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

Abstract

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.