Bromelain treatment leads to maturation of monocyte-derived dendritic cells but cannot replace PGE2 in a cocktail of IL-1ß, IL-6, TNF-α and PGE2.

Immunotherapy using dendritic cells (DC) has shown promising results. However, the use of an appropriate DC population is critical for the outcome of this treatment, and the search for an optimal DC subset is still ongoing. The DC used in immunotherapy today are usually matured with a cytokine cocktail consisting of TNF-α, IL-1ß, IL-6 and PGE(2). These cells have deficits in their cytokine production, particularly IL-12p70, mainly because of the presence of PGE(2). Bromelain is a pineapple stem extract containing a mixture of proteases that has been used clinically in adjuvant cancer treatment. In this study, we analysed the effect of bromelain on human monocyte-derived DC. We added bromelain to the cytokine cocktail and modified cytokine cocktails with either no PGE(2) or reduced amounts of PGE(2), respectively. Combining bromelain with the cytokine cocktails containing PGE(2) resulted in an increased surface expression of CD83, CD80 and CD86. The chemokine receptor CCR7 was also considerably upregulated in these DC populations compared with DC treated with the cytokine cocktail alone. Removal or reduction of PGE(2) from the cytokine cocktail did not increase the IL-12p70 secretion from stimulated DC, and addition of bromelain to the different cytokine cocktails resulted in only a minor increase in IL-12p70 production. Moreover, combining bromelain with the cytokine cocktails did not improve the T cell stimulatory capacity of the generated DC populations. In conclusion, bromelain treatment of monocyte-derived DC does not improve the functional quality compared with the standard cytokine cocktail.

Highly infiltrative brain tumours show reduced chemosensitivity associated with a stem cell-like phenotype.

AIMS: Cancer stem-like cells might have important functions in chemoresistance. We have developed a model where highly infiltrative brain tumours with a stem-like phenotype were established by orthotopic transplantation of human glioblastomas to immunodeficient rats. Serial passaging gradually transformed the tumours into a less invasive and more angiogenic phenotype (high-generation tumours). The invasive phenotype (low-generation tumours) was characterized by an increase in stem cell markers and increased phosphorylation of kinases in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. These markers were reduced in the serially passaged vascular tumours. The present study was aimed at investigating how the two phenotypes responded in vitro to doxorubicin, a clinically potent cytotoxic drug for solid tumours. METHODS: Biopsy spheroids were implanted and passaged intracranially in nude rats. Gene expression and protein analyses were performed, and drug sensitivity was assessed. RESULTS: Microarray analysis revealed gene ontology categories connected to developmental aspects and negative regulators of differentiation, especially in the infiltrative stem cell-like tumours. The highly invasive stem-like phenotype was chemoresistant compared with the angiogenic phenotype. By interfering with the PI3K it was possible to sensitize tumour spheroids to chemotherapy. Real-time quantitative polymerase chain reaction showed downregulation of the stem cell markers Nestin and Musashi-1 in low-generation biopsy spheroids following PI3K inhibition. CONCLUSIONS: Highly invasive tumours with a stem-like phenotype are more chemoresistant than angiogenic tumours derived from the same patients. We suggest that treatment resistance in glioblastomas can be related to PI3K/AKT activity in stem-like tumour cells, and that targeted interference with the PI3K/AKT pathway might differentiate and sensitize this subpopulation to chemotherapy.

The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signaling.

Chemoresistance represents a major problem in the treatment of many malignancies. Overcoming this obstacle will require improved understanding of the mechanisms responsible for this phenomenon. The progenitor cell marker NG2/melanoma proteoglycan (MPG) is aberrantly expressed by various tumors, but its role in cell death signaling and its potential as a therapeutic target are largely unexplored. We have assessed cytotoxic drug-induced cell death in glioblastoma spheroids from 15 patients, as well as in five cancer cell lines that differ with respect to NG2/MPG expression. The tumors were treated with doxorubicin, etoposide, carboplatin, temodal, cisplatin and tumor necrosis factor (TNF)alpha. High NG2/MPG expression correlated with multidrug resistance mediated by increased activation of alpha3beta1 integrin/PI3K signaling and their downstream targets, promoting cell survival. NG2/MPG knockdown with shRNAs incorporated into lentiviral vectors attenuated beta1 integrin signaling revealing potent antitumor effects and further sensitized neoplastic cells to cytotoxic treatment in vitro and in vivo. Thus, as a novel regulator of the antiapoptotic response, NG2/MPG may represent an effective therapeutic target in several cancer subtypes.

The glial precursor proteoglycan, NG2, is expressed on tumour neovasculature by vascular pericytes in human malignant brain tumours.

Glial precursor cells express NG2 and GD3 in the developing brain. These antigens are both over-expressed during neoplasia, which suggests they may have specific functions in the malignant progression of human brain tumours. This study describes the expression of NG2 and GD3 in 28 paediatric and adult brain tumours. Glioblastoma biopsy spheroids were also implanted into nude rats to assess the regional distribution of the molecules within the tumour. These xenografts showed extensive infiltration and growth that mimicked the growth patterns of human gliomas in situ. NG2 was identified in 20 out of 28 brain tumours, where the expression was confined to the main mass of the tumour, and was reduced towards the tumour periphery. NG2 was mainly associated with blood vessels on both the pericyte and basement membrane components of the tumour vasculature. Ki67 (MIB-1) labelling indicated that NG2 expression was associated with areas of high cellular proliferation. Conversely, all the tumours expressed GD3, which was present both in the tumour main mass and throughout the periphery. Thus, the expression of NG2 may be indicative of tumour progression and might be an amenable target for future therapeutic interventions.

Biological mechanisms of glioma invasion and potential therapeutic targets.

The current understanding of glioma biology reveals targets for anti-invasive therapy which include manipulations of extracellular matrix and receptors, growth factors and cytokines, proteases, cytoskeletal components, oncogenes and tumor suppressor genes. A better understanding of the complex regulation and the signalling molecules involved in glioma invasion is still needed in order to design new and effective treatment modalities towards invasive tumor cells. Representative and valid in vitro experimental systems and animal models of gliomas are necessary for the characterization of the invasive phenotype and further development of anti-invasive therapy. In the future, it will probably be important to move from comparative genomic modelling through protein characterization based on advanced proteomic techniques to analyse tissue samples, where the aim for gliomas should be to compare invaded and non-invaded tissue. This will hopefully render promising new therapeutic targets for gliomas.

The glioma-associated gangliosides 3'-isoLM1, GD3 and GM2 show selective area expression in human glioblastoma xenografts in nude rat brains.

This work describes the in vivo expression and distribution of glioma-associated gangliosides (GD3, GM2, 3'-isoLM1) in a novel human brain tumour nude rat xenograft model. In this model, the tumours, which are established directly from human glioblastoma biopsies, show extensive infiltrative growth within the rat brain. This model therefore provides an opportunity to study ganglioside expression not only within the macroscopic tumour, but also in brain areas with tumour cell infiltration. The ganglioside expression was studied by confocal microscopy of immunostained brain sections using antiganglioside monoclonal antibodies. Xenografts from four human glioblastoma multiformes were established in rats and the brains removed after 3-4 months. Ganglioside GD3 was expressed in the tumour parenchyma while ganglioside 3'-isoLM1 was more abundantly expressed in the periphery of the tumour associated with areas of tumour cell invasion. GM2 expression was only seen in one tumour, where it was located within the main tumour mass. Double staining with a pan antihuman monoclonal antibody (3B4) and the antiganglioside monoclonal antibodies confirmed that the ganglioside expression was associated with tumour cells. This work supports the concept of different biological roles for individual gangliosides and indicates that antibodies or ligands directed against GD3 and 3'-isoLM1 might be complementary when applied in the treatment of human glioblastomas.

Bromelain reversibly inhibits invasive properties of glioma cells.

Bromelain is an aqueous extract from pineapple stem that contains proteinases and exhibits pleiotropic therapeutic effects, i.e., antiedematous, antiinflammatory, antimetastatic, antithrombotic, and fibrinolytic activities. In this study, we tested bromelain's effects on glioma cells to assess whether bromelain could be a potential contributor to new antiinvasive strategies for gliomas. Several complementary assays demonstrated that bromelain significantly and reversibly reduced glioma cell adhesion, migration, and invasion without affecting cell viability, even after treatment periods extending over several months. Immunohistochemistry and immunoblotting experiments demonstrated that alpha3 and beta1 integrin subunits and hyaluronan receptor CD44 protein levels were reduced within 24 hours of bromelain treatment. These effects were not reflected at the RNA level because RNA profiling did not show any significant effects on gene expression. Interestingly, metabolic labelling with 35-S methionine demonstrated that de novo protein synthesis was greatly attenuated by bromelain, in a reversible manner. By using a transactivating signaling assay, we found that CRE-mediated signaling processes were suppressed. These results indicate that bromelain exerts its antiinvasive effects by proteolysis, signaling cascades, and translational attenuation.

The effect of heat- and auto-polymerized denture base polymers on clonogenicity, apoptosis, and necrosis in fibroblasts: denture base polymers induce apoptosis and necrosis.

Eluates from poly(methyl methacrylate)-based denture base polymers have recently been found to enhance death by apoptosis and necrosis in U-937 human monoblastoid cells. The present study investigated the potential of such polymers to induce apoptosis and/or necrosis and to alter clonogenicity in L929 murine fibroblasts. A fibroblast cell line was chosen because the impairment of fibroblasts subjacent to denture bases may result in a weaker or more permeable mucosa. Two aspects were addressed: the effect of direct contact with the denture base polymers and the effect of eluates extracted from the polymers. For this purpose L929 fibroblasts were seeded on disks manufactured from three heat-polymerized and four autopolymerized denture base polymers or in different concentrations of their eluates. The effects were evaluated by light, fluorescent, confocal and electron microscopy, counting of colonies, and flow cytometry. Disks and eluates of all polymers enhanced cell death by apoptosis and necrosis in L929 cells and decreased their clonogenic potential in a dose-dependent manner. Apoptosis was the main form of cell death. In general, the deleterious effects were stronger when cells were plated directly on the polymer disks than in the eluates. The autopolymerized polymers, except one, yielded higher percentages of apoptosis and necrosis than the heat-polymerized polymers. The results of the study indicated that poly(methyl methacrylate)-based denture base polymers trigger death-signals in L929 fibroblasts and open doors for possible modulation of the cell/biomaterial interaction.

Alginate-encapsulated producer cells: a potential new approach for the treatment of malignant brain tumors.

In recent years gene therapy has evolved as a new treatment for brain tumors, where genetically engineered cells can be used to deliver specific substances to target cells. However, clinical success has been limited due to insufficient gene transfer, lack of prolonged gene expression, and immunorejection of producer cells. These obstacles may be overcome by encapsulating producer cells into immunoisolating substances such as alginate. This may provide a stable in situ delivery system of specific proteins, which can interfere with tumor growth and differentiation. This article represents a fundamental study describing the in vitro and the in vivo behavior of alginate-encapsulated producer cells. The viability and cell cycle distribution of encapsulated NIH 3T3 cells was studied by confocal laser scanning microscopy (CLSM) and by flow cytometry. The CLSM study showed a high viability of the encapsulated NIH 3T3 cells during 9 weeks in culture. The flow cytometric analysis revealed a change in cellular ploidy after 1 week in culture, with normalization in ploidy after 3 and 9 weeks. The production of the bacterial E. coli beta-galactosidase in alginate-encapsulated BT4CnVlacZ cells was studied by x-gal staining, and the cells expressed prolonged beta-galactosidase activity. H528 hybridoma cells producing monoclonal antibodies (mAbs) against the human epidermal growth factor receptor (EGFR) were encapsulated in alginate, and the mAb release was determined. The release of mAbs stabilized around 400 ng/ml/h after 12 days in vitro. To actually demonstrate that alginate-encapsulated H528 cells potentially inhibit a heterogeneous glioma cell population, cell migration from human GaMg glioma spheroids was studied during stimulation with EGF in the presence of encapsulated H528 cells. The migration in vitro was totally inhibited in the presence of H528 encapsulated cells. Alginate beads with H528 cells were also implanted into rat brains, and after 9 weeks the distribution of mAbs within the brain was studied by immunohistochemistry. It is shown that the alginate entrapped H528 cells produce mAbs inside the brain for prolonged periods and that the mAbs are distributed within all CSF compartments. Encapsulated producer cells represent a potential delivery system for specific proteins to brain tumors. Different producer cells may be encapsulated in alginate to target phenotypic features and microenvironmental factors, which may influence the progressive growth of brain tumors.

Laminin expression by glial fibrillary acidic protein positive cells in human gliomas.

Extracellular matrix components are regarded as important substrates for invasive tumor cells. The present work focuses on the expression of laminin in the brain in response to invading brain tumors. Biopsies obtained from tissue macroscopically evaluated as the border zone between tumor and normal brain, in 5 patients undergoing surgery for glioblastoma multiforme, were examined by immunocytochemistry and scanning confocal microscopy for the expression of laminin and glial fibrillary acidic protein. Laminin was mainly found in all the specimens associated with the basal lamina of blood vessels, but a variable degree of punctate laminin deposits were also observed in the parenchyma not associated with blood vessels. In the specimens with substantial deposits, scanning confocal microscopy showed that some of the laminin co-localized with intracellular glial fibrillary acidic protein. Punctate deposits of laminin were also seen in an intracranial BT4C rat glioma model, where it was particularly abundant in the brain/tumor confrontation zone. Previous in vitro studies have shown that laminin, among several extracellular matrix components, represent a highly permissive substrate for glioma cell migration. The presented results indicate that laminin can be produced by glial fibrillary acidic protein positive cells during glioma cell invasion in humans. This glycoprotein may thus represent one important substrate among many, which contribute to the invasive phenotype of gliomas.

Extracellular matrix-induced cell migration from glioblastoma biopsy specimens in vitro.

The present knowledge about the interaction between the extracellular matrix (ECM) and gliomas is mostly based on studies of permanent cell lines. Since such cultures have undergone an extensive clonal selection in vitro, the experimental results obtained may be quite different from those obtained from studies on true biopsy specimens. The present work demonstrates how different ECM components affect tumor cell migration from human glioblastoma specimens grown as biopsy sample spheroids. Biopsy specimens from 12 glioblastomas and 1 gemistocytic astrocytoma were included in this study. Spheroids were directly initiated from the biopsy specimens, and after 3-4 weeks in culture, they were used in a migration assay. A custom-made filtered medium, where the high molecular weight (>100 kDa) proteins were removed, was supplemented with the following ECM components: laminin, fibronectin, collagen type IV and vitronectin. The cell migration was negligible when spheroids were propagated in the filtered medium. The ECM components as well as complete DMEM evoked strong stimulatory effects on different biopsy specimens. Opposed to that observed earlier for permanent glioma cell lines, highly variable responses were observed between the different biopsy samples on the various ECM components. In general, correlation analyses revealed that specimens that were strongly stimulated by laminin were also stimulated strongly by fibronectin, collagen type IV and vitronectin. This suggests that the capacity to migrate as a response to ECM was confined more to each biopsy specimen than to any specific ECM component. Since biopsy sample spheroids, as original tumors, consist of different cell types, an immunohistochemical characterization of the migrating cells was also performed. Anti-glial fibrillary acidic protein (GFAP) staining revealed both GFAP-positive and -negative migrating cells. Immunostaining for von Willebrand factor and CD11b indicated that the migrating cells were neither endothelial nor microglial cells. This study, therefore, indicates that migratory responses of glioma biopsy specimens to different ECM components is much more heterogeneous than that observed earlier for cell lines. Furthermore, the presented findings support the notion that gliomas may utilize different cell surface receptors for their migration, depending on the cell substrates available.

Stimulation of extracellular matrix components in the normal brain by invading glioma cells.

Malignant gliomas are characterized by an extensive invasion of tumor cells into the normal brain parenchyma. A substantial amount of data indicates that cell movement in general is regulated by specific interactions between extracellular matrix components and specific cell-surface receptors. In the present work, multicellular spheroids from 4 human glioma cell lines (U-373Mg, A-172Mg, U-251Mg and HF-66) were confronted with normal rat brain cell aggregates in vitro, which resulted in a progressive invasion of tumor cells into the brain aggregates. The co-cultures were then sectioned and immuno-stained for specific extracellular matrix components (laminin, fibronectin and collagen type IV) and for specific cell-surface receptors which bind to these components (integrins beta1, beta4, alpha3, alpha6). In addition, flow-cytometric measurements and Northern blot analyses showed expression of several different integrins within the cell lines. The alpha3 subunit was expressed strongly in all cell lines. Whereas the beta1 subunit was expressed weakly in exponentially growing monolayer cultures, it showed a pronounced expression in multicellular spheroids, indicating that the integrin expression may vary depending on the micro-environment within a tumor. Furthermore, normal brain tissue was able to produce laminin when confronted with the glioma cells, which also was observed for fibronectin and collagen type IV. The relevance of our observations to the in vivo situation was investigated further by immuno-staining 5 human glioma biopsy samples for laminin. In some areas of the tumors, specific deposits of laminin were observed. In conclusion, we have shown that normal brain tissue has the ability to produce extracellular matrix components, such as laminin, collagen type IV and fibronectin, when confronted with invading glioma cells. Our results show that the glioma cells express specific integrins which can interact with these extracellular matrix components. Such interactions may facilitate tumor cell migration and invasion.

Role of high molecular weight extracellular matrix proteins in glioma cell migration.

Malignant human gliomas are characterized by an uncontrolled cell proliferation and infiltrative growth within the brain. Complete surgical removal is difficult due to disseminated tumour cells, and the fundamental mechanisms responsible for this spread are poorly understood. An extensive tumour cell movement along blood vessels is frequently observed and this may be due to specific interactions between tumour cell surface receptors and specific extracellular matrix (ECM) components present in conjunction with vascular elements. In order to investigate the influence of ECM on glioma cell migration, three different human glioma cell lines (U-373 MG, A-172 MG and HF-66) were exposed to known ECM components of the basement membrane (laminin, fibronectin and collagen type IV). Cell migration from multicellular spheroids was studied, using a custom-made medium which was prepared by removing the high molecular weight protein fraction (>100 kDa) from newborn calf serum by ultrafiltration. To this medium, the specific ECM components were added. For two of the cell lines (A-172 MG and U-373 MG), laminin was the most potent stimulator of glioma cell migration; the effect of laminin exceeded that evoked by ordinary serum-supplemented medium. For the HF-66 cell line, fibronectin was the most potent stimulator of migration. Western blot analysis showed that the A-172 MG and HF-66 cell lines expressed low amounts of laminin compared with U-373 MG, which showed extensive intrinsic synthesis of this ligand. U-373 MG was the only cell line that migrated in pure filtered medium. The cells stimulated by fibronectin expressed a different morphology from those stimulated by laminin suggesting that specific ECM-receptor binding may activate different cytoskeletal components within the cells. Furthermore, it was shown that there was no difference in the amount of protein synthesis between cells grown in filtered medium and in filtered medium supplemented with different ECM components. This suggests that ECM-induced cell migration is not dependent on a high level of protein synthesis. It is also shown that alpha3 integrin, which is a receptor-subunit for laminin, fibronectin and collagen type IV, was highly expressed in all cell lines. This study indicates that glioma cells need serum proteins with a molecular weight >100 kDa to migrate in vitro, and that laminin and fibronectin play an important role in this process.

Adhesion and migration of human glioma cells are differently dependent on extracellular matrix molecules.

To analyse the interactions between glioma cells and extracellular matrix (ECM) proteins, the adhesive and migratory capacity of five human glioma cell lines (D37MG, D54MG, GaMG, U118MG and U251MG) were studied. The expression of integrins was analysed and correlated to the adhesive and migratory abilities of the cells. All cell lines were able to adhere to and migrate on the extracellular matrix proteins collagen IV, fibronectin, laminin and vitronectin. Laminin was superior in propagating adhesion and migration in all five cell lines. As analysed by flow cytometry, the expression of the integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, beta 3, beta 4, alpha v, and integrin alpha v beta 5 proved to be uniform between the cell lines. All integrins except alpha 4, alpha 6, beta 3 and beta 4 were expressed on more than 85% of the cells. Inhibition of adhesion with synthetic peptides and antibodies directed against integrins demonstrated that adhesion on laminin was independent of integrins and the 67 kD laminin receptor. On the other hand, migration was shown to be integrin-dependent on all substrates. The results indicate that the mechanism responsible for cell adhesion in human gliomas differ from those present during migration, and that integrins play an important role in regulating these two mechanisms.

Epidermal growth factor and laminin receptors contribute to migratory and invasive properties of gliomas.

Gliomas are characterized by their extensive invasion into the brain parenchyma. Recently it has been shown that normal brain cells can produce laminin, fibronectin and collagen type IV when confronted by invading glioma cells. Laminin stimulates cell migration of several human glioma cell lines in vitro. This migration can be inhibited by adding blocking monoclonal antibodies (MAbs) against the most expressed integrin subunits, alpha3 and beta1. Previous studies have shown that glioma cell migration, invasion and growth are stimulated by epidermal growth factor (EGF). However, MAb directed against the EGF receptor (EGFR) did only partly inhibit the invasive process in vitro. Since laminin has regional peptide homology with EGF (EGF-like repeats), the present work was aimed at studying how two human glioma cell lines exposed to antibodies to the EGFR, reacted to laminin stimulated migration. Furthermore, we wanted to study which role the EGFR and the laminin receptor integrin subunits alpha3 and beta1 play during glioma cell invasion. EGFR expression of two glioma cell lines, AN1/lacZ and U-251/lacZ was studied by flow cytometry and immunofluorescence microscopy. A cell migration assay was used to study effects of MAbs against EGFR on migration from laminin-stimulated tumor spheroids. Tumor cell invasion was evaluated by using an in vitro co-culture model, where normal fetal brain cell aggregates were confronted with multicellular tumor spheroids. The results show that both cell lines expressed EGFR, AN1/lacZ 4-fold more than U-251/lacZ. MAb against EGFR inhibited the laminin-stimulated migration only from AN1/lacZ spheroids. MAbs against alpha3 and beta1 integrin subunits inhibited glioma cell invasion in vitro. The present work indicates possible connections between laminin-stimulated cell migration and the EGFR expression on glioma cells. These elements contribute to the characteristic features of glioma cells and may be an important part of the complex relationships between growth factors, integrins and extracellular matrix during glioma cell invasion.

Brain tumor cell invasion, anatomical and biological considerations.

Gliomas exhibit diffuse infiltration into the normal brain parenchyma, and the tumor cells often show morphological features similar to reactive glia cells, making it difficult to discriminate tumor cells from other neural cell populations both in vitro and in vivo. Several methods have therefore been developed in order to observe migrating tumor cells in experimental tumor models. These include labeling of tumor cells with vital dyes as well as by using genetic markers. Despite the fact that these malignancies are highly invasive in the brain, they rarely metastazise out of the central nervous system (CNS). The dissemination of tumor cells is probably mediated both by passive cell displacement and by active cell migration. Tumor cells may be displaced within the brain by the passive flow of cerebrospinal fluid (CSF) within the perivascular space and along ventricular linings. Tumor growth and invasion occur in a micromillieu that is regulated both by cancer cells and normal cells. The biological attributes of invasion and cell migration include cell adhesion to extracellular matrix components, cell locomotion, and the ability to create space into which to move. This process is characterized by the degradation and turnover of ECM components, which implies the production of specific proteases and inhibitors. Tumor progression is also influenced by numerous growth factors which may stimulate the malignant cells both by paracrine and autocrine mechanisms. Tumor growth requires the persistent formation of new blood vessels and the induction of angiogenesis is most likely occurring during early stages of tumor development. This process is regulated both by several inducers and inhibitors of endothelial cell proliferation and migration.

Stimulation of glioma-cell migration by laminin and inhibition by anti-alpha3 and anti-beta1 integrin antibodies.

An induction of laminin in the confrontation zone between tumor cells and normal brain tissue has been observed in our model systems in vivo and in vitro. In order to study the effects of ECM components on glioma-cell migration and invasion, we have used 2 lacZ-transfected glioma cell lines, AN1/lacZ and U-251 /lacZ. Cell migration from multicellular spheroids was studied using different types of media: DMEM with 10% serum, Ultra Culture medium, and filtrated DMEM with serum in which the protein fraction > 100 kDa had been removed by ultrafiltration. Laminin, fibronectin and collagen type-IV were individually added to the different media, and cell migration from the spheroids was studied. The results show that cell migration in both cell lines, was stimulated by laminin and fibronectin. Collagen type-IV stimulated only cell migration of U-251/lacZ cells. Scanning electron microscopy revealed an extensive change in cell shape as a result of laminin stimulation. Flowcytometric studies showed that both AN1/lacZ and U-251/lacZ strongly express the alpha3 beta1 integrin receptor, which can bind to several ECM components (laminin, fibronectin, collagen). Immunofluorescence microscopy demonstrated that the same integrin sub-units were expressed in multicellular spheroids. When monoclonal antibodies to alpha3 and beta1 were added to the laminin-stimulated cultures, cell migration was significantly reduced. This indicates that the alpha3 beta1 integrin receptor plays an important role during glioma-cell migration.

The lac-z reporter gene: a tool for in vitro studies of malignant glioma cell invasion.

A reporter gene (lac-z) was introduced into rat (BT4C) and human (D-54 MG) proliferating glioma cell lines by means of liposomal transfection. Lac-z-transfected glioma cells were first cultured as multicellular spheroids and then confronted with fetal brain aggregates. After various intervals the lac-z reporter gene product, bacterial beta-galactosidase, was histochemically detected in the cocultures. beta-Galactosidase was only detected in the glioma cells which showed an intense blue staining, which made them easily distinguishable from fetal tissue. Both glioma cell lines showed a clear pattern of migration and increasing invasion with time as the tumor cells infiltrated and destroyed the brain aggregates. Spheroid growth curves showed no significant differences between transfected and nontransfected cell lines. Likewise, flow cytometry measurements revealed no significant changes in ploidy between transfected and nontransfected rat glioma cells. In comparison, a shift in ploidy was observed in the human glioma cells after lac-z transfection. Stable integration of the lac-z gene into tumor cells was verified by Southern blot analysis. The results indicate that transfection of the lac-z reporter gene into glioma cells lines does not affect their growth or invasion potential in vitro. The lac-z reporter gene can thus be exploited to facilitate visualization of single migrating tumor cells and quantification of tumor invasion in in vitro coculture systems.