Macrophages are important innate immune cells with the ability to adapt their phenotype to environmental cues. Research on human macrophages often uses monocyte‐derived macrophages cultured in vitro, but it is unclear if culture medium affects macrophage phenotype. The objective of this study was to determine the impact of culture medium composition on monocyte‐derived macrophage phenotype. Monocyte‐derived macrophages were generated in different formulations of culture media (RPMI 1640, DMEM, MEM, McCoy's 5a and IMDM). Viability, yield and cell size were monitored, and RT‐qPCR, flow cytometry or ELISA was used to compare levels of phenotype markers (CD163, CD206, CD80, TNFα, IL‐10, SIRPα, LILRB1 and Siglec‐10). Yield, cell size, gene expression, membrane protein levels and release of soluble proteins were all affected by changes in culture medium composition. The most pronounced effects were observed after culture in DMEM, which lacks the non‐essential amino acids asparagine, aspartic acid, glutamic acid and proline. Supplementation of DMEM with non‐essential amino acids either fully or partly reversed most effects of DMEM on macrophage phenotype. The results suggest culture medium composition and amino acid availability affect the phenotype of human monocyte‐derived macrophages cultured in vitro.

Most soft tissue sarcoma (STS) patients do not respond to traditional checkpoint inhibitor treatment, which may be due to infiltrating immunosuppressive tumour-associated macrophages. This study investigated the prognostic value of four serum macrophage biomarkers. Methods: Blood samples were taken from 152 patients with STS at the time of diagnosis; clinical data were prospectively collected. The concentrations of four macrophage biomarkers (sCD163, sCD206, sSIRPα, sLILRB1) were measured in serum, dichotomised based on median concentration, and evaluated either individually or when combined with established prognostic markers. Results: All macrophage biomarkers were prognostic of overall survival (OS). However, only sCD163 and sSIRPα were prognostic for recurrent disease (sCD163: hazard ratio (HR): 1.97 (95% CI:1.10–3.51) and sSIRPα: HR: 2.09 (95% CI: 1.16–3.77)). A prognostic profile was made based on sCD163 and sSIRPα; it also included c-reactive protein and tumour grade. Patients with intermediate- or high-risk prognostic profiles (adjusted for age and tumour size) had a higher risk of recurrent disease compared to low-risk patients (HR: 2.64 (95% CI: 0.97–7.19)) and (HR 4.3 (95% CI: 1.62–11.47)), respectively. Conclusion: This study demonstrated that serum biomarkers of immunosuppressive macrophages were prognostic for OS; when combined with well-established markers of recurrence they allowed for a clinically relevant categorising of patients.