Mapping Functional Heterogeneity in Tissue

摘要 摘要：质谱学成像(MSI)提供了前所未有的分子分布细节 活体组织样本。尽管MSI有能力映射从大蛋白到小分子的各种分子 代谢物，这些数据只描述了组织在单个时间点的状态，缺乏任何关于如何 这些分子相互作用，提供新陈代谢功能。典型的功能研究从以下方面获得动力学数据 组织样本中同位素标记底物的管理和标记转运的监测 指定的时间进程。然而，MSI跟踪的同位素动力学有一些独特的要求，以便 执行类似的功能研究并充分利用该技术的功能，以提供特定于位置的 代谢物流量。这项提议将开发一种基于MSI的方法，通过 用定时输注甘氨酸同位素来测量谷胱甘肽和丝氨酸代谢流量的比率 在每个50x50x10m图像体素内。这种定时输液允许逐个体素地确定代谢 速率，即其他方法不可能实现的功能图像。此外，由于代谢流量和代谢途径 选择受到组织灌流和氧合的影响，我们还将开发基于MSI的方法来 测量同一样本中的组织灌注量和缺氧量。将使用匹莫硝唑的同位素制剂来绘制地图 慢性和循环缺氧区可以直接与谷胱甘肽代谢活动联系在一起。至 演示这些技术在不同组织类型中的可行性，我们将绘制功能异质性图 横跨小鼠肝脏和乳腺4T1肿瘤。由于MSI方法使用的是冰冻切片、组织化学数据 可用于将传统的组织标志物与代谢功能联系在一起。所描述的方法 本文将在谷胱甘肽和丝氨酸途径上进行演示，但可用于研究功能 任何组织和代谢网络中的异质性，只要底物选择得当。

ABSTRACT Abstract: Mass spectrometry imaging (MSI) provides unprecedented detail of molecular distributions across ex vivo tissue samples. Although MSI has the capability to map molecules ranging from large proteins to small metabolites, these data only describe the tissue status at a single time point and lack any information on how these molecules interact to provide metabolic function. Typical functional studies derive kinetic data from administration of isotope-labeled substrates and monitoring label transit in tissue samples harvested over a specified time-course. Isotope kinetics tracked by MSI, however, have some unique requirements to in order to perform a similar functional study and exploit the full power of the technique to provide location-specific metabolite flux. This proposal will develop an MSI-based method to track isotope metabolic activity through the use of a timed infusion of isotopologues of glycine to measure the rate of glutathione and serine metabolic flux within each 50 x 50 x 10 m image voxel. This timed infusion allows a voxel-by-voxel determinations of metabolic rates, i.e. functional images that are not possible by other methods. In addition, since metabolic flux and pathway selection is influenced both by tissue perfusion and oxygenation, we also will develop MSI-based methods to measure tissue perfusion and hypoxia in the same samples. Isotopologues of pimonidazole will be used to map regions of both chronic and cycling hypoxia that can be tied directly to glutathione metabolic activity. To demonstrate the feasibility of these techniques in different tissue types, we will map functional heterogeneity across mouse liver and mammary 4T1 tumors. As the MSI method uses frozen thin-sections, histochemical data from adjacent sections can be used to tie traditional tissue markers to metabolic function. The methods described herein will be demonstrated on the glutathione and serine pathways but can be used to study functional heterogeneity in any tissue and any metabolic network with proper selection of substrates.