Cancer-associated neutrophils retain functional plasticity and can undergo alternative activation when exposed to different cues found in the tumor microenvironment (TME) [1], [2], [3]. Several studies have reported the presence of at least two distinct neutrophil phenotypes in the disease setting, including LDNs and HDNs [4].

LDNs are different from HDNs, and they have a density below 1.077 g/mL and precipitate in the PBMC layer after density gradient centrifugation of whole blood [4], [5]. Morphologically, LDNs have a ring-shaped nucleus, whereas HDNs have a segmented nucleus [6], [7]. LDNs consist of granulocyte myeloid-derived suppressor cells (G-MDSCs) and mature large neutrophils [8]. The phenotype of LDNs may vary depending on the context of the disease [9]. In sepsis, LDNs proliferate and exhibit characteristics of G-MDSCs [10]. The LDNs in systemic lupus erythematosus (SLE) trigger rapid cell death via the immune complex, and this cell death results in the extracellular release of nuclear DNA and oxidized mitochondrial DNA [11]. In patients with rheumatoid arthritis (RA), low-density neutrophils (LDNs), present in high numbers in the blood of both RA and SLE patients, have opposing phenotypes contributing to clinical manifestations of each disease [12]. In cancer patients, several differences occur between these two neutrophil subsets, such as the ability to inhibit the activation of CD8+ T cells [4], [7]. In the breast cancer metastasis model, immature LDNs, but not mature LDNs, were found to accumulate and acquire a higher proportion in metastatic sites and peripheral blood at a later stage of liver metastasis [2]. Additionally, the transition of HDNs to LDNs was found to occur spontaneously in several tumor models [2], [5]. Tumor growth factor-β (TGF-β) might act as a major regulator in inducing this transition from HDNs to mature LDNs in a dose-dependent manner [2].

Cancer immunotherapy, mainly including immune checkpoints-targeted therapy and the adoptive transfer of engineered immune cells, has revolutionized the oncology landscape as it utilizes patients' own immune systems in combating the cancer cells [13]. Immunosuppressive tumor microenvironment disturbs cancer immunotherapy process [13]. CD molecular regulates immune cell functions within tumor microenvironment. In 2020, CD73 was found to be up-regulated in tumor-infiltrating NK cells and induced immune exhaustion. CD73+ NK cells displayed impaired anti-tumor function and undergo transcriptional reprogramming and upregulated IL-10 production [14]. CD47 is the most thoroughly studied and has emerged as a rising star among targets for cancer treatment. CD47-targeting antibodies and inhibitors have been investigated in various preclinical and clinical trials [13]. In 2023, Kaijun Zhao showed that Glutaminyl-peptide cyclotransferase-like protein (QPCTL) catalyzes the pyroglutamylation of CD47 and is crucial for the binding between CD47 and SIRPα. High QPCTL expression predicts high grades of gliomas and poor prognosis with impaired infiltration of adaptive immune cells in the tumor microenvironment as well as higher cancer stemness. Moreover, targeting QPCTL will be a promising immunotherapy in glioma cancer treatment [15].

The CCDC25 protein, is a 25 kDa coiled protein present in various mammalian cells, and a membrane-bound protein identified as a receptor of neutrophil extracellular traps (NETs) DNA, and it forms a link between neutrophils and cancer metastasis [16]. Several studies showed that CCDC25 is expressed in cancer cells as a transmembrane protein and correlated with poor disease prognosis in patients with liver cancer [17], [18]. Eosinophilic extracellular traps activate pulmonary neuroendocrine via the CCDC25-ILK-PKCα-CRTC1 pathway, and inhibition of CCDC25 alleviates allergic inflammation [19]. However, the association between LDNs and CCDC25 during the metastasis of HCC remains largely unknown.

In this study, we determined the correlation between the abundance of LDNs and tumor metastasis in HCC. We found that a subset of CD61+ LDNs can act as an important pro-metastatic factor, and the CD61+ LDNs promoted HCC metastasis by upregulating the expression of CCDC25 in tumor cells. Next, we showed that CD61+ LDNs can promote cancer metastasis via the TLR9-NF-κB-CCDC25 signaling pathway. The findings of various experiments revealed the pro-metastatic effect of the subset of CD61+ LDNs, which might act as a suitable target for the treatment of HCC in the clinical setting.