Bioimaging of Fluorescence-Labeled Mitochondria in Subcutaneously Grafted Murine Melanoma Cells by the “In Vivo Cryotechnique”

Abstract

Mitochondrial functions play important roles in metabolism of cancer cells and are affected by the microenvironment including blood circulation. To overcome the technical artifacts of conventional fixation and dehydration methods, the in vivo cryotechnique (IVCT) was combined with fluorescent protein expression and used to examine the distribution of mitochondria in tumor tissues obtained from melanoma-grafted mice. Quick-freezing followed by freeze-substitution (FS) could well retain the fluorescence intensity of fluorescent proteins including mitochondria-targeted DsRed2 (mitoDsRed) in cultured B16-BL6 cells. In the subcutaneous tumor tissues produced by injection of mitoDsRed-expressing B16-BL6 cells, the melanoma cells scattering throughout the tumor tissues prepared with IVCT followed by FS had clear fluorescence of mitoDsRed, and individual tumor cells expressing hypoxia markers, including carbonic anhydrase IX and hypoxia-inducible factor-1α, had decreased volume of mitoDsRed. The cytoplasm and processes of the cancer cells extended along the collagen type IV-immunopositive basement membranes and often contained mitoDsRed. Combination of fluorescent protein expression and IVCT would be a powerful tool to examine cells and organelles with fluorescent protein expression along with histochemical/immunohistochemical labelings. Furthermore, the results suggest that mitochondrial volume and distribution could be modulated by the hypoxic microenvironment and attachment to basement membranes.