To address these limitations, we developed the Hydrogel-Encapsulated Microchannel Array (HEMICA), a tunable polyacrylamide-based microfluidic platform that replicates physiologically relevant stiffness (8-35 kPa) and confining geometries (3-60 µm wide, 200 µm long). HEMICA enables direct comparisons between migration mechanisms in compliant versus stiff microenvironments while maintaining defined architecture.
Using HEMICA, we uncovered a stiffness-dependent switch in the mode of confined breast cancer cell migration. In stiff microchannels, whether composed of PDMS (≥1.3 MPa) or polyacrylamide (35 kPa), migration requires front-rear polarization of the Na⁺/H⁺ exchanger 1 (NHE1) and aquaporin-5 (AQP5), consistent with the osmotic engine model. However, in softer (8 or 15 kPa) microchannels, this polarization is disrupted, and cells instead rely on a distinct strategy driven by Arp3, integrin-linked kinase (ILK), and integrin-β1. Disruption of any of these components markedly impairs migration in these softer microchannels. Moreover, cell entry into soft microchannels increases membrane tension and triggers calcium influx through a mechanosensitive ion channel, which in turn activate RhoA and drives myosin-II-dependent contractility. Migration inside soft microchannels also depends on myosin-II, as its knockdown markedly impairs cell motility. In our talk, we will describe how this actomyosin-based mode of migration in compliant environments is metabolically distinct from the osmotic engine-driven migration observed in stiffer settings.
Through targeted molecular interventions, cells confined within soft channels can be reprogrammed to adopt a phenotype that is observed inside stiff microchannels. Under such interventions, canonical cytoskeletal regulators, such as Arp3, ILK, and myosin-II become dispensable, and cell motility persists even when the expression of the aforementioned molecules is disrupted.
Together, our findings reveal a substrate stiffness-dependent transition between fundamentally distinct migration strategies, shaped by the interplay between ion channels, cytoskeletal remodeling, and cellular metabolism. Importantly, our findings indicate that, in vivo, cancer cells are capable of modulating their migratory behavior based on the surrounding tissue environment. HEMICA offers a powerful, biomimetic platform for uncovering physiologically relevant mechanisms of cancer cell dissemination and provides new insights into how mechanical cues influence metastatic potential.