Mapping Functional Heterogeneity in Tissue

摘要 翻译后摘要：质谱成像（MSI）提供了前所未有的细节分子分布在整个前 体内组织样本。尽管MSI有能力绘制从大蛋白到小蛋白的分子图谱， 这些数据仅描述了单个时间点的组织状态，缺乏关于代谢物的任何信息。 这些分子相互作用以提供代谢功能。典型的功能研究从以下方面获得动力学数据： 施用同位素标记的底物并监测在一个月内收获的组织样品中的标记物转运 特定的时间进程。然而，MSI跟踪的同位素动力学有一些独特的要求， 进行类似的功能研究，并充分利用该技术的力量， 代谢物通量该提案将开发一种基于MSI的方法，通过 使用甘氨酸的同位素体的定时输注来测量谷胱甘肽和丝氨酸代谢通量的速率 在每个50 x 50 x 10 μ m图像体素内。这种定时输注允许逐体素地确定代谢产物。 速率，即其他方法无法实现的功能图像。此外，由于代谢通量和途径 选择受到组织灌注和氧合的影响，我们还将开发基于MSI的方法， 测量相同样本中的组织灌注和缺氧。将使用哌莫硝唑的同位素体绘制 慢性和周期性缺氧的区域，可以直接与谷胱甘肽代谢活动。到 为了证明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 在小鼠肝脏和乳腺4T1肿瘤中。由于MSI方法使用冷冻薄切片， 可用于将传统的组织标记物与代谢功能联系起来。描述的方法 本文将证明谷胱甘肽和丝氨酸途径，但可用于研究功能 在任何组织和任何代谢网络中的异质性与适当的选择底物。

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.