Preliminary Study On The Effect And Mechanism Of IL-21R Signaling On The Growth Of Hepatocellular Carcinoma

Background and ObjectiveMany studies have confirmed the antitumor role of IL-21 in animal tumor models,such as models of melanoma,pancreatic carcinomas,mammary adenocarcinoma and bladder cancer.In contrast,other studies have shown that IL-21 can promote a protumorigenic inflammatory circuit to induce colitis-associated colon cancer(CAC)development.However,the role of IL-21 R signaling in HCC development is unclear.Hepatocellular carcinoma(HCC)is a typical inflammation-associated cancer.The interaction between HCC antigenicity and the immunological microenvironment and the immunosuppressive microenvironment in HCC jointly drive he initiation and development of HCC.Therefore,the balance between immunological response factors and immunosuppressive response factors is critical for HCC growth,progression and metastasis in patients.However,little is known about the mechanism that controls the balance of immune responses and immunosuppressive responses in HCC growth.In this study,we investigated the role of IL-21R signaling in HCC growth,maintaining the balance of the systemic immune system and memory immune response to tumor challenge by using the IL-21R knockout(KO)mice and two mouse models of HCC.MethodsFirst,we established two murine HCC models:subcutaneous HCC model and orthotopic HCC model.To investigate the role of IL-21R signaling in HCC growth,we comparatively analyzed the mice survival time,morphology and histopathology of liver tumor in IL-21R KO and WT mice by using these two HCC models.In order to determine the mechanism that IL-21R signaling affects HCC growth,MTT test was firstly performed to examine whether IL-21 have an impact on the proliferation of Hepal-6 cells in vitro;Then we investigated the infiltration of immune cells and their functions in liver tumors by immunohistochemical method and flow cytomry;To observe integrally a status of the systemic immune system,we determined the change of immune cells in the spleen and lymph node in IL-21 R KO and WT orthotopic HCC mice.Second,to investigate the effects of IL-21 R signaling on immune responses to tumor rechallenge,we performed two-time subcutaneous inoculation in IL-21R KO and WT mice(the second inoculation at day 25)and measured the two-time subcutaneous tumor growth;We monitored the dynamic change of immune cells in the peripheral blood after the first tumor inoculation and determined the change of immune cells in the spleen and bone marrow after the second tumor inoculation.At last,we downloaded a human HCC GEO RNAseq dataset(GSE14520)from PubMed site and analyzed the exppression levels of IL-21 and IL-21 R in tumor and non-tumor tissues;Through Kaplan-Meier and log-rank test,we also analyzed the associations between IL-21/IL-21R exppression level in tumor or non-tumor tissues and overall survival time or recurrence in HCC patients.ResultsFirst,we successfully establised subcutaneous and orthotopic HCC murine model in the current study.Through comparatively analysis of mice survival time,weight,tumor morphological and pathological change in these two HCC mice models,we demonstrated that IL-21R signaling deficiency promote the HCC rapid growth in the tumor-bearing mice.After an in-depth study,our results showed that IL-21R signaling deficiency result in decreased infiltration of antitumor immune cells(CD4+T cells,CD8+ T cells,NK cells and NKT cells)and increased accumulaiton of protumor immunsuppressive cells(MDSCs,TAM and Treg cells)in liver tumor,spleen and lymph node in tumor-bearing mice;CD4+ T cell,CD8+ T cell and NK cell from IL-21R KO mice have a decreased capacity to produce IFN-y.In addition,we found that antigen presenting cells(APCs)(B cells,Cillc+ DCs,conventional DCs(cDC)and plasmatoid DCs(pDC))were markedly decreased and their antigen presentting ability markedly reduced in the spleen in IL-21R KO mice.These data demonstrate that IL-21R signaling defects impair the antitumor function of antitumor immune cells in tumor microenvirnment and result in the disequilibrium of systemic immune system in tumor-bearing mice.Second,after two-time subcutaneous tumor inoculation,compared with the first subcutaneous tumor in WT mice,the second subcutaneous tumor showed slower growth,and tumor volume decreased and then almost disappeared at last.However,no significant differences were found in the growth speed and tumor volume between the first and second subcutaneous tumors in IL-21R KO mice,indicating that IL-21R KO mice failed to generate tumor-specific memory responses to tumor rechallenge;The results from analysis of immune cells in the peripheral blood,spleen and bone marrow after the two second inoculation(day 25-43)showed that IL-21R KO mice have a decreased population and activation of antitumor immune cells and an increased accumulation of protumor immunosuppresive cells compared to WT mice.These results demonstrate that IL-21R signaling is important for the generation of tumor-specific memory responses to tumor rechallenge.At last,after analyzing an human HCC GEO RNAseq dataset(GSE14520),we found that IL-21 and IL-21R expression levels in non-tumorous tissues were significantly higher than those in tumorous tissues and there are positive correlations between IL-21 expression in non-tumorous tissues or IL-21R in tumorous tissues and overall survival or recurrence in HCC patients.These results demonstrate that IL21/IL-21 R signaling is a protective factor in controlling HCC development in HCC patients.ConclusionsIn this study,we established two HCC murine models and demonstrated that IL-21 R signaling have a protective role in controlling HCC growth by increasing the activation and function of T cells,NK cells and NKT cells and decreasing the accumulation of protumor immunosuppressive cells(MDCSs and Treg cells)in tumor microenvironment.Moreover,we demonstrated that IL-21 R signaling plays a critical role in maitaining the balance of systemic immune system and immunological memory response to tumor challange.