The development of necrosis and apoptosis in glioma: experimental findingsusing spheroid culture systems

Cell death in gliomas may occur either by apoptosis, or, in the case of hig h grade tumours, by necrosis, but questions remain as to the pathogenesis a nd relationship between these processes. The development of cell death was investigated in multicellular glioma spheroid cultures. Spheroids model the development of cell death due to diffusion gradients in a three-dimensiona l system without confounding influences of immune response, pressure gradie nts, etc. Spheroid cultures were established from four malignant glioma cel l lines: U87, U373, MOG-G-CCM and A172; harvested from culture at weekly in tervals and stained with Haematoxylin and Eosin (H&E), TdT-mediated dUTP-X nick end labelling (TUNEL) and by immunohistochemistry for vimentin, Glial Fibrillary Acidic Protein (GFAP) and Ki67. Annexin V flow cytometry and cou nts of apoptotic cells on H & E stained sections were performed to assess l evels of apoptosis. Modes of cell death were also characterized by electron microscopy. Spatially separate zones of proliferation, differentiation and central cell death developed with increasing spheroid diameter. Central ce ll death developed at a predictable radius (300-400 mum) for each cell line . Ultrastructural examination showed this to be necrotic in type. Apoptosis was most reliably assayed by morphological counts using H & E. Basal level s of apoptosis were low (<0.5%), but increased with increasing spheroid dia meter (>2% in U87). In particular, levels of apoptosis rose following devel opment of central necrosis and apoptoses were most abundant in the peri-nec rotic zone. There were quantitative differences in the levels of apoptosis and necrosis between glioma cell lines. The predictable onset of necrosis i n the spheroids will allow us to investigate the pathogenesis of necrosis a nd events in prenecrotic cells. There is a relationship between the develop ment of necrosis and apoptosis in this model and these processes can be sep arately assayed. Further in vitro and genetic studies will enable us to stu dy these events and interactions in greater detail than is possible using o ther cell culture and in vivo systems.