| Objective To establish 4T1-induced metastatic breast cancer model in BALB/C mice. Tumor antigen peptides were obtained from 4T1 cells and HSP70 peptides complexs (HSP70-PC) were reconstructed in vitro. To investigate the effect of HSP70-PC induce specific immune response to activate and proliferation T cell. Further, to investigate the specific antitumor effect of dendritic cell (DC ) modified by reconstructed HSP70-PC in vitro and in vivo. Methods 30 BALB/c mice were injected s.c. in the mammary fatpad with 1×105 4T1 cells . Tumor growth was determined by multiplying the measured perpendicular diameters of the tumor. and counting the number of metastasized tumor colonies on the surface of the lungs. 2,3 and 4 weeks after injection , the mice were killed and the tissues of mammary fatpad , lung, and liver were taken, fidxed in 10% neutral bufered formalin, embedded in paraffin and observed microscopically respectively. 4T1 antigen peptides were obtained using techniques including freezing and thawing heat precipitation and acid precipitation. The stimulating effect of the in vitro HSP70 binding 4T1 peptides on spleen cells from BALB/C mice and the proliferation of stimulated spleen cells were observed by T cell activation test. The cytotoxicity of proliferated T cell was detected by incubating 4T1 cells. The levels of IL-12 and TNFa secreted by DC from BALB/C mouse modified by HSP70-4T1 peptides complex was determined. The activation of lymphocytes by modified DC and the cytotoxicity of the activated lymphocytes to 4T1 cell were tested in vitro. The inhibition of 4T1 tumor in BALB/C mouse by injection modified DC was observed in vivo. Results Challenge with 1×105 4T1 cells led to progressive tumor growth rapidly at the primary site in all BALB/c mice. Multiple metastases can be found in lungs 3 weeks after fatpad injection of the 4T1 cell line in most animals, Liver metastases can be found in most animals 4 weeks after orthotopic injection. The obtained tumor antigen peptides were a peptides mixture. The mixed peptides could activate T cell and cause T cells proliferation in vitro after presented by HSP70 .The proliferated T cells showed specific cytotoxicity to 4T1 cells[4T1 kill rate(64.2±8.70)%(P<0.05) ]. The BALB/C mice immunized with HSP70-4T1 peptides complexs are significently more resistant to 4T1 tumor cells compare with the navie BALB/C mice 0.75μg of 4T1 peptide bound to HSP70 could mature 1×10~6 DC. 5×10~3 modified DC could activate 2×10~6 lymphocytes. The dose of peptides could be reduced to 1/40 if the modified DC was used to instead of direct use HSP70-4T1 peptides complex stimulated the same amount of lymphocyte. The activated lymphocytes could specifically kill 4T1 cells in vitro, the kill rate is (60.24±6.23)%. 4T1 tumor growth in BALB/C mouse could be inhibited significantly by injection DC modified by HSP70-4T1 peptide complexes. Conclusion The 4T1 matastatic breast cancer model is an excellent mouse model for the study of metastatic progression of breast cancer in humans. The method for preparting of 4T1 tumor antigen peptides used in this paper is simple and easy; the obtained antigen peptides can induce specific immun response in vitro and in vivo. HSP70 -4T1 peptide complexes could effectively modify DC from BALB/C mouse; these modified DC can specifically activate lymphocytes and effectively inhibit 4T1 tumor growth in BALB/C mouse. The dose of peptides could be reduced significantly by DC presenting to activated lymphocytes.
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