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标志，由细胞外基质（ECM）的理化改变介导；然而，增强ECM硬度促进肿瘤血管生成从而促进肿瘤生长的机制尚不清楚。本项目研究的假设是，乳腺癌细胞的旁分泌信号可以增加脂肪源性干细胞（ASCs）的纤维连接蛋白（Fn）基质组装，从而增强ASCs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，我们提出了生物化学和物理科学方法的结合，这将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基质的构象和硬度的影响。具体来说，我们将使用荧光共振能量转移（FRET）成像和表面力仪（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。这项工作将有三个具体目标：在目标1中，我们将评估ASCs在存在或不存在肿瘤细胞条件培养基下的Fn基质组装，并识别有助于这些变化的信号分子。在Aim 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Aim 3中，我们将确定asc调节的Fn基质组装是否促进肿瘤血管生成、僵硬和体内生长，并评估Aim 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 (tgf - β)信号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，我们预计将发现与Fn力学有关的新因素，但在肿瘤血管化中尚未确定其作用。通过将Fn构象和机制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刚度和血管形成之间关系的理解，并可能导致新的抗血管生成靶点的鉴定和改进治疗方法。虽然拟议研究的重点是确定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，培养系统和机械测试策略为研究这些过程引入了全新的方法。

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.