Integrative Spatial Transcriptomics and Machine Learning Reveal NFκB1-Linked Tumor-Associated Macrophage Polarization Driving Anti-Tumor Immunity in Colorectal Cancer

Background Tumor-associated macrophages (TAMs) influence the tumor microenvironment (TME) of the colorectal cancer (CRC) within various intratumoral niches. However, the spatial arrangement of macrophage polarization and the related alterations in genes remained unknown. Methods We conducted a comprehensive analysis of spatial transcriptomics (ST) and bulk RNA-sequencing data to explore the spatial environments of M1 and M2 macrophages in CRC. A machine learning framework including LASSO and stepwise Cox regression, was employed to identify strong prognostic genes related to macrophages. The importance of the core gene, NFκB1, was confirmed through TCGA and GEO datasets, as well as HPA immunohistochemistry, with subsequent functional enrichment and drug sensitivity analyses. Results Spatial profiling delineated a compartmentalized immune architecture: M2 macrophages preferentially accumulated within the hypoxic tumor core and at the invasive front, whereas M1 macrophages were mainly distributed along the tumor periphery, where they formed a barrier-like host-defense interface. Consistent with this spatial separation, a higher presence of M1 was associated with earlier tumor stages and better survival outcomes, whereas M2 macrophage levels offered limited prognostic insight. Through machine-learning-based feature selection, NFκB1 was defined as a protective marker within the M1 transcriptional alteration. Similarly, lower NFκB1 expression was linked to higher TNM stages and poor outcomes in other cohorts. Mechanistically, the reduction of NFκB1 was associated with 'cold' TME, characterized by impaired tertiary lymphoid structures, reduced cytolytic activity, and diminished chemokine-dependent T-cell movement, such as CXCL9 and CXCL10. Furthermore, tumors with low NFκB1 levels exhibited increased autophagy and extracellular matrix remodeling pathways, aligning with a more invasive phenotype. Conclusion Our research provides a detailed status and distribution of macrophage polarization in CRC and identifies NFκB1, a prognostic marker reflecting the TME status of CRC, as a key regulator of antitumor immunity.