Supporting data fo"Tumor-derived exosomes impair CD8+ T cells anti-tumor ability via ferroptosis in hepatocellular carcinoma"
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 were injected.
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.
Charpt 1-RNA sequence data, PowerPoint/Excel/PDF format.In this part, we identified the protein and RNA expression of ferritin heavy chain 1 (FTH1) in both tumor and non-tumor tissues.We used flow cytometry to characterize the secreted cytokines and the immunosuppressive phenotype of ferroptotic CD8+ T cells.
Charpt 2-wester blot data, JPG format.In this part,we also observed the difference in the expression of exosomal-FTH1 in the serum of HCC patients and healthy donors.We examined the correlation between exosomal-FTH1 in serum and T cells in tumor tissues, as well as the correlation between FTH1 and ferroptotic CD8+ T cells in tumor tissues. Statistical analyses were conducted to determine the prognostic potential of FTH1 and the expression of ferroptotic CD8+ T cells in HCC.
Charpt 3-IHC data, jpg format and files can be opened in software Imaris.In this part,we investigated the effect of tumor-derived exosomes on CD8+ T cells function.We identified exosomes by three different methods: western blot, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Then, we isolated CD8+ T cells from peripheral blood mononuclear cells (PBMCs) of healthy donors and established a co-culture system of exosomes and CD8+ T cells. RNA-sequencing analysis was performed in tumor-derived exosomes (TDEs) treated CD8+ T cells and the corresponding control cells to unravel the molecular mechanisms, which was subsequently validated using various experiments, such as cell death, iron level, lipid peroxidation, and finally, short hairpin RNAs (shRNAs) targeting FTH1 were designed.
Charpt 4-Flow cytometry data, files can be opened in software Flow Jo.In this part,the therapeutic effect of FTH1 was explored.Lentivirus-mediated stable cell lines and the matched control cell lines were generated. In the mechanism study part, we first used CRISPR interference to identify the pathway of TDEs release, and further confirmed it by immunofluorescence observation. The exosome release inhibitor GW4869 was added to the co-culture system to confirm the essential role of exosomes in the ferroptosis process. Finally, we verified the binding site of FTH1 by co-immunoprecipitation
