Porphyromonas gingivalis Induced Neutrophil Extracellular Traps Promote Metastasis of Esophageal Squamous Cell Carcinoma

QI Yijun; YU Yilin; WU Dangrou; ZHANG Heqian; LIU Qiwei; WU Xiaoshuang; KANG Yongan; GAO Shegan

doi:10.15926/j.cnki.issn2096-7381.2026.02.002

Abstract：Objective To investigate the effect of Porphyromonas gingivalis(Pg)infection on lung metastasis of esophageal squamous cell carcinoma(ESCC)and the role of neutrophil extracellular traps(NETs)during this metastatic process.Methods A nude mouse model of ESCC lung metastasis was established by tail vein injection of luciferase-labeled KYSE150 cells(LKC).In the direct infection group(Pg group),LKC pre-co-cultured with Pg were inoculated;in the oral exposure group(OR group),Pg bacterial suspension was applied to the oral cavity after inoculation;the control group(CON group)was not infected.At the 3rd week after inoculation,each group was further divided into two subgroups,GSK484(+)and GSK484(-),the former was intraperitoneally injected with the NETs inhibitor GSK484,and the latter was injected with drug-free solvent.In vivo fluorescence imaging was performed at the 3rd week(before intervention)and weekly from the 4th to the 8th week(experimental termination);ex vivo fluorescence imaging of the lungs was performed at the end of the 8th week.The culture supernatant of Pg-infected neutrophils was collected,and its effect on the invasive ability of KYSE30 cells was assessed using Transwell chambers.Immunohistochemistry was performed to detect Ki67 and Ly6G expression;immunofluorescence was used to detect myeloperoxidase(MPO)and citrullinated histone H3(Cit-H3)expression,and DAPI counterstaining was performed to evaluate NETs formation;flow cytometry was used to detect the proportion of CD11b+and Ly6G+neutrophils.Results Pulmonary fluorescence signals appeared in the Pg group as early as the 3rd week,gradually increased in intensity,and were the most prominent thereafter,followed by the OR group,whereas the CON group showed no obvious signals.The culture supernatant of neutrophils infected with Pg significantly enhanced the invasive ability of KYSE30 cells.Ly6G+neutrophils were significantly enriched within and around lung metastatic lesions in both the Pg and OR groups,the proportion of neutrophils in lung tissue was elevated,and MPO and Cit-H3 expression was enhanced with increased co-localization.GSK484 intervention reversed the above changes,and the proportion of neutrophils in lung tissue after intervention showed no significant difference compared with the CON group.Conclusion Pg infection promotes ESCC lung metastasis that depends on neutrophil recruitment and NETs formation in lung tissue,suggesting that targeting NETs formation may be a potential strategy to block ESCC lung metastasis.