Bio‐Orthogonally Click‐Assembled Macrophage Spheroids on Micropatterned Scaffolds for Sustained M1 Polarization and Antitumor Function

ABSTRACT

Macrophages are central effectors of innate immunity and cancer immunotherapy; however, their application is limited by poor spheroid‐forming capacity and phenotypic instability in immunosuppressive tumor microenvironments. Herein, we report a bio‐orthogonal click‐chemistry‐based strategy for engineering mechanically robust and functionally durable macrophage spheroids without exogenous polarizing stimuli. Macrophage membranes were functionalized via cholesterol‐mediated hydrophobic insertion of azide and dibenzocyclooctyne (DBCO) groups, enabling strain‐promoted alkyne–azide cycloaddition (SPAAC)‐mediated intercellular coupling. Combined with micropatterned nanofiber scaffolds, this approach generates compact, spatially confined spheroids from inherently low‐adhesion macrophages. The engineered spheroids exhibit spontaneous M1‐skewed polarization, associated with increased hypoxia‐inducible factor‐1α (HIF‐1α) signaling and elevated inducible nitric oxide synthase (iNOS) expression, pro‐inflammatory cytokine secretion, and suppressed IL‐10 production. In paracrine co‐culture assays, click‐assembled spheroids display time‐dependent, contact‐independent antitumor activity against melanoma cells. Proteomic profiling revealed activation of innate immune signaling, cytokine‐associated pathways, and redox‐related metabolic programs, consistent with sustained pro‐inflammatory functionality. Collectively, this work demonstrates that bio‐orthogonal surface engineering and three‐dimensional spheroid architecture can stabilize macrophage phenotype and function, providing a versatile proof‐of‐concept platform for engineering immune spheroids with durable M1‐skewed functionality in vitro. While these findings remain to be validated in vivo, they highlight the potential of this approach for cancer immunoengineering applications.