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

• 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
• 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

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）的组成和结构决定了肿瘤细胞的转移结果，特别是肿瘤细胞是否会从非侵入性表型转变为侵入性表型。然而，细胞外的生物分子在很大程度上是未知的。识别和理解特异性ECM蛋白在肿瘤进展中的作用和修饰需要更全面和准确地表征肿瘤微环境中的ECM。ECM蛋白通常是共价交联的，使它们抵抗溶解和蛋白水解裂解。因此，传统的蛋白质组学技术对组织样本的分析不能反映实际的蛋白质组成。缺乏合适的样品制备和定量方法来准确表征ECM的分子特征仍然是该领域进展的主要障碍。该R33申请的重点是开发和验证鉴定和量化支持或抑制肿瘤发生事件的基质之间不同的ECM蛋白和交联肽的方法。我们在“肿瘤相关细胞外基质详细表征的样品制备方法”R21资助中概述的目标方面取得了相当大的进展。这项工作的亮点包括1)开发用于评估ECM驱动表型的细胞培养模型，2)定义一个强大的ECM提取方案，最大限度地减少细胞污染，并允许对强混沌可溶性和不溶性ECM组分进行蛋白质组学分析，以及3)应用无标记相对定量方法来估计蛋白质丰度变化。最终，这项工作产生了一种蛋白质组学方法，与所有其他测试相比，该方法可以更准确地表示组织蛋白质组成。这项R33扩展资助提出的工作建立在这些进展的基础上，提供了深入表征肿瘤进展过程中ECM的必要方法，以提供以前无法获得的细胞微环境重塑事件的分子细节。为了便于绝对定量，我们开发了一个稳定同位素标记的报告肽库，为ECM蛋白提供了一个敏感的代理。我们将为含有羟脯氨酸残基的纤维性胶原生成额外的肽，以便这些重标记文库可以添加到感兴趣的ECM样品中进行蛋白质定量。这种方法将允许更高的通量分析，提高灵敏度，准确性，以及样品内和样品间蛋白质水平的比较。我们将生成一种交联肽鉴定策略，以表征赖氨酸氧化酶（LOX）介导的与肿瘤进展相关的交联的类型和位点。该方法包括对蛋白水解肽进行混合亲和标记，然后进行串联亲和纯化，以选择性地富集交联肽。该方法将首先使用对照蛋白和组织进行开发，然后应用于我们的癌症模型和使用+/- LOX抑制剂的对照小鼠的组织。最后，我们将通过应用稳定同位素脉冲标记方法，结合内部标准肽库，研究肿瘤前病变、早期和晚期肿瘤之间ECM组成和交联的变化。含有chow的稳定同位素Lys提供的30天脉冲标记将允许测量纳入小鼠组织和肿瘤ECM的新蛋白质。蛋白质丰度和时间点之间的降解差异将使用我们的报告肽含有精氨酸的子集来解释。这些实验应该允许我们获得分子分辨率，以前无法实现的，在肿瘤进展过程中发生的ECM沉积，加工和降解之间的动态和相互关系。这些ECM特异性技术的绝对定量和验证方法的发展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基础生物学、癌症进展和纤维化疾病的一个鲜为人知的领域。在这个历史上被忽视的领域的发现可能会为我们对这些疾病的理解提供范式转变，以及考虑治疗靶向的蛋白质类型，并作为诊断标记的来源。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（方案和数据集），以推动该领域实现其最终目标，使发现能够改善患者的生活。