Spatial SILAC: Examining the Proteome in 3D Cell Cultures

空间 SILAC：检查 3D 细胞培养物中的蛋白质组

基本信息

批准号：
10021670
负责人：
Amanda B. Hummon
金额：
$ 37.72万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10021670
关键词：
Adherent Culture Affect Amino Acids Animal Testing Antineoplastic Agents Antitumor Drug Screening Assays Apoptotic Biochemical Biological Models Calcineurin Cell Culture Techniques Cell Nucleus Cells Cetuximab Chemicals Clinical Code Colon Carcinoma Complex Data Drug Design Drug Screening Epithelial Epithelium Evaluation Failure Feedback Fluorouracil Future Goals Human body Image In Vitro Individual Isotope Labeling Isotopes KRAS2 gene Label Light Location Maps Mass Spectrum Analysis Measures Metabolic Methodology NF-kappa B Nutrient Oxygen Pathway interactions Peptides Periodicity Permeability Pharmaceutical Preparations Pharmacodynamics Pharmacotherapy Physiological Population Primary Neoplasm Problem Solving Process Proliferating Proteins Proteome Proteomics Radial Reproducibility Research Resolution Sampling Solid Structure Testing Therapeutic Therapeutic Uses Time Tissues Work analytical method cancer therapy cell type cost cost effectiveness cost estimate design drug candidate drug development drug distribution drug metabolism improved in vivo irinotecan mass spectrometer monolayer novel novel anticancer drug novel therapeutics peptide drug pharmacokinetics and pharmacodynamics phosphoproteomics pre-clinical professor research and development response screening therapeutic candidate three dimensional cell culture tool tumor two-dimensional

项目摘要

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.

项目摘要药物开发过程效率低下且昂贵，部分是因为大多数治疗剂最初是用二维细胞培养物进行筛选。这些细胞培养物不能准确反映组织结构人体。为了改善药物筛查，应使用更准确的模型系统。三- 尺寸细胞培养物，也称为球形植物，填补了这一空白。与肿瘤相似，结肠癌球形素含有根本的对称营养素和氧梯度。球体发展出不同的细胞种群反映了这些化学梯度。在此提案中，我们正在开发一种分析方法，称为空间 SILAC，专门标记使用不同同位素在球体内存在的不同细胞种群。在AIM 1中，我们将证明这些同位素可以通过质谱仪很容易区分，从而提供细胞在球体内的同位素“邮政编码”。在AIM 2中，我们将结合成像质量光谱法药物分布研究通过评估蛋白质组学变化对治疗的响应，使用同位素标签。我们将像以前一样评估Irinotecan，5-氟尿嘧啶和西妥昔单抗通过成像质谱法检查了这三种药物在球体中的分布。我们将比较通过成像分布的治疗治疗响应的空间局部蛋白质组学变化地图。在AIM 3中，我们将应用空间SILAC方法来筛选新开发的循环穿透由Co-I教授Dehua Pei设计的肽（CPP）。这些有希望的癌症候选者需要球体中的临床前筛查和空间Silac提供了理想的测试床，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CPPS及其代谢物在小节中的分布。然后，我们将评估这些药物由空间SILAC引起的靶向和不靶向蛋白质组学变化。结果产生在此目标中，将用于优化未来CPP的设计。总而言之，该提议描述了一本小说质谱方法将简化药物开发过程，同时也提供丰富有关新治疗候选者的体外药代动力学和药效学信息。