The interplay of ion transporters and cytoskeleton in breast cancer migration and metastasis

无法在临床上治疗肿瘤转移是大多数癌症患者死亡的原因。细胞迁移是癌细胞从原发肿瘤向体内远处器官转移扩散的关键步骤。正如渗透引擎模型(OEM)所解释的那样，细胞的运动受细胞-基质相互作用、肌动蛋白细胞骨架和离子转运体(如Na[+]/H[+]交换器1(NHE1)等离子转运体参与的细胞体积调节的控制。细胞骨架和离子转运体在细胞运动中的作用通常是孤立地研究的。该项目的总体目标是采用多学科方法，包括最先进的生物工程和成像工具、定量分析和体内模型，以确定离子转运体和细胞骨架在乳腺癌细胞体内迁移和转移中的相对作用和潜在的串扰。这一应用将检验这一假设，并得到有趣的初步数据的支持，即NHE1和SWELL1分别介导的前沿局部等渗膨胀和后缘收缩的协调作用支持禁闭中的迁移。我们进一步假设NHE1和SWELL1与细胞骨架协同作用，介导有效的迁移和转移。鉴于缺乏针对三重阴性乳腺癌(TNBC)的靶向治疗，我们将优先选择TNBC细胞系和患者来源的异种移植(PDX)肿瘤细胞作为模型。在目标1中，我们将确定NHE1和SWELL1在不同硬度的限制µ通道内的细胞迁移、在3D凝胶中的细胞迁移以及在3D乳腺癌细胞有机体的细胞扩散中的功能作用。我们还将阐明NHE1和SWELL1分别在细胞前端和后端极化分布的机制，并使用新型光遗传工具来改变它们的空间极化，并测试这些改变如何影响细胞迁移的方向和效率。同时，我们将开发一种创新的数学模型，以确定使OEM介导的细胞运动成为可能的关键变量。在目标2中，我们将描述OEM与各种细胞骨架成分之间的相互作用，包括β1整合素、肌球蛋白II、肌动蛋白和微管。重要的是，我们将定义负责NHE1和SWELL1沿纵向细胞表面穿梭的细胞内转运机制。我们还将引入一个全面的数学模型来破译OEM和细胞骨架组件在调节迁移效率方面的串扰。在目标3中，我们将利用原位移植到小鼠第四乳房脂肪垫上的TNBC细胞株和PDXs，在活体内演示NHE1和SWELL1沉默对自然乳腺组织轨迹中细胞迁移的影响，并研究它们在乳腺癌生长和转移中的作用。我们将用斑马鱼的实验来补充小鼠的研究，这使我们能够以非凡的光学清晰度成像其整个血管系统，以描绘离子转运体在转移级联的不同步骤中的作用。这一应用程序汇集了一支拥有生物工程、成像、细胞和分子生物学、定量分析、PDX、体内研究和乳腺癌生物学专业知识的研究团队。

Summary- The inability to clinically treat tumor metastasis is responsible for the majority of cancer patient deaths. Cell migration is a pivotal step in the metastatic dissemination of cancer cells from a primary tumor to distant organs in the body. Cell motility 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), as explained by the Osmotic Engine Model (OEM). The roles of cytoskeleton and ion transporters in cell locomotion have been typically studied in isolation. 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 define the relative roles and potential crosstalk between ion transporters and the cytoskeleton in breast cancer cell migration and metastasis in vivo. This application will test the hypothesis, supported by intriguing preliminary data, that the coordinated action of local isosmotic swelling at the leading edge and shrinkage at the trailing edge mediated by NHE1 and SWELL1, respectively, supports migration in confinement. We further hypothesize that NHE1 and SWELL1 act in concert with cell cytoskeleton to mediate efficient migration and metastasis. Given the lack of targeted therapies for triple negative breast cancer (TNBC), we will prioritize TNBC cell lines and patient-derived xenograft (PDX) tumor cells as models. In Aim 1, we will establish the functional roles of NHE1 and SWELL1 in cell migration inside confining µ-channels of different stiffnesses, in 3D gels and in cell dissemination from 3D breast cancer cell organoids. We will also elucidate the mechanism responsible for the polarized distribution of NHE1 and SWELL1 at the cell front and rear, respectively, and use novel optogenetic tools to alter their spatial polarization and test how these alterations affect the direction and efficiency of cell migration. In parallel, we will develop an innovative mathematical model to identify the key variables that enable OEM-mediated cell motility. In Aim 2, we will delineate the interplay between OEM and the various cytoskeletal constituents, including Beta1 integrins, myosin II, actin and microtubules. Importantly, we will define the intracellular transport mechanisms responsible for NHE1 and SWELL1 shuttling along the longitudinal cell surface. We will also introduce a comprehensive mathematical model to decipher the crosstalk of OEM and cytoskeletal components in regulating migration efficiency. In Aim 3, we will demonstrate the effects of NHE1 and SWELL1 silencing on cell migration in natural mammary tissue tracks in vivo and examine their roles in breast cancer growth and metastasis, using TNBC cell lines and PDXs orthotopically transplanted to the 4th mammary fat pad of mice. We will complement mouse studies with experiments in zebrafish, which enables us to image its entire vasculature at exceptional optical clarity, in order to delineate the roles of ion transporters in different steps of the metastatic cascade. This application brings together a team of investigators with expertise in bioengineering, imaging, cell & molecular biology, quantitative analysis, PDXs, in vivo studies and breast cancer biology.