| Background As an alternative treatment for prostate cancer, cryoablation has achieved a certain curative effect and has been widely used in the control of the disease. In previously clinical work, we found that there are some patients pulmonary metastases regressed spontaneously after using cryoablation for prostate cancer. Subsequent studies have found that a cryo-immunologic response may participate in this process. Bystander effect is derived from gene therapy, and was defined as an action at a distance from the treatment area. Bystander effect has been reported in gene therapy, radiation therapy and antitumor drugs. In recent years, bystander effect has been widely reported in animal models of gene therapy and radiation therapy. The possible mechanism is as follows: local treatment can induce the body immune inflammatory reaction, that lead to the damage of untreatment cells through compound oxidative stress, ROS, NO and inflammation related signaling pathways, or through intracellular cells release dangerous signal may induce the body's ability to identify tumor, thus affecting the body's immune microenvironment and stimulate the body's anti-tumor immune response, achieve the tumor cell killing effect of the distance area. Accordingly, this study was to verify whether cryoablation can induce the bystander effect of prostate cancer, and to investigate whether the immune inflammatory mechanisms involved in the bystander effect, in order to provide new treatments for malignant tumors and further provide theoretical and experimental basis of tumor therapy.ObjectivesBilateral inguinal transplantation tumor model of prostate cancer in mice model is constructed, then the experimental treatment for unilateral transplantation tumor, survival of mice, pulmonary metastasis and the contralateral transplantation tumor growth change was observed. From the animal model of to test whether cryoablation can induce bystander effect, and observe dynamically the process of the changes of immune cells and inflammatory signal(Hsp70), and give a preliminary interpretation of the relevant mechanism of bystander effect after cryoablation.Materials and Methods1. To establish bilateral RM-1 prostate cancer xenograft mice model. Randomly divided into 3 groups: control group, operation group and cryoablation groups. The right side of the transplanted tumor was applying the treatment with sham-operation,operation and cryoablation respectively, and the left transplanted tumor without any treatment. To observe the changes of the growth of the left transplanted tumor and record the survival of the mice of each group. 2. After 7 days and 14 days, the mice were sacrificed, and the peripheral blood, spleen, lung tissue and tumor tissue samples was reserved. HE staining was used to observe the lung metastasis. The number and proportion of CD4+T, CD8+T and NK cells were detected by flow cytometry, and the percentage changes of Th1/Th2 ratio was also analyzed. Hsp70 changes of serum were detected by ELISA in each group. The opposite side tumor tissue immunohistochemistry observe the proliferation changes(Ki67) of tumor cells. Hsp70 expression in tumor tissues was detected by Western blotting.Results 1. Compared to Group A(Control group) and Group B(Surgery group), the diameter of opposite side tumor in the group C(Cryoablation group) decreased significantly(P<0.05,P<0.05) on day 7 and day 14. Group B(Surgery group) compared with Group A(Control group), there was no statistical difference(P>0.05) on day 7, the diameter of opposite side tumor of group A(Control group) was greater than group B(Surgery group)(P<0.05) on day 14. 2. Compared to Group A(Control group) and Group B(Surgery group), the median overall survival(OS) of Group C(Cryoablation group) significantly prolonged(P < 0.001). Group A(Surgery group) compared with Group B(Control group), there was no statistical difference(P>0.05). 3. As time goes on, lung metastasis rate showed an increasing trend in each group, there was no statistical difference on day 7(P=0.562). However, compared to Group A(Control group) and Group B(Surgery group), lung metastasis rate in the Group C(Cryoablation group) decreased significantly(P<0.05,P<0.05) on day 14. 4. As time goes on, the percent of CD4+ T cells of Group C(Cryoablation group) had raised trend. While that of Group A(Control group) and Group B(Surgery group) had declined trend. Compared to Group A(Control group) and Group B(Surgery group), the percent of CD4+ T cells of Group C(Cryoablation group) increased significantly(P<0.05,P<0.05) on day 7 and day 14. 5. As time goes on, the percent of CD8+ T cells of Group C(Cryoablation group) and Group B(Surgery group) had raised trend, While Group A(Control group) had declined trend. Compared to Group A(Control group) and Group B(Surgery group),the percent of CD8+ T cells of Group C(Cryoablation group) significantly increased(P<0.05,P<0.05) on day 7 and day 14. 6. As time goes on, the percent of NK cells of all the three groups had the same trend which declined at first, then raised. Compared to Group B(Surgery group), the percent of NK cells in the Group A(Control group) and Group C(Cryoablation group) increased slightly(P=0.002, P<0.001) on day 7. On day 14, the percent of NK cells in Group C(Cryoablation group) increased significantly comparing to Group A(Control group) and Group B(Surgery group)(P=0.003, P=0.001). 7. As time goes on, the ratio of Th1/Th2 of Group C(Cryoablation group) had raised trend, Group A(Control group) and Group B(Surgery group) had declined trend. Compared to Group A(Control group) and Group B(Surgery group), the ratio of Th1/Th2 of Group C(Cryoablation group) significantly increased(P<0.05, P<0.05) on day 7 and day 14. 8. As time goes on, the killing activity of CTL cells of Group C(Cryoablation group) had raised trend, Whiled Group A(Control group) and Group B(Surgery group) had declined trend. Compared to Group A(Control group) and Group B(Surgery group), the killing activity of CTL cells of Group C(Cryoablation group) significantly increased(P<0.001,P<0.001) on day 7 and day 14. 9. As time goes on, the proliferation activity(Ki67) of opposite side tumor tissue of Group C(Cryoablation group) had decline trend at first, then raised trend. Group A(Control group) and Group B(Surgery group) had raised trend at first, then declined trend. Compared to Group A(Control group) and Group B(Surgery group), the proliferation activity(Ki67) of opposite side tumor tissue of Group C(Cryoablation group) decreased significantly(P<0.05,P<0.05) on day 7 and day 14. Compared to Group A(Control group) and Group B(Surgery group), and there was no significant change in the other two groups(P>0.05). 10. As time goes on, the Hsp70 changes of serum of all each group had the same trend which raised at first, then declined. Compared to Group A(Control group) and Group B(Surgery group), the Hsp70 changes of serum of group C(Cryoablation group) increased significantly(P<0.01,P<0.01) on day 7 and day 14. There was no significant change in the other two groups(P>0.05). 11. As time goes on, the expression of Hsp70 of Group C(Cryoablation Group) in the cytoplasm of tumor cells had down regulated trend, while Group A(Control group)and Group B(Surgery group) had up regulated trend. Compared to Group A(Control group) and Group B(Surgery group), the expression of Hsp70 of Group C(Cryoablation Group) in the cytoplasm of tumor cells of group C(Cryoablation group) significantly decrease trend on the 7th day and 14 th day.Conclusion1. Cryoablation can retard the rate of opposite side tumor growth, reduce the the incidence of lung metastasis in mice and significantly prolong the survival time of mice. It is suggested that cryoablation for prostate cancer can induce the bystander effect. 2. Cryoablation induced immune response by CD4+ Th1 cell, enhanced the cytotoxic activity of CD8+ T cell and resulted in the inhibition of opposite tumor. 3. Elevated serum Hsp70 after cryoablation may involve in the cryo-immune response. |
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