Neutrophil-macrophage crosstalk via NETs–IL-17/VEGF/S100A9 axis promotes hepatocellular carcinoma progression

Abstract Background Tumor-associated neutrophils and macrophages are key components of the hepatocellular carcinoma (HCC) microenvironment. However, the interplay between them and its contribution to HCC progression remain unclear. Methods Bioinformatic analysis of TCGA datasets and clinical HCC samples was used to evaluate neutrophil extracellular trap (NETs) levels and macrophage polarization. Co-culture of neutrophils, macrophages, and HCC cells, along with molecular analysis and in vivo mouse models, were employed to dissect the mechanisms underlying NETs-mediated macrophage reprogramming and tumor progression. Results NETs were significantly elevated in HCC patients, particularly in advanced and metastatic stages, which were positively correlated with intrahepatic M2 macrophage infiltration and M2d subset-associated cytokines in blood. In vitro, NETs promoted M2d polarization in the presence of HCC cells via IL-17R/NF-κB signaling activated by IL-17 carried within NETs, which subsequently enhanced angiogenesis, migration, invasion, and epithelial-mesenchymal transition; these effects were partially reversed by IL-17R inhibition. In vivo, NETs-induced M2d polarization accelerated tumor growth, angiogenesis, and metastasis, whereas IL-17R blockade attenuated these pro-tumor effects. Moreover, M2d macrophages indirectly promoted NETs formation by upregulating HCC cell-derived S100A9 through VEGF-NF-κB signaling, establishing a positive feedback loop between neutrophils and macrophages. Furthermore, IL-17 carried by NETs (NETs-IL-17) demonstrated strong predictive value for extrahepatic metastasis in HCC, with an area under the ROC curve (AUC) of 0.89. Conclusions A positive feedback loop between neutrophils and macrophages via the NETs-IL-17/VEGF/S100A9 axis accelerates HCC progression and metastasis. More importantly, NETs-IL-17 exhibited potential as an alternative biomarker for predicting extrahepatic metastasis in HCC.