3D chemotaxis chip for investigating natural killer cell migration mechanisms.

INTRODUCTION: Natural killer (NK) cells are a promising tool for cancer immunotherapy, as they can rapidly recognize and kill cancer cells without prior knowledge of tumor-specific antigens while leaving healthy cells unharmed. However, a major challenge in NK cell-based therapies is their inadequate infiltration and function within solid tumors. Advancements in NK cell therapies for solid malignancies require an understanding of the various factors that influence NK cell migration to and within the tumor microenvironment. METHODS: In this study, we developed a chemotaxis chip with a tunable 3D hydrogel that enabled the spatiotemporal analysis of NK cell migration. Through live-cell imaging and cell-tracking analysis, we quantitatively assessed NK cell migration in engineered hydrogels in real time. This platform enabled precise control over matrix composition and physical properties, allowing systematic interrogation of microenvironmental features that regulate NK cell migration. RESULTS: Our findings revealed that NK cells rely heavily on proteasedependent infiltration but can leverage alternative mechanisms for faster migration, and that the inclusion of hyaluronic acid, a tumorrelated extracellular matrix component, promotes NK cell migration in 3D. DISCUSSION: This study established an adaptable hydrogel-based platform for studying immune cell migration in defined 3D environments, providing foundational tools for future mechanistic and translational investigations.