| Cancer immunotherapy was initiated by William Coley more than a century ago. Recently, major advances in cellular and molecular immunology have allowed a comprehensive understanding of the complex and high rate of interactions between the immune system and tumor cells.In 1986, Srivastavereportedthatheatshock protein (HSP) preparations derived from tumor cells had ability to induce protective immune reaction against tumor cells,but many results showed tumor size can be only partially reducted .Because tumor antigen is polyvalence,we assess that the tumor-derived mixed HSP/p vaccine tumor-derived should have a better treatment efficacy. Among all previously studied cytokines and biologic response modifiers, IL-12 has demonstrated the most potent antitumor effects in multiple experimental tumor models. In the current study we investigate the immune response against long term established, large S180 tumors induced by combined treatment with mHSPs ,IL-12 and cyclophosphamide (Cy), an agent known to decrease the number Tregs and enhance the immunostimulatory effect.To verify if mHSP/P compound vaccine can be used as a new methods for immunotherapy of sarcoma . Firstly, we established a stable animal model with mice cancer S180 ,MCA207 and Lewis cell line. the mHSP/Pcomplexes obtained with chromatography of sephacryl S-200HR, characterized each HSP by SDS-PAGE and Western blot. Secondly, Studies of eliciting prophylactic immunity with mHSPs from MCA207 sarcoma in C57B1/6 mice. Thirdly, examined the immunotherapy effects using those purified mHSP/P complexes combined with IL-12 and Cy in mice model of established S180. Fourthly, examined immunomechanism of immuned mice tested by LDH, ELISPOT,and flow cytometry assay in vitro.Fifthly, examined the immunotherapy effects using single mHSPs/P or mHSPs/P combined with IL-12 and Cy in mice model of Lewis lung cancer cell line.The results were: 1, with modified chromatography we obtained mHSP/P complexes contained HSP70, HSP60 , gp96 components identified by SDS-PAGE and Western-bloting assay. 2, mHSPs/P from MCA207 showed obviously prophylactic immune effects to MCA207, they can markedly decease the tumor occurrence and inhibit the tumor growth, as well as prolonged survival. Some mice could completely reduct growing tumor and survival in good health. Amongthree groups vaccine, mHSPs/P 30ug/time×3 give the best results, tumor growth inhibition rate was 91.29%, 70% tumor were totally retarded. The effective treatment doses was 5ug～30ug/time×3. 3, mHSPs/P from S180 sarcoma combined with IL-12 and Cy showed obviously active immunotherapy effects to S180 sarcoma. Eight of 10 mice could completely fight off growth tumor and survival in good health. 4, The spleen lymphocytes of survival mice immuned with mHSP/P and mHSPs +Cy+ IL-12 were sensitized and expanded in vitro, cytotoxicity of this lymphocytes against S180 was 33-66.39%, NK cells and IFN-γ were both increased after immunization, The induced immunity with mHSPs +Cy+ IL-12 were stronger than mHSP/P. 5, mHSPs/P from Lewis cancer as vaccine and combined with IL-12 and Cy treatment showed both prophylactic immune and therapeutic immune effects was not satisfected as it in sarcoma. This different results was discussed and the improvement of immunotherapy was suggested.Conclusion: The mHSP/Pcomplexes isolated from tumor cells may be a promising vaccine for treatment of orthopedic sarcoma . Especially for prevent tumor recurrence and metastasis after surgery. mHSPs/P +Cy+ IL-12 may be a more efficient immunotherapy for treatment of tumor bearing patients.
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