Fibronectin and its role in tumor stiffness and vascularization

描述（由申请人提供）：组织硬度增加是乳腺癌的一个标志，这是由细胞外基质（ECM）的物理化学改变介导的；然而，增强的 ECM 硬度促进肿瘤血管生成进而促进肿瘤生长的机制尚不清楚。该项目研究了这样的假设：乳腺癌细胞的旁分泌信号传导会增加脂肪干细胞 (ASC) 的纤连蛋白 (Fn) 基质组装，从而增强 ASC 和内皮细胞的促血管生成能力，从而促进肿瘤血管化。为了研究这一假设，我们提出了生化和物理科学方法的结合，使我们能够量化肿瘤来源的可溶性因子信号传导对 ASC 沉积的 Fn 基质的构象和刚性的影响。具体来说，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/组织水平上测量Fn力学，并将评估这些参数对体外和体内促血管生成信号的影响。这项工作将实现三个具体目标：在目标 1 中，我们将在存在或不存在肿瘤细胞条件培养基的情况下评估 ASC 的 Fn 基质组装，并鉴定导致这些变化的信号分子。在目标 2 中，我们将分析 ASC 调节的 Fn 基质特征对 ASC 和内皮细胞的肿瘤相关、促血管生成表型的贡献。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血管生成、硬度和生长，并评估目标 1 中确定的信号分子在此发病机制中的贡献。转化生长因子 β (TGF-β) 信号传导将是拟议研究的最初重点，因为该因子调节肿瘤发生、细胞收缩性和 Fn 组装。此外，我们预计会鉴定出与 Fn 力学相关但在肿瘤血管化中作用尚不明确的新因素。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硬度和血管化之间联系的理解，并可能导致新的抗血管生成靶点的识别和改进的治疗方法。虽然拟议研究的重点是确定 ASC 在此过程中的作用，但各种其他生理和病理情况严重依赖 ECM 力学（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研究这些过程的全新方法。 公共健康相关性：持续的血管生成是乳腺癌的一个标志，受细胞外基质 (ECM) 力学的影响；然而，目前尚不清楚肿瘤相关脂肪干细胞（ASC）的纤连蛋白（Fn）基质组装是否在此过程中发挥作用。这项研究将整合生化和物理科学工具，以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肿瘤血管化。这种跨学科策略不仅有可能彻底改变我们对肿瘤血管生成的理解，而且还可以提供广泛适用的方法来研究依赖于 Fn 力学的其他生理和病理过程。

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.