title: Bioimaging of fluorescence-labeled mitochondria in subcutaneously grafted murine melanoma cells by the "in vivo cryotechnique".
abstract: The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes.
authors.affiliation: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi, Japan (TL, ZH, NO, BW, TS, YS, SO).
authors.forename: Ting
authors.initials: T
authors.lastname: Lei
authors.forename: Zheng
authors.initials: Z
authors.lastname: Huang
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Ohno
authors.forename: Bao
authors.initials: B
authors.lastname: Wu
authors.forename: Takashi
authors.initials: T
authors.lastname: Sakoh
authors.forename: Yurika
authors.initials: Y
authors.lastname: Saitoh
authors.forename: Ikuo
authors.initials: I
authors.lastname: Saiki
authors.forename: Shinichi
authors.initials: S
authors.lastname: Ohno
mesh_terms: D000818:Animals; D015687:Cell Hypoxia; D015925:Cryopreservation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008546:Melanoma, Experimental / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism*; D057054:Molecular Imaging; D009368:Neoplasm Transplantation; D014407:Tumor Cells, Cultured
chemical_list: D008164:Luminescent Proteins
keywords: blood vessels; cancer; fluorescent protein; in vivo cryotechnique; mitochondria