Distinct Mucosal Microbiota Signatures Associated with Lymph Node Involvement in Colon Cancer

Background: The gut microbiota has been increasingly implicated in colorectal cancer development and progression. However, whether mucosal microbiota composition differs according to clinicopathological features of colon cancer, remains incompletely defined. Methods: Paired mucosal biopsies from tumor tissue and non-tumoral mucosa were collected from 32 patients undergoing surgery for colon cancer (64 planned samples). Three samples failed amplification, leaving 61 samples for downstream analysis. DNA was sequenced targeting the V3-V4 regions of the 16S rRNA gene. Alpha and beta diversity metrics were analyzed, and differential abundance analysis was performed using linear mixed-effects models accounting for paired sampling and clinical covariates. Results: Alpha diversity analysis showed significantly higher microbial richness in patients aged ≥70 years compared with younger patients (observed richness estimate −47.19, p = 0.035; Faith's phylogenetic diversity estimate −6.34, p = 0.013) and in MMR-proficient compared with MMR-deficient tumors (estimate 57.18, p = 0.039). Beta diversity analysis showed that tumor location was the only clinical variable significantly associated with overall microbial community composition (PERMANOVA R2 = 0.030, p = 0.021). Differential abundance analysis identified two amplicon sequence variants associated with nodal status: ASV96, assigned to Fusobacterium nucleatum (F. nucleatum), was enriched in node-positive patients (q = 0.025), whereas ASV330, assigned to Alistipes finegoldii (A. finegoldii), was enriched in node-negative patients (q = 0.032). Presence-absence analysis confirmed a higher prevalence of F. nucleatum in tumor tissues from node-positive compared with node-negative patients (88% vs 26%, p = 0.004). Conclusions: Global microbial diversity and community structure were not associated with lymph node involvement; however, taxon-specific mucosal signatures differed according to nodal status. Enrichment of F. nucleatum characterized in node-positive tumors, whereas A. finegoldii was more abundant in node-negative cases. These findings suggest that mucosal microbial taxa may reflect tumor aggressiveness in colon cancer and warrant validation in larger, clinically annotated cohorts.