(341c) Ion Channel-Mediated Regulation of Confined Cell Migration Under Elevated Extracellular Viscosity

Cell migration is a fundamental process governing a wide array of physiological and pathological events, including cancer metastasis. So far, most of the in vitro experiments including migration assays are performed in viscosity similar to water (0.77 centipoise (cP) at 37^oC), however viscosity of bodily fluids are markedly higher with physiological values ranging from 4–6 cP in whole blood and exceeding 8 cP under pathological conditions. Recent findings demonstrate that elevated extracellular viscosity, paradoxically enhances cell motility on 2D surfaces and in confined microenvironments. This enhanced migration is associated with increased actin polymerization via actin-related protein 2/3 (Arp2/3) at the leading edge.

At baseline water-like viscosity of 0.77cP, the Osmotic Engine Model (OEM) of cell migration in confined channels demonstrates that the sodium-hydrogen exchanger 1 (NHE1) polarizes at the leading edge of migrating cells, facilitating localized water influx via Aquaporin 5 (AQP5) and promoting cell protrusion. On the other hand, the volume-regulated anion channel SWELL1 localizes to the trailing edge, where it mediates water efflux through Aquaporin 4 (AQP4) and facilitates rear retraction through cell shrinkage. This polarization of NHE1 at leading edge and SWELL1 at the trailing edge orchestrates cell migration in confined channels at basal viscosity. It has also been shown that elevated viscosity leads to increased NHE1 polarization and activity through actin-binding partner ezrin leading to hyperactivation of OEM.

This prompted us to explore effect of elevated viscosity on SWELL1. Interestingly, under high-viscosity conditions, inhibition or knockdown of SWELL1 does not impair migration. Instead, the potassium-chloride cotransporter (KCC4) emerges as a key mediator. KCC4, a membrane protein responsible for the coupled efflux of potassium (K⁺) and chloride (Cl⁻) ions, plays a vital role in cell volume regulation. To investigate its function, we performed single and dual knockdowns of KCC4 and NHE1 in MDA-MB-231 cells. At baseline viscosity (0.77 cP), KCC4 knockdown had no significant effect on cell motility, whereas SWELL1 knockdown markedly reduced migration. However, at elevated viscosity (8 cP) in confined microchannels, KCC4 knockdown unlike SWELL1 knockdown significantly reduced motility. Furthermore, single knockdowns of KCC4 or NHE1 at 8 cP resulted in reduced migration compared to control, with the dual knockdown exhibiting an even greater decrease in motility. These findings were corroborated using a 3D collagen-based migration assay, where both single and dual knockdowns led to reduced motility, supporting a synergistic role for NHE1 and KCC4 at elevated viscosity.

Cell volume measurements in confinement revealed complementary roles: NHE1 knockdown led to decreased volume, KCC4 knockdown increased volume, while dual knockdown restored volume to control levels. Live-cell imaging with a KCC4 reporter revealed increased rearward polarization of KCC4 at 8 cP compared to 0.77 cP. Using optogenetic tools, we further demonstrated the ability to control the directionality and efficiency of migration by spatiotemporally modulating KCC4 polarization.

Together, our findings uncover a critical, viscosity-dependent role for KCC4 in confined cell migration. In coordination with NHE1, KCC4 regulates cell volume dynamics and polarity to facilitate movement in physiologically and pathologically relevant environments. These insights position KCC4 as a key player in the study of cancer cell migration and metastasis under elevated extracellular viscosity.