Fig. 6

D-2HG induces ANGPTL4 secretion to enhance macrophage M2 polarization in the tumor microenvironment. A-C 4T1 shNT cells or shAngptl4 cells were subcutaneously injected into nude mice, with D-2HG administered intraperitoneally on days 7, 9, 11, 13, and 15. Tumors were dissected on day 17 and photographed (A). Tumor weight (B) and growth curve (C) were analyzed (n = 5/group). D Correlation between macrophage infiltration and ANGPTL4 expression (TIMER database). E Flow cytometry analysis of macrophage subpopulations in 4T1 xenografts. The percentages of M1-like (F4/80 + CD86 +) and M2-like (F4/80 + CD206 +) tumor-infiltrating macrophages were calculated (n = 3/group). F. Representative images of CD86 and CD206 immunohistochemical staining in tumor sections. Scale bar: 50 μm. G Inflammation-related marker genes of peritoneal macrophages were analyzed by qPCR after stimulation with recombinant mouse ANGPTL4 protein (mANGPTL4) or PBS for 24 h. H Inflammation-related marker genes of peritoneal macrophages were analyzed by qPCR after treatment with E0771 conditioned media (CM) and 100 ng/mL mANGPTL4 or PBS for 6 h. I Inflammation-related marker genes of peritoneal macrophages were analyzed by western blot after E0771 CM treatment with 100 ng/mL mANGPTL4 or PBS for 24 h. J Schematic diagram of RNA-seq for exploring the role of ANGPTL4 in regulating macrophage polarization. K GSEA analysis of RNA-seq results showed the enhanced enrichment of the macrophage M1 vs. M2 down pathway in E0771 CM-treated peritoneal macrophages combined with mANGPTL4. L KEGG analysis showed ANGPTL4 was involved in the regulations of pathways related to cytokine and chemokine binding. M Activation of the integrin pathway in peritoneal macrophages was analyzed by western blot after E0771 CM treatment with 100 ng/mL mANGPTL4 or PBS for the indicated duration. *P < 0.05, **P < 0.01, ***P < 0.001