The Tumor-Promoting Role of Neutrophil Extracellular Traps in Esophageal Squamous Cell Carcinoma and Their Interaction with the Gut Microbiota

Background and Objectives In recent years, the role of neutrophil extracellular traps (NETs) in the tumor microenvironment has garnered increasing attention, yet their relationship with esophageal squamous cell carcinoma (ESCC) remains poorly understood. Additionally, the interplay between gut microbiota and the tumor immune microenvironment may influence the progression of ESCC. This study aims to investigate the diagnostic value of NETs-related markers (CitH3, MPO, and NE) in ESCC patients, their correlation with clinical characteristics, and the impact of NETs levels on patient prognosis. Furthermore, we seek to elucidate the pro-tumorigenic mechanisms of NETs in ESCC. By analyzing gut microbiota composition, we also aim to uncover differences in microbial communities between ESCC patients and healthy individuals and explore their association with NETs levels, thereby providing novel theoretical foundations for the early diagnosis and treatment of ESCC. Methods Peripheral blood, surgical specimens, fecal samples, and clinical data were collected from 60 ESCC patients undergoing surgical treatment, along with peripheral blood and fecal samples from 60 healthy controls. ELISA was employed to measure plasma levels of CitH3, MPO, and NE in both groups, and their correlations with clinical features were analyzed. The diagnostic efficacy of NETs markers was evaluated using ROC curves, and the 3-year survival rates of patients with high versus low CitH3 levels were compared. Changes in NETs levels pre- and post-surgery, as well as the impact of different surgical approaches on NETs, were assessed. 16S rDNA gene sequencing was utilized to analyze differences in gut microbial composition, and its correlation with plasma NETs levels was explored. Immunohistochemistry, Western blot (WB), and qRT-PCR were performed to detect the expression of CitH3, MPO, and NE in surgical specimens. In vitro experiments involved stimulating neutrophils with phorbol esters to generate NETs, followed by functional assays and pathway analyses to evaluate the effects of NETs on ESCC cells. A subcutaneous xenograft model in nude mice was established to validate the pro-tumorigenic mechanisms of NETs. Results The plasma levels of CitH3, MPO, and NE in ESCC patients were significantly elevated compared to those in healthy controls and were correlated with clinical characteristics. The AUC value for diagnosing ESCC using NETs was 0.981, demonstrating high sensitivity and specificity. Elevated CitH3 levels were indicative of lower survival rates. Postoperative levels of CitH3, MPO, and NE increased, with robot-assisted minimally invasive esophagectomy (RAMIE) showing lower levels of these markers compared to video-assisted minimally invasive esophagectomy (VAMIE). Dysbiosis of the gut microbiota in ESCC patients was associated with NETs levels. In vitro experiments revealed that NETs promoted ESCC cell proliferation, migration, invasion, and angiogenesis. WB analysis indicated that NETs facilitated epithelial-mesenchymal transition (EMT) and angiogenesis by upregulating the protein expression levels of N-Cadherin, Vimentin, MMP2, MMP9, HIF-1α, TNF-α, VEGF, VEGFA, Ang-1, and Ang-2. In vivo experiments confirmed that NETs promoted tumor growth, and DNase1 partially reversed this effect. Conclusions This study elucidates the tumor-promoting role of NETs in ESCC and their association with gut microbiota. NETs markers (CitH3, MPO, and NE) were significantly elevated in ESCC patients, offering diagnostic and prognostic value. NETs promote tumor progression by regulating EMT and angiogenesis pathways, with DNase1 partially reversing this effect. Dysbiosis of the gut microbiota in ESCC patients is linked to NETs levels. These findings provide novel insights into the early diagnosis and targeted therapy of ESCC, warranting further exploration into the regulatory mechanisms of NETs and microbiota.