Abstract LB135: Deferiprone alters macrophage function and metabolism

Abstract

Abstract We studied the effects of the intracellular, clinically approved, iron chelator deferiprone (DFP) on macrophage characteristics, function, and metabolism. This research expands on our previous studies investigating the ability of DFP to alter metabolism of breast and prostate cancer tumors and enhance treatment response. We used the murine macrophage cell line RAW 264.7, unpolarized (M0) & polarized (to M1 by LPS, to M2 by IL-4), in vitro to evaluate the effects of DFP on macrophage (i) doubling times (cell count assay), (ii) polarization (flow cytometry), (iii) functional response by measuring cytokine (IL-12, IL-10, TNFα), secretion (Luminex multiplex cytokine detection system, Millipore), phagocytosis (IgG-coated latex beads), and reactive oxygen species production (fluorometric detection), (iv) glycolytic and oxidative metabolism (Agilent xFe96 analyzer), and (v) glycolytic metabolism (measuring incorporation of 1-13C-glucose by 1H & 13C magnetic resonance spectroscopy (MRS) of cell extracts and supernatants). The EC50s of 48 h DFP exposure are 79±5 µM, 93±3 µM, and 114±5 µM for M0, M1, and M2-polarized RAW 264.7 cells, respectively. The effect of DFP is cytostatic and increases cell doubling times. Exposure to 100 µM DFP for 48 h increases the number of M1-polarized cells in M0 and M1 cultures, but not in M2-polarized cells. While M2-polarized cells secreted more anti-inflammatory IL-10 and less pro-inflammatory TNFα than M0 cells, their cytokine secretion was unaffected by DFP. M1-polarized cells stimulated the secretion of TNFα and IL-10. The IL-10 secretion may be a feedback mechanism to curtail the pro-inflammatory effects of LPS. DFP decreased IL-10 secretion by ~33% in M1-polarized cells, enhancing their M1 activation state. IL-12 secretion was minimal and inconsistent. The ~21% phagocytosis in M0 cells remained unchanged by M2 polarization and increased to ~37% in M1-polarized cells. DFP exposure increased phagocytosis efficiency in M0, M1, and M2-polarized cells but highest in M1 cells. While no detectable H2O2 was produced, RAW 264.7 cells make detectable nitric oxide (NO), with M1-polarized cells yielding the most NO. NO yield was unchanged by DFP. Like the results above, we found the greatest effect of DFP to be on mitochondrial metabolism in M1 macrophages, specifically, significant reductions of maximal, basal, and ATP-linked respiration. The increased suppression of oxidative metabolism in M1-polarized macrophages by DFP conforms with the observed reduction of anti-inflammatory cytokines. The DFP increased phagocytosis, specifically dominant in M1 macrophages, suggests an increased reliance on glycolysis. To support the above observations, we currently study the incorporation of 1-13C-Glucose into cellular metabolites of M0, M1, and M2-polarized cells without & with DFP. Our data support a pro-inflammatory effect of DFP on macrophages, which may enhance their anti-tumor effect in vivo through altering tumor and macrophage metabolism. Citation Format: Ellen Ackerstaff, Sadna Budhu, Dov P. Winkleman, Soe S. Min, Jenny N. Ijoma, Inna S. Serganova, Ronald G. Blasberg, Taha Merghoub, Jason A. Koutcher. Deferiprone alters macrophage function and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB135.