Surface Of Glioma Cells Research Articles

Cytolysis of malignant glioma cells by lymphokine-activated killer cells combined with anti-CD3/antiglioma bifunctional antibody and tumor necrosis factor-α

With the aim of developing an effective immunotherapy for malignant glioma, glioma cells were incubated with tumor necrosis factor-alpha (TNF-alpha) to increase their susceptibility to lysis by lymphokine-activated killer (LAK) cells. Treatment with exogenous TNF-alpha induced the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of glioma cells. In addition, the cytolytic activity of LAK cells toward exogenous TNF-alpha treated glioma cells was significantly greater than LAK cell activity toward untreated glioma cells. This increase in cytolytic activity was blocked by anti-ICAM-1 monoclonal antibodies (MAb). Furthermore, co-treatment with a bifunctional antibody (BFA) composed of anti-CD3 (UCHT1) and antiglioma (G-22) antibodies synergistically increased the cytolytic activity of LAK cells towards TNF-alpha-treated glioma cells. These results indicate that a combination of exogenous TNF-alpha and anti-CD3/antiglioma BFA may provide an effective modified adoptive immunotherapy for patients with malignant glioma.

モノクローナル抗体結合リポソームによる悪性脳腫ようのターゲティング療法へのアプローチ

Malignant glioma is one of the most lethal tumors arising in the central nervous system. The patients with malignant glioma have poor prognosis with an average survival of less than 2 years even if they receive multimodality treatment including surgery and post operative adjuvant therapy. Therefore, we need to develop a new form of therapy that can inhibit the growth and invasion of glioma cells. Here, we have evaluated the efficacy of liposomes conjugated or associated with a monoclonal antibody for malignant glioma. We prepared two types of monoclonal antibodies for the targeting therapy. One is a G-22 monoclonal antibody reactive with the CD44 that overexpresses on the surface of human glioma cells and another is a 3C10 monoclonal antibody reactive with the fusion junction of a deletion-mutant epidermal growth factor receptor (EGFR) on their cells. The deletion-mutant (Type III) was reported to be most prevalent, being approximately 17% of the patients with malignant glioma. Using these antibodies, we prepared the liposomes conjugated or associated with a monoclonal antibody (Immunoliposomes). We were successful with in vitro and in vivo transfer of anticancer drugs or biological response modifiers (BRM) or genes using the immunoliposomes. The immunoliposomes containing methotrexate, cisplatin or adriamycin have selective cytotoxicity toward glioma cells. Also, the immunoliposomes entrapping the BRM genes (ex. interferon-β, tumor necrosis factor-α) increased the growth inhibition of glioma cells much more than the liposomes with no antibody. These results suggest that liposomes conjugated or associated with a monoclonal antibody would be a useful clue for the tumor targeting of malignant gliomas.

Reinforced cytotoxicity of lymphokine-activated killer cells toward glioma cells by transfection of the killer cells with the gamma-interferon gene.

Lymphokine‐activated killer (LAK) cells generated from peripheral blood lymphocytes incubated with recombinant interleukin‐2 were transfected with the human γ‐interferon (HuIFN‐γ) gene by means of liposomes having a positive charge on their surface. The cells secreted significant amounts of HuIFN‐γ (reaching more than 5 U/ml) into the culture medium. The HuIFN‐γ produced by the cells induced intercellular adhesion molecule‐1 (ICAM‐1) and enhanced the expression of Fas antigen on the surface of human glioma cells. Also, LAK cells transfected with HuIFN‐γ gene exhibited reinforcement of cytotoxicity toward human glioma cell lines (U251‐MG and SK‐MG‐1). Furthermore, the reinforcement was significantly quenched by anti‐ICAM‐1 and/or anti‐TNF‐α monoclonal antibody.

Transfection‐induced Tumor Necrosis Factor‐α Increases the Susceptibility of Human Glioma Cells to Lysis by Lymphokine‐activated Killer Cells: Continuous Expression of Intercellular Adhesion Molecule‐1 on the Glioma Cells

To develop more effective adoptive immunotherapy, we transfected the human tumor necrosis factor‐α (TNF‐α) gene into human glioma cells (U251‐SP), which were used as target cells. TNF‐α is known to increase both the expression of intercellular adhesion molecule‐1 (ICAM‐1) on the surface of glioma cells and the susceptibility of glioma cells to lymphokine‐activated killer (LAK) cell cytolysis. We compared the expression of ICAM‐1 induced by TNF‐α generated by the TNF‐α gene‐transfected cells with that induced by exogenously added TNF‐α. When the TNF‐α gene was transfected into U251‐SP cells, the expression of ICAM‐1 was detected on the cell surface from 3 days after the transfection and continued until at least 9 days. In contrast, it was expressed only transiently in the case of exogenously added TNF‐α. Also, the cytolytic activity of LAK cells induced by transfection‐induced TNF‐α was significantly stronger than that induced by exogenously added TNF‐α. The increased susceptibility was quenched by anti‐ICAM‐1 monoclonal antibody. These data indicated that continuous expression of ICAM‐1 induced by TNF‐α gene transfection of glioma cells resulted in higher cytolytic activity of LAK cells.

Liposomal transfection of human γ-interferon gene into human glioma cells and adoptive immunotherapy using lymphokine-activated killer cells

The authors evaluated the effect of liposomal transfection of human gamma-interferon (HuIFN-gamma) gene into human glioma cells and lymphokine-activated killer (LAK) cells, alone and in combination. An HuIFN-gamma gene inserted in a eukaryotic expression vector was entrapped in liposomes bearing positive surface charges. Liposomal gene transfection induced production of HuIFN-gamma and its secretion in culture medium of human glioma cell lines (SK-MG-1 and U-251 MG). At 4 days after transfection, the cells produced 10 to 50 U/ml of HuIFN-gamma in the medium, whereby the major histocompatibility complex (MHC) class I and II antigens, as well as intercellular adhesion molecule-1 (ICAM-1), were induced on the glioma cell surface. The growth-inhibiting effect of transfection-induced HuIFN-gamma was much stronger in comparison with control cultures exposed to 500 U/ml of exogenously added HuIFN-gamma. In addition, 20% to 40% growth inhibition was obtained in the glioma cells when they were treated with LAK cells alone at a 5:1 ratio of effector to target cells. Liposomal transfection of HuIFN-gamma gene into human glioma cells combined with immunotherapy using LAK cells was more effective than either technique alone. The reinforcement of growth inhibition in the case of combined therapy was quenched by anti-ICAM-1 monoclonal antibody, but not by anti-MHC class I or II monoclonal antibodies. These results suggest that the combined effect of liposomal transfection of HuIFN-gamma gene plus LAK cells into human glioma cells is a potentially useful therapy for malignant glioma, and that the mechanisms of the reinforcement of growth inhibition are closely related to the expression of ICAM-1 on the glioma cell surface.

Polysialosyl glycoconjugates defined by monoclonal antibody M6704 are expressed in human gliomas and embryonic neurons.

The distribution of polysialosyl glycoconjugates defined by a monoclonal antibody M6704 (M6704 antigen) and of its O-acetylated counterpart (O-Ac-M6704 antigen) was investigated in human gliomas and in normal adult and fetal central nervous system. Among 32 gliomas, M6704 antigen was confined to the glioma cell surface and cytoplasmic processes in 21 cases, whereas O-Ac-M6704 antigen was expressed in the Golgi region in 22 cases and at the cell surface in seven cases. In adult tissues M6704 antigen was barely detectable, whereas in fetal tissues it was spatially and temporally expressed at the cell surface and along processes of post-mitotic neurons. The O-Ac-M6704 antigen first appeared in the Golgi regions of neurons at 66 gestational days and increased gradually with further maturation to adult levels. Several polysialoglycoproteins, the most dominant being 51 KD, and C-series polysialogangliosides were isolated from gliomas. These results indicate that M6704 antigen is a distinctive component expressed selectively by embryonic neurons during development and by glioma cells.

Cellular processing and proteoglycan nature of amyloid precursor proteins.

Amyloid beta protein (beta/A4 or A beta), the main proteinaceous component of the amyloid depositions of the Alzheimer's brain, derives from the proteolytic processing of the amyloid precursor protein (APP). Cleavage of the amyloid precursor by at least two distinct secretase activities produces soluble secreted APP. The major secretase cleavage (site I) takes place between A beta 16 and 17, while the minor cleavage (site II) takes place after A beta Lys 28 and may produce potentially amyloidogenic secreted APP. Full-length cellular APP is cleaved by secretase intracellularly in the Trans-Golgi Network (TGN) or in post-Golgi vesicles. The resultant soluble APP is transported to the plasma membrane and exocytosed. The biological activity of the APP is still not completely understood, although it seems to act as a cell adhesion molecule. Recent studies have shown that in glioma cells, most of the soluble secreted APP occurs as a chondroitin sulfate proteoglycan (CSPG). In addition, full length APP CSPG has been detected in neuroblastoma and fibroblast cells as well as on the surface of glioma cells, and in human brain. These results suggest that the proteoglycan nature of the APP proteins may be important for their biological function.

Chondroitin sulfate proteoglycan form of cellular and cell-surface Alzheimer amyloid precursor

The biological function of the amyloid precursor protein (APP) is still not fully understood. Recently, we reported that secreted truncated APP occurs in a chondroitin sulfate proteoglycan form. Here we present evidence that full length APP-chondroitin sulfate proteoglycan is present on the cell surface of C6 glioma cells. In addition, densitometric quantitation of Western blots showed that approximately 50% of the mature cell-associated full length APP is in the proteoglycan form. These findings suggest that the proteoglycan nature of APP may be important for the implementation of its biological function.

Cell‐surface expression of glia maturation factor β in astrocytes

Glia maturation factor beta (GMF-beta) is a 17-kDa acidic protein isolated from the brain. When added to cultured cells, GMF-beta promotes the phenotypic expression of glia and neurons and inhibits the proliferation of their respective tumors. Although astrocytes produce GMF-beta and store it inside the cells, they do not secrete the protein into the cultured medium. This poses a question as to how GMF-beta mediates intercellular communication. This paper provides an answer by demonstrating the presence of GMF-beta on the surface of astrocytes, using gold-labeled antibody enhanced with silver. It appears that cell-surface GMF-beta acts on the target cells at close range when cells are in direct contact. In contrast to astrocytes, we failed to detect GMF-beta on the surface of C6 glioma cells, although these cells, like astrocytes, possess endogenous intracellular GMF-beta and are also responsive to GMF-beta added to the medium. The lack of cell-surface expression of GMF-beta in C6 cells may reflect a breakdown in intercellular communication in these malignant cells.

Radioimaging of human malignant gliomas using indium-labelled monoclonal antibodies

A monoclonal antibody (MUC 2-63) was raised against a neuroectodermal antigen expressed on human malignant gliomas, neuroblastomas and melanomas. The mouse monoclonal antibody MUC 2-63 was of IgG1 isotype, it binds the antigenic determinants with the molecular weight of 32,000 Dalton present on the cell surface of native glioma cells. Seven patients with brain tumours, five with biopsy proven malignant gliomas, received an intravenous injection of the 111In-DTPA coupled monoclonal antibody MUC 2-63. Six patients had an uptake of 0.01-0.04% of the injected dose at the site of the tumour, which correlated to the computerized tomography (CT) images. The half-lives in the tumours of 111In were 38-259 h. The maximal uptake in the tumours were noticed between 40 and 67 h. One patient failed to demonstrate any intracranial uptake of the radioactivity. This patient had been treated with surgery and radiotherapy for a stage 2 testicular seminoma four years ago. He recently developed clinical signs of a primary brain tumour and the CT diagnosis was malignant glioma. All patients had nonspecific uptake in the liver, half-lives were between 60 and 84 h, the corresponding maximal uptake was detected between 22 and 45 h. No side effects were observed by administration of the mouse monoclonal antibody MUC 2-63.

Immunohistochemical Detection and Correlation Between MHC Antigen and Cell-mediated Immune System in Recurrent Glioma by APAAP Method

As part of an on-going clinical trial of immunotherapy for recurrent malignant gliomas, using alkaline phosphatase-anti-alkaline phosphatase method with monoclonal antibodies, we investigated the correlation between expression of the major histocompatibility complex (MHC) and the subpopulation of tumor-infiltrating lymphocytes (TILs) in 38 glioma specimens (20 grade IV, 11 grade III, and 7 grade II) from 33 patients. Thirty specimens (78.9%) were positive to class I MHC antigen and 20 (52.6%) were positive to class II MHC antigen. The correlations between class I MHC antigen expression and the number of infiltrating T8 (p less than 0.01), and also between class II MHC antigen expression and the number of infiltrating T4 (p less than 0.05) were significant. We conclude that TILs are the result of immunoreaction (host-defense mechanism). 31.6% of specimens had perivascular infiltration of T cells. The main infiltrating lymphocyte subset in moderate to marked perivascular cuffing was T4. Our results may indicate that lack of MHC antigen on the glioma cell surface has a share in the poor immunogenicity in glioma-bearing patients. In addition, considering the effector/target ratio, the number of infiltrating lymphocytes against glioma cells was too small, so the immunological intervention seems to be essential in glioma therapy. Previous radiation therapy and chemotherapy, including steroid therapy, did not influence lymphocyte and macrophage infiltration.

Monoclonal antibodies in neuro-oncology

Many studies have suggested the possible existence of tumor-associated antigens in brain gliomas. Strong evidence for the existence of such cell determinants was provided by recent investigations using hybridoma technology. The possibility of obtaining monoclonal antibodies (MAbs) against glioma-associated antigens should help to allow their identification, purification, and characterization. Utilizing MAbs as reagents of predefined specificity, a number of central and peripheral nervous system antigens could be detected. The molecules recognized by MAbs in glioma cells can be subdivided into four categories: [1] biochemical defined proteins, [2] specificities shared by nervous system-lymphoid cells, [3] oncoembryonic-oncofetal determinants, and [4] tumor-restricted antigens. Of greater significance is the heterogeneity of antigen expression among various individual glioma cells observed in frozen sections of tumor biopsies. Using a panel of MAbs, the phenotypic heterogeneity, i.e., the variation in antigen expression can be documented within and among malignant gliomas and cell lines derived from them. In spite of this the characteristic pattern of antibody binding to brain tumors makes MAbs the potentially best reagents for immuno-histochemical application in surgical neuropathology. Moreover, immuno-cytological screening of tumor cells in the cerebrospinal fluid has also proved to be valuable. The localization of radio-labelled MAbs in experimental and human gliomas growing subcutaneously and intracranially in athymic nude mice were explored by radioscintigraphy and autoradiography. Imaging experiments with 131I-labelled MAbs recognizing epitopes on the glioma cell surface showed high levels of specific activity in xenografts. Preliminary data indicate that administration of 131I-MAbs as well as drug conjugates (daunomycin-MAbs) causes a depression of glioma cell proliferation in vitro as well as delayed tumor growth and thus prolonged survival time of tumor-bearing mice. The mechanisms of antibody delivery and transport of "immunotoxins" from the vascular compartment to intracerebral tumor tissue are presently a subject of discussion. The complexity of this area necessitates comprehensive experimental work in order to define the factors involved in the delivery of MAbs to brain to tumor tissue and thus optimize the rate of blood-to-tumor transport. Current investigations have shown that it is possible to image malignant human gliomas using radio-labelled antibodies. The next step will be to attain target immunotherapy. The use of MAbs as carrier molecules for clinical applications might soon be possible.

Immunocytochemical Localization of Glia-Derived Nexin, Laminin and Fibronectin on the Surface or Extracellular Matrix of C6 Rat Glioma Cells, Astrocytes and Fibroblasts.

The expression and cellular distribution of glia-derived nexin (GDN), laminin and fibronectin on C6 rat glioma cells, rat brain astrocytes and rat fibroblasts were investigated by immunoblotting and immunocytochemistry. Western blot analysis of C6 cell homogenates confirmed the specificities of the antibodies. Immunocytochemical staining of C6 cells, astrocytes and fibroblasts showed that laminin, fibronectin and GDN were abundant on the surface of glioma cells and astrocytes whereas on fibroblasts fibronectin was abundant though only traces of GDN and laminin could be detected. The light microscopy data were confirmed by ultrastructural studies showing that each antigen was present on the surface of the C6 rat glioma cells as numerous spots with slightly different distribution patterns for each of the antigens. In fibroblast cultures, the antigens were also localized in the extracellular matrix in the vicinity of the cells. Migrating fibroblasts but not migrating glioma cells or astrocytes deposit the matrix-proteins onto the substratum leaving behind a track of GDN, laminin and fibronectin. When the cells were treated with heparin prior to antibody incubation, the GDN immunoreactivity completely disappeared, whereas the distribution and abundance of laminin and fibronectin was not affected. Our data show that GDN binds, possibly by a heparin-like molecule, to the outer surface of cells or to the extracellular matrix and may protect cells and matrix proteins against proteolytic degradation.

Detection of human glioma-associated antigen by rat monoclonal antibody raised against syngeneic rat glioma cells.

A monoclonal antibody termed "FR77" was obtained from a hybridoma clone established by fusion between P3x63Ag8.653 mouse myeloma cells and spleen cells of a Fischer F344 rat hyperimmune to syngeneic 9L/R3 glioma cells. Immunoperoxidase staining of various cultured cells showed that FR77 was reactive to both rat and human glioma cells, but was not reactive with other nonglioma cells. Immunohistochemical examination of paraffin-embedded or cryostat-frozen sections of various human tissues revealed that FR77 was strongly reactive with glioblastoma, grade III astrocytoma, and craniopharyngioma; partially reactive with intracerebral primitive neuroectodermal tumor, pineoblastoma, and desmoplastic medulloblastoma; and weakly reactive with low-grade astrocytoma. It was not reactive with other types of brain tumors and normal human tissues tested. The FR77-defined antigen was observed to be predominantly localized in the cytoplasm of antigen-bearing cells as suggested by the immunostaining pattern, but part of it was also expressed on the cell surface of glioma cells as demonstrated by a complement-mediated cytotoxic test. Fractionation of the antigenic component and periodic acid treatment of tumor tissue bearing the FR77-defined antigen indicated that the antigen is of a neutral glycolipid nature and that the antigenic determinant to FR77 is present on its sugar portion.

In vitro studies on the cell-mediated immune response to human brain tumors. II. Leukocyte-induced coats of glycosaminoglycan increase the resistance of glioma cells to cellular immune attack.

We have examined the ability of cultured human glioma cells to elicit allogeneic cytolytic lymphocyte responses in vitro in order to delineate properties of glioma cells that may contribute to their ability to escape cellular immune attack. When glioma cells were cultured together with allogeneic peripheral blood mononuclear cells (PBMC) in mixed lymphocyte-tumor cultures (MLTC), it was observed that cells from eight of 12 glioma lines were surrounded by clear pericellular "halos," which appeared to impede contact between PBMC and the glioma cells. Enzymatic, histochemical, and immunochemical studies indicated that these halos represented glycosaminoglycan (GAG) coats that contained hyaluronic acid (HA) as a major constituent. Electron microscopic studies demonstrated the presence of many thin microvillous processes spanning the width of the halos. The presence of GAG coats around glioma cells in MLTC reduced the generation of cytolytic T lymphocytes specific for antigens on the glioma cells. Likewise, these cell coats decreased the lysis of glioma cells by cytolytic lymphocytes, once generated. The production of thick coats of GAG by glioma cells was induced by interaction of glioma cells with a nondialyzable factor produced by PBMC in culture. This factor did not cause glioma cells to release increased amounts of HA into the medium, but rather increased the production of HA that remained associated with the glioma cell surface. The formation of thick, protective GAG coats by glioma cells as a result of their interaction with the PBMC-derived factor constitutes a nonspecific suppressor mechanism that may contribute to the ability of this class of human solid tumors to evade cellular immune attack.

Electron-microscopic visualization of binding of antibodies from sera of glioma patients on cultured glioma cells.

Several human cell lines (normal and neoplastic glia, cerebral metastases from adenocarcinoma, fibroblasts) were incubated with sera from patients with well and poorly differentiated glioma and with sera from healthy donors and then stained with PAP complex to define and localize the antibody reaction with cell surface antigens by means of electron microscopy. The sera of glioma patients proved to contain antibodies which bound the tumor-associated antigenic determinants on the cell membranes of gliomas and of cerebral metastases from adenocarcinoma in tissue cultures. Further, absorption testing of the reactive sera on normal brain, well-differentiated astrocytoma and cultured glioblastoma cells, together with cross-reactivity experiments suggests that at least two antigens or groups of antigens are expressed on the glioma cell surface: one shared by well and poorly differentiated glioma cells and the other by poorly differentiated glioma cells and the cells of cerebral metastases from adenocarcinoma.

Complement fixation by IgM and IgG autoantibodies on cultured human glial cells.

The humoral immune response to autologous gliomas has been investigated in 16 patients using immune adherence and anti-C3 immunofluorescence assays of serum samples. Significant antiglioma antibody activity was detected in 56% of these sera. Fractionation experiments demonstrated that IgM class antibodies were more effective that IgG in their ability to fix complement on cultured glioma cell membranes. In addition, autologous IgG was shown to inhibit IgM complement-fixing activity in some experiments. "Capping" of immune complexes on the glioma cell surface is also described.

Cell-Mediated Cytotoxicity and Serum-Mediated Blocking: Evidence that Their Associated Determinants on Human Tumor Cells are Different

The preceding paper showed that patients with gliomas may have lymphocyte-mediated cytotoxic activity (LMC) directed against at least two determinants on the glioma cell surface. The present study showed that serum from patients with gliomas could block this LMC. The blocking activity, however, was specific for different determinants on the glioma cell than those to which the LMC was directed. Blocking activity was specific for tumor cells homotypic to those of the serum donor. It was effective, however, in blocking the cytotoxic activity against these cells of lymphocytes from patients with tumors either homotypic or heterotypic to that of the serum donor. Likewise, although patients with glioblastomas or melanomas had LMC against fetal glial cells, sera from such patients were unable to block the LMC against these fetal glial targets. The specificity of the blocking activity was confirmed by absorption of the sera with various normal and neoplastic cells. These studies have thus shown an immunologic functional dichotomy among different determinants on the glioma cell surface.

On the availability of certain metabolites at the outer surface of normal and malignant cells for the membranous de novo synthesis of ATP and other nucleotides.

The formation of ATP at the cell surface of intact glia and glioma cells in culture has been established. The ATP-forming capacity at the surface of the malignant cells was several times greater than that of the normal glia cells. The ATP-forming capacity was about the same on reincubation one hour after the first incubation. The cells were kept in Eagle's medium in the meantime. ADP, NAD+ and 3-phosphoglyceraldehyde could all be available from a postulated intramembranous metabolic pool and take part in biochemical reactions at the cell surface, provided that albumin was not present in the incubation medium. An incubation medium which was complete except for 3-phosphoglyceraldehyde was only slightly less effective as regards ATP formation at the surface of both glia and glioma cells, compared with the complete incubation medium. The presence of nucleoside diphosphate kinase at the glioma cell surface was confirmed. When intact cells were incubated with only the phosphoryl group donor (ATP) of the reaction but with the acceptor nucleoside diphosphates (CDP, GDP, UDP) ommitted, only CTP and GTP were formed. No UTP was found. Thes latter results indicate that both CDP and GDP are available from the postulated intramembranous metabolic pool, while UDP is not.

(32-P) Phosphoryl transfer by endogenous protein kinase at the glia and glioma cell surface in culture into extrinsic acceptor proteins.

(32-P) Phosphoryl transfer by endogenous protein kinase at the glia and glioma cell surface in culture into extrinsic acceptor proteins.