Methods for the Analysis of Tumor Extracellular Matrix

肿瘤细胞外基质的分析方法

• 批准号: 7941682
• 负责人: KIRK C HANSEN
• 金额: $ 19.97万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7941682
• 关键词: Area; Arts; Attention; Biological Markers; Cancerous; Cell Communication; Cell Culture Techniques; Cell Line; Cells; Cellular Structures; Chemicals; Clinical Trials; Deposition; Development; Diagnostic; Digestion; Effectiveness; Epithelial Cells; Evaluation; Excision; Extracellular Matrix; Extracellular Matrix Proteins; Foundations; Grant; Human; Individual; Ions; Label; Lead; Liquid Chromatography; Malignant Neoplasms; Mammary gland; Mass Spectrum Analysis; Methods; Modeling; Modification; Molecular; Morphologic artifacts; Neoplasm Metastasis; Outcome; Patient Care; Peptides; Phenotype; Play; Preparation; Property; Proteins; Proteolysis; Proteomics; Protocols documentation; Recombinants; Relative (related person); Resistance; Role; Sampling; Source; Techniques; Testing; Therapeutic; Tissues; Transglutaminases; Western Blotting; Work; base; cancer cell; crosslink; extracellular; human tissue; improved; in vivo; inhibitor/antagonist; neoplastic cell; novel therapeutics; numb protein; programs; protein crosslink; public health relevance; research study; tandem mass spectrometry; tumor; tumor progression

DESCRIPTION (provided by applicant): Recent key studies have shown that the composition of the extracellular matrix (ECM) can determine metastatic outcome, specifically whether a tumor cell will transition from a non-invasive to an invasive phenotype. However, the study of ECM-cell interactions has been limited to reductionist methods that focus on one or a few ECM proteins. To date, the extracellular biomolecules that are responsible for influencing this change in cell phenotype are largely unknown. A better understanding of the ECM role in promoting tumor cell metastasis will be facilitated by a more global characterization of ECM composition. Proteomic approaches to study ECM have been hindered by the proteolytic and solubilization resistant properties of highly cross-linked matrix components, making study by more traditional proteomic techniques difficult, if not impossible. A major barrier to progress in this field is the lack of suitable sample preparation methods for effective molecular characterization of ECM. The focus of this grant is the optimization of ECM sample preparation methods. To develop effective sample preparation methods we will establish a reproducible source of ECM. A three- dimensional cell culture model will be used to evaluate the tumor promotional attributes of ECM secreted by two pairs of isogenic human mammary epithelial cell lines. The first areas of sample preparation development will be in the optimization of cell removal from its underlying ECM, with emphasis on eliminating contaminate intracellular proteins that co-purify with the ECM. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) profiling and Western blots will be used to evaluate the level of cellular contaminants. Next, ECM solubilization strategies and effective cleavage methods will be explored using as endpoints the number of distinct ECM protein identified and percent sequence coverage. Utilization of a label-free mass spectrometry based quantification method will provide relative ranking of the strategies and assist in further method optimization. We hypothesize that protein-protein crosslinks influence the effectiveness of matrix sample preparation methods for ECM. The two major classes of proteins that catalyze the cross-linking of ECM proteins show aberrant expression and activity in neoplastic cell lines and various cancerous tissues. We will use recombinant human tissue transglutaminase and its inhibitors to determine how crosslinking influences cell removal, solubilization and digestion efficiencies with emphasis on protein quantification. Cancer cells deposit ECM proteins into their microenvironment that can program non-metastatic cells to a phenotype consistent with metastasis. The development of the proposed cell culture model and state-of the- art ECM specific proteomic techniques will advance our ability to explore and characterize the role of the extracellular matrix in metastasis. These studies will be the foundation for translational experiments, clinical investigations and should ultimately lead to biomarkers and therapeutic approaches that will improve patient care and survivability. PUBLIC HEALTH RELEVANCE: Despite the fundamental role that the cell microenvironment plays in tumor progression, methods to study the underlying molecular mechanisms are lacking. This work is aimed at developing the sample preparation methods necessary to characterize the matrix component of the cell microenvironment so that we may ultimately develop new therapeutic strategies and identify early diagnostic markers of cancer.

描述（由申请人提供）：最近的关键研究表明，细胞外基质（ECM）的组成可以确定转移性结果，特别是肿瘤细胞是否会从非侵入性转变为侵入性表型。但是，对ECM细胞相互作用的研究仅限于重点是一种或几种ECM蛋白质的还原主义方法。迄今为止，负责影响这种细胞表型变化的细胞外生物分子在很大程度上未知。通过更全球的ECM组成表征，可以更好地了解ECM在促进肿瘤细胞转移中的作用。蛋白质组学研究ECM的方法受到高度交联基质组件的蛋白水解和溶解性抗性特性的阻碍，从而使更传统的蛋白质组学技术变得困难，即使不是不可能的话。该领域进步的主要障碍是缺乏有效的ECM分子表征的合适样品制备方法。该赠款的重点是优化ECM样品制备方法。为了开发有效的样品制备方法，我们将建立可再现的ECM来源。三维细胞培养模型将用于评估由两对等源性人类乳腺上皮细胞系分泌的ECM的肿瘤促进属性。样品制备开发的第一个领域将是优化细胞从其潜在的ECM中去除，重点是消除与ECM共纯化的细胞内蛋白质。液相色谱串联质谱法（LC-MS/MS）分析和蛋白质印迹将用于评估细胞污染物的水平。接下来，将使用AS端点探索ECM溶解策略和有效的切割方法。基于无标签质谱的定量方法的利用将提供策略的相对排名，并有助于进一步的方法优化。我们假设蛋白质 - 蛋白质交联影响基质样品制备方法对ECM的有效性。催化ECM蛋白质的两种主要类别的蛋白质在肿瘤细胞系和各种癌组织中表现出异常的表达和活性。我们将使用重组人组织转谷氨酰胺酶及其抑制剂来确定交联如何影响细胞去除，溶解和消化效率，重点是蛋白质定量。癌细胞将ECM蛋白沉积到其微环境中，可以将非转移细胞编程为与转移一致的表型。所提出的细胞培养模型和最新的ECM特异性蛋白质组学技术的发展将提高我们探索和表征细胞外基质在转移中的作用的能力。这些研究将是转化实验，临床研究的基础，并最终应导致生物标志物和治疗方法，以改善患者护理和生存能力。 公共卫生相关性：尽管细胞微环境在肿瘤进展中起着基本作用，但仍缺乏研究潜在的分子机制的方法。这项工作的目的是开发出表征细胞微环境的基质组成部分所需的样品制备方法，以便我们最终可以制定新的治疗策略并确定癌症的早期诊断标志物。