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

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.