Objective The study was designed to test the hypothesis that halofuginone enhances treatment by improve bad tumor microenvironment caused by radiation,and try to explore the role of TGF-β in this progress.Methods Lewis cells(2×106)were were injected s.c.in the right hind limb of C57BL/6 mice,tumor-bearing mice were randomly divided into five groups.Control group(NC),without any treatment; Halofuginone group(HF),HF was injected intraperitoneally from day 6 to day 15;Radiotherapy group(RT),Radiation was delivered using the clinical linear accelerator(6MV photons; Elekta Synergy linear accelerator),10Gy×2 was delivered on day 8 to day 9; Radiotherapy combine Halofuginone group(RT+HF),halofuginone was administered as in the halofuginone group,and radiation was delivered as in the irradiation group; Radiotherapy combine TGF-β inhibitor(SB431542)group(RT+SB431542),SB431542 was injected intraperitoneally from day 6 to day 15,and radiation was delivered as in the irradiation group.3 mice in each group were euthanized on day 15.Tumors were harvested and divided into two parts,one part were analyzed for tumor-infiltrating regulatory T cells(Treg),TAMs(tumor accosiated marcophages),CD8 and MDSCs via flow cytometry,and another was used to test the change of TGF-β signal pathway by Elisa,Immunohistochemistry,and Western blotting.Spleens were also harvested for analysis of the proportion of Tregs.The remaining mice were followed for tumor growth.Results he FACS results show that mice in RT group had a statistically significant increase in Tregs both in tumor and spleen than in NC group(P<0.05),and the proportion of Tregs in RT+HF group and in RT+SB431542 group significantly decreased than in RT group(P<0.05),the proportion of Tregs in NC group and in HF group had no significant difference(P>0.05).Elisa results show that the level of TGF-β 1 in tumor increased after irradiation(P=0.037),and it can be inhibited by HF or TGF-β inhibitor SB431542 as low level of TGF-β 1 was detected in RT+HF group and in RT+SB431542 group(P<0.05),but there is no different between RT+HF group and RT+SB431542 group(P=0.733).The expression of TGF-β 1 in each group tested by Immunohistochemistry showed similar trend as tested by Elisa.The results of Western Blot show that the TGF-β/smad signaling was activated by radiotherapy,and this effect was reversed by HF or TGF-β inhibitor SB431542 as up-regulated expressions of p-Smad 2 and down-regulated of smad 6 was observed in RT group while reduced expressions of p-Smad 2 and enhanced smad 6 showed in RT+HF group and RT+SB431542 group.The followed results demonstrate mice treated with HF and those without treatment had similar growth kinetics and survival,but mice treated with radiotherapy began showing significant tumor growth delay at 5th day after irradiation and exhibit favorable survival,and whats more,promoted tumor growth delay and prolonged survival was observed in RT+HF group and RT+SB431542 group.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 cant 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-βinhibitor,the proportion is elevated.The subtypes expression of TAMs within tumors were different.The M1 subtype macrophage and the ratio of M1/M2 in NC group was significantly decreased than HF group while the M2 subtype macrophage had no difference between the two groups.Conclusions Generally speaking,our study demonstrated that Halofuginone combine with Irradiation synergistically has greater effect than single treatment.Moreover,We have initially explored the halofulginone changes the immunosuppression caused by radiotherapy.In addition,The antitumor effects of halofuginone could contributed to its inhibitory effects on the TGF-β signaling pathway.