Imaging Mitochondrial Redox States In Vivo by Hyperpolarized MR

描述（由申请人提供）：在许多病理条件下，包括癌症、糖尿病、衰老和心血管疾病，已经鉴定出包括氧化还原状态在内的线粒体代谢异常。线粒体 NAD（烟酰胺腺嘌呤二核苷酸）耦合的氧化还原电位 NAD /NADH 调节代谢中的许多氧化还原反应，并且还介导对细胞生长、存活和凋亡重要的关键信号事件。为了开发疾病诊断和治疗的有效方法，非常需要一种非侵入性方法来量化和/或测量体内组织中线粒体NAD/NADH氧化还原电位。在该项目中，我们将开发并验证一种临床可转化的非侵入性超极化碳 13 磁共振波谱成像 (HP-13C-MSI) 方法，用于使用乳腺癌动物模型对体内线粒体氧化还原电位 NAD /NADH 进行成像。 HP-13C-MR 的灵敏度比传统 MR 高 10,000 倍以上，已用于组织代谢成像。其基本原理是仅使用线粒体中酶促反应的平衡常数来量化 NAD 偶联氧化还原电位，即 -羟基丁酸 NAD ` 乙酰乙酸 NADH H ，前提是乙酰乙酸/-羟基丁酸的比率和线粒体 pH 值可以通过 MR 方法进行量化。所提出的方法专门针对线粒体中的 NAD /NADH 配对。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 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/黄素蛋白荧光成像离体的研究已将线粒体氧化还原状态与小鼠异种移植物中人类黑色素瘤和乳腺癌的转移潜力联系起来，因此该项目还将建立线粒体NAD/NADH与乳腺癌转移潜力之间的相关性，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以及治疗靶标。我们将研究通过 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) 和高度转移 (MDA-MB-231) 乳腺癌细胞系。我们将对不同线粒体代谢条件下的灌注细胞模型和小鼠异种移植物进行氧化还原电位测量，并将结果与​​生化测定相关联。我们还将在患有低度或高度恶性乳腺肿瘤的人类患者身上验证该技术，以证明其临床可转化性。

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.