Journal: Science translational medicine

Article Title: Metabolic adaptations of ovarian tumors in patients treated with an IDO1 inhibitor constrains antitumor immune responses

doi: 10.1126/scitranslmed.abg8402

Figure Lengend Snippet: (A) Cumulative data of the fold change in human healthy donor CD8+ T cell proliferation stimulated with anti-CD3/CD28 beads in the presence of the indicated combinations of NAD and antagonists to A2BR (PSB 1115), A2AR (SCH 58261), P2XR (oATP), P2X1R (NF 449), P2X3R (NF 110), A1R (PSB 36), P2X4R (5-BDBD) and P2Y1R (MRS 2279) respectively. In all box charts, the median and min-max of the values are presented; *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001. Wilcoxon and one-way ANOVA tests. (B) Survival data of IE9mp1-mIDO1 tumor-bearing mice treated with EPA, A2a, A2b receptor antagonist, or combination of EPA, A2a, and A2b receptor antagonist (n= 5 animals per group). Mice were treated by oral gavage with 300mg/kg EPA in 200μl 0.5% methylcellulose, i.p. injection 5 days on and two days off from d7-d21, with or without (i) NAMPT inhibitor FK866 (500 μg per mouse i.p.) once every week for 3 weeks (on day 13, 20, 27); and (ii) with or without A2a, A2b or A2a/A2b antagonists (1mg/kg, i.p. 5 days on two days off from d7-d21). *, P<0.05 compared to vehicle. Statistical analysis was calculated using a log-rank analysis. (C). Heatmap representing the Log2FC of core set genes of immune pathways that are significantly differentially expressed (FDR < 0.05) in the treatment groups compared with the vehicle group of mice. Blue and red indicate down- and up-regulation, respectively.

Article Snippet: C57BL/6 mice were challenged intraperitoneally (i.p.) with 1×10 7 IE9mp1-mIDO1 or IE9mp1-Empty-Vector tumor cells in a final volume of 500μl Dulbecco’s PBS (Corning Cellgro®) and treated by oral gavage with 300mg/kg IDO1 inhibitor INCB023843 (Incyte Corp.) in 200ul 0.5% methylcellulose, i.p. injection 5 days on and two days off from d7-d21, with or without (i) NAMPT inhibitor FK866 (Sigma-Aldrich, 500 μg per mouse i.p.) once every week for 3 weeks (on day 13, 20, 27); (ii) purinergic receptor A2a or A2b antagonists (Sch 58261 from Sigma-Aldrich or PSB 1115 from Tocris Bioscience, 1mg/kg, i.p. 5 days on two days off from d7-d21).

Techniques: Injection

Journal: Pharmaceutics

Article Title: Mesenchymal Stromal Cell Exosomes Mediate M2-like Macrophage Polarization through CD73/Ecto-5′-Nucleotidase Activity

doi: 10.3390/pharmaceutics15051489

Figure Lengend Snippet: Time schedule for treatment of primary macrophages with MSC exosomes and inhibitors in vitro. ( A ) Primary macrophages were treated with MSC exosomes and harvested at 24 and 48 h for qPCR and IF. ( B ) Primary macrophages were co-treated with MSC exosomes and PSB12379 (CD73 inhibitor), before being harvested at 30 min for WB, and at 24 and 48 h for qPCR and IF analyses. ( C ) Primary macrophages were pre-treated with ZM241385 (A 2A receptor inhibitor), PSB1115 (A 2B receptor inhibitor), wortmannin (AKT inhibitor) or U0126 (ERK inhibitor) prior to exosome treatment, before being harvested at 30 min for WB, and at 24 and 48 h for qPCR and IF analyses. WB, western blot; qPCR, quantitative reverse transcription polymerase chain reaction; IF, immunofluorescence.

Article Snippet: The role of adenosine receptors in the activation of AKT and ERK pathways was investigated by pre-treating the cells with 2 μM of ZM241385 (A 2A receptor antagonist; Tocris), 200 nM PSB1115 (A 2B receptor antagonist; Tocris), 1 μM wortmannin (AKT inhibitor; Cell Signaling Technology, Danvers, MA, USA), or 10 μM U0126 (ERK inhibitor; Cell Signaling Technology) for 1 h, before treatment with 10 μg/mL exosomes ( C).

Techniques: In Vitro, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Immunofluorescence

Journal: Pharmaceutics

Article Title: Mesenchymal Stromal Cell Exosomes Mediate M2-like Macrophage Polarization through CD73/Ecto-5′-Nucleotidase Activity

doi: 10.3390/pharmaceutics15051489

Figure Lengend Snippet: Exosomal CD73 mediates activation of AKT and ERK signaling through specific adenosine receptors A 2A and A 2B . ( A ) Western blotting and ( B ) semi-quantitative analysis of AKT and ERK phosphorylation in primary macrophages treated with MSC exosomes in the presence or absence of CD73 inhibition by PSB12379. Representative images ( n = 3). ( C ) Western blotting and ( D ) semi-quantitative analysis of AKT and ERK phosphorylation in primary macrophages pre-treated with A 2A receptor inhibitor (ZM241385) and A 2B receptor inhibitor (PSB1115) before exosome treatment. Representative images ( n = 3). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to M0, # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to M0 + Exo. n = 3–4/group.

Article Snippet: The role of adenosine receptors in the activation of AKT and ERK pathways was investigated by pre-treating the cells with 2 μM of ZM241385 (A 2A receptor antagonist; Tocris), 200 nM PSB1115 (A 2B receptor antagonist; Tocris), 1 μM wortmannin (AKT inhibitor; Cell Signaling Technology, Danvers, MA, USA), or 10 μM U0126 (ERK inhibitor; Cell Signaling Technology) for 1 h, before treatment with 10 μg/mL exosomes ( C).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Inhibition

Journal: Pharmaceutics

Article Title: Mesenchymal Stromal Cell Exosomes Mediate M2-like Macrophage Polarization through CD73/Ecto-5′-Nucleotidase Activity

doi: 10.3390/pharmaceutics15051489

Figure Lengend Snippet: MSC exosomes modulate macrophage polarization through specific adenosine receptors A 2A and A 2B . ( A ) IF staining at 48 h for CD206 and iNOS in primary macrophages treated with MSC exosomes, following pre-treatment with adenosine A 2A receptor inhibitor (ZM241385) and A 2B receptor inhibitor (PSB1115). Representative images ( n = 3). Scale bar: 100 μm. Quantitative MFI of ( B ) CD206 and ( C ) iNOS at 48 h. ( D ) Gene expression analysis of M2-associated genes at 24 and 48 h. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to M0, # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to M0 + Exo. n = 3–4/group.

Article Snippet: The role of adenosine receptors in the activation of AKT and ERK pathways was investigated by pre-treating the cells with 2 μM of ZM241385 (A 2A receptor antagonist; Tocris), 200 nM PSB1115 (A 2B receptor antagonist; Tocris), 1 μM wortmannin (AKT inhibitor; Cell Signaling Technology, Danvers, MA, USA), or 10 μM U0126 (ERK inhibitor; Cell Signaling Technology) for 1 h, before treatment with 10 μg/mL exosomes ( C).

Techniques: Staining, Gene Expression