Fibronectin and its role in tumor stiffness and vascularization

描述(申请人提供)：组织硬度增加代表乳腺癌的一个特征，它是由细胞外基质(ECM)的物理化学变化所介导的；然而，ECM硬度增加促进肿瘤血管生成从而促进生长的机制尚不清楚。该项目研究的假设是，乳腺癌细胞的旁分泌信号增加了脂肪干细胞(ASCs)对纤维连接蛋白(FN)基质的组装，从而增强了ASCs和内皮细胞的促血管生成能力，促进了肿瘤的血管形成。为了研究这一假设，我们提出了一种生化和物理科学相结合的方法，使我们能够量化肿瘤来源的可溶性因子信号对ASC沉积的FN基质的构象和刚性的影响。具体地说，我们将使用荧光共振能量转移(FRET)成像和表面力装置(SFA)分别在大分子和细胞/组织水平测量FN力学，并将评估这些参数在体外和体内对促血管生成信号的影响。这项工作将在三个具体目标中完成：在目标1中，我们将评估ASCs在存在或不存在肿瘤细胞条件介质的情况下组装FN矩阵，并识别与这些变化有关的信号分子。在目标2中，我们将分析ASC调节的FN基质特征对肿瘤相关的、促血管生成的ASCs和内皮细胞表型的贡献。在目标3中，我们将确定ASC调节的FN基质组装是否促进体内肿瘤的血管生成、僵硬和生长，并评估目标1中确定的信号分子在这一发病机制中的作用。转化生长因子β信号转导将是拟议研究的最初焦点，因为该因子调节肿瘤发生、细胞收缩和纤维连接蛋白组装。此外，我们期待着识别已经涉及FN机制的新因素，但在肿瘤血管形成中的作用尚不明确。通过将FN构象和力学与肿瘤微环境中的促血管生成信号联系起来，这项工作将广泛影响我们对肿瘤僵硬和血管形成之间联系的理解，并可能导致识别新的抗血管生成靶点和改进的治疗方法。虽然拟议的研究重点是确定ASCs在这一过程中的作用，但其他各种生理和病理情况严重依赖于ECM机制(例如，器官发生、动脉粥样硬化)。作为该项目的一部分开发的培养系统和机械测试策略引入了全新的方法来研究这些过程。 公共卫生相关性：持续的血管生成是乳腺癌的一个特征，它受到细胞外基质(ECM)机制的影响；然而，尚不清楚肿瘤相关脂肪干细胞(ASCs)组装的纤维连接蛋白(FN)基质是否在这一过程中发挥作用。这项研究将结合生化和物理科学工具来确定肿瘤来源的可溶性因子对ASC沉积的FN基质刚性的影响，并评估这些变化是否促进了肿瘤血管的形成。这种跨学科的策略不仅有可能彻底改变我们对肿瘤血管生成的理解，而且还为研究依赖于FN机制的其他生理和病理过程提供了广泛适用的方法。

DESCRIPTION (provided by applicant): Increased tissue stiffness represents a hallmark of breast cancer that is mediated by physicochemical alterations of the extracellular matrix (ECM); however, the mechanisms through which enhanced ECM stiffness promotes tumor angiogenesis, and hence growth, are poorly understood. This project investigates the hypothesis that paracrine signaling by breast cancer cells increases fibronectin (Fn) matrix assembly by adipose-derived stem cells (ASCs), thereby enhancing the pro-angiogenic capability of both ASCs and endothelial cells to promote tumor vascularization. To investigate this hypothesis we propose a combination of biochemical and physical science approaches that will enable us to quantify the impact of tumor-derived soluble factor signaling on the conformation and rigidity of ASC-deposited Fn matrices. Specifically, we will use Fluorescence Resonance Energy Transfer (FRET) imaging and the Surface Forces Apparatus (SFA) to measure Fn mechanics at the macromolecular and cell/tissue level, respectively, and will assess the impact of these parameters on pro-angiogenic signaling in vitro and in vivo. This work will be accomplished in three specific aims: In Aim 1, we will evaluate Fn matrix assembly by ASCs in the presence or absence of tumor cell- conditioned media and identify signaling molecules contributing to these changes. In Aim 2, we will analyze the contributions of ASC-regulated Fn matrix characteristics towards a tumor-associated, pro-angiogenic phenotype of ASCs and endothelial cells. In Aim 3, we will determine whether ASC-regulated Fn matrix assembly promotes tumor angiogenesis, stiffness, and growth in vivo and evaluate the contributions of the signaling molecules identified in aim 1 in this pathogenesis. Transforming growth factor beta (TGF-beta) signaling will be the initial focus of the proposed studies, as this factor modulates tumorigenesis, cell contractility, and Fn assembly. Additionally, we anticipate identification of novel factors already implicated in Fn mechanics yet with an undefined role in tumor vascularization. By correlating Fn conformation and mechanics with pro-angiogenic signaling in the tumor microenvironment this work will broadly impact our understanding of the connection between tumor stiffness and vascularization and may lead to the identification of novel anti- angiogenic targets and improved therapies. While the emphasis in the proposed studies is to determine the role of ASCs in this process, a variety of other physiological and pathological situations critically rely upon ECM mechanics (e.g., organogenesis, atherosclerosis). The culture systems and mechanical testing strategies developed as part of this project introduce radically new approaches to investigate these processes. PUBLIC HEALTH RELEVANCE: Sustained angiogenesis is a hallmark of breast cancer that is influenced by extracellular matrix (ECM) mechanics; however, it remains unclear whether or not fibronectin (Fn) matrix assembly by tumor-associated adipose-derived stem cells (ASCs) may play a role in this process. This research will integrate biochemical and physical science tools to determine the effect of tumor-derived soluble factors on the rigidity of ASC-deposited Fn matrices and evaluate if these changes promote tumor vascularization. This interdisciplinary strategy has the potential to not only revolutionize our understanding of tumor angiogenesis, but also to provide widely applicable approaches to study other physiological and pathological processes that depend on Fn mechanics.