Tumor-derived exosomes promote CD8+ T cells ferroptosis by modulating FTH1/NCOA4 axis the in HCC

Abstract

Background & aims Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with dreadful clinical outcome. Tumor cells may induce dysfunction in CD8+T cells by secreting various bioactive substances, including exosomes. Ferroptosis, a unique form of cell death characterized by iron-dependent lipid peroxide accumulation, may contribute to T cell dysfunction. Ferritin heavy chain 1 (FTH1), a known regulator of ferroptosis, has gained attention. However, the role of tumor-derived exosomes (TDEs) in regulating ferroptosis of CD8+T cells remain unexplored. Methodology To evaluate the expression level of FTH1, exosomal FTH1 and ferroptotic CD8+T cells, PCR, western blot, Elisa and immunohistochemical staining assays were performed on our local clinical cohort. Flow cytometry was used to characterize the phenotype of ferroptotic CD8+T cells. The clinical significances of FTH1 and ferroptotic CD8+T cells were evaluated by Cox proportional hazards model. To investigate the functional roles and molecular mechanism of exosomal FTH1, isolating T cells were cultured with TDEs in vitro, RNA sequencing analysis was employed, and results were further validated with the use of and CRISPR interference and inhibitors. To assess the treatment effect, the efficacy of FTH1 knockdown in augmenting antitumor immunity in vivo was evaluated using mouse liver tumor models, some mice were given intraperitoneal injections of antibodies against CD4 or CD8 before the cancer cells wereinjected. Results Our clinical findings revealed a significant increase in FTH1 expression in HCC tissues, inversely correlated with overall survival. Specifically, serum exosomal FTH1 was inversely proportional to the proportion of CD8+T cells in tumor tissue, with high levels of FTH1 expression positively correlating with an abundance of ferroptotic CD8+T cells within HCC tissue. Ferroptotic CD8+T cells displayed general immunosuppressive marker relative to non-ferroptosis CD8+T cells. In in vitro experiments, FTH1 was highly expressed in exosomes of HCC cell lines. TDEs mediated uptake of FTH1 by CD8+T cells induced lipid peroxidation, which could be reversed by a ferroptosis inhibitor, subsequently impairing the cytotoxic functions of CD8+T cells and exacerbating their exhaustion status. FTH1 knockdown reduced the ferroptosis phenotype and enhanced the antitumor activity of CD8+T cells. Mechanistically, we observed TSG101 facilitating the association of FTH1 with CD63-containing vesicles and the NCOA4-mediated ferritin selective autophagy (ferritinophagy) during ferritin degradation, promoting labile iron accumulation and subsequent ferroptosis in CD8+T cells. Using an exosome release inhibitor to block exosome secretion greatly reduced TDE-caused ferroptosis in CD8+T cells. In in vivo experiments, mice with FTH1 knockdown formed smaller tumors, inhibited ferroptosis in CD8+T cells in animal models, and effectively restored their antitumor activity; these effects were not observed in mice with CD8+T cells depletion. Conclusions Our findings suggest that upregulation of FTH1 expression is positively associated with the prevalence of ferroptotic CD8+T cells. FTH1 is secreted via exosomes, promoting CD8+T cells ferroptosis through NCOA4-mediated ferritinophagy. Targeting FTH1 to prevent CD8+T cells ferroptosis may restore T cell function and enhance the efficacy of cancer therapy.