Macrophage phagocytosis and efferocytosis: Implications for therapeutic modulation in the tumor microenvironment

Macrophages play a central role in the tumor microenvironment. Depending on their activation state and local signals, they can exert both pro- and anti-tumorigenic effects. Tumor-associated macrophages (TAMs) often exhibit an immunosuppressive, pro-tumoral phenotype that promotes tumor growth, angiogenesis, metastasis, and therapy resistance. Due to their plasticity and abundance in solid tumors, TAMs are attractive therapeutic targets: either by reprogramming them toward an anti-tumoral state or depleting their tumor-supportive functions. Understanding macrophage behavior in cancer is critical for developing effective immunotherapies, given their dual roles. This requires reliable, physiologically relevant in vitro assays modeling key functions, such as phagocytosis, efferocytosis, cytokine secretion, and tumor cell interaction. Macrophage phagocytosis and efferocytosis assays assess the impact of test compounds on the uptake activity of human M0, M1, and M2 macrophages. CD14+ monocytes are isolated from cryopreserved PBMCs and differentiated into M0 macrophages using M-CSF for six days, then polarized into M1 (IFN-gamma + LPS), M2 (IL-4 + IL-13) or TAM-like (IL-4 + IL-10 + TGF-beta) phenotypes. After polarization, cells are treated with test compounds at defined concentrations and time points, followed by incubation with either pHrodo™ bioparticles or apoptotic pHrodo™ labelled tumor cells (camptothecin-treated Raji cells) to assess phagocytosis or efferocytosis, respectively. Quantification is performed by measuring red fluorescence over 12 - 24 hours using a Cytation 5 reader. Our experiments indicate that M0, M2 and TAM-like macrophages have a strong capacity for phagocytosis and efferocytosis, while M1 macrophages have minimal particle or apoptotic tumor cell uptake. Time-resolved analysis reveals distinct uptake kinetics. M0 and M2 macrophages rapidly internalize targets within 2-4 hours. However, M0 cells subsequently decline in activity, while M2 macrophages maintain a sustained plateau for up to 12 hours with an overall higher uptake. Repolarizing M2 macrophages to an M1 phenotype using LPS and IFN-gamma markedly reduces their phagocytic capacity. Blocking the "don't eat me" signal with an anti-CD47 antibody significantly enhances the efferocytosis of Raji cells. We anticipate that cytokine profiling will reveal mechanistic differences between phagocytosis mediated by pattern recognition receptors and efferocytosis, which is critical for tissue homeostasis. These findings suggest that the phenotype of macrophages strongly influences their phagocytic and efferocytic behavior, with M2 cells exhibiting the highest capacity. Strategies that modulate macrophage polarization or relieve inhibitory signals could enhance these functions and support further investigation into their therapeutic potential.