Tumor-on-a-chip with single-cell microfluidics reveals distinct NK cell immunosuppression by hypoxia versus lipid...

Immunosuppression induced by metabolic stress within the solid tumor microenvironment (TME) critically limits the efficacy of cancer immunotherapies. Here, we develop a tumor microenvironment-on-a-chip (TMoC) platform integrated with single-cell microfluidic analysis to precisely model both hypoxic and lipid-enriched conditions commonly found in solid tumors. Utilizing this system, we elucidate the distinct effects of these two metabolic stressors on natural killer (NK) cell function. Hypoxia broadly suppresses NK cell cytotoxicity and downregulates multiple functional molecules. In contrast, lipid accumulation exerts a selective inhibitory effect, reducing granzyme B and IL-10 expression while relatively preserving IFN-γ mRNA expression. Single-cell analysis reveals stress-induced functional heterogeneity and secretory synergy among NK cells. Notably, we identified a relatively high-IFN-γ-secreting subpopulation retained under lipid-rich conditions despite an overall reduction in IFN-γ secretion at the single-cell level. Furthermore, single-cell pairing assays dissociate global killing efficacy from killing kinetics, revealing that hypoxia and lipid accumulation impair NK cells through distinct kinetic modes. Using the TMoC platform to evaluate combination therapies targeting both hypoxia and lipid metabolism, we show that dual intervention synergistically restores NK cell cytolytic function. The platform demonstrates superior screening sensitivity compared to conventional in vitro models. Collectively, this work highlights the potential of integrating engineered microphysiological systems with single-cell analysis to decipher complex TME interactions and guide the development of immunotherapeutic strategies.