Elevated neutrophil extracellular traps formation driven by the HPV-mediated CXCL8-CXCR1/2-ROS-p21 axis promotes the growth and metastasis of cervical cancer.

BACKGROUND: The role of neutrophil extracellular traps (NETs) in cervical cancer (CC) progression, particularly their association with human papillomavirus (HPV) infection, remains unclear. METHODS: We analyzed public single-cell RNA sequencing data to explore neutrophil heterogeneity in the HPV-associated CC microenvironment and quantitatively assessed NETs in clinical tissues and serum from normal subjects, HPV-negative, and HPV-positive CC patients. In vitro, CC cell lines and normal epithelial cells were treated with neutrophil-conditioned medium, followed by phenotypic evaluation using EdU, Transwell, and vasculogenic formation assays. In vivo validation was conducted using subcutaneous xenograft and tail-vein metastasis models. Potential mechanisms of HPV-regulated NETs formation were further investigated through co-culture, immunoblotting, and ROS detection. RESULTS: Single-cell transcriptomic analysis revealed a characteristic neutrophil subpopulation (N4) in HPV-positive tumor microenvironments, whose gene expression signature is highly correlated with NETs formation capability. NETs were elevated in CC tissues and sera, especially in HPV-positive tumors. High tissue levels of the NETs-specific marker Cit-H3 correlated with larger tumor diameter, deep stromal invasion and poor differentiation. Functionally, NETs promoted proliferation, angiogenesis, invasion and migration of CC cells; in vivo, experiments showed NETs drove tumor growth and pulmonary metastasis, effects abolished by DNase 1-mediated NETs degradation. Mechanistically, HPV-positive cancer cells secrete substantial amounts of CXCL8, which activates CXCR1/2 receptors, thereby inducing reactive oxygen species production and p21 upregulation to drive neutrophil extracellular trap formation. Targeting this axis reversed NETs generation. CONCLUSION: The HPV-triggered CXCL8-CXCR1/2-ROS-p21 pathway drives NETs formation that accelerates CC growth and metastasis. Circulating NETs represent a potential prognostic biomarker.