Establishment And Application Of Subcutaneous Transplantation Tumor Model Of Human Breast Cancer In Mice

Animal models of human cancer have become an essential instrument for anticancer drug's research.Nude and severe combined immunodeficiency(SCID) mice are the most common xenograft animals,they're appropriate for drug's screening and development but not for tumor vaccine's research.Tumor vaccines mainly boost the body's active immunity for tumor rejection.Therefore,for studying tumor vaccine activities a immunocompetent animal model of human cancer is very necessary.In present study,we have successfully established a human breast cancer model in ICR mice and used this model to study MUC1-based vaccine,MUC1-MBP anti-tumor effect.MUC1 is a member of mucin family and has been considered as the perfect target molecule in the study of tumor vaccine.Our group has been successfully developed a human MUC1-MBP fusion protein vaccine by gene recombiant. MUC1-MBP can induce humoral and cellular immune responses in mice and suppress the growth of mouse Lewis lung cancer cells.But it's still not clear that the anticaner effect for the human breast cancer.In this study,we established the subcutaneous transplantation tumor model of human breast cancer in ICR mice and using it investigate the anticancer effect of fusion protein MUC1-MBP.Methods and results:1.Establishment of a subcutaneous transplantation tumor model of human breast cancer in miceFirstly,healthy ICR mice were selected and irradiated by X-ray with different doses for immunosuppression;Secondly,human breast MCF-7 cancer cells were inoculated s.c.into ICR mice.After that,to maintain mouse immunosuppression for a short time,FTY-720 was administrated by lavage.We screened optimal experimental conditions for tumor growth in mice:The irradiated dose is 5 Gy;3 X 10~6 human breast MCF-7 cancer cells were inoculated into each mouse;ICR mice were given FTY-720 for 4～6 days by gavage.Through the above experimental conditions,ICR mice can grow tumors with the lowest mortality,the best stability and the incidence of tumor of 100%.The tumors could be observed for 21-24 days.2.Identification of human breast cancer tissuesIn order to identify that growing tumors in ICR mice are human breast cancer tissues,we dissected the mice and the tumors are obvious visual,pale,surrounding rich blood vessels.Pathological sections of those tumors were made and stained by hematoxylin and eosin staining and revealed infiltrating breast cancer.The incidences of tumor were 100%,100%and 80%with irradiated doses of 6 Gy,5 Gy and 4 Gy respectively,which were in accordance with the anatomic observation results.As MUC1 was highly expressed on human breast cancer cells,to further confirm that the growed tumors in mice were human breast cancer cells,we detected expression of MUC1 by immunohistochemistry with polyclonal antibody of anti-MUC1.The result showed that MUC1 expressions were positive on these tumor cells.3.Expression and identification of recombinant MUC1-MBP fusion protein and MBP proteinRecombinant MUC1-MBP fusion protein and MBP protein were purified with amylose affinity chromatography,and identified by SDS-PAGE.We got purified MUC1-MBP and MBP proteins.4.Preventive effect of MUC1-MBP and MBP protein on human breast cancerWe investigated effects of MUC1-MBP and MBP protein on human breast cancer using this breast cancer model.MUC1-MBP and MBP protein were subcutaneous injected into ICR mice for three times once a week.To monitor MUC1-specific immunity we determined anti-MUC1 antibody titers in serum from immunized mice by ELISA.When MUC1-specific antibody titers rose to 1/8000,we began to make the tumor model.MCF-7 breast cancer cells were inoculated s.c.into mice after mouse immune functions were suppressed according to the above described.We observed the tumor growth and the results showed that mean tumor sizes in MUC1-MBP,MBP and PBS groups were 17.2±18.0 mm~3,22.3±21.5 mm~3 and 91.9±56.3 mm~3 respectively. Tumors were not found in two mice of MUC1-MBP group and one mouse in MBP group but occured in all the mice of PBS group.It suggests that both MUC 1-MBP and MBP have preventive effects on human breast cancer and MUC1-MBP anticancer activity is more significant,compared with MBP.5.Rejection of MUC1-MBP and MBP proteins to human breast cancerFirstly,we established tumor model of human breast cancer in mice in according to the above described,and then divided the mice into three groups and inoculated MUC1-MBP,MBP,PBS s.c.for twice at 1-week interval and seven days after the second immunization,the mice were sacrificed.The results showed that the mean tumor sizes of MUC1-MBP,MBP and PBS groups were 32.6±32.3 mm~3,56.9±56.7 mm~3,101.7±73.6 mm~3 respectively.It suggests that both MUC1-MBP and MBP can reject human breast cancer and MUC1-MBP anti-cancer activity is more significant, compared with MBP group.In this study,we established a subcutaneous transplantation tumor model of human breast cancer in mice which is more economic,convenient and higher tumor incidence.We can study anticancer activites of MUC1-MBP fusion protein using this model.Our data demonstrated this human breast cancer animal model is a useful tool for cancer immunotherapeutic research.