The Role Of Radiotherapy Combined With Halofuginon In TAMs Of Tumor-bearing Mice

Objective The objective of this study was to research the influence of combination of radiotherapy with halofuginone to the tumor-associated macrophages(TAMs) within tumor microenvironment or mice spleen of tumor-bearing mice. And on this basis, to explore the effects of radiotherapy to body immunity, in which TAMs involved.Methods We first establish a subcutaneous transplanted cancer model in C57BL/6J mice,which were identified Specific Pathogen Free(SPF). We injected Lewis lung cancer cells into right hind limb of healthy femal mice. The mice were experienced local radiotherapy or medication after the transplantation tumor mass reaches 5mm, about at the sixth day after inoculated. The second step was to select 25 mice, which had the similar tumor burden, and then they were randomly assigned to five groups. They are negtive control(NC) group, which means without any treatment modality,halofuginone(HF) group, which means halofuginone injection alone, local radiotherapy(RT) group, which means local irradiation, and two combined group,one is RT combined HF, and the other is RT combined SB431542, a selective inhibitor of transform growth factor beta(TGF-?). The HF group and RT combined HF group mice were injected halofuginone by intraperitoneal administered in a 2ug/0.1ml/d dose between the sixth day and two weeks. The RT combined SB431542 group were intraperitoneal administered with SB431542 in a dose of 0.1ml/d at the same time. The two combined groups and the RT group were administered radiotherapy and the radiation was delivered using 6MV photons(Elekta Synergy linear). The total dose was 20 Gy in two days after medication. After two weeks, these experimental mice were took off the neck to death from the humanitarian considerations and the study requirenments. The third step was to evaluate the TAMs within tumor microenvironment and spleens. Tumors and spleens were made into single cell suspension to be analyzed by flow cytometry analysis(FCA) to test the expression of TAMs subtypes within tumors and spleens. The other tumors and spleens were made into frozen section and examined with direct immunofluorescece. The immunofluorescece was to check the functional expression of TAMs subtypes.Results 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. The percentage of M1 and M2 subtypes and the ratio of M1/M2 had no difference between the NC group and RT group, while the M1 percentage had a downward trend and M2 subtype had a uptrend in RT group. The RT combined HF group had no significant difference compared to NC group and HF group in the percentage of M1, M2 and the radio of M1/M2. But compared to RT group, the RT combined HF group had a uptrend in M1 percentage and a downtrend in M2 percentage, and the results was a uptrend of the ratio of M1/M2. The percentage of M1 and the ratio of M1/M2 in HF group had a significant improvement than the NC group and RT group, while there had no difference between the M2 percentage. The M2 subtype percentage in the RT combined SB431542 group had a uptrend compared to RT group and RT combined HF group. Also, the ratio of M1/M2 had a significant decrease than HF group(p=0.01). From the above results, we can get the priliminary result was that the application of HF could improve the differentiation of TAMs within the tumor microenvironment, skewing the TAMs differentiated to M1 subtype.RT had the effect of decrease the ratio of M1/M2, making TAMs differentiated into pro-tumorigene M2 subtype. The combined application of RT and HF had a negtive effect compared to the application of HF alone, but still be better than RT alone, that means inhibiting TAMs skewing to M2 subtype effectively. The combination of RT and SB431542 will lead to a increase of M2 subtype percentage than HF group. The immunofluorescece revealed that RT group had a lower expression of i NOS and a higher expression of Arg-1 than NC group, indicating the downtrend of ratio of M1/M2. The expression of i NOS was higher and Arg-1 was lower in RT+HF group than RT group and HF group, indicating RT+HF group made the TAMs differentiated into M1 subtype. In addition to tumor microenvironment, there also had some changes in tumor-bearing mice. We had not found significant difference of M1 and M2 proportion in each group, while the ratio of M1/M2 in HF group is significantly higher than RT+SB431542 group. The expression of M2 biomarker showed decreasing trend in RT group rather than NC group and HF group. The two combined groups showed a increase tendency in the ratio of M1/M2 than RT group and HF group, suggesting radiotherapy combined with HF or SB431542 maybe promote TAMs differentiated into antitumorigion M1 subtype. The functional hallmarker of TAMs in mice spleen was also different. The expression of i NOS in RT group, RT+HF group and RT+SB431542 was higher than NC group and HF group, while the expression of Arg-1 showed a declining trend in the two combining groups. These data indicated that radiotherapy combined with HF or SB431542 will lead to the TAMs skew to differentiate to M1 subtype.Conclusion Radiotherapy will inhibit TAMs differentiated to antineoplastic M1 subtype, which result in a immunosupprressive state within tumor microenvironment. Radiotherapy combined with halofuginone resulted in the down-regulation of pro-tumorigen M2 macrophage, which maybe related to the inhibition of TGF-?. Radiotherapy combined with SB431542 did not improve the local immunosupprression resulted by TAMs,which meant inhibtion of TGF-? alone maybe not an effective method for differentiation of TAMs. Radiotherapy had little influence on TAMs within spleen while combined halofuginone or SB431542 would promote TAMs differentiated to M1 macrophage.