放疗联合抗CEACAM1免疫疗法治疗小鼠原位胶质瘤的实验研究

Experimental study of radiotherapy combined with anti-CEACAM1 immunotherapy in the treatment of glioma in situ in mice

目的观察放疗与单克隆抗体封闭免疫检查点癌胚抗原相关细胞黏附分子(CEACAM1)联合治疗对小鼠原位胶质瘤的抑制作用及小鼠免疫应答的影响。方法收集2019年3—12月于山西医科大学第一医院神经外科行手术治疗并于1个月内进行放疗的原发脑胶质瘤患者。分别检测放疗前、后脑胶质瘤患者外周血中T淋巴细胞表面CEACAM1的表达;将GL261胶质瘤细胞(稳定转染荧光素酶基因)种植于C57BL/6小鼠右侧尾状核,并采用小动物活体成像系统观察肿瘤的生长情况,同时将小鼠分为同型对照组、放疗组、抗CEACAM1组和联合治疗组(各组n=5);观察小鼠治疗后的生存情况;免疫组织化学检测放疗前、后小鼠脑胶质瘤上CEACAM1的表达水平;流式细胞术检测小鼠脑浸润T淋巴细胞中CD4^(+)、CD8^(+)及调节性T细胞(Tregs)的表达水平;酶联免疫吸附试验(ELISA)检测小鼠血清干扰素γ(IFN-γ)和白细胞介素10(IL-10)的表达水平。结果与放疗前相比,放疗后1周脑胶质瘤患者的外周血中CD4^(+)T淋巴细胞表面CEACAM1的阳性细胞比率明显升高(P<0.05),放疗后1个月其比率进一步升高(P<0.05);放疗后1周CD8^(+)T淋巴细胞表面CEACAM1的阳性细胞比率的差异无统计学意义(P>0.05),放疗后1个月其比率明显升高(P<0.05)。联合治疗组能明显抑制小鼠脑胶质瘤的生长,可延长其生存期,且该组部分小鼠(2/5)的生存期>90 d(P<0.05)。免疫组织化学检测结果显示,与放疗前相比,放疗后1周小鼠CEACAM1的表达差异无统计学意义(P>0.05),但与放疗前及放疗后1周相比,放疗后1个月其表达水平均显著升高(均P<0.05)。流式细胞术检测结果显示,与同型对照组相比,联合治疗组小鼠脑内浸润CD8^(+)T淋巴细胞比率升高,Treg细胞比率减少,CD8^(+)T/Treg比值升高(均P<0.05)。ELISA检测结果显示,与同型对照组相比,联合治疗组小鼠外周血中IFN-γ的表达水平升高,而IL-10的表达水平降低(均P<0.05)。结论放疗联合CEACAM1抑制剂的治疗方法可以对小鼠脑胶质瘤产生强大而持久的抗肿瘤免疫反应,并可延长部分小鼠的生存期。

Objective To observe the inhibitory effect of radiotherapy and monoclonal antibody-targeted blocking immune checkpoint molecule CEACAM1(carcinoembryonic antigen-associated cell adhesion molecule)on glioma in situ in mice and its effect on immune response in mice.Methods We collected the clinical data of primary glioma patients who consecutively underwent operation and radiotherapy within 1 month in the Department of Neurosurgery of the First Hospital of Shanxi Medical University from March to December 2019.The expression of CEACAM1 on the surface of T lymphocytes in the peripheral blood of glioma patients before and after radiotherapy was detected respectively.GL261 glioma cells(stably transfected with luciferase gene)were implanted in the right caudate nucleus of the brain of C57BL/6 mice,and a small animal in vivo imaging system was used to observe the growth of the tumor.At the same time,the mice were divided into the isotype control group,the radiotherapy group,the anti-CEACAM1 group and the combined treatment group(n=5 in each group);the survival of the mice after treatment was observed.Immunohistochemical test was performed to detect the expression level of CEACAM1 in mouse brain gliomas before and after radiotherapy.Flow cytometry was performed to detect the expression levels of CD4+,CD8+and regulatory T cells(Tregs)in mouse brain infiltrating T lymphocytes.The expression levels of serum interferonγ(IFN-γ)and interleukin 10(IL-10)in mice were detected by enzyme-linked immunosorbent assay(ELISA).Results Compared with before radiotherapy,the expression level of CEACAM1 on the surface of CD4+T lymphocytes in the peripheral blood of glioma patients was significantly increased at 1 week after radiotherapy(P<0.05),and the expression level was further increased at 1 month after radiotherapy(P<0.05).There was no significant change in the expression level of CEACAM1 on the surface of CD8+T lymphocytes 1 week after radiotherapy(P>0.05),and its expression level increased 1 month after radiotherapy(P<0.05).The combined treatment group could significantly inhibit the growth of gliomas in mice and prolong their survival period,and the survival period of some mice(2/5)in this group was more than 90 days(P<0.05).The results of immunohistochemistry showed that compared with before radiotherapy,there was no significant difference in the expression of CEACAM1 in mice 1 week after radiotherapy(P>0.05),but compared with before radiotherapy and 1 week after radiotherapy,the expression level of CEACAM1 increased significantly at 1 month after radiotherapy.high(both P<0.05).The results of flow cytometry showed that compared with the control group of the same type,the ratio of infiltrating CD8+T lymphocytes in the brain of the mice in the combined treatment group increased,the ratio of Treg cells decreased,and the ratio of CD8+T/Treg increased(all P<0.05).The ELISA results showed that the expression level of IFN-γin the peripheral blood of mice in the combined treatment group increased,while the expression level of IL-10 decreased(both P<0.05).Conclusion Radiotherapy combined with CEACAM1 inhibitor therapy can generate a strong and durable anti-tumor immune response against gliomas in mice,and prolong the survival of some mice.