Viscotaxis: Novel cell migration mechanisms regulated by microenvironmental viscosity

Viscotaxis：微环境粘度调节的新型细胞迁移机制

• 批准号: 10379292
• 负责人: Konstantinos Konstantopoulos
• 金额: $ 46.44万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10379292
• 关键词: 1-Phosphatidylinositol 3-Kinase; 3-Dimensional; Actins; Actomyosin; Acute; Affect; Anterior; Biological Assay; Biomedical Engineering; Biophysics; Blood; Breast Cancer Cell; Breast Cancer cell line; Breast cancer metastasis; Cancerous; Cations; Cell Volumes; Cell membrane; Cells; Complement; Cytoplasm; Cytoskeleton; Data; Distant; Elements; Epithelial Cells; Equilibrium; Event; Exhibits; Exposure to; Extracellular Matrix; Extracellular Matrix Degradation; Feedback; Fiber; Focal Adhesions; Goals; Growth; Human body; Image; Imaging Device; In Vitro; Injections; Integrins; Intercellular Fluid; Intuition; Ion Channel; Ions; Light; Liquid substance; Lung; Mechanics; Mediating; Memory; Migration Assay; Modeling; Motion; Motor; Mucins; Mus; Neoplasm Metastasis; Neoplasm Transplantation; Nuclear Translocation; Optics; Organ; Organoids; Patients; Pattern; Physiological; Plasma; Primary Neoplasm; Process; Prognostic Marker; Proteins; Publishing; Regulation; Resolution; Role; Signal Transduction; Site; Speed; Stretching; Sum; Survival Analysis; Swelling; Tail; Testing; Tissues; Travel; Veins; Viscosity; Water; Zebrafish; breast cancer survival; cancer cell; cell motility; chloride-cotransporter potassium; confocal imaging; extracellular; in vivo; in vivo Model; interdisciplinary approach; interstitial; macromolecule; malignant breast neoplasm; mammary; mechanotransduction; member; migration; mouse model; multiphoton microscopy; neoplastic cell; new therapeutic target; novel; particle; patient derived xenograft model; preconditioning; receptor; triple-negative invasive breast carcinoma; tumor

Cell motility is a key step in the metastatic cascade of events, as it enables cancerous cells dissociating from a primary tumor to navigate through interstitial tissues and ultimately colonize distant organs. Cell locomotion is governed by cell-matrix interactions, the actomyosin cytoskeleton, and cell volume regulation via the involvement of ion transporters, such as the Na+/H+ exchanger 1 (NHE1). To date, most cell motility assays are performed in medium with a viscosity close to that of water (0.77 cP). However, the viscosity of the interstitial fluid varies up to 2-3 cP, and can be further augmented by the presence of macromolecules secreted not only by resident epithelial cells in various tissues but also by tumor cells. Cancer cell plasticity is a key feature in metastasis, as tumor cells need to adapt to and navigate through diverse tissue microenvironments presenting different stiffness, degrees of confinement, viscosity and extracellular matrix (ECM) composition. It is currently unknown how tumor cells sense and respond to (patho)physiologically relevant levels of viscosity. The overarching goal of this project is to employ a multidisciplinary approach involving state-of-the-art bioengineering and imaging tools, quantitative analysis and in vivo models to elucidate the effects of extracellular viscosity on breast cancer cell migration, invasion and metastasis. This application will test the hypothesis, supported by intriguing preliminary data, that elevated extracellular viscosity (≥3cP) promotes NHE1-dependent cell swelling, which triggers the activation of the mechanosensitive ion channel TRPV4, thereby initiating downstream signaling. In Aim 1a, we will establish that TRPV4 is the key mechanosensor of elevated viscosity, which initiates RhoA activation, and delineate the presence of a potential feedback loop between NHE1-dependent TRPV4 activation and RhoA. In Aim 1b, we will demonstrate that the coordinated action of local isosmotic swelling at the leading edge and shrinkage at the trailing edge mediated by NHE1 and potassium-chloride cotransporter 4, KCC4, respectively, supports confined migration at elevated viscosities. Cells, as active mechanical objects upon sensing elevated extracellular viscosity, respond by balancing forces in the cell cytoplasm with those in the extracellular microenvironment, thus resulting in increased cytoskeletal tension, higher RhoA-dependent cell contractility and actin reorganization, which ultimately precipitate nuclear translocation of YAP (Aim 1c). We will characterize the roles of viscosity-sensing mechanisms in discrete steps of metastatic dissemination in a live zebrafish model that affords the unique advantages of optical transparency and exceptionally high-resolution along with high-speed imaging of transplanted tumor cells (Aim 2a). We will complement these studies with mouse models to characterize the localization patterns and functional roles of TRPV4, NHE1, KCC4 and YAP in cell migration in natural mammary tissue tracks in vivo (Aim 2b) and in breast cancer growth and metastasis (Aim 2c), using triple-negative breast cancer cell lines and patient-derived xenografts (PDXs). In sum, this project will define how cells sense and respond to extracellular viscosity and identify novel targets to reduce metastasis.

细胞运动是转移级联事件中的关键步骤，因为它使癌细胞能够 从原发性肿瘤中分离出来，穿过间质组织并最终定殖于远处的器官。 细胞运动由细胞-基质相互作用、肌动球蛋白细胞骨架和细胞体积调节控制 通过离子转运蛋白的参与，例如 Na+/H+ 交换器 1 (NHE1)。迄今为止，大多数细胞的运动 测定在粘度接近水 (0.77 cP) 的介质中进行。然而，该物质的粘度 间质液变化高达 2-3 cP，并且可以因分泌的大分子的存在而进一步增加 不仅通过各种组织中的常驻上皮细胞，而且还通过肿瘤细胞。癌细胞的可塑性是一个关键特征 在转移中，由于肿瘤细胞需要适应并穿越不同的组织微环境 不同的刚度、限制程度、粘度和细胞外基质 (ECM) 成分。目前是 未知肿瘤细胞如何感知和响应（病理）生理相关的粘度水平。这 该项目的总体目标是采用涉及最先进生物工程的多学科方法 和成像工具、定量分析和体内模型来阐明细胞外粘度对 乳腺癌细胞的迁移、侵袭和转移。该应用程序将测试该假设，并得到以下支持： 有趣的初步数据表明，细胞外粘度升高 (≥3cP) 会促进 NHE1 依赖性细胞肿胀， 触发机械敏感离子通道 TRPV4 的激活，从而启动下游 发信号。在目标 1a 中，我们将确定 TRPV4 是粘度升高的关键机械传感器，它启动 RhoA 激活，并描绘了 NHE1 依赖性 TRPV4 之间潜在反馈回路的存在 激活和 RhoA。在目标 1b 中，我们将证明局部等渗肿胀的协调作用 由 NHE1 和氯化钾协同转运蛋白 4 介导的前缘和后缘收缩， KCC4 分别支持高粘度下的限制迁移。细胞作为活跃的机械物体 感知细胞外粘度升高，通过平衡细胞质中的力与细胞中的力来做出反应 细胞外微环境，从而导致细胞骨架张力增加，RhoA依赖性细胞更高 收缩性和肌动蛋白重组，最终促成 YAP 的核转位（目标 1c）。我们将 表征粘度传感机制在活体内转移传播的离散步骤中的作用 斑马鱼模型具有光学透明和高分辨率的独特优势 以及移植肿瘤细胞的高速成像（目标 2a）。我们将补充这些研究 小鼠模型来表征 TRPV4、NHE1、KCC4 和 YAP 的定位模式和功能作用 天然乳腺组织中的细胞迁移跟踪体内（目标 2b）以及乳腺癌生长和转移 （目标 2c），使用三阴性乳腺癌细胞系和患者来源的异种移植物（PDX）。综上所述，本项目 将定义细胞如何感知和响应细胞外粘度，并确定减少转移的新靶点。