| Objective: This study was undertaken to evaluate the abscopal effect of halofuginone combining with irradiation. And try to explore the possible role of immune microenvironment and TGF-? signaling pathway in this response.Methods: Lewis lung cancer cells(2×10/0.2ml) were injected s.c. in the right hind limb of C57BL/6 mice, mice with tumor were randomly divided into two groups, one is for observation of abscopal effect, the other is used to test the immunologic function. In the abscopal effect group, two days after injection of right hind limb, 2×106/0.2ml Lewis lung cancer cells were injected s.c. in the left hind limb of C57BL/6 mice as the right hind limb. Both groups continued to divide into five treatment groups. Control group(NC), without any treatment; Halofuginone group(HF), halofuginone was injected intraperitoneally of 2ug/ml from day 7 to day 15;Irradiation group(RT), irradiation was delivered with the 6MV linear accelerator of 10Gy×2 from day 8 to day 9; Irradiation combined halofuginone group(RH), halofuginone was administered as in the halofuginone group, and irradiation was delivered as in the irradiation group; Irradiation combined TGF-? inhibitor group(RS), TGF-? inhibitor was injected intraperitoneally from day 7 to day 15, and irradiation was delivered as in the irradiation group. Seven days after the irradiation, tumors and spleen were harvested and analyzed for CD8+T cell via flow cytometry. Ten days after the irradiation, tumors and spleen were harvested and analyzed for myeloid-derived suppressor cells(MDSCs) via flow cytometry. And in the other group of abscopal effect, the tumor diameter was measured every three day, at the same time, the survival time were also observed.Results: After 3 weeks of drug toxicity observation, drug concentration used in this experiment was 2?g/day as the experimental dose. The FACS results show that mice in NC?HF and RT group had no differences in MDSC proportion both in tumor and spleen, and the proportion of MDSC in RH group and in RS group significantly decreased than in RT group, especially the proportion of MDSC in RH group had a statistically decrease compared with RT group(P=0.039) in spleen. In the tumor tissue, it is a pity that we can't explore some results among five treatment groups. However, in the spleen tissue, the proportion of CD8+T cell in HF group and RT group were higher than NC group. And after the administration of halofuginone and TGF-? inhibitor, the proportion is elevated. Tumor volume of both legs in each group had no statistical difference before treatment(P=0.331). For the irradiated right hind limb, after 4 days of irradiation, tumors of RT group?RH group and RS group were significantly smaller than NC group(P=0.016?0.007 and 0.000, respectively) and HF group(P=0.027?0.002 and 0.001, respectively). After 10 days of irradiation, tumor volume of RH group was smaller than RT group(P = 0.046). After 14 days of irradiation, tumor of RS group was smaller than RT group(P=0.017). There is no difference between RH group and RS group during the observation(P>0.05). For the non-irradiated left hind limb, after 7 days of irradiation, tumor of RH group was smaller than HF group(P=0.04). After 10 days of irradiation, tumor of RH group was smaller than NC group(P=0.031) and RT group(P=0.038). At last, we can't measure tumor volume of two mice in RH group. RT group and RS group survival time were longer than NC group and HF group. RH group had the longest survive time among all the groups. In the end of observation, there were two mice alive in RH group, while others were all dead.Conclusion:Halofuginone could induce the abscopal effect of xenograft combining with irradiation and its effect on immune microenvironment and TGF-?/smad signaling may play crucial roles. |
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