Tanaka Shotaro, Harada Hiroshi, Hiraoka Masahiro
Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, Shinjyuku-ku, Tokyo 162-8666, Japan.
Department of Radiation Oncology and Image-applied Therapy, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Saitama 332-0012, Japan.
Biochem Biophys Res Commun. 2015 Sep 4;464(4):1151-1156. doi: 10.1016/j.bbrc.2015.07.095. Epub 2015 Jul 22.
The alkalization of intracellular pH (pHin) advances together with enhancement of aerobic glycolysis within tumor cells (the Warburg effect), and that is responsible for the progression of tumor malignancy together with hypoxia and angiogenesis. But how they correlate each other during tumor growth is poorly understood, partly due to the lack of suitable imaging methods. In present study, we propose a novel method to visually determine the pHin of tumor xenograft model from fluorescent image ratios. We utilized tandemly-linked two fluorescent proteins as a pH indicator; yellow fluorescent protein (YFP, pH sensitive) as an indicator, and red fluorescent protein (RFP, pH insensitive) as a reference. This method can eliminate the influence of optical factors from tissue as well as of the diverse expression level of pH indicator in the grafted cells. In addition, that can be operated by filter-based fluorescent imagers that are generally used in small animal study. The efficacy of the pH indicator, RFP-YFP, was confirmed by studies using recombinant protein in vitro and HeLa cells expressing RFP-YFP in vivo. Furthermore, we prepared nude mice subcutaneously xenografted HeLa cells expressing RFP-YFP cells as tumor model. The image ratios (YFP/RFP) of the tumor at the day 5 after surgery clearly showed the heterogeneous distribution of diverse pHin cells in the tumor tissue. Concomitantly acquired angiography using near-infrared fluorescence (680 nm for emission) also indicated that the relative alkaline pHin cells located in the region far from tumor vessels in which tumor aerobic glycolysis would be facilitated by progression of hypoxia and nutrient starvation. Applying the present method for a multi-wavelength imaging concerning pO2 and/or nutrient starvation states in addition to pHin and angiogenesis would provide valuable information about complicated alteration of tumoral cell states during tumorigenesis.
细胞内pH值（pHin）的碱化与肿瘤细胞内有氧糖酵解的增强（瓦伯格效应）同步进展，这与缺氧和血管生成共同导致肿瘤恶性程度的进展。但在肿瘤生长过程中它们如何相互关联却知之甚少，部分原因是缺乏合适的成像方法。在本研究中，我们提出了一种从荧光图像比率直观测定肿瘤异种移植模型pHin的新方法。我们利用串联的两种荧光蛋白作为pH指示剂；黄色荧光蛋白（YFP，对pH敏感）作为指示剂，红色荧光蛋白（RFP，对pH不敏感）作为参照。该方法可以消除组织光学因素以及移植细胞中pH指示剂不同表达水平的影响。此外，它可以通过小动物研究中常用的基于滤光片的荧光成像仪来操作。pH指示剂RFP-YFP的功效通过体外使用重组蛋白和体内表达RFP-YFP的HeLa细胞的研究得到证实。此外，我们制备了皮下移植表达RFP-YFP细胞的HeLa细胞的裸鼠作为肿瘤模型。术后第5天肿瘤的图像比率（YFP/RFP）清楚地显示了肿瘤组织中不同pHin细胞的异质分布。同时使用近红外荧光（发射波长为680nm）进行的血管造影也表明，相对碱性pHin细胞位于远离肿瘤血管的区域，在该区域缺氧和营养饥饿的进展会促进肿瘤有氧糖酵解。将本方法应用于除pHin和血管生成外还涉及pO2和/或营养饥饿状态的多波长成像，将为肿瘤发生过程中肿瘤细胞状态的复杂变化提供有价值的信息。
