CD61 + LDNs drive cancer metastasis by upregulating CCDC25 expression via DNA-TLR9-NF-kB axis in HCC

Abstract

Abstract Background A subset of neutrophils isolated from peripheral blood mononuclear cells (PBMC) has recently been described in cancer patients. Methods Double gradient centrifugation was used to separate neutrophil subset. Western blot assay was performed to assess CCDC25 expression level. Results In this study, we found that low density neutrophils(LDNs) were highly enriched in metastatic hepatocellular carcinoma (HCC) patients compared to non-metastatic HCC patients. Then, we showed that the most prominent marker on LDNs was CD61, compared to high density neutrophils(HDNs). The CD61+LDNs subset displayed an increased ability in triggering metastasis, producing free DNA, and synthesizing cytokine CCL5, and a decreased ability in forming NETs, synthesizing cytokine TNF-α and producing ROS, as compared with CD61−LDNs or HDNs. Transcriptomic analysis revealed that CD61+LDNs displayed distinct gene transcribe from CD61−LDNs and HDNs. The abundance of circulating CD61+LDNs was negatively correlated with disease prognosis, and positively correlated with the expression of CCDC25 within tumor. These CD61+LDNs increased the invasion of HCC cells by upregulating CCDC25. Mechanistically, the CD61+LDN-derived free DNA, excluding the NETs-DNA, enhanced the invasiveness of HCC cells and triggered their metastatic potential, which was mediated by TLR9-NF-κB-CCDC25 signaling. Blocking this signaling reversed the invasion of the CD61+LDNs-evoked HCC cells. In vivo, we consistently showed that CD61+LDNs enhanced HCC metastasis to the lungs. Conclusions Overall, our findings showed that a subset of CD61+LDNs has pro-metastatic effects on HCC, which might be used for targeting HCC in the clinical setting.