Integration of single-cell sequencing and experimental validation identified P2RY13/NF-κB as an immunological factor of patients with colorectal cancer

Background Colorectal cancer (CRC) is a leading cause of mortality worldwide. Nevertheless, the functional heterogeneity of macrophages in the microenvironment of CRC and the corresponding mechanism during inflammation–cancer transition have not been fully elucidated. Methods Single-cell transcriptome sequencing (GSE144735), spatial transcriptome (10X Genomics), and The Cancer Genome Atlas multi omics data were integrated to systematically analyze the molecular characteristics and functional regulatory network of tumor-associated macrophages (TAMs) in CRC. The Seurat process was utilized to identify cell subpopulations and classify TAMs. A least absolute shrinkage and selection operator (LASSO) Cox model was constructed to predict the survival risk of patients with CRC. Moreover, its predictive value for response to immunotherapy was validated based on the Tumor Immune Dysfunction and Exclusion (TIDE) score. Further in vitro experiments were performed to investigate the effect of feature genes on CRC and the underlying mechanisms. Results We identified 12 cellular subpopulations and classified TAMs into pro-inflammatory (296 cells, TNF superfamily member 10 [TNFSF10 ⁺ ]) and pro-tumor (446 cells, secreted phosphoprotein 1 [SPP1 ⁺ ]) subtypes. Functional analysis revealed that pro-inflammatory TAMs activate anti-tumor immunity via interferon signaling, whereas pro-tumor TAMs drive epithelial–mesenchymal transition and angiogenesis. Spatial transcriptomics confirmed the enrichment of SPP1 ⁺ TAMs at the tumor-invasive front, showing negative correlation with immune checkpoint genes (r = − 0.68, p < 0.001). An eight-gene prognostic signature derived from the LASSO Cox modeling (area under the curve = 0.83) effectively stratified the survival risk of patients (hazard ratio = 3.21, p = 1.2×10 ^− 5 ), with TIDE scores validating its predictive value for response to immunotherapy. Notably, purinergic receptor P2Y13 (P2RY13) acted as a novel prognostic marker. Furthermore, in vitro assay results demonstrated that P2RY13 attenuated the tumor property of CRC and M2 macrophage polarization through inhibiting the nuclear factor-kappa B (NF-κB) pathway. Conclusion This study revealed the key role of TAM polarization in inflammation–cancer transition of CRC, providing new biomarkers and potential intervention targets for precision immunotherapy.