Multi-omics Approaches to CCAAT/Enhancer-Binding Protein Beta in Oral Squamous Cell Carcinoma: Crosstalk Between Tumor Cells and Tumor-Associated Macrophages Driving Disease Progression

Background: CCAAT/Enhancer-Binding Protein Beta (CEBPB) is an important transcription factor that regulates tumor progression. However, the mechanism by which CE-BPB regulates the progression of Oral Squamous Cell Carcinoma (OSCC) remains incom-pletely understood. Tumor progression depends on complex intercellular interactions within the tumor microenvironment. The purpose of this study was to investigate the role and epige-netic regulatory mechanisms of CEBPB in interactions between OSCC cells and tumor-infil-trating immune cells. Methods: Bulk RNA-seq, ChIP-seq, and scRNA-seq data were obtained from The Cancer Ge-nome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) database. The HOMER algorithm was employed to identify enhancers and predict the CEBPB-binding mo-tif. Cell cluster analysis, functional enrichment, and intercellular interaction analysis were per-formed using the "Seurat" R package. H3K27ac enrichment at GAS6 enhancers was validated by ChIP-qPCR. Metastatic OSCC cells with CEBPB knockdown or GAS6 overexpression were established and co-cultured with THP-1 cells. IL-10 and IL-6 secretion from co-cultured THP-1 cells was detected via ELISA. Chemotaxis of OSCC cells toward THP-1 cells was assessed through a Transwell assay. Results: CEBPB was upregulated in OSCC and correlated with poor prognosis. By integrating H3K27ac ChIP-seq and bulk RNA-seq data, 131 CEBPB-regulated enhancer-controlled genes were identified in lymph node metastatic OSCC cells. scRNA-seq analysis revealed eight ma-jor cell clusters in primary foci and lymph node metastases, including T/NK cells, malignant epithelial cells, B/plasma cells, macrophages, fibroblasts, dendritic cells, endothelial cells, and mast cells, with the malignant epithelial cells stratified into distinct sub-clusters. CEBPB ex-pression was elevated in malignant epithelial cells of lymph node metastases compared to pri-mary foci. Furthermore, 15 pairs of enhanced ligand-receptor interactions were identified in lymph node metastases relative to primary foci. GAS6 was a CEBPB-regulated enhancer-con-trolled gene, primarily mediating interactions between malignant cells and macrophages. CE-BPB knockdown in metastatic OSCC cells significantly impaired their chemotaxis toward co-cultured THP-1 cells, and downregulated IL-10/IL-6 secretion and CD206 expression in co-cultured THP-1 cells. Conversely, GAS6 overexpression reversed these inhibitory effects. Conclusion: CEBPB activated GAS6 transcription in metastatic OSCC cells. The CE-BPB/GAS6 axis in metastatic OSCC cells enhanced their chemotaxis toward macrophages and promoted the M2 polarization of macrophages, thereby facilitating the establishment of an immunosuppressive microenvironment.