To investigate the characteristics of the tumor immune microenvironment in colon cancer, identify key immune-related genes, and evaluate their prognostic value, thereby providing evidence for immunotherapy target screening and prognostic stratification.

Based on transcriptomic and somatic mutation data from 514 colon cancer patients in the TCGA database, CIBERSORT and ESTIMATE algorithms were used to calculate immune score, stromal score, and ESTIMATE score. The associations between these scores and patients’ clinical outcomes (e.g., overall survival) as well as various clinicopathological parameters were statistically evaluated. Differentially expressed genes (DEGs) were identified using the limma package and functionally annotated through GO/KEGG enrichment analyses. Somatic mutation analysis was used to identify differentially mutated genes (DMGs), which were cross-analyzed with DEGs to identify key genes associated with tumor-infiltrating immune cells and prognosis. Clinical data and cellular experiments were used to validate the results.

A high immune score was significantly positively correlated with overall survival in colon cancer patients (low/high immune score groups: 140, 51, 17, 7, 3 vs. 309, 93, 15, 5, 2; χ²=6.45, P=0.011), and the immune score significantly decreased with tumor stage progression. A total of 627 immune-related DEGs were identified, mainly enriched in immune pathways such as cytokine–cytokine receptor interaction. Combined with mutation analysis, FN1 and DOCK2 were identified as intersecting key genes with both differential expression and mutation: high expression of FN1 was associated with poor prognosis (P=0.011), increased with tumor stage, and was significantly positively correlated with lymph node and distant metastasis; high expression of DOCK2 had a protective effect (P=0.075), decreased with stage progression, and was negatively correlated with distant metastasis. Experiments showed that FN1 was significantly overexpressed in colon cancer tissues and peripheral blood (P=0.0257, 0.0004), while DOCK2 was significantly under expressed (P=0.0260, P<0.0001). Functional experiments demonstrated that knockdown of FN1 significantly inhibited colorectal cancer cell proliferation (48 h: t=3.58, P=0.0006; 60 h: t=3.66, P=0.0005), invasion (t=3.27, P=0.0404), and colony formation (t=10.98, P=0.0001). In contrast, DOCK2 knockdown promoted these malignant phenotypes (t=−1.94, P=0.0401; t=−9.14, P=0.0002; t=−3.95, P=0.0149).

FN1 and DOCK2 regulate colon cancer progression and prognosis by modulating the tumor immune microenvironment. FN1 serves as a risk factor, while DOCK2 acts as a protective factor, potentially serving as novel targets for immunotherapy and biomarkers for prognostic stratification.