Nicotinamide phosphoribosyltransferase (NAMPT) plays a pivotal intracellular role in maintaining nicotinamide adenine dinucleotide (NAD) metabolism, but it is also secreted into the extracellular space, where it functions as a cytokine (eNAMPT). Elevated circulating eNAMPT levels have been reported in inflammatory disorders and malignant diseases. In breast cancer patients, plasma eNAMPT concentrations are increased and show a strong association with disease stage and patient prognosis. On this basis, we explored the involvement of eNAMPT in the progression of triple-negative mammary tumors by assessing the impact of its inhibition using a specific neutralizing monoclonal antibody (C269). Female BALB/c mice were inoculated with 4T1 clone 5 cells, while female C57BL6 mice received EO771 cells. Tumor growth, spleen enlargement and metastatic dissemination were monitored. The anti-eNAMPT neutralizing antibody was administered twice weekly, and animals were sacrificed after 28 days. Excised tumors were examined using histopathological techniques, flow cytometry, western blotting, immunohistochemistry, immunofluorescence and RNA sequencing. These analyses enabled detailed characterization of tumor properties, including the isolation of tumor-infiltrating lymphocytes and cancer cells, and allowed investigation of the molecular pathways responsible for the observed effects. Additionally, three-dimensional (3D) co-culture systems were employed to analyze functional interactions between tumor cells and T lymphocytes. Treatment with C269-mediated eNAMPT blockade significantly reduced primary tumor burden and lowered the incidence of lung metastases. Transcriptomic profiling combined with functional experiments demonstrated that eNAMPT modulated T-cell activity through the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) signaling axis. Neutralization of eNAMPT restored effective antitumor immunity, characterized by enhanced activation of CD8+IFNγ+GrzB+ T cells and a marked attenuation of the immunosuppressive phenotype of regulatory T cells. Together, these findings provide the first evidence that eNAMPT represents a previously unrecognized immunotherapeutic target in triple-negative breast cancer.
