Comprehensive analytical approach identifies a subtype of CTCF+ tumor-associated neutrophils associated with CRC development and as a biomarker for immunotherapy.

Background: Colorectal cancer (CRC) is an aggressive and heterogeneous tumor with limited therapeutic options. Tumor-associated neutrophils (TANs) play multifaceted roles in the tumor microenvironment (TME) depending on their polarization. Understanding TAN heterogeneity and the mechanisms underlying their tumor-promoting activities is critical for advancing CRC treatment. Methods: This study integrated single-cell RNA sequencing, spatial transcriptomics, bulk RNA sequencing, and in vivo/ex vivo experiments to characterize TANs in CRC. Results: We identified a distinct subpopulation of TANs characterized by high CCCTC-binding factor (CTCF) expression (CTCF+ TANs) enriched in hypoxic tumor regions. CTCF+ TANs exhibit enhanced migratory capacity and IL-1β secretion, correlating with poor prognosis and resistance to immunotherapy. Mechanistically, CTCF regulates the expression of Migration and Invasion Enhancer 1 (MIEN1), which promotes TAN recruitment and migration without triggering inflammation. Functional studies revealed that CTCF+ TANs suppress T-cell immunity, facilitate epithelial-to-mesenchymal transition (EMT), and contribute to CRC progression and metastasis. In vivo, targeting CTCF-MIEN1-IL-1β signaling rescued the immunosuppressive microenvironment and improved the efficacy of anti-PD-L1 therapy. Conclusions: CTCF+ TANs represent a novel TAN subtype that drives CRC progression and immunosuppression via the CTCF-MIEN1-IL-1β axis. These findings highlight the potential of targeting CTCF+ TANs to overcome immunotherapy resistance and improve patient outcomes.