Spatial SILAC: Examining the Proteome in 3D Cell Cultures

项目摘要 药物开发过程是低效和昂贵的，部分原因是大多数治疗药物最初是 用二维细胞培养筛选。这些细胞培养物不能准确地反映组织结构， 人体的为了改善药物筛选，应使用更准确的模型系统。三个... 三维细胞培养物（也称为球状体）填补了这一空白。与肿瘤相似，结肠癌球状体 含有径向对称的养分和氧气梯度。球状体发育出不同的细胞群 反映了这些化学梯度。在这个提案中，我们正在开发一种分析方法，称为空间 SILAC，使用不同的同位素特异性标记球状体内存在的不同细胞群体。 在目标1中，我们将证明这些同位素可以很容易地通过质谱仪区分，从而提供 一个球状体内细胞起源的同位素“邮政编码”。在目标2中，我们将结合联合收割机成像质量 光谱法药物分布研究，评估对治疗剂的反应中的蛋白质组学变化， 使用同位素标记。我们将评估伊立替康，5-氟尿嘧啶和西妥昔单抗，因为我们以前 通过成像质谱法检查了这三种药物在球体中的分布。我们将比较 空间定位的蛋白质组学变化对成像分布的治疗性治疗的响应 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 肽（CPPs），由Co-I教授Dehua Pei设计。这些有前途的癌症候选药物需要 球状体中的临床前筛选和空间SILAC提供了一个理想的测试平台，以评估其体外 药代动力学和药效学。我们将首先进行成像质谱研究， 确定CPP及其代谢物在球状体中的分布。然后我们将评估 由Spatial SILAC的这些药物引起的靶向和脱靶蛋白质组学变化。生成的结果 在这一目标将被用来优化未来的CPP的设计。总而言之，这篇文章描述了一部小说。 质谱方法，将简化药物开发过程，同时还提供丰富的 新治疗候选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.