Purification and amino acid analysis of two human glioma-derived monocyte chemoattractants.

Two chemoattractants for human monocytes were purified to apparent homogeneity from the culture supernatant of a glioma cell line (U-105MG) by sequential chromatography on Orange A-Sepharose, an HPLC cation exchanger, and a reverse phase HPLC column. On SDS-PAGE gels under reducing or nonreducing conditions, the molecular masses of the two peptides glioma-derived chemotactic factor 1 and 2 were 15 and 13 kD, respectively. Amino acid composition of these molecules was almost identical, and differed from other cytokines that have been reported. The NH2 terminus of each peptide was apparently blocked. When tested for chemotactic efficacy, the peptides attracted approximately 30% of the monocytes added to chemotaxis chambers, at the optimal concentration of 10(-9) M. Potency and efficacy were comparable with that of FMLP, which is often used as a reference attractant. The activity was chemotactic rather than chemokinetic. In contrast to their interaction with human monocytes, the pure peptides did not attract neutrophils. These pure tumor-derived chemoattractants can now be compared with attractants produced by normal cells and evaluated for their biological significance in human neoplastic disease.

Receptors for B cell stimulatory factor 2. Quantitation, specificity, distribution, and regulation of their expression.

B cell stimulatory factor 2 receptors (BSF-2-R) were studied using radioiodinated recombinant BSF-2 with a specific activity of 6.16 X 10(13) cpm/g. Kinetic studies showed that binding of 125I-BSF-2 to CESS cells reached maximum level within 150 min at 0 degrees C. There was a single class of receptors with high affinity (Kd 3.4 X 10(-10) M) on CESS, and the number of receptors was 2,700 per cell. Binding of 125I-BSF-2 to CESS was competitively inhibited by unlabeled BSF-2 but not by IL-1, IL-2, IFN-beta, IFN-gamma, and G-CSF, indicating the presence of the receptors specific for BSF-2. EBV-transformed B lymphoblastoid cell lines (CESS, SKW6-CL4, LCL13, and LCL14) expressed BSF-2-R, whereas Burkitt's lines did not. EBV or EBNA2 did not induce the expression of the receptors on Burkitt's cells. The plasma cell lines (ARH-77 and U266) expressed BSF-2-R, fitting the function of BSF-2 as plasma cell growth factor. Several other cell lines, the histiocytic line U937, the promyelocytic line HL60, the astrocytoma line U373 and the glioblastoma line SK-MG-4, in which BSF-2 was inducible with IL-1 or TPA, displayed BSF-2-R with Kd in the range of 1.3-6.4 X 10(-10) M, suggesting the autocrine mechanism in BSF-2 function. The four T cell lines (CEM, HSB, Jurkat, and OM 1) did not express a detectable number of receptors, but normal resting T cells expressed 100-1,000 receptors per cell. BSF-2-R were not present on normal resting B cells but expressed on activated B cells with a Kd of 3.6-5.0 X 10(-10) M, fitting the function of BSF-2, which acts on B cells at the final maturation stage to induce immunoglobulin production.

Hexabrachion proteins in embryonic chicken tissues and human tumors.

Cell cultures of chicken embryo and human fibroblasts produce a large extracellular matrix molecule with a six-armed structure that we called a hexabrachion (Erickson, H. P., and J. L. Iglesias, 1984, Nature (Lond.), 311:267-269. In the present work we have determined that the myotendinous (M1) antigen described by M. Chiquet and D. M. Fambrough in chicken tissues (1984, J. Cell Biol., 98:1926-1936), and the glioma mesenchymal extracellular matrix protein described by Bourdon et al. in human tumors (Bourdon, M. A., C. J. Wikstrand, H. Furthmayr, T. J. Matthews, and D. D. Bigner, 1983, Cancer Res. 43:2796-2805) have the structure of hexabrachions. We also demonstrate that the M1 antigen is present in embryonic brain, where it was previously reported absent, and have purified hexabrachions from brain homogenates. The recently described cytotactin (Grumet, M., S. Hoffman, K. L. Crossin, and G. M. Edelman, 1985, Proc. Natl. Acad. Sci. USA, 82:8075-8079) now appears to be identical to the chicken hexabrachion protein. In a search for functional roles, we looked for a possible cell attachment activity. A strong, fibronectin-like attachment activity was present in (NH4)2SO4 precipitates of cell supernatant and sedimented with hexabrachions in glycerol gradients. Hexabrachions purified by antibody adsorption, however, had lost this activity, suggesting that it was due to a separate factor associated with hexabrachions in the gradient fractions. The combined information in the several, previously unrelated studies suggests that hexabrachions may play a role in organizing localized regions of extracellular matrix. The protein is prominently expressed at specific times and locations during embryonic development, is retained in certain adult tissues, and is reexpressed in a variety of tumors.

beta-Internexin, a ubiquitous intermediate filament-associated protein.

In this article we show a Triton-insoluble, intermediate filament-associated protein of approximately 70 kD to be expressed ubiquitously in diverse mammalian cell types. This protein, assigned the name beta-internexin, exhibits extreme homology in each of the various cell lines as demonstrated by identical limited peptide maps, similar mobilities on two-dimensional gels, and detection in Triton-soluble and -insoluble extracts. beta-Internexin also shares some degree of homology with alpha-internexin, an intermediate filament-associated protein isolated and purified from rat spinal cord, which accounts for the immunologic cross-reactivity displayed by these polypeptides. Light microscopic immunolocalization of beta-internexin with a monoclonal antibody (mAb-IN30) reveals it to be closely associated with the vimentin network in fibroblasts. The antigen is also observed to collapse with the vimentin reticulum during the formation of a juxtanuclear cap induced by colchicine treatment. Ultrastructural localization, using colloidal gold, substantiates the affinity of beta-internexin for cytoplasmic filaments and, in addition, demonstrates its apparent exclusion from the intranuclear filament network. We examine also the resemblance of beta-internexin to a microtubule-associated polypeptide and the constitutively synthesized mammalian heat shock protein (HSP 68/70).

The human laminin receptor is a member of the integrin family of cell adhesion receptors.

A receptor for the adhesive basement membrane protein, laminin, was isolated from human glioblastoma cells by affinity chromatography on laminin. This receptor has a heterodimeric structure similar to that of receptors for other extracellular matrix proteins such as fibronectin and vitronectin. Incorporation of the laminin receptor into liposomal membranes makes it possible for liposomes to attach to surfaces coated with laminin. The receptor liposomes also attached to some extent to surfaces coated with fibronectin, but not with other matrix proteins. These properties identify the laminin receptor as a member of the integrin family of cell adhesion receptors.

Immunohistochemical localization of apolipoprotein E in human glial neoplasms.

Immunocytochemical analyses revealed the presence and distribution of apolipoprotein E (apo E) in normal human brain tissue as well as in 77 human intracranial neoplasms. In normal brain tissues, the perikarya of astrocytes exhibited a strong positive reaction, whereas the Bergmann glia were stained to a moderate degree. However, no immunoreactivity was observed with neurons, oligodendrocytes, ependymal cells, and choroidal epithelium. Among the intracranial neoplasms, oligodendroglioma, choroid plexus papilloma, hemangioblastoma, primary malignant lymphoma, neurinoma, meningioma, pituitary adenoma, and craniopharyngioma were all negative. Immunoreactivity in the peripheral neuroblastoma was nil. However, the perikarya of astrocytomas and glioblastomas showed a positive reaction. Analyses on the degree of anaplasia and the amount of apo-E as an intensity of immunostaining showed a negative correlation. The astrocytic elements were stained in mixed oligoastrocytomas and medulloblastomas with glial differentiation. A few cases of ependymomas showed weak perikaryal immunostaining. Western blot analyses with anti-apo E antibody of a freshly prepared surgical specimen with astrocytomas revealed a single band with a molecular weight of approximately 37,000. The well differentiated cultured human astrocytoma cells secreted apo E into the medium. These lines of evidence suggest that apo E may serve as a potential marker specific for astrocytomas and glioblastomas, as well as an indicator of astrocytic tumor cell differentiation. The apo E localization in human brain tumors could be clinically relevant and diagnostically useful.

Amount of satellite DNA in four experimentally induced tumors of the central nervous system. Quantitative changes in a glioblastoma producing C-type particles.

The quantitative preservation of satellite NA was studied in several central nervous system (CNS) neoplasms; four tumor lines deriveo from 3-methylcholanthrene implantation into the CNS of mice were compared with brain and tissue cultures of normal mouse cells by analytical centrifugation in cesium chlorie. Three tumors showed no detectable difference from normal cells; nuclear and whole cell preparations were comparable. Only a glioblastoma line proucing C-type particles (TC509) revealed a significant difference from normal cells and exhibited a decrease of approximately 20% in satellite DNA or 2% of the total DNA on repeated examination for 1 year. C-type RNA virus may be related to relative decreases in satellite DNA observed in TC509.

Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate.

The activity of the serine protease plasminogen activator (PA), which correlates with tumorigenicity and metastatic capacity, was examined using the 125I-labeled fibrin plate assay in cell extracts from four human glioma lines as a function of growth in vitro. Cell-associated inhibitory activity to plasmin and urokinase-type PA was also measured concurrently. The relative PA activities differed markedly among the lines, whereas inhibitory activities did not. Two lines, SNB-19 and SNB-75, exhibited maximal PA activities (1-6 m Plough units/micrograms protein) as cultures approached confluence, whereas two other lines, SNB-56 and SNB-78, expressed low PA activity at all times (less than 0.2 m Plough units/micrograms protein). The PA of SNB-19 cell extracts was predominantly urokinase-type PA. In addition to having the highest PA levels, SNB-19 and SNB-75 were the most clonogenic in soft agar and tumorigenic in nude mice. In contrast, SNB-56 and SNB-78 were poorly clonogenic in soft agar and were not tumorigenic in nude mice. Measured directly, inhibitory activities to plasmin, urokinase-type PA, and tissue-type PA were detected in SNB-19 (high PA) and SNB-56 (low PA) cell extracts. However, there were no qualitative or quantitative differences in inhibitor effects between SNB-19 and SNB-56 suggesting that the differences in PA activity between these lines resulted from changes in PA activity and were not due to differential plasminogen activator inhibitor effects. The ability of the differentiating agent sodium butyrate (NaB) to modulate total PA activity was also examined. Peak SNB-19 cell PA activity was decreased in a concentration (Ki, 0.75 mM) and time-dependent manner by the addition of nontoxic amounts of NaB. The dose-dependent decrease in PA activity induced by NaB was most likely due to an effect on PA itself, since the action of inhibitor on urokinase was unchanged in response to NaB. These results suggest that net cellular PA activity in glioma cells is a balance between relative PA activity and inhibitor(s) effects and that this balance can be modulated by sodium butyrate.

Expression of two types of receptor for insulin-like growth factors in human malignant glioma.

Two types of receptors for insulin-like growth factors (IGFs) were characterized in glioma cell lines established from different human brain tumors of glial origin (astrocytoma grades III and IV) by competitive binding assay, affinity labeling, and protein phosphorylation. Type I IGF receptor is a heterodimer composed of alpha-subunits (Mr 130,000), which bind IGF I and II with equal affinity, and of beta-subunits (Mr 98,000), which show tyrosine kinase activity and autophosphorylation stimulated by IGF I and II with equal potency. The type II IGF binding site is a monomer (Mr 250,000) which binds IGF II with 10 times higher affinity than IGF I. The cellular concentration of type II IGF binding site is about 2- to 5-fold higher than the amount of type I IGF receptor. The characteristics of the two types of IGF receptors in human glioma cell lines are similar to those described recently in fetal rat astrocytes. In contrast the type I IGF receptor in glioma cells is different from that studied previously in normal adult brain regarding the equal affinity for IGF I and II, and the higher molecular size of the alpha-subunit (130,000 versus 115,000). It is suggested that glioma cells may represent a fetal cell type in tumor development of adult human brain. A role of IGF in malignant glioma has not yet been determined, but the presence of IGF receptors is a prerequisite for cellular actions of IGF.

Characterization of the gamma-aminobutyric acid receptor system in human brain gliomas.

The properties of [3H]-gamma-aminobutyric acid [( 3H]GABA) binding were studied in biopsied specimens from normal human brain and from 18 cases of human brain gliomas, made up of 6 astrocytomas, 6 glioblastomas, 3 oligodendrogliomas, and 3 medulloblastomas. In fresh membranes obtained from normal gray and white matter one population of Na+-dependent GABA receptors was observed, while in the frozen Triton X-100-treated membranes two distinct populations of Na+-independent binding sites were detected. Specific GABA binding sites in brain gliomas were shown only in frozen Triton X-100-treated membranes. As in normal tissue, these receptors are Na+-independent and bind [3H]GABA with two distinct affinity components. The biochemical profiles of [3H]GABA binding to membranes obtained from different tumors of glial origin are quite similar and cannot be related to the degree of malignancy of the neoplasia.

Effects of mycophenolic acid on detection of glial filaments in human and rat astrocytoma cultures.

Expression of glial fibrillary acidic protein (GFAP) was assayed in 11 glioma-derived cell cultures. Treatment of cells with an inhibitor of guanine nucleotide biosynthesis, mycophenolic acid, enhanced detection of GFAP by indirect immunofluorescence microscopy. Quantitation of GFAP and vimentin demonstrated that enhanced fluorescence occurs without an increase in the level of intermediate filament proteins. Immunoblots provided the most sensitive method for monitoring GFAP expression and showed the limitations of using immunofluorescence detection methods. GFAP was detectable in cultures derived from malignant Grade IV astrocytomas and its expression was stable during the course of the study. While mycophenolic acid has been reported to induce differentiation in leukemia cells at low concentration (D.L. Lucas et al., J. Clin. Invest., 72: 1889-1990, 1983), its effect on glioma cultures at concentrations of 100 microM was consistent with a role as an inhibitor of DNA synthesis, and as an effector of altered intermediate filament organization.

Immunocytochemical characterization of extracellular matrix proteins expressed by cultured glioma cells.

The immunolocalization of type I, III and IV collagens and fibronectin in two rat glioma cell lines in vitro (BT4C and BT4Cn) is described. In addition, antibodies against denatured type I and III collagens were used to study breakdown products of native type I and III collagens. For the BT4C cells, the extracellular matrix expression in monolayer cultures and in multicellular tumor spheroids was compared. Type IV collagen was strongly expressed in BT4C tumor spheroids but was negative in the corresponding monolayer cultures. Denatured type I collagen was found both in monolayers and in spheroids of BT4C, suggesting either a rapid turnover (i.e., synthesis and immediate breakdown) of type I collagen or an altered collagen gene transcription. Both cell lines were negative for native type I and III and denatured type III collagen. Fibronectin was strongly expressed in both cell lines. Supporting the immunofluorescence data, the hydroxyproline content in the tumor spheroids was twice the amount found in monolayer cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunoblotting also verified the immunostaining experiments, showing that glioma spheroids and injected tumor cells have the potential for fibronectin and collagen production, given the appropriate growth conditions.

Radioimmunodetection of human glioma xenografts by monoclonal antibody to epidermal growth factor receptor.

Murine IgG2a monoclonal antibody (MAb) 425 specifically detects epidermal growth factor receptor, which is expressed on human gliomas and tumors of other tissue origin but rarely on normal brain tissues, and not at all on bone marrow and peripheral blood cells. 131I-labeled F(ab')2 fragments of this MAb injected into nude mice grafted with U-87 MG glioma cells preferentially localized in tumor tissue compared to normal mouse tissues, as determined by differential tissue counting of radioactivity. The mean tumor-to-tissue ratios of radioactivity ranged between 8.2 (blood) and 55.8 (muscle) at 2 days after the injection of 15 muCi of 131I-425 F(ab')2/mouse. Radiolabeled fragments of an anti-hepatitis virus IgG2a MAb did not localize in tumors. The localization index derived from the ratios of specific antibody to indifferent antibody in tumor tissue relative to blood was 9.94 at 2 days following the MAb injection. The labeled MAb did not localize in a xenograft of colorectal cancer tumor, which does not express the epidermal growth factor receptor. Tumors could be located by whole-body gamma-scintigraphy without background subtraction following the injection of 100 muCi of radiolabeled MAb 425 F(ab')2 fragments. The data suggest that MAb 425 is a likely candidate for clinical diagnostic and radioimmunotherapy trials.

Immunolocalization of extracellular matrix proteins during brain tumor invasion in BD IX rats.

The distribution of several native extracellular matrix proteins (type I, III, and IV collagens and fibronectin) using immunofluorescent localization is described for in two different malignant gliomas (BT4A and BT4An). In addition, antibodies against denatured forms of type I and III collagens were used to localize areas of active degradation within the tumors. We have shown that both tumors express the native connective tissue components studied, although the distribution of these components within and between the tumors was different. In addition, native type I and III collagens and fibronectin were overexpressed in the tumors compared to the normal brain. Morphometry on immunostained type IV collagen sections showed an increase in vascular elements in both tumors compared to normal brain tissue. The BT4A tumor, which by light microscopy showed a degradative mode of invasion, expressed denatured type I and III collagens at the tumor-brain border zone, suggesting that this tumor has collagenolytic activity. The present article suggests that the distribution and changes in extracellular matrix protein synthesis and degradation may play an important role in the progressive growth of brain tumors in vivo.

Altered expression and distribution of heparan sulfate proteoglycans in human gliomas.

The expression of heparan sulfate proteoglycans (HSPGs) by human glioma cells was examined by biochemical and immunological methods in vitro and in vivo. Chondroitin sulfate was shown to represent the major [3H]glucosamine-labeled glycosaminoglycan synthesized by cultured normal brain cells. However, high-grade glioma-derived cells were shown to express significantly increased quantities of hyaluronic acid and heparan sulfate and approximately equal amounts of chondroitin sulfate compared with normal glial cells. To investigate further the differential expression of HSPGs, proteoglycans were isolated from glioma cells and were used as an immunogen to generate monoclonal antibodies (MAbs). One of these MAbs, 39H (an IgM), was shown to bind more to high-grade glioma-derived cells then to low-grade glioma or normal brain cells in vitro. MAb 39H was also observed to bind to isolated HSPGs but not to heparan sulfate glycosaminoglycan chains or trypsin-treated cells. Immunofluorescence staining of the cultured high-grade glioma cells revealed an intense diffuse cell surface staining pattern over the entire cell and also isolated footpads. In contrast, the low-grade tumor or normal glial cells showed a distinctive punctated staining. A similar differential staining of MAb 39H was most prominent between tissue sections of glioblastoma multiforme and anaplastic astrocytomas versus low-grade astrocytomas and normal brain. The low grade gliomas exhibited a weak punctated staining, whereas the high-grade gliomas showed significantly more intense staining, particularly along the apical regions of the cells. These results suggest that altered expression of HSPGs may be related to the malignant transformation or growth potential of glial-derived cells.

Differential spectrum of expression of neural cell adhesion molecule isoforms and L1 adhesion molecules on human neuroectodermal tumors.

A series of four medulloblastomas, seven neuroblastomas (Nb), two ependymomas, and three gliomas, human neuroectodermal tumors, were screened for their expression of adhesion molecules L1, carcinoembryonic antigen, neural cell adhesion molecule isoforms (N-CAM) and HNK1 epitope by Western blotting and double immunofluorescence labeling. All seven neuroblastomas, whatever their differentiated state, expressed L1, a neural cell surface developmental antigen, whereas all other tumors tested were negative. All tumors expressed N-CAM; however, a large diversity was observed among the isoforms. Low sialylated N-CAM 140 was present, with different intensity, in ependymomas and astrocytomas. High sialylated isoforms were detected by a monoclonal antibody (anti-MenB) specifically recognizing high polymers of alpha 2-8 linked neuraminic acid. They were expressed in all medulloblastomas studied (4 of 4), and in 4 of 7 Nbs examined. Negative cases corresponded to tumors having undergone chemotherapeutic treatment or to ganglioneuroma. The interconversion from high to low sialylated forms might reflect changes which are critical for the conversion of Nbs into benign ganglioneuromas. HNK1 epitope was expressed on a large diversity of molecules by nearly all tumors except two Nbs which were also negative with anti-MenB antibody. This simultaneous loss of carbohydrate epitopes could correlate with higher maturation states of the tumors. None of the tumors expressed carcinoembryonic antigen. Therefore, anti-L1 and anti-MenB antibodies define differentiation-related antigens that could differentiate between Nbs and other tumors and may prove helpful in diagnosis and understanding of the biological nature of neuroectodermal tumors. An immunodot assay was set up and allowed to titrate the presence of polysialic acid units in cerebrospinal fluid from patients presenting meningeal spread of medulloblastomas. It could help to assess metastasis and to monitor the effects of chemotherapeutic treatment on polysialylated N-CAM positive tumors.

A two-dimensional polyacrylamide gel electrophoresis system for the analysis of mammalian cell surface proteins.

A detailed analysis of mammalian cell surface proteins is described by a new two-dimensional polyacrylamide gel electrophoresis technique. The first dimension gel contains 2% acrylamide, 0.1% sodium dodecyl sulfate, 0.3% Triton CF10 and 9 M urea. A combination of the detergents and urea permits the separation of poorly soluble, hydrophobic cell surface proteins. Under these conditions, the molecular size of proteins has a limited contribution to the fianl separation due to a low acrylamide concentration. Differences in charge properties, hydrophobicity, and glycosylation are the elements determining the resolution. In the second dimension, the proteins are separated primarily according to molecular weights, by a conventional polyacrylamide gel system in the presence of 0.1% sodium dodecyl sulfate. In this study, proteins of C6 rat glioma cell line are characterized. Cell surface proteins are specifically radio-labeled with 125I by a lactoperoxidase method, and compared with presumptive integral surface proteins which are resistant to extraction with 0.1 M NaOH. Also studied are total cellular proteins, fucose- and glucosamine-containing glycoproteins, and protein species with variable susceptibility to weak trypsin digestion. The electrophoresis system allows an unambiguous identification of each protein species.

Glial fibrillary acidic protein synthesized in vitro using messenger RNA from a human glioma cell line.

Messenger RNA (mRNA) extracted from a continuous human glioma cell line grown in culture or as a solid tumor was translated in an mRNA-dependent reticulocyte lysate system. Translation products labeled with [35S]methionine were immunoprecipitated with antiserum specific for glial fibrillary acidic (GFA) protein, separated by one- and two-dimensional polyacrylamide gel electrophoresis and analyzed fluorographically. Immunoprecipitates from both cell culture and tumor mRNA translations had a molecular weight of 49,000 daltons, consistent with GFA protein extracted from human tissue. In two dimensions, the 49,000-dalton band resolved into two to three spots at pH 5.7-5.9, the isoelectric point of GFA protein. Minor lower molecular weight products were detected in fluorographs of heavily overloaded gels or in film exposed for extended periods of time. These data indicate that the GFA protein produced by this glioma cell line is chemically and immunologically similar to normal human GFA protein, which suggests that the primary phenotypic expression of GFA protein in this tumor cell line is not altered by the neoplastic process.

Granular cell glioblastoma: a malignant granular cell neoplasm of astrocytic origin.

Granular cell tumors in the cerebral hemispheres are rare and of unknown origin. Two large aggressive cerebral neoplasms composed mainly of granular cells and also containing neoplastic astrocytes were studied histologically, ultrastructurally and immunohistochemically. In both instances, transitional forms between astrocytes and typical granular cells were demonstrated. In the second case which consisted of a central core of typical glioblastoma surrounded by a thick shell of granular cells, the latter spread in a fashion that is characteristic of intrinsic tumors of the central nervous system by forming abundant secondary structures of Scherer, particularly under the pia and around cortical blood vessels. All the evidence indicates that in these two cases granular cells were transformed neoplastic astrocytes. Paradoxically, many granular cells exhibited benign cytological features that belied their malignant nature.

Application of flow cytometry to analyses of cultured human glioma and fetal brain cells.

Methods are described to study cell surface and cytoplasmic antigens of cultured human glioma, fetal brain cells and fibroblasts using flow cytometry. This required harvesting the cultured cells with Versene or mild trypsin treatment and fixation in 4% paraformaldehyde prior to staining for glial fibrillary acidic protein (GFAP) and fibronectin using indirect immunofluorescence. At passage 10, 38% of fetal brain cells [CHII] were GFAP-positive but at passage 14 only 3.5% expressed GFAP. Two glioblastomas and an anaplastic astrocytoma had 38.8%, 6.7% and 81.3% GFAP-positive cells, respectively. Of the 10(4) cells studied, 91.6%, 79.1% and 40.8% were fibronectin-positive for glioblastoma multiforme [12-18], oligodendroglioma [12-10] and fetal brain [CHII] cells, respectively. Two fibroblast lines had 33.5% and 43.1% of the cells expressing fibronectin. The validity of these results was confirmed by staining for GFAP and fibronectin using peroxidase-antiperoxidase and immunofluorescence microscopy. Using low angle forward light scatter to estimate cell size and gating techniques it was found that GFAP-positive CHII and anaplastic astrocytoma cells were generally larger than GFAP-negative cells of the same type. No correlation between cell size and fibronectin expression was found for glioblastoma [12-18] cells. These results demonstrate the validity of the described methods and illustrate some specific applications and the potential value of flow cytometry to neurooncology.