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

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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.