Methods for the Analysis of Tumor Extracellular Matrix

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入性表型转变为侵入性表型。然而，对ECM-细胞相互作用的研究仅限于专注于一种或几种ECM蛋白的还原方法。迄今为止，影响这种细胞表型变化的细胞外生物分子在很大程度上是未知的。更好地了解ECM在促进肿瘤细胞转移中的作用将有助于更全面地表征ECM的组成。研究ECM的蛋白质组学方法一直受到高度交联基质组分的蛋白质水解和抗溶解特性的阻碍，这使得更传统的蛋白质组学技术的研究变得困难，如果不是不可能的话。该领域进展的主要障碍是缺乏合适的样品制备方法来有效地表征ECM的分子特征。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，我们将建立一个可重复的ECM来源。我们将利用三维细胞培养模型来评估两对等基因人乳腺上皮细胞系分泌的ECM对肿瘤的促进作用。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Western blots将用于评估细胞污染物的水平。接下来，将以鉴定的不同ECM蛋白的数量和序列覆盖率百分比为终点，探索ECM的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，并有助于进一步优化方法。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，该微环境可以将非转移性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。这些研究将成为转化实验、临床研究的基础，并最终导致生物标志物和治疗方法的出现，从而改善患者的护理和生存能力。

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.