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 (IFNγ)-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.