Zingiberen Newsaponin Enhances Antitumor Immunity in Anaplastic Thyroid Carcinoma Via Induction of Autophagy-Dependent Pyroptosis

Background: Anaplastic thyroid carcinoma (ATC) is a lethal malignancy with a dismal prognosis and limited therapeutic options. Although immunotherapy has revolutionized the oncological landscape, the efficacy of Programmed Cell Death Protein 1 (PD-1) blockade as a monotherapy remains underwhelming in ATC. Consequently, identifying novel agents capable of triggering immunogenic cell death (ICD) to sensitize ATC to immune checkpoint inhibitors is a critical clinical priority. Materials and Methods: Through high-throughput screening of 3200 compounds, Zingiberen newsaponin (ZN) was identified as a lead anti-ATC agent. Its therapeutic efficacy was evaluated using ATC cell lines, patient-derived organoids, and xenograft models. Mechanistic investigations were conducted via pharmacological inhibition, genetic knockout, and Western blotting to examine the autophagy-pyroptosis axis. Furthermore, an immunocompetent murine ATC model was employed to assess the synergistic effects of ZN and anti-PD-1 therapy on the tumor immune microenvironment through immunohistochemical analysis of T-cell infiltration. Results: ZN exhibited potent antiproliferative activity against ATC cells with half-maximal inhibitory concentration values ranging from 1.95 to 2.98 μM. Morphological assessment and biochemical assays (Lactate dehydrogenase, interleukin-1 beta, and high mobility group box 1 release) confirmed that ZN induces Gasdermin E (GSDME)-mediated pyroptosis via the activation of caspase-3 and caspase-8. Importantly, genetic knockout of GSDME significantly attenuated the pyroptotic phenotype, corroborating the central role of GSDME in ZN-induced pyroptosis. Mechanistically, ZN-induced pyroptosis was found to be autophagy-dependent, as pharmacological inhibition of autophagy with chloroquine significantly reversed GSDME cleavage and cell death both in vitro and in vivo . Consistently, ATG5 knockdown markedly rescued ZN-induced cell pyroptosis. Furthermore, ZN functioned as a potent inducer of ICD. In immunocompetent murine models, ZN treatment remodeled the tumor immune microenvironment by significantly increasing the infiltration of CD8 + T cells. Notably, the combination of ZN and anti-PD-1 therapy demonstrated superior synergistic antitumor efficacy compared with monotherapy. Conclusions: Our study identifies ZN as a novel inducer of autophagy-mediated GSDME pyroptosis in ATC. By triggering ICD and recruiting CD8 + T cells, ZN sensitizes ATC to PD-1 blockade, offering a promising therapeutic strategy for this lethal disease.