APOE+ macrophages induce tumor cell metastatic characteristics via TNFSF12/TNFRSF12A signaling, correlating with poor patient prognosis.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, with metastasis representing a major determinant of poor prognosis. Epithelial-mesenchymal transition (EMT) promotes metastatic progression by enhancing tumor cell migration and invasion. This study aimed to identify EMT-associated malignant epithelial programs in CRC and define the macrophage-tumor cell signaling mechanisms contributing to EMT and apoptosis resistance. We integrated single-cell RNA sequencing, bulk RNA-seq, spatial transcriptomics, immunohistochemistry, and functional validation experiments. Malignant epithelial subpopulations were identified using inferCNV and reclustering analyses, and EMT-related programs were evaluated by gene set scoring. Transcriptional regulation was assessed using pySCENIC, public ChIP-seq data, ChIP-qPCR, and TNFRSF12A promoter luciferase reporter assays. Cell-cell communication and ligand activity were analyzed using CellPhoneDB and NicheNet. Functional validation included western blotting, conditioned-medium assays, TNFSF12 neutralization, and Transwell migration assays. We identified an EMT-associated malignant epithelial subgroup enriched for TNFRSF12A expression. FOSL2 was highly active in this subgroup and positively associated with TNFRSF12A expression. ChIP-qPCR and promoter luciferase assays supported direct FOSL2-mediated transcriptional activation of TNFRSF12A. APOE+ macrophages were identified as a potential source of TNFSF12 in the tumor microenvironment. TNFSF12 acted on TNFRSF12A-expressing CRC cells and was associated with BCL2L1 upregulation, EMT-like changes, and enhanced migration. TNFSF12 overexpression increased BCL2L1 expression, whereas APOE+ macrophage-conditioned medium promoted E-cadherin downregulation, Vimentin and BCL2L1 upregulation, and tumor cell migration. These effects were attenuated by anti-TNFSF12 neutralization. Clinically, combined high TNFSF12 and TNFRSF12A expression was associated with advanced disease stage and poorer survival. Together, these findings identify a macrophage-tumor cell signaling axis in which FOSL2 upregulates TNFRSF12A in CRC cells, while APOE+ macrophage-derived TNFSF12 activates downstream BCL2L1-associated survival and EMT-related programs. The TNFSF12-TNFRSF12A-BCL2L1 pathway may represent a potential therapeutic vulnerability in metastatic CRC. NOVELTY AND IMPACT: This study identifies a previously undercharacterized macrophage-tumor cell signaling mechanism in colorectal cancer (CRC), in which APOE+ macrophage-derived TNFSF12 acts on TNFRSF12A-expressing malignant epithelial cells to promote EMT-associated progression. By integrating single-cell transcriptomics, bulk RNA-seq, spatial transcriptomics, and functional validation, we show that FOSL2 directly enhances TNFRSF12A transcription in EMT-associated tumor cells, thereby increasing their responsiveness to TNFSF12. Functionally, TNFSF12/TNFRSF12A signaling promotes BCL2L1-associated survival signaling, EMT-like changes, and tumor cell migration, while combined high TNFSF12 and TNFRSF12A expression is associated with advanced disease stage and poor patient survival. These findings reveal a clinically relevant macrophage-tumor cell communication axis and highlight the TNFSF12-TNFRSF12A-BCL2L1 pathway as a potential therapeutic vulnerability in metastatic CRC.