Imaging Mitochondrial Redox States In Vivo by Hyperpolarized MR

描述(申请人提供)：线粒体代谢异常，包括氧化还原状态，已在许多病理条件下被发现，包括癌症、糖尿病、衰老和心血管疾病。线粒体NAD(烟酰胺腺嘌呤二核苷酸)偶联的氧化还原电位NAD/NADH调节代谢中的一系列氧化还原反应，并介导对细胞生长、存活和凋亡至关重要的关键信号事件。为了开发有效的疾病诊断和治疗方法，迫切需要一种非侵入性的方法来定量和/或测量体内组织中线粒体的NAD/NADH氧化还原电位。在这个项目中，我们将开发和验证一种临床可翻译和非侵入性的超极化碳13磁共振波谱成像(HP-13C-MRSI)方法，用于在体内使用乳腺癌动物模型对线粒体氧化还原电位NAD/NADH进行成像。HP-13C-MR的灵敏度是传统MR的1万多倍，并已被用于组织新陈代谢成像。其基本原理是仅使用线粒体中酶反应的平衡常数来定量NAD偶联的氧化还原电位，即-羟基丁酸酯NAD‘乙酰乙酸酯NADH H，前提是乙酰乙酸酯/羟基丁酸酯的比率和线粒体的pH可以通过MR方法来定量。该方法专用于线粒体中的NAD/NADH对。我们还将建议使用13C标记的酯探针(例如羟基丁酸乙酯)来区分细胞外和细胞内的MR信号，以获得更高精度的氧化还原电位。由于我们之前使用低温NADH/黄素蛋白荧光成像的体外研究已经将线粒体氧化还原状态与人黑色素瘤和乳腺癌在小鼠异种移植瘤中的转移潜能联系起来，本项目还将建立线粒体NAD/NADH与乳腺癌转移潜能之间的相关性，这支持线粒体氧化还原潜力作为癌症诊断和治疗反应的生物标志物和治疗靶点。我们将研究用HP-13C-MR方法测量的线粒体氧化还原电位NAD/NADH是否可以区分惰性(MCF-7)和高转移(MDA-MB-231)乳腺癌细胞系。我们将在不同线粒体代谢条件下对灌流细胞模型和小鼠异种移植细胞进行氧化还原电位测量，并将结果与生化分析相关联。我们还将在患有低级别或高级别乳腺肿瘤的人类患者身上验证这些技术，以证明其临床可译性。

DESCRIPTION (provided by applicant): Abnormalities in mitochondrial metabolism including redox state have been identified under many pathological conditions including cancer, diabetes, aging, and cardiovascular disease. Mitochondrial NAD (nicotinamide adenine dinucleotide)-coupled redox potential NAD+/NADH regulates a number of oxidation-reduction reactions in metabolism, and also mediates key signaling events important for cell growth, survival and apoptosis. There is a great need for a non-invasive method of quantifying and/or measuring mitochondrial NAD+/NADH redox potential in tissues in vivo for the purpose of developing effective approaches for disease diagnosis and treatment. In this project we will develop and validate a clinically translatable and non-invasive hyperpolarized-carbon-13 magnetic resonance spectroscopic imaging (HP-13C-MRSI) method for imaging the mitochondrial redox potential NAD+/NADH in vivo using breast cancer animal models. HP-13C-MR is over 10,000 times more sensitive than conventional MR and has been used to image tissue metabolism. The rationale is to quantify the NAD-coupled redox potential using the equilibrium constant for an enzymatic reaction in mitochondria only, i.e., -hydroxybutyrate + NAD+ ` acetoacetate + NADH + H+, provided that the ratios of acetoacetate/- hydroxybutyrate and the mitochondrial pH can be quantified by MR methods. The proposed method is specific for the NAD+/NADH couple in mitochondria. We will also propose to use 13C-labeled ester probes (e.g. ethyl- hydroxybutyrate) to differentiate between extracellular and intracellular MR signals to obtain the redox potential with higher accuracy. Since our previous studies using cryogenic NADH/flavoprotein fluorescence imaging ex vivo have linked mitochondrial redox state to the metastatic potential of human melanoma and breast cancer in mouse xenografts, this project will also establish a correlation between mitochondrial NAD+/NADH and the metastatic potential of breast cancer, which supports mitochondrial redox potential as a biomarker for cancer diagnosis and treatment response, and a target for therapy. We will investigate whether the mitochondrial redox potential NAD+/NADH measured by the HP-13C-MR method can differentiate an indolent (MCF-7) and a highly metastatic (MDA-MB-231) breast cancer cell line. We will perform redox potential measurements on perfused cell models and mouse xenografts under different mitochondrial metabolic conditions and correlate the results with biochemical assays. We will also validate the techniques on human patients with low or high grade breast tumors to demonstrate its clinical translatability.