Phosphorothioate modification of the TLR9 ligand CpG ODN inhibits poly(I:C)-induced apoptosis of hepatocellular carcinoma by entry blockade.

Toll-like receptors (TLRs) play a crucial role in the innate immune response and subsequent induction of adaptive immune responses. Recently, it has been noted that TLRs on tumor cells are involved in tumor development, and several TLR agonists, such as the TLR3 agonist poly(I:C) and the TLR9 agonist CpG ODN, are being developed as vaccine adjuvants and cancer immunotherapeutics. In this study, we investigated whether combining poly(I:C) with a TLR9 agonist CpG ODN would result in a stronger anti-tumor effect on hepatocellular carcinoma cells (HCCs). Surprisingly, we found that simultaneous transfection of poly(I:C) and ODN M362 exhibited a lower pro-apoptotic effect on HCCs than transfection with poly(I:C) alone. Simultaneous co-transfection was accompanied by down-regulation of poly(I:C)-related innate receptors, pro-inflammatory cytokines and apoptotic genes induced by poly(I:C), indicating that ODN M362 blocked the activation of poly(I:C)-triggered intrinsic immune responses and cellular apoptosis. Further studies indicated that these effects were partly due to the phosphorothioate-modification of CpG ODN, which blocked the entry of poly(I:C) into tumor cells. This entry blockade was avoided by administering poly(I:C) after CpG ODN. Moreover, poly(I:C)-mediated pro-apoptotic effects were enhanced in vitro and in vivo by pre-treating HCC cells with CpG ODN. Our findings thus suggest that when combining poly(I:C) and CpG ODN for cancer therapy, these agents should be used in an alternating rather than simultaneous manner to avoid the blocking effect of phosphorothioate-modified TLR9 ligands.

NK cells are the crucial antitumor mediators when STAT3-mediated immunosuppression is blocked in hepatocellular carcinoma.

STAT3 is highly activated in a wide variety of cancers and functions to promote tumor survival. We previously reported that blocking STAT3 activation inhibited human hepatocellular carcinoma (HCC) growth in vitro, but whether this treatment also triggered antitumor immune responses in vivo remained unknown. In this study, we found that blocking the STAT3 pathway in HCC cells dramatically inhibited murine HCC growth in vivo and prolonged survival of tumor-bearing mice. Importantly, the presence of STAT3-blocked HCC augmented NK cell cytotoxicity against HCC and increased expression of molecules associated with NK cell activation and cytotoxicity. In T cell-deficient nude mice, a unique NK cell-mediated antitumor function against STAT3-blocked HCC was suggested. NK cells were shown to be necessary and sufficient in NK or T cell depletion experiments, or by adoptively transferring NK cells. Furthermore, regulatory T cells and immunosuppressive IL-10 and TGF-ß cytokines were reduced in mice bearing STAT3-blocked HCC cells, suggesting that these factors may be involved in HCC-induced NK cell suppression. These findings indicate that blocking STAT3 in HCC cells can initiate innate immunity in vivo.

Targeting blockage of STAT3 in hepatocellular carcinoma cells augments NK cell functions via reverse hepatocellular carcinoma-induced immune suppression.

STAT3 is an important transcriptional factor for cell growth, differentiation, and apoptosis. Although evidence suggests a positive role for STAT3 in cancer, the inhibitory effects of tumor STAT3 on natural killer (NK) cell functions in human hepatocellular carcinoma are unclear. In this study, we found that blocking STAT3 in hepatocellular carcinoma cells enhanced NK-cell antitumor function. In the case of STAT3-blocked hepatocellular carcinoma cells, NKG2D ligands were upregulated, which promoted recognition by NK cells. Importantly, the cytokine profile of hepatocellular carcinoma cells was altered; in particular, TGF-ß and interleukin 10 (IL-10) expression was reduced, and type I interferon (IFN) was induced, thus facilitating NK-cell activation. Indeed, the cytotoxicity of NK cells treated with supernatant from STAT3-blocked hepatocellular carcinoma cells was augmented, with a concomitant elevation of molecules associated with NK cytolysis. Further experiments confirmed that the recovery of NK cells depended on the downregulation of TGF-ß and upregulation of type I IFN derived from STAT3-blocked hepatocellular carcinoma cells. These findings demonstrated a pivotal role for STAT3 in hepatocellular carcinoma-mediated NK-cell dysfunction, and highlighted the importance of STAT3 blockade for hepatocellular carcinoma immunotherapy, which could restore NK-cell cytotoxicity in addition to its direct influence on tumor cells.