Spatial SILAC: Examining the Proteome in 3D Cell Cultures

项目摘要 药物开发过程效率低，费用昂贵，部分原因是大多数治疗方法最初是 用二维细胞培养进行筛选。这些细胞培养物不能准确地反映 人体。为了改进药物筛选，应该使用更准确的模型系统。三个- 立体细胞培养，也被称为球体，填补了这一空白。类似于肿瘤，结肠癌球体 含有径向对称的营养和氧气梯度。球体发育出不同的细胞群。 它们反映了这些化学梯度。在本提案中，我们正在开发一种分析方法，称为空间 SILAC，使用不同的同位素专门标记球体内存在的不同的细胞群体。 在目标1中，我们将展示这些同位素可以通过质谱计很容易地区分开来，从而提供 一个椭球体内细胞起源的同位素“邮政编码”。在目标2中，我们将结合成像质量 光谱学药物分布研究与对治疗反应的蛋白质组变化的评估， 使用同位素标签。我们将像以前一样评估伊立替康、5-氟尿嘧啶和西妥昔单抗 用成像质谱仪检测了这三种药物在球体中的分布。我们会比较一下 治疗反应的空间定位蛋白质组学变化及其影像分布 地图。在目标3中，我们将应用我们的空间SILAC方法来筛选新开发的循环穿透 多肽(CPPs)，由Co-I教授裴德华设计。这些有希望的抗癌药物候选药物需要 临床前筛选和球体中的空间SILAC为体外评估其提供了一个理想的试验台 药代动力学和药效学。我们将首先进行成像质谱学研究，以 测定CPPs及其代谢物在球体中的分布。然后，我们将评估两个 这些药物引起的靶向和非靶向蛋白质组学变化。所产生的结果 在这一目的中，将用于优化未来CPP的设计。总而言之，这项提议描述了一部小说 质谱学方法，将简化药物开发过程，同时还提供丰富的 新候选治疗药物的体外药代动力学和药效学信息。

Project Summary The drug development process is inefficient and expensive, in part because most therapeutics are initially screened with two-dimensional cell cultures. These cell cultures do not accurately reflect tissue structures in the human body. To improve drug screening, more accurate model systems should be used. Three- dimensional cell cultures, also known as spheroids, fill this gap. Similar to a tumor, colon cancer spheroids contain radially symmetric nutrient and oxygen gradients. The spheroid develops distinct cellular populations that reflect these chemical gradients. In this proposal, we are developing an analytical approach, called Spatial SILAC, to specifically label the divergent cellular populations present within spheroids using different isotopes. In Aim 1, we will show that these isotopes can be readily distinguished by a mass spectrometer, thus providing an isotopic “zip code” of the cell’s origin within a spheroid. In Aim 2, we will combine imaging mass spectrometry drug distribution studies with an evaluation of proteomic changes in response to therapeutics, using the isotopic labels. We will evaluate irinotecan, 5-fluorouracil and Cetuximab as we have previously examined the distribution of these three drugs in spheroids by imaging mass spectrometry. We will compare the spatially localized proteomic changes in response to therapeutic treatment with the imaging distribution maps. In Aim 3, we will apply our Spatial SILAC approach to screen newly developed cyclic penetrating peptides (CPPs), designed by Co-I Professor Dehua Pei. These promising cancer drug candidates require preclinical screening and Spatial SILAC in the spheroids provides an ideal testbed to evaluate their in vitro pharmacokinetics and pharmacodynamics. We will first perform imaging mass spectrometry studies to determine the distribution of the CPPs and their metabolites in the spheroids. We will then evaluate both the targeted and off-targeted proteomic changes caused by these drugs by Spatial SILAC. The results generated in this aim will be used to optimize the design of future CPPs. In summary, this proposal describes a novel mass spectrometric approach that will streamline the drug development process, while also providing rich in vitro pharmacokinetic and pharmacodynamic information on new therapeutic candidates.