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

临床上无法治疗肿瘤转移是大多数癌症患者的原因。 死亡。细胞迁移是癌细胞从原发肿瘤向外扩散的关键步骤 身体里遥远的器官。细胞的运动受细胞-基质相互作用、肌动蛋白细胞骨架和 通过离子转运体参与细胞体积调节，如Na+/H+交换器1(NHE1)，AS 由渗透引擎模型(OEM)解释。细胞骨架和离子转运体在细胞运动中的作用 通常都是孤立研究的。该项目的总体目标是采用多学科的 涉及最先进的生物工程和成像工具、定量分析和体内模型的方法 明确离子转运体和细胞骨架在乳腺癌中的相对作用和潜在的串扰 体内的细胞迁移和转移。这个应用程序将检验这一假说，并得到耐人寻味的初步支持 数据表明，前缘局部等渗膨胀和后缘收缩的协调作用 分别由NHE1和SWELL1介导，支持封闭条件下的迁移。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。给定 缺乏针对三重阴性乳腺癌(TNBC)的靶向治疗，我们将优先考虑TNBC细胞系和患者- 来源异种(PDX)肿瘤细胞作为模型。在目标1中，我们将确定NHE1和NHE1的功能角色 SWELL1在不同硬度的限制通道内、在3D凝胶中和在细胞扩散中的细胞迁移 来自3D乳腺癌细胞的有机体。我们还将阐明导致极化的机制 NHE1和SWELL1分别在细胞的前部和后部的分布，并使用新的光遗传学工具改变 并测试这些变化如何影响细胞迁移的方向和效率。在……里面 同时，我们将开发一个创新的数学模型，以确定使OEM- 介导的细胞运动。在目标2中，我们将描述OEM和各种细胞骨架之间的相互作用 成分，包括b1整合素、肌球蛋白II、肌动蛋白和微管。重要的是，我们将定义细胞内 负责NHE1和SWELL1沿纵向细胞表面穿梭的运输机制。我们会 还介绍了一个综合的数学模型来破译OEM和细胞骨架之间的串扰 在调节迁徙效率方面的组成部分。在目标3中，我们将演示NHE1和SWELL1的影响 体内自然乳腺组织中细胞迁移的沉默及其在乳腺癌中的作用 利用TNBC细胞系和PDX原位移植到第四乳房脂肪垫的生长和转移 老鼠的世界。我们将用斑马鱼的实验来补充小鼠的研究，这使我们能够想象它的整个 具有特殊光学清晰度的血管系统，以描绘离子转运体在不同步骤中的作用 转移级联反应。这一应用程序汇集了一支拥有生物工程专业知识的研究团队， 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。

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 b1 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.