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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.

项目总结