Experimental Study Of IL-15and MTOR Inhibitor In The Treatment Of Breast Cancer Of Mice

Objective:To investigate the effects of everolimus and interleukin-15(IL-15) on the treatment of breast cancer in mice independently or in combination.To determine the growth of tumor, the animal survival, tumor metastasis, the immune response and the tumor cell apoptosis. To explore the mechanism underlining the therepeutic effects and to provide a theoretical basis for the treatment of breast cancer.Methods:Plasmids pHi2-spIL15-CMV-TAT (L3) and pHi2-EGFP-CMV-TAT (L6) were tran-sfected into4T1mouse breast cancer cells. The transfection efficiency was an-alyzed by the FCM and fluorescene microscope. IL-15expression24and48hours after cells transfection was determined by ELISA, to confirm the efficien-t transgene expression in transfected4T1cells.Everolimus and IL-15were applied for the treatment of breast cancer in mice. In order to assess the effect of different treatments on mice, tumor size, metastasis, and animal survival were monitored, Ki-67, CD4, CD8and NKG2D expression in tumor tissue were determined. The flow cytometry was also used to analyze the CD4+,CD8+T cells subsets and CD4+, CD25+regulatory T cells and of CD16+CD32+cells in peripheral bolld and spleen of mice. Cell apoptosis in tumor tissue were detected using Tunel assay. To explore the mechanism of IL-15and mTOR inhibitors on the treatment of breast cancer.Results:1. Compared to the control group, the tumor size of everolimus treatment group and IL-15treatment group were significantly reduced, the differences w-ere statistically significant (P<0.05);compared to IL-15treatment group and everolimus treatment group, tumor size of the combination group was also red-uced, but the difference was not statistically significant(P>0.05). Although bot h of the two treatments reduced the tumor volume, the difference between the everolimus group and IL-15group was not statistically significant (P>0.05). 2.Compared with combination plus sequential therapy, the tumor size of continuous treatment of everolimus were smaller in the first20days, the tumo-r size of the sequential therapy were smaller than the immunotherapy group i-n the first20days, but in the long-term there was no significant difference; t-here was no difference of the tumor growth trend between immunotherapy gro-up and the combination plus sequential therapy group;there was also no signif-icant difference between the combination therapy group and the immunotherap-y or the evrolimus group.3. Both everolimus and IL-15effectively prolonged the survival of tumor-baring mice. Compared with the control group, the differences was not statistic-ally significant (P>0.05). The difference between the two treatment groups w-as not statistically significant (P>0.05).Compared with the control group, co-mbination treatment slightly extended the survival time of animal, but the survi-val time was relatively short compared with the everolimus and IL-15treatme-nt group, while the difference was not statistically significant (P>0.05).4. CD4、NKG2D expression of tumor tissue in IL-15treatment group wer-e higher than both everolimus treatment group and the control, the expression of CD8was higher than the everolimus treatment group, the expression of Ki-67is higher than the control group, and the difference was statistically sig-nificant (P<0.05); the expression of CD4, CD8, of NKG2D and Ki-67expres-sion of everolimus treatment group were lower than the control group, and th e difference was statistically significant (P<0.05); but CD4, CD8and NKG2D expression compared with IL-15treatment group were decreased, and the diff-erence was statistically significant (P<0.05); Ki-67expression compared with IL-15treatment group was slightly low, but the difference was not statisticall-y significant (P>0.05):the expression of CD4of the combination group is hi-gher than the control group and the expression of CD8is lower than control group and IL-15treatment group, the expression of Ki-67was lower than the control group,the expression of NKG2D was higher than the control and the everolimus group, while lower than the IL-15treatment group, the differences were statistically significant (P<0.05). 5. IL-15significantly increased the CD4+、CD8+T cell ratio and reduce the CD25+regulatory T cell ratio in peripheral blood lymphocytes, the differen-ce was statistically significant (P<0.05) compared with the control and the ev-erolimus group, it also increased the CD16+T cell ratio, the difference was sta-tistically significant (P<0.05) compared with the other groups; while, everoli-mus reduced the CD4+, CD8+and CD16+T cell ratio and increase the CD25+cell ratio, but the difference was not stastistically significan (P>0.05) compare-d with the control.6. IL-15significantly increased the CD4+、CD8+T cell ratio of the spleen cell, reduced the CD25+regulatory T cell ratio, the difference was statistically significant (P<0.05) compared with the control and the everolimus group; ever-olimus reduced the CD4+、CD8+and CD16+T cell ratio and increased the CD25+cell ratio,but the difference was not stastistically significan (P>0.05) co-mpared with the control.7. IL-15significantly increased the CD16+T cell ratio of the lymphocyte of the liver, the difference was statistically significant (P<0.05) compared with the control and the everolimus group. It also increased the CD4+、CD8+T cell ratio, reduced the CD25+cell ratio, the difference was not stastistically sig-nifican (P>0.05) compared with the control; everolimus reduced the CD4+CD8+and CD16+T cell ratio and increase the CD25+cell ratio, but the differ-ence was not stastistically significan (P>0.05) compared with the control.8.Compared with the control,the number of apoptotic cell of the IL-15an-d the everolimus groups are significantly increasing, the difference was statistic ally significant (P<0.05).Conclusion:IL-15plays anti-tumor activity by enhancing the activity of immune cells, to inhibit the growth of tumor cell and prolong the survial of the animal. mTOR inhibitor everolimus has a direct inhibitory effect on tumor growth, promotes apoptosis, prolongs survival time of mouse. The mechanism and therapeutic target of IL-15and everolimus are different. In this study, the combination drug therapy does not produce a synergistic effect, and does not enhance the antitumor effect. The reason may be that the mTOR inhibitor also inhibits the immune systerm directly. The study provieded the experimental basis for the treatment of breast cancer in the future.