2.2 Differentiation and polarization of THP-1 monocytes into unpolarized, M1-like, and M2-like macrophages and macrophage-derived conditioned medium formation

The protocol used to differentiate and polarize human THP-1 monocytes into unpolarized, M1-like, and M2-like macrophages was set up in (20) and used as in (9). Briefly, THP-1 monocytes were incubated with 150 nM Phorbol 12-myristate 13-acetate (PMA) for 24 h and were then left untreated for 24-h period in complete RPMI medium. This allowed for the differentiation of monocytes into unpolarized macrophages characterized by a decrease in monocyte marker expression and an increase in macrophage marker expression (20). The unpolarized macrophages were left untreated or were polarized into M1-like macrophages via 24-h incubation with LPS (10 pg/ml; Sigma; L8630) and IFNγ (20 ng/ml; R&D Systems) or were polarized into M2-like macrophages via 48-h incubation with IL-4 (20 ng/ml; R&D Systems) and IL-13 (20 ng/ml; R&D Systems). The proportion of M1 macrophages in the cell population after polarization with LPS and IFNγ has been quantified by fluorescence activated cell sorting (FACS), using human leukocyte antigen (HLA) as the marker for M1 phenotype. There were 83.1% of cells positive for HLA in the M1-like population compared with 17% in unpolarized macrophages and to 5.9% in the M2-like population (data not shown).

Unpolarized, M1-like, and M2-like macrophages were then exposed to normoxia, chH, and cyH for 6 h and were then left for 16 h in normoxic air (21% O₂). chH incubation corresponded to 6 h of 1% O₂, whereas cyH corresponded to four cycles of 1% O₂ for 1 h and 21% O₂ for 30 min. The incubation of cells in hypoxia was performed as in (9). In this study, normoxia and the cyH reoxygenation were performed by exposing cells to atmospheric air (21% O₂) and not physioxia. Nonetheless, the hypoxia value that we used was physiologically relevant because O₂ saturation in tumor is comprised between 1% and 2% (8–15 mmHg) O₂ in a majority of solid tumors (32).

During this 22-h (6 h + 16 h) incubation period, macrophages were incubated in CO₂-independent medium (18015, Gibco) supplemented with 4 mM glutamine (G8540, Sigma) and glucose (3.75 g/L; 6877, Roth). After incubation, conditioned media were harvested, and the pH of each medium was adjusted to that of CO₂-independent medium pH ± 0.02. As shown previously, the polarization state of macrophages incubated during this period of time (6 h + 16 h) in N, chH, or cyH was not modified. Indeed, unpolarized, M1-like, and M2-like macrophages remained, respectively, unpolarized, M1-like, and M2-like macrophages after this incubation period (9). Conditioned media were either stored at 4°C for a maximum period of 3 days and used for experiments or were frozen in liquid nitrogen (snap-freeze) and stored at −70°C and were used afterward for experiments. The media that were stored at 4°C no more than 3 days or that were snap-frozen and stored at −70°C showed the same biological effects (data not shown).