic protein per cell compared with glioma cells growing on plastic. Gelatin gel analysis showed that U 343 MG-A glioma cells growing on plastic secreted a 65,000-D metalloproteinase that was not secreted by glioma cells grown on the leptomeningeal extracellular matrix. We conclude that in this system, the extracellular matrix of a normal human leptomeningeal culture substantially inhibited the proliferation of and induced differentiation in an anaplastic glioma cell line. Our analysis of single components of the extracellular matrix suggests that these effects may be mediated in part by type I and IV collagen. The mechanism by which the leptomeningeal extracellular matrix inhibits glioma cell proliferation may be by diminishing tumor-associated protease secretion so that the degradation of extracellular matrix macromolecules in the tumor cell microenvironment is prevented and tumor cell migration becomes less likely.

Distribution of extracellular matrix proteins in primary human brain tumours: an immunohistochemical analysis.

Using immunohistochemical techniques, we localized several glycoproteins of the extracellular matrix in paraffin-embedded sections of 4 normal brain and 38 primary intracranial tumour specimens. All specimens were positively immunostained to various degrees by monoclonal antibodies to type IV collagen and procollagen III and by antisera to laminin and fibronectin. Staining was consistently most intense at sites of contact between neuroepithelial and mesenchymal or leptomeningeal elements; there was no demonstrable staining within or between neuroepithelial elements in the neuropil. Tumour cells from meningiomas and from the sarcomatous portion of a gliosarcoma were positively immunostained for fibronectin and laminin. The integrity of the glial limitans externa was demonstrated by the positive linear reaction product produced by immunostains for type IV collagen and laminin, even in the most malignant gliomas. The deposition of extracellular matrix glycoproteins at the glial-mesenchymal interface observed in this study of primary human brain tumours is a manifestation of one of the interactions between tumour and stromal cells in the central nervous system. A loss of coordination and an alteration in the interactions between epithelial cells and stromal cells across extracellular matrices such as basement membranes are thought to be fundamental steps in the development and progression of cancer. Further characterization studies focusing on other markers of the extracellular matrix are needed to elucidate completely the function of this structure in the central nervous system.

Characterization of fetal human brain cultures. Development of a potential model for selectively purifying human glial cells in culture.

Seven fetal human brain and three fetal human leptomeningeal cultures were characterized according to cell morphology, ultrastructural features, antigen expression, and collagen biosynthesis capabilities. Primary cultures derived from mechanically and enzymatically dissociated samples of fetal human brain consisted of a heterogeneous cell population in which astrocytes, oligodendrocytes, neurons, mesenchymal (leptomeningeal) cells, and macrophages were identified by light and electron microscopy. With progressive subcultivation, a homogeneous, leptomeningeal cell-derived population predominated. Fetal human brain and leptomeningeal specimens embedded in paraffin were analyzed immunohistochemically for the distribution of glial fibrillary acidic protein (GFAP), vimentin, factor-VIII-related antigen, fibronectin, laminin, type IV collagen, and procollagen III. Only GFAP and vimentin identified astrocytes and radial glia in the developing human brain; fibronectin, laminin, and the collagen types were immunolocalized largely to the leptomeninges and to the cerebral vasculature. The percentage of cells positively identified by antiserum to GFAP was greatest in primary cultures of fetal human brain; by the fourth passage, none of the fetal brain cultures were GFAP positive. The progressive decrease in the percentage of GFAP-positive cells was accompanied by an increase in the percentage of cells identified by collagen immunomarkers. Furthermore, in double immunolabeling experiments, antibodies to GFAP recognized a population of cells that was not identified by antibodies to laminin, fibronectin, type IV collagen, or procollagen III. SDS-PAGE and DEAE-cellulose chromatography of [3H]-proline-labeled early-passage fetal human brain cultures revealed collagen profiles identical to those obtained from direct cultures of the leptomeninges. The characteristics of later-passage fetal human brain cultures were identical in all respects to those of the fetal human leptomeningeal cultures. The proliferation of leptomeningeal cells could be inhibited by exposing the cells to cis-hydroxyproline (200 micrograms/ml). Primary fetal human brain cultures similarly treated with the proline analogue were found to be highly enriched for glial cells; these cultures were more than 90% GFAP positive. We conclude that primary fetal human brain cultures consist of a heterogeneous population of cells, most of which under the present culture conditions can be identified as glial cells. Subcultivation of human fetal brain cultures results in the overgrowth of mesenchymal cells, which are presumably derived from the leptomeninges.(ABSTRACT TRUNCATED AT 400 WORDS)

Establishment and characterization of five cell lines derived from human malignant gliomas.

We established and characterized five cell lines derived from human malignant gliomas (four glioblastomas multiforme and one highly anaplastic astrocytoma). All cell lines exhibited tumor cell morphology and growth kinetics, and anchorage-independent growth in soft agar. Cytogenetic analysis revealed significant aneuploidy in all five cases as well as clonal chromosomal alterations unique to each cell line. No cell line was tumorigenic in athymic mice. Two of the cell lines were sensitive to carmustine (BCNU) in monolayer and soft-agar cultures. Electron microscopy showed marked variability between cell lines in the number and structure of intracytoplasmic organelles; SF-126 formed collagen fibers in vitro. Immunohistochemical analysis of the surgical specimens showed variable expression of glial fibrillary acidic protein (GFAP) in malignant astrocytes; positive immunostaining for glycoproteins of the extracellular matrix was found predominantly in perivascular regions. In early-passage cultures, only cell line SF-295 expressed GFAP; at establishment, none of the cell lines expressed GFAF or glutamine synthetase. Fibronectin and laminin were expressed by all cell lines in early-passage culture, but expression of these glycoproteins at establishment was variable. Only SF-126 was positively identified by immunostains for procollagen III; this was also the only cell line in which DEAE-cellulose chromatography and SDS-PAGE demonstrated interstitial collagen synthesis. These well-characterized glioma-derived cell lines may now serve as useful tools with which to study the cell biology of gliomas. The synthesis of interstitial collagen by a glioma-derived cell line may suggest a derivation from vascular mesenchymal elements, either reactive or transformed, in the original heterogeneous malignant glioma, rather than from a glial precursor cell.

Establishment and characterization of a cell line from a human gliosarcoma.

A human gliosarcoma culture was characterized from the time of inception to the time of establishment of the cell line (SF-539 BT). Immunohistochemical analysis of the original tumor showed 2 distinct regions of cells. The gliomatous regions were identified by immunostains for glial fibrillary acidic protein and the sarcomatous regions by immunostains for laminin, collagen type IV, procollagen type III, and fibronectin. In early-passage culture, both types of cells maintained their characteristic immunohistochemical profiles; however, after the fourth subcultivation in monolayer culture, no cells expressing glial fibrillary acidic protein could be identified. All cells had become morphologically uniform and expressed laminin, collagen type IV, procollagen type III, and fibronectin only. The immunostaining profile of clones grown in soft agar was similar to that of cells in monolayer culture. At establishment, SF-539 BT has a saturation density of 1.3 X 10(6) cells/25 sq cm, a doubling time of 32 h, and a plating efficiency of 22% in monolayer culture. The tumor cell line is resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, has an abnormal karyotype, grows anchorage independently, and forms a tumor that most closely resembles a spindle cell sarcoma in athymic mice. Its ultrastructure in monolayer culture consists of large cells with an expanded rough endoplasmic reticulum and abundant multivesicular bodies; in athymic mice, extracellular collagen fiber formation is prominent. DEAE-cellulose chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cultures labeled with [3H] proline demonstrated interstitial collagen formation. We conclude that the cell line at establishment is a collagen-producing spindle cell sarcoma that resembles the sarcomatous regions of the original mixed tumor. Further cell separation and characterization studies are needed to determine the pathogenesis of mixed tumors such as gliosarcoma.

Characterization of normal human brain cultures. Evidence for the outgrowth of leptomeningeal cells.

To establish the histogenetic identity of the predominant cell type in monolayer cultures of normal human adult brain, eight brain specimens were placed into culture and characterized according to cell kinetics, karyotype, antigenic expression, and ultrastructural features. The protein profiles of both the cell layer and the medium were analyzed in selected cultures using sodium dodecyl sulfate polyacrylamide gel electrophoresis and diethylaminoethyl cellulose chromatography. All cultures displayed a limited life span in vitro; marked contact inhibition at confluence; a normal karyotype; an intracytoplasmic and extracellular glycoprotein profile consisting of fibronectin, procollagen type III, laminin, and collagen type IV; specialized intercellular junctions; and interstitial collagen chain synthesis. All of these features were identified in our previous study of human leptomeningeal cultures. The results of immunocytochemical staining for glial fibrillary acidic protein were negative in all cultures of normal human brain, except in early passages in two cultures, which lost the glial cell marker during subsequent passages; immunostains for vimentin were positive in all cells in all cultures. These results support the hypothesis that, in this study, cultures derived from normal human brain are not of glial origin. Our findings also suggest that glial cells are less well-suited to monolayer growth under our culture conditions than are other cell types in enzyme-dissociated brain tissue placed in culture, especially leptomeningeal cells. The identification of leptomeningeal cells as the predominant cell type in normal human brain cultures may prove useful in attempts to foster the growth of human glial cells by culturing brain samples under conditions that prohibit the growth of leptomeningeal cells. Under such conditions, astrocytes, oligodendroglia, and ependymal cells could be isolated with greater ease and cultured separately. These purified cultures of different glial cell types would then provide a more relevant in vitro model for studying human neurological diseases.