(CANCER RESEARCH 50. 6039-6044. September 15. I990| Effect of Epidermal Growth Factor on Glioma Cell Growth, Migration, and Invasion in Vitro1 Morten Lund-Johansen,2 Rolf Bjcrkvig, Peter A. Humphrey, Sandra H. Bigner, Darell D. Bigner, and Ole-Didrik Laerum The (jade Institute, Department of Pathology, L niversity of Bergen, llaukelanil Hospital, \-5U2l Bergen, \orway f.\l. I.-J., K. B., O-I). I../, and Department of Pathology ¡P. A. //., .V.//. B., n. D. R.I, antl the Preuss Laboratory for Brain Tumor Rest-arch fD. I). fÃ-.¡. Duke I'nirersily Medical (enter, Durham, \orth ('anilina 277IO ABSTRACT Effects of epidermal growth factor (KGK) and an antibody (Ab-528) reactive against the binding site for I ( .1 on human I ( ,1 receptors were studied on multicellular tumor spheroids obtained from three human glioma cell lines with high (D-37 MG), medium (D-247 MG), and low (D-263 MG) levels of EG F receptor expression. The D-247 MG and D- 263 MG spheroids grew slowly or not at all in the absence of I (.I , while in the presence of EGF they were growth stimulated. Tumor cell migra tion, as measured by the spread of cells from spheroids on a plastic substratum, was increased by the addition of KGF for all three cell lines. Stimulation of migration could be blocked by a subsequent addition of Ab-528 to the medium at a concentration of 50 MS/ml. Invasiveness of glioma cell spheroids into fetal rat brain aggregates was related to FGF receptor expression; the two lines with medium to high receptor expres sion (D-247 MG and D-37 MG) were invasive, while the line with low EGF receptor expression (D-263 MG) was noninvasive, as assessed by an ¡nvitro coculture assay. In the D-247 MG cell line, morphometry revealed EGF-enhanced invasiveness of the tumor cells. The addition of the Ab-528 to EGF-treated cocultures reduced invasion in both Ãœ-247 MG and D-37 MG cell lines. Antibody Ab-528 alone did not affect glioma cell growth or migration but did inhibit invasiveness. The present study suggests that, in brain tumors with an increased number of normal- sized M, 170,000 EGF receptors, EGF or an EGF-like ligand such as transforming growth factor-a may selectively facilitate expansive tumor growth and tumor cell invasion. This effect may in part be blocked or retarded by specific antibodies to the EGF receptor. INTRODUCTION EGF,' a 6045-Da polypeptide, has been demonstrated to stimulate the in vitro growth of both nonneoplastic and neo- plastic glial cells (1-5). Neoplastic glial cells also exhibited migration in response to EGF (6). EGF transduces its prolif- erative signal by specific binding to the EGF-r, a M, 170,000 transmembrane glycoprotein with intrinsic tyrosine kinase ac tivity. Glioma cells in vivo often overexpress the EGF-r due to EGF-r gene amplification (7-9). Often associated with this amplification is structural rearrangement of the gene (8-10). which results in the expression of truncated EGF-rs (10-12). In vitro, gliomas only rarely amplify the EGF-r gene (13), and a wide range of EGF-r expression from IO4 to IO6 EGF-r molecules per cell has been observed (14-17). High expression of the EGF-r in culture in the absence of an amplified gene may be related to increased gene dosage due to extra copies of chromosome 7, to changes in transcriptional or translational control, or to changes in EGF-r mRNA and/or protein turn over. A few variant EGF-r proteins have been detected in glioma Received 3/9/90; accepted 6/19/90. The costs of publication of this article were defrayed ¡npart b\ the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 I'.S.C. Section 1734 solely to indicate this fact. 1Supported by the Norwegian Cancer Society. Grant 88-47; the NIH. Cirants CA 11898. NS 20023. and C A 32672: the American Cancer Society. Grant 89- 171; and by a grant from the McDonnell Foundation. 2 To whom requests for reprints should be addressed, at the Gade Institute of Pathology. Haukeland Hospital. N-5021 Bergen. Norway. •' The abbreviations used arc: EGF. epidermal growth factor; EGF-r. epidermal growth factor receptor; DMEM. Dulbccco's modified Eagle's medium. cells in vitro (18), but most EGF-rs in cultured glioma cells are of apparently normal molecular weight (17). EGF and its receptor have been implicated in bladder carci noma invasiveness (19, 20). but to date the role of EGF and EGF-r in glioma cell invasiveness has not been studied. Here we report the effect of EGF on glioma growth, glioma cell migration, and glioma invasiveness by using an in vitro spheroid model system. Three different glioma cell lines expressing low, medium, and high levels of normal-sized EGF-r were studied. Medium or high levels of EGF-r were required for invasiveness to occur. EGF stimulated the growth of the glioma spheroids with the greatest stimulation observed at the lowest EGF-r concentration and the least stimulation in the cell line with the highest EGF-r levels. EGF stimulated cell migration in all 3 lines and enhanced invasiveness of 1 cell line. An antibody reactive against the EGF-binding domain on the EGF-r blocked these EGF-mediated effects; alone the antibody did not affect growth or migration but did slow invasiveness. These studies suggest that EGF, or a similar ligand such as transforming growth factor-it, via binding to the normal-sized A/r 170,000 EGF-r. plays a significant role in glioma cell growth, migration, and invasiveness. The level of EGF-r expres sion in the cells influenced these biological behaviors, but a general effect was of EGF stimulation of glioma cell growth and migration at all levels of EGF-r expression. MATERIALS AND METHODS Tissue Culture Conditions and Chemicals. The cells were cultured in a standard tissue culture incubator (37°C,5% CO2, 95% air, 100% humidity). All tissue culture plastic articles were from Nunc (Roskilde, Denmark). The cells were cultured in DMEM supplemented with 10% heat-inactivated newborn calf serum, 4 times the prescribed concentra tion of nonessential amino acids, i.-glutamine. penicillin (100 lU/ml), and streptomycin (100 ug/m\) (all from Flow Laboratories, Glasgow, Scotland). The nonadhesive base coating of culture plastic for 3-dimen- sional cultures consisted of 0.75% Noble agar (Difco Laboratories, Detroit. MI) dissolved in medium. Tissue culture grade EGF (Grand Island Biological Co., Grand Island. NY) was reconstituted with Hanks' balanced salt solution (Gibco, Paisley. Scotland) to 100 ng/ml. The EGF-r antibody Ab-528 (Oncogene Science. Mineóla. NY) was dis solved in 0.9% NaCl solution to 500 ug/m\ before use. All experiments were done in triplicate. Tumor Cells. Three different human glioblastoma multiforme cell lines with a different density of EGF-rs were chosen for the experiments: D-263 MG (2.9 x 10* EGF receptors/cell), D-247 MG (1.5 x IO5 receptors/cell), and D-37 MG (1.59 x IO6receptors/cell) were studied (5. 21-23). The EGF-r in these 3 lines is normal sized at M, 170,000 and is fully functional in the high affinity binding of EGF and auto- phosphorylation (17). The quantitative expression of EGF-r in these cell lines has been shown to be stable during several passages (5). The cells were maintained as monolayer cultures in 80-cnr tissue culture flasks in 20 ml DMEM and passed 1:5 (v/v) with 0.05% trypsin in 0.02% EDTA (Flow Laboratories) by confluence. The formation of multicellular spheroids was obtained as described (24) by seeding 3 x IO6single cells in 20 ml DMEM into 80-cm2 culture flasks base coated with 10 ml medium agar. Within 2 to 3 days, spheroids were formed. 6039 Research. on January 27, 2016. © 1990 American Association for Cancer cancerres.aacrjournals.org Downloaded from