Experimental Study Of Mixed Heat Shock Proteins/peptides Complexes For Immunotherapy Of Mice Sarcoma

In 1986, Srivastave reported that heat shock protein (HSP) preparations derived from tumor cells had ability to induce protective immune reaction against tumor cells. Since then, isolated HSP from tumor cells as a vaccine has been tested in mice with different tumors, results showed that tumor size was reduction in all, some even complete regression. Recently, gp96( a kinds of HSP) has been used as a vaccine for treatment of cancers and has begun phase 3 clinical trails, results showed that HSP vaccine is efficient, safe, manufacturability and ethical acceptable.This experimental study was aimed to verify if HSPs/peptides compound vaccine can be used as a new methods for immunotherapy of bone sarcoma . First, we observed the expression of many subtype of HSP (HSP60, HSP70, HSP90 and gp94) in some orthopedic sarcoma tissues, include osteosarcoma, chondrosarcoma, fibrosarcoma, liposarcoma, malignant synovial sarcoma and Ewing's sarcoma by immunohistochemical methods. Secondly, we established a stable animal model with mice sarcoma SI80 cell line. Thirdly, the HSP60 and HSP70/peptides were isolated and purified from SI80 tumor cells with affinity chromatography, obtained mixed HSPs/peptides complexes with molecular shave chromatography by sephacryl S-200HR, characterized each HSP by SDS-PAGE and Western blot. Fourthly, Examined the immunotherapy effects using those purified HSP70/peptides, isolated HSP60, and mixed HSPs/peptides complexes in mice model xenografed SI80. Fifthly, examined CTL of immuned mice tested by MTTassay in vitro.The results were: 1, All four different subtype of HSP were expressed in different bone tumors. HSP60 and gp94 expressed in a high degree, as well as positive cases were 95-100%, HSP90 expressed in a moderate degree, positive cases were 95-100%, while HSP70 expressed in a low or moderate degree, positive rate were 0-95%. 2, with modified molecular shave chromatography and affinity chromatography, we obtained purified HSP70/peptides and HSP60 separately, also with modified molecular shave chromatography we obtained mixed HSPs/peptides complexes, The yield of mixed HSPs/peptides complexes is 20-25mg/g wet weight of tumor tissues, HSP70/peptides is 100-150ug/g, and HSP60 is 400-500ug/g. 3, All three kinds vaccine (HSP60, HSP70/peptides, mixed HSPs/peptides compound) showed obviously active immunotherapy effects in prophylactic treatment group, They can markedly decease the tumor occurrence and inhibit the tumor growth, the tumor growth inhibition rate were 51.52%- 90.39 %, as well as prolonged survival. Some mice could completely fight off growth tumor and survival in good health. Mixed HSPs/peptides compound used in treatment of pre-established tumor mice also got the same results, tumor growth inhibition rate was 71%. Among three type vaccine mixed HSPs/peptides complexes 20ug/time X 3 give the best results, tumor growth inhibition rate was 73.26%-90.39%, 27% tumor were totally retarded. The effective treatment doses was 2ug~30ug/time X3. 4, The spleen lymphocytes of survival mice immuned with mixed HSPs/peptides compound was sensitized, it can be expanded in vitro, cytotoxicity of this lymphocytes against SI80 was 9.43-17.39%, and against liver tumor cells was 6.12-10.23%, results showed it might be that this vaccine has induced both specific (adaptive) and non-specific (innate) immune activity.Conclution: The mixed HSPs/peptides complexes isolated from tumor cells may be a promising more efficient vaccine for treatment of orthopedic sarcoma . Especially for prevent tumor recurrence and metastasis after surgery.