Characterizing Traction Forces in Upstream-Migrating Hematopoietic-like KG1a Cells Under Shear Flow.

Unlabelled: 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 which enables leukocytes to move against the direction of fluid flow on Intercellular Adhesion Molecule-1 (ICAM-1) surfaces. Upstream migration is mediated by the leukocyte integrin, Lymphocyte Function-Associated Antigen-1 (LFA-1). While this behavior 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 (TFM) to quantify spatiotemporal patterns of force generation during upstream migration of KG1a cells, a hematopoietic progenitor cell line that exhibits robust upstream migration on ICAM-1 functionalized hydrogels. Under static (no-flow) conditions, KG1a cells displayed random motility and traction profiles that varied with time. In contrast, cells exposed to shear flow generated persistent, polarized tractions aligned with the axis of migration. Population analysis showed that maximum RMS traction forces were significantly elevated during upstream migration compared to static conditions (mean: 428.5 ± 63.0 nN vs 220.8 ± 22.2 nN, p = 0.0078), as were average RMS forces (mean: 82.6 ± 12.9 nN vs 45.9 ± 4.4 nN, p = 0.0184), while minimum force values remained comparable between groups. These findings indicate a specific amplification of stresses required to overcome applied forces during upstream migration. By integrating single-cell and population-level force analyses, this study defines upstream migration as a mechanically reinforced state characterized by amplified, directionally coherent traction dynamics. Our methods enable future dissection of the molecular regulators that coordinate force generation with migration under flow.

Significance: Upstream migration is a critical yet poorly understood mode of immune cell motility. Here, we use traction force microscopy under physiologically relevant shear stresses to provide the first quantitative characterization of traction dynamics during upstream migration. We show that upstream migrating cells generate significantly greater average and peak forces than cells under static conditions, revealing a distinct mechanical program for directed migration against flow. These findings establish a platform for dissecting the molecular regulators of force generation in immune cells and set the stage for future perturbation-based studies aimed at understanding how mechanical forces shape immune cell trafficking and vascular navigation.