Engineered Deoxyribonucleic Acid-Tunable Spheroid Platform for Analysis of Tumor-Macrophage Interactions.

Tumor architecture strongly influences immune cell behavior, yet the individual contributions of cell-cell spacing and hypoxia remain difficult to isolate in vivo. Here, we use a DNA-programmable 3D tumor spheroid platform to generate 4T1 spheroids with tunable intercellular spacing and predictable hypoxia gradients, enabling structural and biochemical cues to be decoupled. Using this system, we compared infiltration and polarization dynamics of immortalized RAW 264.7 macrophages and primary BMDMs. Macrophages readily infiltrated preformed spheroids across all DNA lengths, indicating that the packing differences achievable here do not impose a major physical barrier. Instead, infiltration kinetics were cell-specific, where BMDMs penetrated more rapidly and reached maximal infiltration by 24 h, while RAW macrophages infiltrated more slowly. Soluble factors released by spheroids induced a modest pro-inflammatory shift in 2D controls, confirming contact-independent activation. Within spheroids, RAW macrophages exhibited hypoxia-dependent polarization at 24 h, with lower hypoxia promoting higher CD80/CD206 ratios. This effect equalized by 48 h. BMDMs showed more muted and hypoxia-insensitive responses, with inflammation peaking around 24 h and gradually resolving. These results establish DNA-engineered spheroids as a powerful system for dissecting how cell spacing and hypoxia regulate macrophage behavior.