Macrophages often pervade solid tumors, but their spatial distribution is nonuniform and might relate to the heterogeneity of tumor-associated macrophages (TAMs) evident from single-cell analyses. Any effects of spatial heterogeneity on macrophage functions including phagocytosis are unknown. We observed that macrophages form clusters in tumors under immunotherapeutic conditions that maximize cancer cell phagocytosis and in reductionist tumoroid models of co-cultured melanoma cells and macrophages. Clustering on low-adhesion substrates also occurs within hours of activating macrophages to an M1 state with interferon-γ while activation to an M2 state with interleukin-4 favors dispersion. Increased expression of adhesion receptors (integrin αL and intercellular adhesion molecule-1) and decreased cellular contractility both contribute to clustering of M1 macrophages. In tumor spheroids, neighboring macrophages extend intrusive pseudopodia or ‘intrudopodia’ through cancer cell junctions, and coordinated intrudopodia help detach and individualize cancer cells for phagocytosis. Importantly, recently identified anti-tumor TAM subtypes in human cancers that cluster at the interface between tumor cell nests and the tumor stroma share features with phagocytic clustering macrophages from our reductionist studies. Therefore, macrophage clusters and their cooperative advantage for phagocytosis of solid tumor cells could be important effectors of macrophage immunotherapies.
