(74a) Osmotic Regulation of Cell Migration and Volume in Microfluidic Channels

Introduction: Cell homeostasis and diverse processes, including migration, are tightly regulated by cell volume. During migration through tissues, metastatic cancer cells experience varying degrees of physical confinement. We hypothesized that cell volume regulation by osmolarity is especially important for migration in confined microenvironments, due to recent work from our lab suggesting that actomyosin-based motility and adhesion are dispensable for confined migration, in contrast to 2D planar migration. We engineered a microfluidic-based chemotactic device with 3 μm-wide extracellular matrix-coated channels to maintain distinct extracellular osmolarities at the leading and trailing edges of the cell. Using phase contrast timelapse microscopy, we imaged live cancer cell migration in isotonic medium everywhere, or with hypo/hypertonic medium in the top, bottom, or top and bottom of the chamber.

Results: Upon switching from isotonic to hypotonic medium at only the top of the chamber, we observed a rapid reversal of cell migration direction (away from the hypotonic medium and chemoattractant). The speed of migration in the opposite direction decreased as the osmolarity of the medium approached isotonic. Meanwhile, switching to hypotonic medium at only the bottom of the chamber allowed cells to continue migrating towards the chemoattractant. Finally, switching to hypotonic medium in both the top and bottom of the chamber caused cells to migrate twice as fast towards the FBS. Similar responses were observed in wider channels (6, 10, 20, and 50 μm) and also in multiple cancer cell lines (AB3 mouse sarcoma, HTB-94 human sarcoma, and MDA-MB-231 breast cancer), though the threshold osmolarity necessary to produce a response varied between cell types. We also evaluated the effects of hypertonic shock at the top, bottom, or top and bottom of the chamber.

Changes in cell migration velocity were accompanied by a decrease in length (and therefore volume), the kinetics and final value of which were dependent on the polarity of the osmotic shock (top, bottom, or top and bottom of the chamber), osmolarity, and cell type. To confirm the decreases in volume and verify that cells occupied the entire channel, we used reduced-height channels (6 μm) and confocal imaging of F-actin-stained cells. Importantly, inhibition of actin polymerization (via latrunculin-A) stalled cell reversal after switching medium to hypotonic at the top of the chamber, even though latrunculin-A-treated cells migrated normally during confined migration in isotonic medium. In future work, we expect that microtubule dynamics will be important for cell polarity change, while ion channels and aquaporins will regulate cell volume changes that occur in these confined spaces upon osmotic shock.

Conclusions: Our results indicate that cancer cell migration in confined microenvironments is sensitive to fluctuations in extracellular osmolarity, and the location of osmotic shock (leading vs. trailing edge of the cell) is extremely important in determining the cellular response. Importantly, actin polymerization is a key process driving cell migration away from a hypotonic medium, even though it is dispensable for normal migration in isotonic medium in confined spaces.