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).
J Histochem Cytochem. 2014 Apr;62(4):251-64. doi: 10.1369/0022155413520313. Epub 2014 Jan 6.
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.
血流器官的微环境会影响癌细胞的代谢特征，而这些特征受到线粒体功能的影响。然而，传统固定和脱水技术的技术伪影（包括缺血/缺氧）阻碍了对这些方面的组织病理学分析。本研究旨在将体内冷冻技术（IVCT）与荧光蛋白表达相结合，并检查移植黑素瘤肿瘤中荧光标记的线粒体。在经过冷冻技术和冷冻置换（FS）后，培养的 B16-BL6 细胞中荧光蛋白的强度保持良好。在靶向线粒体的 DsRed2（mitoDsRed）表达细胞的皮下肿瘤中，发现更多的癌细胞围绕着广泛开放的血管，这些血管中含有大量红细胞。这些血管对免疫球蛋白 M 呈阳性免疫染色，并被基底膜包裹。使用 IVCT-FS 方法可以检测到散射黑素瘤细胞中的 mitoDsRed 荧光，并且随着缺氧标志物的表达，单个癌细胞中的总 mitoDsRed 体积显著减少。MitoDsRed 经常分布在细胞质中，并沿着基底膜延伸的过程中分布。将 IVCT 与荧光蛋白表达相结合是一种有用的工具，可以检查荧光标记细胞和细胞器的行为。我们提出，线粒体体积在缺氧微环境中是动态调节的，线粒体的分布是由癌细胞与基底膜的相互作用调节的。
