Predatory Phagocytosis Between Macrophages and Primary & Metastatic Melanoma

Postponing metastasis of melanoma is crucial for patient survival given that post-metastatic survival rates can be as low as 15%. Macrophages, critical innate immune cells, are key to survival in metastatic melanoma due to their functions in homeostasis and phagocytosis. This makes them prime targets for exploitation by cancer. Exposure to IFN-γ or IL-4 polarizes macrophages into M1 (pro-inflammatory) or M2 (tissue repair) phenotypes, respectively, but the relative ability of these phenotypes to phagocytose melanoma remains unclear. This study hypothesizes that macrophage polarization enhances melanoma phagocytosis and examines the capacity of these phenotypes to phagocytose primary versus metastatic cell lines over 16 h. THP-1 monocyte-derived macrophages were differentiated into M0 phenotypes using 50 ng/ml PMA for two days, followed by four days of rest in RPMI 1640 with 10% heat-inactivated FBS. M1 and M2 phenotypes were induced via exposure to 20 ng/ml IFN-γ and IL-4, respectively, for 24 hours. Phenotypes were verified via previous cell surface marker assays. Melanoma cells (primary or metastatic) were treated with 0.5 mM staurosporine for 24 h to induce apoptosis before phagocytosis assays. Phagocytosis was analyzed by co-culturing melanoma cells (primary or metastatic) with macrophages for 16 h. To quantify the resulting phagocytosis, we used flow cytometry to examine the resulting staining of macrophages that had been pre-stained with CellTracker® Orange CMRA and melanoma that had been pre-stained with CellTrace® Violet. Predatory macrophages were identified as orange-stained macrophages with violet fluorescence above the cutoff, while predatory melanoma cells were violet-stained melanoma with orange fluorescence above the cutoff. In a separate protocol we qualitatively monitored the phagocytic process using time-lapse imaging every 15 min for 16 h with a Zeiss Axiovert 200M microscope and a 10x objective. Phagocytosis of metastatic (LM45) melanoma cells by macrophages averaged 0.9% (M0), 1.6% (M1), and 1.1% (M2). For primary (WM278) melanoma cells, phagocytosis averaged 28.8% (M0), 32.6% (M1), and 34.1% (M2). Surprisingly, melanoma cells also demonstrated the ability to phagocytose macrophages, with predatory melanoma phagocytosis averaging 15.2% (M0), 18.4% (M1), and 13.1% (M2). Time-lapse imaging revealed phagocytosis occurred through “nipping” behavior, or trogocytosis, rather than traditional engulfment. In summary, IFN-γ or IL-4 treatment does not enhance macrophage phagocytosis of metastatic melanoma but increases activity against primary melanoma, supporting the hypothesis. Primary melanoma cells demonstrated a unique ability to phagocytose macrophages, something not observed in metastatic cell lines. M2 macrophages exhibited the highest capacity for tumor microenvironment cleanup, while primary melanoma cells were least predatory against this phenotype. Future studies exploring mechanisms behind LM45 resistance to phagocytosis and primary melanoma’s macrophage phagocytosis could inform development of improved immunotherapies. Project funded through KCOM Graduate Program Grant #851-068 and ATSU Research Support. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.