锰佐剂通过重塑免疫微环境促进放疗的抗肿瘤作用

Manganese promotes anti-tumor effect of radiotherapy through reshaping the immune microenvironment

目的探索锰佐剂在放疗抗肿瘤作用中的增敏效应及其可能机制。方法选取6~8周龄的雄性C57BL/6小鼠(体质量为20~23 g)300只,通过皮下接种肿瘤细胞构建动物模型,按随机数字表法分为4组:对照组、放疗组、锰佐剂治疗组、放疗与锰佐剂联合治疗组;荷瘤第9天起每隔1 d通过滴鼻给予锰佐剂(10μg/只)治疗,共4次;第10天给予肿瘤局部单次20 Gy放疗,定期监测小鼠的肿瘤生长及生存状况。通过构建单侧皮下荷瘤模型明确锰佐剂对放疗抗肿瘤作用的增敏效应;构建双侧皮下荷瘤模型观察锰佐剂对放疗诱导的远端效应;采用流式细胞术检测单侧皮下荷瘤模型(MC38-OVA)肿瘤浸润免疫细胞的改变,采用免疫组化染色检测单侧皮下荷瘤模型小鼠的脾脏功能。结果基于多瘤种单侧皮下荷瘤模型重复验证,显示锰佐剂联合放疗可显著抑制肿瘤生长,延长小鼠生存期(P<0.05);双侧皮下荷瘤模型结果显示,锰佐剂可增强放疗引起的远端效应,无论是否进行放疗,两侧肿瘤均出现明显缩退(P<0.05);流式细胞术检测发现,锰佐剂可进一步增加放疗诱导的CD8^(+)T细胞浸润(P<0.05),且可降低放疗引起的Treg细胞和MDSC细胞浸润(P<0.05);小鼠脾脏的病理观测结果显示锰佐剂可增加脾脏的淋巴细胞储备,改善脾脏功能。结论锰佐剂可作为放疗的增敏剂,通过改善脾脏功能重塑肿瘤微环境协同抗肿瘤。

Objective To explore the sensitizing effect of manganese for radiotherapy against tumors and its possible mechanisms.Methods A total of 300 male C57BL/6 mice(6~8 weeks old,weighing 20~23 g)with subcutaneous tumor were randomly divided into 4 groups:control group,radiotherapy group,manganese treatment group,and combined radiotherapy and manganese treatment group.Nasal drip with 10μg manganese adjuvant was applied to the mice from the latter 2 groups on day 9 of tumor bearing,then single dose of 20 Gy radiation was locally administered on day 10.Tumor growth and mouse survival were monitored regularly.The sensitizing effect of manganese on radiotherapy was determined by monitoring and comparing the tumor growth among different unilateral mouse models.Bilateral tumor-bearing model was used to examine the effect of manganese on abscopal effects induced by radiotherapy.Flow cytometry was used to illustrate the changes in tumor-infiltrating immune cells in unilateral tumor bearing model.Immunohistochemical staining was employed to evaluate the spleen function in unilateral tumor bearing mice.Results Based on repeated validation of 3 different unilateral tumor-bearing models,radiotherapy combined with manganese therapy significantly inhibited tumor growth and prolonged survival of mice(P<0.05).The results of bilateral tumor-bearing model showed that manganese therapy enhanced abscopal effects of radiotherapy,and significant regression was observed in both side of tumor under radiotherapy or not(P<0.05).Flow cytometry revealed that manganese further increased radiation-induced CD8^(+)T infiltration(P<0.05)and decreased radiation-induced infiltration of Treg cells and myeloid-derived suppressor cells(MDSCs)(P<0.05).Furthermore,manganese increased lymphocyte reserve pool of the spleen and improved its function.Conclusion Manganese adjuvant could act as a sensitizing agent for radiotherapy,by improving the function of spleen and reprogramming the tumor microenvironment synergistically.