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

Abstract

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-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.

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.

Keywords: CCDC25; Hepatocellular carcinoma; Low-density neutrophils; Orthotopic model; Tumor microenvironment.