Morphological Heterogeneity and Phenotype Modifications during Long Term in Vitro Cultures of Six New Human Glioblastoma Cell Lines

Abstract

Long term in vitro cultures of six human malignant gliomas were established to obtain permanent lines and to assess, under conditions of prolonged culture, changes in morphology and phenotype of neoplastic cells and the extent of these modifications. We analyzed expression of the following markers by immunocytochemistry: glioma-specific antigens (GE2 and CG12), fibronectin, intermediate filaments (GFAP, vimentin, neurofilaments), class I and II histocompatibility antigens (HLA-ABC and HLA-DR), growth factor and receptor (alpha TGF and EGF-receptor), proliferation-associated antigen (Ki-67). Strong and stable staining with the two antiglioma monoclonal antibodies (GE2 and CG12) was seen, with coexpression of GFAP and fibronectin in five of six cell lines (after 20 passages) and presence of vimentin and neurofilaments. HLA-DR expression was heterogeneous, with a peculiar intracellular compartmentation in four of six cell lines. Cells showed clear cytoplasmic positivity for alpha TGF and strong membrane staining for EGF-receptor. In previous studies we showed that these cell lines have increased copies of chromosome 7; therefore we speculate that an autocrine pathway of stimulation may maintain the neoplastic growth. The percentage of Ki-67 positive proliferating cells ranged from 40 to greater than 60%, depending on cell line and passage. A slight decrease in the positivity of some markers (GFAP, vimentin and HLA-DR in 2/6 cell lines) was observed after prolonged in vitro culture (greater than 12 months), but morphophenotypic modifications, established within a few passages after explanation, were maintained with time. A clonogenic assay showed values of plating efficiency (PE) higher than corresponding values of other similar cell lines with a tendency to increase in the late passages. PE and Ki-67 positivity were not associated with tumorigenicity into nude mice (except the Hu 197 cell line). These results indicate that, in culture, all six cell lines acquired stable morphology, a well defined antigenic phenotype and high growth rate. Further studies will be performed on these permanent cell lines to clarify differentiation steps of malignant gliomas.