(175a) Cluster Formation of Macrophages Depends on Their Activation

Macrophages are tissue-resident immune cells that interact with matrix and adjacent tissue cells that include other macrophages. How macrophages or any immune cells organize together within mixtures with other cells is understudied compared to other tissue cell types. Here, we investigated macrophage organization in ‘tumoroid’ co-cultures with cancer cells and also following activation with polarizing cytokines. Mouse melanoma B16 cells lacking the macrophage checkpoint ligand CD47 were cultured as cohesive tumoroids on low adhesion substrates, and mouse bone marrow-derived macrophages were added with or without a tumor-opsonizing monoclonal antibody. Opsonized B16 cells were phagocytosed by macrophages that segregated into a compact central aggregate in the immuno-tumoroids whereas macrophages in unopsonized tumoroids remained dispersed. Macrophages activated with interferon-γ to an M1-state formed cohesive aggregates on their own, whereas macrophages polarized with interleukin-4 to an M2-state dispersed, and M1-M2 co-cultures demixed. Genetic and pharmacological perturbations indicate roles for differential adhesion and contractility that drive organization in other cell mixtures. A ‘phase diagram’ of condensed (M1) and gas-like (M2) macrophage states reflects the strength of cell-cell and cell-substrate interactions and might apply broadly to macrophage immunobiology, cancer immunotherapy, infectious diseases (e.g., granulomas), and biomaterials science (e.g., foreign body response).