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）的组成可以决定转移结果，特别是肿瘤细胞是否会从非侵袭性表型转变为侵袭性表型。然而，ECM-细胞相互作用的研究仅限于专注于一种或几种 ECM 蛋白的还原论方法。迄今为止，影响细胞表型这种变化的细胞外生物分子在很大程度上还是未知的。对 ECM 成分进行更全面的表征将有助于更好地了解 ECM 在促进肿瘤细胞转移中的作用。研究 ECM 的蛋白质组学方法受到高度交联基质成分的蛋白水解和抗溶解特性的阻碍，使得通过更传统的蛋白质组学技术进行研究变得困难，甚至不可能。该领域取得进展的一个主要障碍是缺乏合适的样品制备方法来有效地表征 ECM。本次资助的重点是 ECM 样品制备方法的优化。为了开发有效的样品制备方法，我们将建立可重复的 ECM 来源。三维细胞培养模型将用于评估两对同基因人乳腺上皮细胞系分泌的ECM的肿瘤促进特性。样品制备开发的首要领域是优化从其基础 ECM 中去除细胞，重点是消除与 ECM 共同纯化的污染细胞内蛋白质。液相色谱-串联质谱 (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，将使用已识别的不同 ECM 蛋白的数量和序列覆盖百分比作为终点来探索 ECM 溶解策略和有效的切割方法。利用基于无标记质谱的定量方法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-蛋白质交联会影响 ECM 基质样品制备方法的有效性。催化 ECM 蛋白交联的两大类蛋白在肿瘤细胞系和各种癌组织中表现出异常表达和活性。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，从而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先进的 ECM 特异性蛋白质组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。这些研究将成为转化实验、临床研究的基础，并最终产生可改善患者护理和生存能力的生物标志物和治疗方法。 公共卫生相关性：尽管细胞微环境在肿瘤进展中发挥着重要作用，但仍缺乏研究潜在分子机制的方法。这项工作旨在开发表征细胞微环境基质成分所需的样品制备方法，以便我们最终可以开发新的治疗策略并识别癌症的早期诊断标志物。