Role of CD61+ Low-Density Neutrophils in Promoting Hepatocellular Carcinoma Metastasis Through CCDC25 Upregulation.

Background: A subset of neutrophils isolated from the peripheral blood mononuclear cells (PBMC) layer has recently been described in cancer patients. Methods: Double-gradient centrifugation was used to separate the neutrophil subsets. Western blotting and immunohistochemical assays were performed to assess CCDC25 expression levels. Results: In 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- kappa B-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. Conclusions: Overall, 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.