Advanced Methods to Evaluate Extracellular Matrix and Crosslinking in the Tumor M

评估肿瘤 M 细胞外基质和交联的先进方法

基本信息

批准号：
8845180
负责人：
KIRK C HANSEN
金额：
$ 38.27万
依托单位：
UNIVERSITY OF COLORADO DENVER
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：
2014-05-02 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8845180
关键词：
Accounting Advanced Development Affinity Chromatography Affinity Labels Amino Acids Animal Model Architecture Area Biology Biomechanics Biomedical Research Blood Vessels Breast Carcinoma Cancer Control Cancer Model Cattle Cell Communication Cell Culture Techniques Clinical Data Data Set Deposition Development Diagnostic Diagnostic Neoplasm Staging Digestion Disease Emerging Technologies Enzymes Event Extracellular Matrix Extracellular Matrix Proteins Fibrillar Collagen Fibrosis Goals Grant Growth Health Human Hydroxyproline Intercellular Fluid Label Lead Lesion Libraries Malignant Neoplasms Mass Spectrum Analysis Measurement Measures Mediating Medicine Methods Modeling Modification Molecular Mus Neoplasm Metastasis Normal tissue morphology Organ Outcome Pancreas Patients Peptide Library Peptides Phenotype Physiologic pulse Plasmids Play Post-Translational Protein Processing Preparation Process Protein-Lysine 6-Oxidase Proteins Proteomics Protocols documentation Proxy Reagent Relative (related person)Reporter Research Personnel Resistance Resolution Resources Role Sampling Site Solid Neoplasm Source Specificity Stable Isotope Labeling Staging Techniques Testing Therapeutic Intervention Time Tissue Sample Tissues Trypsin Tumor Cell Invasion Tumor Promotion Tumor Tissue Tumor stage Validation Work affinity labeling anticancer research base crosslink design extracellular high throughput analysis improved inhibitor/antagonist insight interest meetings mouse model neoplastic neoplastic cell novel diagnostics novel therapeutics pressure protein crosslink research study response stable isotope therapeutic target tissue preparation tumor tumor microenvironment tumor progression tumorigenic

项目摘要

DESCRIPTION (provided by applicant): The composition and architecture of the extracellular matrix (ECM) defines the metastatic outcome of tumor cells, specifically whether a tumor cell will transition from a non-invasive to an invasive phenotype. However, the extracellular biomolecules responsible are largely unknown. Identifying and understanding the role of specific ECM proteins and modifications involved in tumor progression requires a more comprehensive and precise characterization of ECM within tumor microenvironments. ECM proteins are often covalently cross-linked rendering them resistant to solubilization and proteolytic cleavage. As a result, analyses of tissue samples by traditional proteomic techniques fail to reflect actual protein composition. Lack of suitable sample preparation and quantification methods for accurate molecular characterization of ECM remains a major barrier to progress in the field. The focus of this R33 application is on development and validation of methods to identify and quantify ECM proteins and crosslinked peptides that differ between matrices that support or suppress tumorigenic events. We have made considerable progress towards the goals outlined in our "Sample Preparation Methods for the Detailed Characterization of Tumor Associated Extracellular Matrix" R21 grant. The highlights of this work involves 1) development of cell culture models for evaluating ECM driven phenotypes, 2) defining a robust ECM extraction protocol that minimizes cellular contamination and permits proteomic analysis of both the strong chaotrope soluble and insoluble ECM fractions, and 3) application of a label- free relative-quantification approach for estimation of protein abundance changes. Ultimately, this work has yielded a proteomic method that results in a more accurate representation of tissue protein composition compared to all others tested. The work proposed in this R33 extension grant builds on these advancements by providing the methods necessary for in-depth characterization of ECM during tumor progression to deliver previously unobtainable molecular detail of cellular microenvironment remodeling events. To facilitate absolute quantification we have developed a library of stable isotope labeled reporter peptides that provide a sensitive proxy for ECM proteins. We will generate additional peptides for fibrillar collagens that contain hydroxyproline residues so that these heavy-labeled libraries can be added to ECM samples of interest for protein quantification. This approach will allow for higher- throughput analysis, improved sensitivity, accuracy, and intra- and inter- sample comparison of protein levels. We will generate a crosslinked-peptide identification strategy to characterize the types and sites of lysyl oxidase (LOX) mediated crosslinks that correlate with tumor progression. The method involves mixed affinity labeling of proteolytic peptides followed by tandem affinity purification for selective crosslinked peptide enrichment. The method will initially be developed using control proteins and tissues, and then applied to tissues from our cancer model and control mice treated +/- LOX inhibitor. Finally, we will examine alterations in ECM composition and crosslinking between pre-neoplastic lesions, early stage, and late stage tumors by applying a stable isotope pulse labeling approach in conjunction with or internal standard peptide library. A 30 day pulse labeling provided by stable isotope Lys containing chow will allow for measurements of new protein incorporated into the ECM of mouse tissue and tumors. Protein abundance and degradation differences between time points will be accounted for using the Arg containing subset of our reporter peptides. These experiments should allow us to obtain molecular resolution, previously unattainable, of the dynamic and reciprocal relationship between ECM deposition, processing and degradation that occur during tumor progression. The development of the proposed methods for absolute quantification and validation of these ECM specific techniques will advance our ability to explore and characterize the role of the ECM in cancer progression. The methods will help investigators understand a poorly understood area of basic biology, cancer progression, and fibrotic diseases in general. Findings in this historically overlooked area are likely to provide paradigm shifts in our understanding of these diseases and the types of proteins considered for therapeutic targeting, and as sources of diagnostic markers. These studies will provide important reagents (reporter peptide library plasmids and SIL-PTM peptides) and resources (protocols and datasets) necessary to move this field toward its ultimate goal of enabling discoveries that improve patients' lives.

描述（由申请人提供）：细胞外基质（ECM）的组成和结构定义了肿瘤细胞的转移性结果，特别是肿瘤细胞是否会从非侵入性过渡到侵入性表型。但是，负责的细胞外生物分子在很大程度上尚不清楚。识别和理解特定的ECM蛋白的作用以及在肿瘤进展中涉及的修饰需要对肿瘤微环境中ECM的更全面和精确的表征。 ECM蛋白通常是共价交联的，使它们具有抗溶性和蛋白水解裂解能力。结果，通过传统蛋白质组学技术对组织样品的分析无法反映实际的蛋白质组成。缺乏适当的样品制备和ECM准确分子表征的定量方法仍然是该领域进步的主要障碍。该R33应用的重点是开发和验证方法，以识别和量化ECM蛋白质和交联的肽，这些肽在支持或抑制肿瘤事件的矩阵之间有所不同。我们已经在“样本制备方法详细表征相关的细胞外基质” R21赠款的“样本制备方法中概述的目标取得了长足的进步”。这项工作的亮点涉及1）开发用于评估ECM驱动表型的细胞培养模型，2）定义强大的ECM提取方案，该方案可最大程度地减少细胞污染并允许蛋白质组学分析强的蛋白质组学分析，并且均可溶于强度和不溶性ECM馏分，以及3）应用。估计蛋白质丰度变化的标签 - 游离相对定量方法。最终，这项工作产生了一种蛋白质组学方法，该方法与所有测试的所有其他方法相比，可以更准确地表示组织蛋白组成。 R33扩展赠款中提出的工作是基于这些进步的基础，它通过提供肿瘤进展过程中ECM的深入表征的方法，以提供先前无法获得的细胞微环境重塑事件的分子细节。为了促进绝对定量，我们开发了一个稳定的同位素标记的记者肽的库，该肽为ECM蛋白提供了敏感的代理。我们将生成含有羟基残基的原纤维胶原蛋白的其他肽，以便可以将这些重标记的文库添加到感兴趣的ECM样品中以进行蛋白质定量。这种方法将允许进行更高的吞吐量分析，提高灵敏度，准确性以及蛋白质水平的样品内和样品间比较。我们将生成一个交联肽鉴定策略，以表征与肿瘤进展相关的赖氨酸氧化酶（LOX）介导的交联的类型和位点。该方法涉及蛋白水解肽的混合亲和力标记，然后进行串联亲和力纯化，以进行选择性交联的肽富集。该方法最初将使用对照蛋白和组织开发，然后应用于我们的癌症模型和对照小鼠+/- LOX抑制剂的组织。最后，我们将通过应用稳定的同位素脉冲标记方法与结合或内标准肽库进行稳定的同位素脉冲标记方法，研究ECM组成的变化和ECM组成和交联的变化。由稳定的同位素LYS提供的30天脉冲标记将允许测量掺入小鼠组织和肿瘤ECM中的新蛋白质。时间点之间的蛋白质丰度和降解差异将被解释，以使用含有我们报告基因肽的亚集的ARG。这些实验应该使我们能够获得ECM沉积，加工和降解过程中肿瘤进展过程中的动态和相互关系的分子分辨率。对这些ECM特定技术的绝对量化和验证提出的方法的开发将提高我们探索和表征ECM在癌症进展中的作用的能力。这些方法将帮助研究人员了解一般的基本生物学，癌症进展和纤维化疾病的知识不足。在历史上被忽视的地区的发现可能会在我们对这些疾病的理解以及认为用于治疗靶向的蛋白质的类型以及作为诊断标记的来源提供范式转变。这些研究将提供重要的试剂（报告肽库质粒和SIL-PTM肽）和资源（协议和数据集），以使该领域朝着其最终的目标迈向改善患者生活的最终目标。