Fig 1: Higher FAO leads to increased levels of PGE2 production through COX2 overexpression(A) PMN-MDSCs were sorted from WT and β2-AR−/− tumor-bearing mice. MDSCs were activated with or without ISO, and COX2 expression was analyzed by western blot.(B) PMN-MDSCs were sorted from WT tumor-bearing mice. MDSCs were activated with ISO, LPS, or ISO plus LPS for 24 h, and PGE2 levels were measured by ELISA (n = 3).(C) Human peripheral blood mononuclear cells (PBMCs) were cultured with IL-6 and GM-CSF for 7 days, treated with ISO, ETO, or ISO plus ETO, and at day 7, the level of PGE2 was measured by ELISA.(D) PMN-MDSCs were sorted from bone marrow of WT EL-4 tumor-bearing mice. MDSCs were treated for 24 h with either PBS or ISO, and ETO, torin 1, chloroquine, or 3-methyladenine and PGE2 levels were measured by ELISA.These data are presented as means ± SDs from 3 biological replicates in all of the graphs. One-way ANOVA was used to analyze the statistical significance between >2 groups, and the Student’s t test was used to analyze the statistical significance between 2 groups. In all of the panels, *p < 0.05, **p < 0.01, and ***p < 0.001. p < 0.05 was considered significant.
Fig 2: β-AR signaling in MDSCs decreases glycolysis and enhances oxidative phosphorylation(A–H) MDSCs were derived from bone marrow in the presence of GM-CSF and IL-6, with or without isoproterenol (ISO).(A) Mitochondrial respiration was measured using a Seahorse Extracellular Flux Analyzer (arrows indicate when reagents were added: (1) oligomycin; (2) FCCP; and (3) antimycin A and rotenone).(B) Basal respiration levels.(C) ATP production.(D) Maximum respiration.(E) FAO was measured using a Seahorse Extracellular Flux Analyzer in media in which palmitate was the only fatty acid source (arrows indicate when etomoxir [ETO] was added).(F) FAO consumption.(G and H) MDSCs were derived from bone marrow in the presence of GM-CSF and IL-6, with or without ISO and (G) fatty acid uptake (Bodipy FL C16) and (H) lipid accumulation (Bodipy FL 493/503) was measured.(I–O) WT and β2-AR−/− mice were orthotopically implanted with 4T1 tumor cells. At day 25, WT or β2-AR−/− PMN- MDSCs were sorted by flow cytometry and the rates of (I–L) oxidative phosphorylation, (M and N) FAO, and (O) fatty acid uptake were measured. Mitochondrial respiration was measured using a Seahorse Extracellular Flux Analyzer (arrows indicate when reagents were added: [1] oligomycin; [2] carbonyl cyanide p-trifluoromethoxy-phenylhydrazone [FCCP]; and [3] antimycin A and rotenone).(J) Basal respiration levels.(K) ATP production.(L) Maximal respiration.(M and N) FAO was measured using a Seahorse Extracellular Flux Analyzer in media in which palmitate was the only fatty acid source (arrows indicate when ETO was added). (N) FAO consumption.(O) WT and β2-AR−/− PMN-MDSCs were sorted from 4T1 tumor-bearing mice, and fatty acid uptake in WT or β2-AR−/− were analyzed by flow cytometry.These data are presented as means ± SDs from 3 biological replicates in all of the graphs, and the Student’s t test was used to analyze statistical significance between 2 groups. In all of the panels, *p < 0.05, **p < 0.01, and ***p < 0.001. p < 0.05 was considered significant.
Fig 3: Flow cytometric confirmation of in vitro derived MDSCs(A) Representative flow cytometric analysis of murine bone marrow MDSCs from tumor bearing mice. Purity before sorting is about 60% and can be increased to about 90% post-sorting.(B) A representative flow cytometric analysis of human PBMCs and pre-sort and post-sorted in vitro derived human MDSCs with IL-6 and GM-CSF. Purity before magnetic sorting should range from 50%. After sorting, purity should be above 60%.
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