(56c) Polarized Ion Channels Regulate Migration Direction and Efficiency in Confinement

Introduction: The primary cause of death among cancer patients is tumor metastasis. Cell migration is a key step in the metastatic cascade of events, as it enables tumor cells dissociating from a primary tumor to navigate through interstitial tissues and ultimately colonize distant organs. Cell migration in confinement can persist even when F-actin is completely disrupted; a process that can be explained by the Osmotic Engine Model (OEM)¹. According to OEM, cell locomotion is mediated by highly coordinated cycles of local isosmotic swelling at the cell leading edge and shrinkage at the trailing edge, driven by the polarization of select ion transporters and aquaporins (AQPs). We determined that the Na⁺/H⁺ exchanger 1 (NHE1) together with AQP5, polarized at the leading edge of migrating cells in the confinement, mediate regulatory volume increase², which is consistent with its role in cell protrusion. However, the ion channel(s) responsible for regulating cell shrinkage at the trailing edge in confined cell migration remain unknown. We aimed to characterize the molecular mechanisms of confined migration in order to identify potential therapeutic targets to block breast cancer migration and metastasis in vivo.

Materials and Methods: To explore the potential role of select ion channels in OEM-based confined cell migration, we applied soft lithography and microfabrication techniques to create the PDMS-based microfluidic channels. Moreover, to directly establish the involvement of polarized ion channels in the direction and efficiency of migration, we developed unique optogenetic tools to regulate their spatiotemporal localization. We combined microfluidic migration assays with molecular biology techniques, high resolution imaging, and mathematical modeling to elucidate the functional roles of polarized ion channels in confined migration of breast cancer cells.

Results and Discussion: We have identified a novel ion channel SWELL1 which is preferentially localized at the cell trailing edge of confined breast cancer cells (Fig. 1A), thereby facilitating the local volume decrease by creating an outflow of water. Dual knockdown of NHE1 and SWELL1 does not alter cell volume in confinement, which is in line with their individual counteracting effects on volume regulation (Fig. 1B). We also demonstrate cooperative and pronounced inhibition of motility in narrow channels (Fig. 1C) as well as in cell dissociation of 3D breast cancer organoids upon dual NHE1 and SWELL1 depletion. Moreover, we applied our unique optogenetic tools to demonstrate that polarization of SWELL1 at the cell rear confers migration direction, and that by optogenetically promoting its enrichment at the cell front, the direction of migration is reversed (Fig. 1D). This ground-breaking discovery represents the first direct demonstration of the osmotic engine model (OEM) in regulating cell motility. This presentation will further discuss the role of these ion channels in breast cancer metastasis using the chick embryo and mouse models.

Conclusions: Our data support a model by which the repeated and coordinated cycle of swelling and shrinkage at the cell poles mediated by NHE1 and SWELL1, respectively, regulate cell motility in confinement. Overall, our findings delineate the role of SWELL1 in migration direction and efficiency in confined microenvironments. To this end, we have developed unique optogenetic tools to either enrich (Fig. 1D) or deplete SWELL1 with exquisite spatiotemporal regulation. These findings may provide the basis for identification of novel therapeutic target to combat breast cancer metastasis.

References: 1. Stroka K.M. et. al. Cell, (2014) 157(3):611-623. 2. Jentsch T.J. Nat Rev Mol Cell Biol., (2016) 17(5):293-307