Fig. 6

FFAR2 upregulates Arg1 in MDSCs through G_αq/Calcium/PPAR-γ signaling pathway. A Volcano plot showed significant differences in gene expression of Ffar2^−/− MDSC compared with Ffar2^+/+ MDSC cells (n = 3, biological replicates). (Downregulated genes, purple; Upregulated genes, orange). Interested differentially expressed genes were shown in triangle mark (downregulated) and square mark (upregulated). B All significant KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis results from the Ffar2^−/− MDSC compared with Ffar2^+/+ MDSC (P ≤ 0.05). NES: normalized enrichment score. C and D BM-MDSCs were pretreated with DMSO (0.1%), FFAR2 Agonist (10 μM), G_αq inhibitor (YM-254890; 1 μM), Ca²⁺ inhibitor (2-APB; 100 μM) and PPAR-γ-inhibitor (GW9662; 2 μM) for 2 h before adding GM-CSF and IL-6. All MDSCs were treated for 24 h, and the percentage of Arg1⁺ cells (C) and representative gating strategy (D) were analyzed by flow cytometry (n = 3, biological replicates). E Quantification of L-Arginine in LLC tumor tissue extracts. F BM-MDSCs were activated by GM-CSF and IL6 with or without NaAc. The expression of PPAR-γ, Arg1 and GAPDH were detected by Western blotting. G BM-MDSCs were activated by GM-CSF and IL6 for the indicated time. The expression of GAPDH, p-STAT1 (Tyr701), STAT1, p-STAT3 (Tyr705), STAT3, p-C/EBPβ (Thr217) and C/EBPβ were detected (n = 3, biological replicates). H Mice bearing LLC tumors (n = 3, biological replicates) and received intraperitoneal (i.p.) injections of PBS or NaAc. Representative images of immunofluorescence staining for PPAR-γ and Arg1 in tumors. I LLC-tumor bearing mice were treated with NaAc (500 mg/kg), NaAc + PPAR-γ inhibitor (1 mg/kg), NaAc + L-Arginine (1.5 mg/kg). Representative images of immunofluorescence staining for CD8 in tumors. J and K Representative images of multicolor immunofluorescence staining of a lung adenocarcinoma patient with high FFAR2 expression in (CD15⁺ARG1^high)-MDSCs (Left, who was still alive 39 months after surgery) and low FFAR2 expression in (CD15⁺ARG1^−/low)-MDSCs (Right, who was still alive 62 months after surgery) (J). Log-rank (Mantel-Cox) test of cumulative survival rates of lung adenocarcinoma patients subdivided by the percentage FFAR2^highArg1^highCD15⁺-MDSC of total cells (with > 3%, high infiltration; with ≤ 3%, low infiltration) in tumor tissues (K). C and E are shown as mean ± SEM, and the experiment was performed three times and a representative example is shown. C and E were determined by unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and NS, not significant)