Expression of the High Affinity Neurotensin Receptor Defines an Immune‑cold, Poor‑prognosis Colorectal Cancer Subtype

Objective: We investigated neurotensin receptor 1 (NTSR1) as a potential mediator of the mechanical immune barrier that contributes to T-cell exclusion. Summary Background Data: The efficacy of immunotherapy in advanced colorectal cancer (CRC) is limited by primary resistance, frequently characterized by an “immune-excluded” phenotype where physical and biological barriers prevent effector T-cell infiltration. Through its high-affinity receptor, NTSR1, neurotensin signaling has been implicated in tumor progression, yet its role in shaping the immune landscape of CRC remains unclear. Methods: We integrated transcriptomic and clinical data from The Cancer Genome Atlas discovery cohort with an independent surgical validation cohort from the University of Kentucky. Random Forest machine learning identified molecular features associated with NTS/NTSR1 signaling, and a 3D tumor spheroid co-culture model was employed to evaluate the impact of NTS/NTSR1 signaling on spatial T-cell distribution in vitro . Results: Dual-cohort analyses revealed high NTSR1 expression as an independent risk factor for progression-free interval in the discovery cohort and overall survival in the validation cohort. Mechanistically, the NTS/NTSR1 axis upregulates a mechano-structural network centered on PLXNB3, FLNC, and AHNAK2, while simultaneously driving epithelial dedifferentiation through downregulation of core transcription factors (CDX2, SATB2). Together, these transcriptional programs are associated with the remodeling of the tumor microenvironment into an immune-cold, suppressive state. Moreover, 3D modeling indicated that ligand-activated NTS/NTSR1 signaling restricts T-cell infiltration, which was significantly reversed by the NTSR1 antagonist SR48692, providing functional support for a mechanical immune barrier proposed based on transcriptomic remodeling. Conclusions: Targeting the NTS/NTSR1 axis may attenuate biological and physical barriers underlying immune exclusion, providing a preclinical rationale for converting immune-excluded CRCs into immune-responsive tumors.