WITHDRAWN: Regulatory T cells enhance stem-like characteristics of hepatocellular carcinoma cells through Wnt/β-catenin pathway

Abstract

Background: Tumor initiating cells (TICs) were confirmed to drive the therapeutic resistance of hepatocellular carcinoma (HCC), but the mechanism by which tumor microenvironment maintains the HCC stemness is not fully understood. This study aims to investigate the effect of regulatory T cells (Tregs) on the TICs characteristics of HCC. Methods: Immunocytochemistry, flow cytometry, real-time PCR, western blot, in vitro sphere-formation, and in vivo tumorigenesis assay were used to detect HCC TIC characteristics. Additionally, the association between FoxP3 expression, Wnt/β-catenin pathway activation, and HCC stemness were analyzed by forced expression or inhibition of FoxP3. Results: It was showed Tregs enhanced the stemness of HCC cells by upregulation of TIC-related markers CD133, Oct3/4, Sox2, c-Myc, Klf4, Nanog, CD13, EpCAM, and induction of epithelial to mesenchymal transition (EMT), increase of TICs ratio, as well as promotion of tumorigenic ability. Moreover, β-catenin and c-Myc were upregulated in HCC cells when co-cultured with Tregs. After Wnt/β-catenin pathway inhibition, HCC stemness was also inhibited. In addition, Tregs-derived exosomes played the same role as Tregs in enhancing HCC TIC properties, and exosome inhibition led to decreased TIC ratio as well as TIC markers expression. Furthermore, Tregs-derived exosomes down-regulate FoxP3 and GSK3β of HCC cells. Forced expression of FoxP3 resulted in increased GSK3β, decreased β-catenin and TIC ratio of HCC. In contrast, FoxP3 interference reduced GSK3β, increased β-catenin and TIC ratio. Conclusions: This study, for the first time, demonstrated Tregs enhanced HCC stemness through Wnt//β-catenin pathway by down-regulating FoxP3.