Discovery of indole analogue Tc3 as a potent pyroptosis inducer and identification of its combination strategy against hepatic carcinoma

Rationale: Hepatic carcinoma, one of the most malignant cancers in the world, has limited success with immunotherapy and a poor prognosis in patients. While pyroptosis is considered as a promising immunotherapy strategy for tumors, it still suffers from a lack of effective inducers. Methods: We designed, synthesized and screened an indole analogue, Tc3, featuring a 2, 4-thiazolidinedione substituted indole scaffold. Western blotting, qPCR and immunofluorescence were employed to detect the levels of pyroptosis pathway induced by Tc3. RNA sequencing was used to identify the mechanisms of Tc3 in hepatic carcinoma. To validate anti-tumor effect of Tc3, we used CDXs and PDXs mouse models in vivo. Then, the syngeneic effects of Tc3 with cisplatin and anti-PD-1 antibody were verified via western blotting, immunofluorescence, flow cytometry and ELISA. Results: Treatment with Tc3 notably inhibited the growth of hepatic carcinoma both in vitro and in vivo. Mechanistically, Tc3 inhibited the function of PRDX1 and up-regulated excessive ROS. Then, Tc3 induced gasderminE-mediated pyroptosis by activating the endoplasmic reticulum stress. Tumor cells with high expression of GSDME achieved better responses to Tc3-therapy. Tc3 also improved the efficacy of cisplatin against hepatic carcinoma. Additionally, superior synergistic treatment was observed when Tc3 was combined with anti-PD-1 antibody. Notably, Tc3 activated the tumor immune microenvironment (TIME) and enhanced CD8⁺ T cell infiltration in hepatic carcinoma. Conclusions: Collectively, we identified Tc3 as a promising and effective compound for treating hepatic carcinoma and established its synergistic therapeutic strategy as a pyroptosis inducer.