Measuring the traction forces of upstream-migrating hematopoietic-like KG1a cells under shear flow

Cell migration is critical to leukocyte function, enabling leukocytes to patrol tissues and respond to inflammatory cues. Upstream migration is a distinct form of cell motility that enables leukocytes to move against the direction of fluid flow on intercellular adhesion molecule-1 surfaces. Upstream migration is mediated by the leukocyte integrin, lymphocyte function-associatedantigen-1. Although upstream migration has been observed across multiple immune cell types, the mechanical forces underlying upstream migration have not been measured. Here, we demonstrate the use of traction force microscopy to quantify spatiotemporal patterns of force generation during upstream migration of KG1a cells, a hematopoietic progenitor cell line that exhibits robust upstream migration on intercellular adhesion molecule-1-functionalized hydrogels. Under static (no-flow) conditions, KG1a cells displayed random motility with dynamic traction forces that fluctuated substantially over time. Cells exposed to shear flow exhibited sustained upstream migration with significantly amplified traction forces. Population analysis showed that maximum RMS traction forces were significantly elevated during upstream migration compared with static conditions (mean: 377.9 ± 55.1 nN vs. 220.1 ± 20.3 nN, p = 0.0177), as were average RMS forces (mean: 75.0 ± 13.5 nN vs. 43.0 ± 4.8 nN, p = 0.0425), whereas minimum force values remained comparable. Quantitative spatial analysis revealed that in the presence or absence of shear flow, cells maintained similar spatial distribution of forces, demonstrating that forces are amplified during upstream migration without spatial reorganization. This study demonstrates that during upstream migration cells amplify their contractile forces while preserving their spatial organization, revealing how cells adapt their mechanical output under flow. Our methods enable future dissection of molecular regulators coordinating force generation during migration under flow.