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

Abstract

BackgroundTumor cells frequently develop immune resistance through interferon-{gamma} (IFN-{gamma})-induced PD-L1 expression, acquisition of cancer stem cell (CSC)-like features, and adaptation to hypoxia within the tumor microenvironment (TME). Although IFN-{gamma} activates both STAT1 and STAT3, how these pathways interact to regulate immune evasion under hypoxia remains unclear. MethodsUsing the MC38 murine colorectal cancer model and T cell-tumor spheroid co-culture assays, we examined how IFN-{gamma} 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. ResultsWe found that IFN-{gamma} 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-1 expression. In co-culture assays, Niclosamide improved T cell infiltration and reduced exhaustion under hypoxic conditions, resulting in improved T cell killing. ConclusionsOur findings identified Niclosamide as a potent dual STAT1/3 inhibitor capable of reversing IFN-{gamma} and hypoxia-driven immune evasion. Repurposing Niclosamide may represent a promising strategy to enhance the efficacy of immune checkpoint blockade in solid tumors. key messagesInterferon-{gamma} (IFN-{gamma}) 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-{gamma}-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-1 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.