P05 acetyl salicylic acid induces polarization of macrophages and cytotoxicity on myeloma cells in vitro

Background and aim: As in many cancers, tumor microenvironment (TME) plays a vital role in progression in Multiple Myeloma (MM). Macrophages, which are members of TME, promote tumor progression by secreting various cytokines and can exist in two different forms as pro-tumorigenic (M2 macrophage) and anti-tumorigenic (M1 macrophage). M2 macrophages are inactive cells that cannot perform tumor cell phagocytosis, while M1 macrophages are active cells with high phagocytosis capabilities of tumor cells. It is known that the M1-M2 polarization observed in macrophages in the TME during myeloma progression is associated with poor prognosis. Therefore, reprogramming of macrophages can result in the formation of anti-tumorigenic TME. Acetyl Salicylic Acid (ASA) is used as an anti-thrombotic agent in MM and has been shown to effect the anti-tumorigenic polarization of macrophages in TME in breast and rectal cancer (Hsieh and Wang 2018; Farrugia, Long et al. 2021) Aim of this study is to investigate molecular effects of ASA on macrophage mediated phagocytosis in MM. Methods: To demonstrate the cytotoxic effects of ASA on MM1S myeloma cells, MTT was performed by applying at different concentrations of ASA. IL10 and IL6 expression level from THP1 derived macrophages and MM1S cells was determined by qRT-PCR. In addition, CD80 gene (M1 marker) expression level was determined by qRT-PCR to monitor the effect of ASA on macrophage polarization from M2 to M1 in THP1 cells. According to MTT analysis results, 2.5mM was selected as the optimal dose of ASA and was applied to M1 and M2 macrophages for 48 hours. DiD-labeled macrophage cells and GFP+ MM1S cells were co-cultured and rates of macrophage-mediated phagocytosis were determined using flow cytometry and confocal microscopy. Statistical software GraphPad Prism6 was used for the assessment of the differences between all treated and control groups. Wilcoxon test and Two-way ANNOVA test are used to compare means of multiple samples. Results: It was determined that ASA has a time (24h,48h,72h) and dose-dependent cytotoxic effect on MM1S cells in vitro (Figure 1). ASA had no effect on the viability of macrophages. It was shown that IL6 and IL10 expression were decreased in relation to cytotoxicity in MM1S cells treated with ASA (p<0.05, p<0.0001;). While ASA did not change CD80 expression in M1 macrophages, it caused an increase in CD80 expression in M2 macrophages (p=0.0003), indicating polarization from M2 to M1 phenotype (Figure 2). Finally, among M1 and M2 macrophages following ASA exposure, the baseline rate of phagocytosis of 63% and 26%, were found to be 69% and 70% confirming anti-tumorigenic effect of M1 and polarized M2 cells (Figure 3). Conclusion: To our knowledge this is the first report on the effects of ASA effects on macrophage mediated phagocytosis and anti-myeloma cytotoxicity. At 2.5mM concentration which is equivalent to 45μg/dl and below the therapeutic range required in the clinic, ASA showed anti-tumorigenic effects by suppressing IL6 and IL10 expression, cytotoxicity in MM1S cells and M2 to M1 polarization mediated increase in phagocytosis. We conclude that ASA has a potential to induce direct and indirect effects targeting both MM cells and TME.