Abstract
BackgroundTumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs share many characteristics with alternatively activated macrophages, also called M2. They have been shown to favor tumor development and a role in chemoresistance has also been suggested. Here, we investigated the effects of M2 in comparison to M1 macrophages on cancer cell sensitivity to etoposide.MethodsWe set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). To mimic the communication between cancer cells and TAMs, M0, M1 or M2 macrophages and HepG2 or A549 cancer cells were co-cultured during respectively 16 (HepG2) or 24 (A549) hours, before etoposide exposure for 24 (HepG2) or 16 (A549) hours. After the incubation, the impact of etoposide on macrophage polarization was studied and cancer cell apoptosis was assessed by western-blot for cleaved caspase-3 and cleaved PARP-1 protein, caspase activity assay and FACS analysis of Annexin V and PI staining.ResultsmRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages, which provide a new, easy and well-characterized model of polarized human macrophages. Etoposide-induced cancer cell apoptosis was markedly reduced in the presence of THP-1 M2 macrophages, while apoptosis was increased in cells co-cultured with M1 macrophages. On the other hand, etoposide did not influence M1 or M2 polarization.ConclusionsThese results evidence for the first time a clear protective effect of M2 on the contrary to M1 macrophages on etoposide-induced cancer cell apoptosis.
Highlights
Tumor associated macrophages (TAMs) are present in high density in solid tumors
M2 macrophage polarization can be induced by different stimuli: IL-4 and/ or IL-13, immune complexes and toll-like receptor, IL-1 receptor ligands or IL-10 [6]
Monocyte differentiation into macrophages Human THP-1 monocytes were differentiated into macrophages by an incubation in the presence of phorbol 12-myristate 13-acetate (PMA)
Summary
0.8 x 106 THP-1 monocytes were differentiated and polarized in 6 well plates They were incubated in CO2 independent medium supplemented with 0.5 mM L-glutamine (Sigma, # G3126) and 3.75 g/l of D-glucose (Sigma, #50-99-7) for 16 h before being incubated with or without 50 μM etoposide (Sigma, #E1383) for 24 h. Cell suspension was centrifuged 5 min at 200 g and 4 °C and the pellet resuspended with 1 ml of PBS 5 % his 0.1 % NaN3 The pellet was resuspended with 50 μl of primary anti-CD206 antibody diluted 5 times in PBS 5 % his 0.1 % NaN3 and incubated 30 min at 4 °C.
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