Novel gliosarcoma cell line expressing green fluorescent protein: A model for quantitative assessment of angiogenesis

Angiogenesis is essential for tumor proliferation and metastasis. The extent of angiogenesis is measured by microvessel density (MVD) which has been identified as an independent prognostic factor for relapse-free survival in cancer patients. Existing methods of MVD assessment measure the microvessel count in the most active area of neovascularization ("hot spots") using antibodies against vascular endothelial antigens. This may produce unreliable results because of tissue volume loss and misshapening during the fixation and dehydration procedures. We report here a genetically engineered 9L cell line constitutively expressing green fluorescent protein that can be visualized using fluorescence microscopy without additional histological staining. The model developed in this study allows for the performance of simple and easy MVD counting, assuming that nonfluorescent "black spots" visible by fluorescence microscopy within the borders of the tumor tissue represent blood vessels. This assumption was confirmed by a comparative study utilizing conventional histological methods, anti-CD31 immunohistology, and Hoechst 33258 dye exclusion. This model is also useful for delineation of the true borders between tumor and normal brain tissue, including microscopic tumor extensions, without multiple histological staining. The suggested model allows quantification of tumor angiogenesis in tissue specimens, thus providing independent prognostic information about tumor growth and regression. It is expected to be most valuable in evaluating the efficacy of anti-angiogenic therapy.