Identifying the therapeutic potential of Niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the Tumor Microenvironment

Background Tumor cells frequently develop immune resistance through interferon-γ (IFN-γ)–induced PD-L1 expression, acquisition of cancer stem cell (CSC)–like features, and adaptation to hypoxia within the tumor microenvironment (TME). Although IFN-γ activates both STAT1 and STAT3, how these pathways interact to regulate immune evasion under hypoxia remains unclear. Methods Using the MC38 murine colorectal cancer model and T cell–tumor spheroid co-culture assays, we examined how IFN-γ signaling through STAT1 and STAT3 influences PD-L1 expression, CSC plasticity, and cytotoxic T cell function under normoxic and hypoxic conditions. Pharmacologic inhibitors and siRNA knockdown were used to dissect pathway function, and Niclosamide, an FDA-approved anthelmintic, was evaluated as a dual STAT1/STAT3 inhibitor. Results We found that IFN-γ primarily induced PD-L1 through STAT1 activation, while CSC plasticity was associated with STAT3 signaling. STAT1 and STAT3 displayed reciprocal regulation—blocking one enhanced activation of the other. Niclosamide effectively inhibited phosphorylation of both STAT1 and STAT3, which led to suppressed PD-L1 upregulation and reduced CSC enrichment. In addition, it also partially inhibited hypoxia-induced HIF-1α expression. In co-culture assays, Niclosamide improved T cell infiltration and reduced exhaustion under hypoxic conditions, resulting in improved T cell killing. Conclusions Our findings identified Niclosamide as a potent dual STAT1/3 inhibitor capable of reversing IFN-γ and hypoxia-driven immune evasion. Repurposing Niclosamide may represent a promising strategy to enhance the efficacy of immune checkpoint blockade in solid tumors. key messages Interferon-γ (IFN-γ) enhances cytotoxic T cell function but also promotes tumor immune evasion by upregulating PD-L1 and inducing cancer stem cell– like properties. Our study identifies a reciprocal regulatory mechanism between STAT1 and STAT3 in IFN-γ-treated tumor cells that shapes immune evasion outcomes. We demonstrate that Niclosamide, an FDA-approved anthelmintic, acts as a dual STAT1/STAT3 inhibitor, effectively suppressing PD-L1 induction, limiting cancer stemness, and reducing HIF-1α expression under hypoxia. Niclosamide also restores T cell infiltration and decreases exhaustion in a 3D tumor spheroid model. By repurposing Niclosamide, this work provides a feasible approach to enhance the efficacy of immune checkpoint blockade and guide future translational and clinical studies in immunotherapies against solid tumors.