Role of CD61+ low-density neutrophils in promoting hepatocellular carcinoma metastasis through CCDC25 upregulation

Abstract

BackgroundA subset of neutrophils isolated from the peripheral blood mononuclear cells (PBMC) layer has recently been described in cancer patients. MethodsDouble-gradient centrifugation was used to separate the neutrophil subsets. Western blotting and immunohistochemical assays were performed to assess CCDC25 expression levels. ResultsIn this study, we found that low-density neutrophils (LDNs) were more highly enriched in metastatic hepatocellular carcinoma (HCC) patients than in non-metastatic HCC patients. We then showed a CD61+ LDNs subset, which displayed distinct functions and gene expression, when compared with high-density neutrophils (HDNs) and CD61− LDNs. Transcriptomic analysis revealed that the CD61+ LDNs were predominantly enhanced in the transcription of glycolysis and angiogenesis associated gene, HMGB1 associated gene and granulation protein gene. These CD61+ LDNs displayed a prominent ability to trigger metastasis, compared with HDNs and CD61− LDNs. Specifically, CD61+ LDN-derived HMGB1 protein increased the invasion of HCC cells by upregulating CCDC25. Mechanistically, the CD61+ LDN-derived HMGB1 protein enhanced the invasiveness of HCC cells and triggered their metastatic potential, which was mediated by TLR9-NF-κB-CCDC25 signaling. Blocking this signaling pathway reversed the invasion of the CD61+ LDN-induced HCC cells. In vivo, we consistently showed that CD61+ LDN-derived HMGB1 enhances HCC metastasis to the lungs. ConclusionsOverall, our findings showed that a subset of CD61+ LDNs has pro-metastatic effects on HCC, and may be used to target HCC in the clinical setting.