High-Dose Acetaminophen with N-acetylcysteine Rescue Inhibits M2 Polarization of Tumor-Associated Macrophages

SIMPLE SUMMARY: High dose acetaminophen (AAP) with N-acetylcysteine (NAC) rescue has demonstrated promising results in pre-clinical and clinical studies. However, the mechanism is not clearly understood. In the present manuscript, we evaluate the effects of high dose AAP/NAC on the tumor immune microenvironment. We demonstrate that high dose AAP inhibits M2 polarization of tumor associated macrophages at the RNA and protein levels. The anti-tumor activity of high dose AAP is lost in macrophage depleted mice, underlining the physiologic relevance of the findings. While at traditional doses AAP is not thought to have anti-inflammatory activity, our study is the first to describe AAP-induced changes on anti-tumor immunity. ABSTRACT: High-dose acetaminophen (AAP) with N-acetylcysteine (NAC) rescue is among the few treatments that has shown activity in phase I trials without achieving dose-limiting toxicity that has not progressed to evaluation in later line studies. While the anti-tumor effects of AAP/NAC appear not to be mediated by glutathione depletion and free radical injury, the mechanism of anti-tumor effects of AAP/NAC has not been definitively characterized. In vitro, the effects of AAP/NAC were evaluated on bone marrow derived macrophages. Effects of AAP on IL-4/STAT6 (M2) or IFN/LPS/STAT1 (M1) signaling and downstream gene and protein expression were studied. NAC reversed the AAP toxicity in the normal liver but did not reverse AAP cytotoxicity against tumor cells in vitro. AAP/NAC selectively inhibited IL-4-induced STAT6 phosphorylation but not IFN/LPS-induced STAT1 phosphorylation. Downstream, AAP/NAC inhibited IL-4 induction of M2-associated genes and proteins but did not inhibit the IFN/LPS induction of M1-associated genes and proteins. In vivo, AAP/NAC inhibited tumor growth in EF43.fgf4 and 4T1 triple-negative breast tumors. Flow cytometry of tumor-associated macrophages revealed that AAP/NAC selectively inhibited M2 polarization. The anti-tumor activity of high-dose AAP/NAC is lost in macrophage-depleted mouse syngeneic tumor models, suggesting a macrophage-dependent mechanism of action. In conclusion, our study is the first to show that high-dose AAP/NAC has profound effects on the tumor immune microenvironment that facilitates immune-mediated inhibition of tumor growth.