Roles of tumor-associated neutrophils and neutrophil extracellular traps in the development and metastasis of colon cancer

Colon cancer is one of the most common malignancies worldwide, and metastatic dissemination in advanced stages significantly limits treatment success. Recent studies have demonstrated that tumor-associated neutrophils and the neutrophil extracellular traps they produce may exert pro-tumoral functions within the tumor microenvironment. This study aimed to investigate the expression levels of genes associated with tumor-associated neutrophils in colon cancer patients, to assess their relationship with clinical parameters such as metastasis, disease stage, and immunotherapy response, and to evaluate the potential role of neutrophil extracellular traps / tumor-associated neutrophils biology in the development and progression of colon cancer at the molecular level. Within this scope, the expression levels of 35 genes associated with tumor-associated neutrophils and tumor-associated neutrophils were evaluated, including ACTB, MAPK1, MAPK14/p38, MTOR, NCF1, NCF2, PAK1, PAK3, CXCR2, CEACAM1, PGC1A, PIK3CA, PAD4, ITGAM (CD11b), CYBA, HMGB1, CXCR1, ILK, PRKCA, NCF2, H3C14, SELPLG (CD162), TLR9, SERPINB1, MPO, TLR4, CEACAM6, MMP9, PLCB1, HDAC1, CCDC25, RAC1, RAC2, FCGR1A (CD64), RELA (P65/NF-κB3), and ELANE. The molecular roles of these genes in colorectal cancer pathogenesis were investigated in detail. The study included 77 patients with pathologically confirmed colon cancer and 63 healthy individuals as a control group. Total RNA was isolated from all samples and reverse transcribed into cDNA. Expression levels of the 35 target genes were analyzed by real-time polymerase chain reaction. Among these genes, those involved in neutrophil extracellular traps formation (e.g., MPO, ELANE, PAD4, CXCR1, ITGAM, SELPLG, MAPK1, MAPK14, PRKCA), matrix degradation (e.g., MMP9, CEACAM1/6, TLR4), integrin-linked kinase signaling (e.g., ILK), and metabolic reprogramming (e.g., PGC1A, MTOR) were included. Gene expression analysis was performed using the ΔΔCt method. Statistical analysis included the Shapiro-Wilk test for normality, the Kruskal-Wallis test for group comparisons, the Wilcoxon test for pairwise comparisons, and Spearman correlation coefficients for evaluating associations. Expression differences were interpreted using log2 fold change (log2FC), and a p-value < 0.05 was considered statistically significant. Data were visualized using heatmaps, decision trees, and receiver operating characteristic (ROC) curves. Gene expression profiles in colon cancer patients were compared with the healthy control group, and the expression levels of genes with statistically significant differences were analyzed in relation to clinical and laboratory parameters such as tumor stage, metastatic status, CEA levels, and neutrophil percentage. Sixteen out of the 35 genes were found to be differentially expressed with adjusted p-values below the 0.05 threshold. Of these, eight genes (PAD4, HAT1, H3C14, CEACAM1, CYBA, ITGAM, MTOR, RELA) were significantly downregulated, while eight genes (HDAC1, ELANE, CXCR1, ILK, MPO, SELPLG, TLR9, PAK3) were significantly upregulated. The expression levels of certain genes showed statistically significant correlations with clinical variables, including disease stage, metastasis, and treatment response. Specifically, ILK and HDAC1 were associated with diagnostic discrimination; PAD4, ILK, and CYBA with metastatic dissemination; H3C14 with tumor stage; MPO and TLR9 with lymph node involvement; ELANE and CYBA with CA19-9 levels; and ELANE, PAD4, and TLR9 with serum CEA levels. Decision tree and ROC analyses suggested that combinations of certain genes may have prognostic value in colorectal cancer. This study reveals that genes associated with tumor-associated neutrophils and neutrophil extracellular traps may play critical roles in colon cancer progression and metastasis. Identifying genes that contribute to the remodeling of the immune microenvironment, treatment resistance mechanisms, and formation of the pre-metastatic niche may be of clinical importance in the development of new immunotherapeutic targets. Molecular targeting strategies focused on the neutrophil extracellular traps / tumor-associated neutrophils axis may offer a promising avenue for the improved management of colorectal cancer.