The Intervention Of Anti-interleukin-33 Active Immunization In A Murine Model Of Breast Cancer

Background Interleukin-33(IL-33)is a kind of multi-functional cytokines,which is closely associated with a variety of tumor dieases,such as breast cancer,lung cancer,gastric cancer,ovarian cancer and other cancers with excessive expression and accumulatiom of IL-33.As a key mediator triggering type 2 immune responses,IL-33 significantly stimulates the differentiation and proliferation of Th2 and type 2 macrophage,and promotes the infiltration of MDSCs and Tregs into tumor tissue.Thus IL-33 may play an important effect on promoting tumor immunosuppression and immune escape.Therefore,IL-33 may be an important target in the immunotherapy of breast cancer.Objective The purpose of this study intends to antagonize over-accumulated IL-33 in tumor-burden mice through a active immunization approach of hepatitis B core antigen(HBcAg)VLPs presenting full-length IL-33 molecules,and investigate the influences on cellular immune responses and the tumor growth,and thus reveal the possible roles of IL-33 in breast cancer and the potentials of anti-IL-33 vaccine as a valuable candidate for the treatment of breast cancer.Methods The recombinant plasmid encoding for a chimeric protein with IL-33 inserted into the immunodominant epitope of HBcAg,was tansformed into E.coli DH5a cells,and the expression of the recombinant proteins were induced by IPTG.The proteins were purified with a procedure consists of ammonium sulfate precipitation and density gradient centrifugation,and the presence of VLPs was detected with electron microscopy.Mice were immunized with the VLPs through preventive and therapeutic strategies separately in a 4T1 grafted tumor model.The studies examined the levels of IL-33-specific IgG antibody in the serum with ELISA.The size of the tumor was measured every 2 days.When the largest tumor reached to 15mm,the mice were sacrificed and the samples waere collected and analyzed.The IgG subtypes IgG1 and IgG2a in serum were detected with ELISAs to reflect Thl/Th2 response bias;moreover,the splenocytes isolated from the experimental mice were stimulated in vitro with PMA and the cytokine production and immune cell responses were analyzed.The expression of IFN-γ and IL-4 was detected by ELISA,the level of IFN-γ secreting splenocyte was measured by ELISPOT,and the number of CD8+IFN-γ+CTL cells in splenocytes and immunosuppressive cells T-regs and MDSCs in tumor tissues were analyzed by flow cytometry.Results The recombinant protein HBcAg-IL-33(named HBcAg-33)was expressed efficiently in E.coli,and the purified protein presents mainly in the form of VLPs;Immunization with VLPs presenting IL-33 induced high titers of IL-3 3-specific IgG,and significantly suppressed the growth of grafted 4T1 tumor using a preventive vaccination strategy.In another study with a therapeutic vaccination strategy employed,the immunization with HBcAg-33 VLPs inhibited the tumor growth in the early stage.Although there was no significant inhibitory effects on tumor growth found at later stage,the pulmonary metasatasis was significantly inhibited.The vaccination elevated the IFN-γ expression of stimulated splenocytes in vitro,and the number of splenocyte cells secreting Th1/CTL-like cytokine IFN-γ.Analyzed by flow cytometry,the level of CD8+IFN-γ+CTL cell in splenocytes was significantly increased.while the number of Th2 in splenocytes and those of MDSCs and Treg in tumor tissue were decreased.Corrsponding to the results of cytokine expression and Th1/Th2 cell bias,the ratio of IgG1/IgG2a was decreased.Conclusion The vaccines of HBcAg VLPs presenting full-length IL-33 stimulates persistent and high tittered IL-33-specific antibody response,and significantly inhibits tumor growth and metastasis in a model of breast cancer.The possible mechanism may account for promoting T cell response to Th1/CTLs bias and suppressing the infiltration of Tregs and MDSCs into tumor tissues.The results help to reveal the roles of IL-33 in the formation,growth and metastasis of tumor of breast,and indicate that the strategy of anti-IL-33 vaccine immunization may hold a potential in the clinical treatment of breast cancer.