Three-dimensional imaging of tumor angiogenesis

OBJECTIVE: To three-dimensionally visualize the microvessel environment of tumor angiogenesis by confocal laser scanning microscopy (CLSM). STUDY DESIGN: To reveal underlying mechanisms of tumor angiogenesis, a 7,12 -dimethylbenz(a) anthracene-induced rat cancer model was used. For demonstr ating tumor vasculature,fluorescence injection method (FITC-conjugated gela tin solution) was employed. FITC gelatin was injected into the left ventric le of the rat heart. After complete perfusion, the mammary glands were rese cted, fixed under ice cold conditions and subjected to immunohistochemistry (IHC) for tumor cells. The LSM-410 (Carl Zeiss, Jena, Germany) was employe d on thick secretions (300-2,000 mu m) to elucidate detailed microvessel ne tworks (MVN) and tumor cells. RESULTS: Tumor vasculature on thick sections was clearly detected by CLSM a t the maximum focus depth of 2,000 mu m. Three-dimensional (3-D), reconstru cted images of normal mammary glands showed regular and linear MVN. In DMBA -induced mammary cancer, vascular density of MVN was markedly increased and showed an anastomosing, irregular MVN pattern. Furthermore, focal segmenta tion and tortuous, branching patterns of microvessels were also seen. CONCLUSION: Application of the fluorescence injection method and LHC using CLSM teas very useful for studying the 3-D relationship between tumor angio genesis and neoplastic epithelial changes. These results suggest that appli cation of this technique is ideal for studying 3-D imaging of tumor angioge nesis.