KRAS抑制剂AMG510联合放疗激活肺癌免疫动物模型研究

KRAS inhibitor AMG510 combined with radiotherapy activates lung cancer immunity in an animal model

目的探讨AMG510与放疗联合能否通过提高抗肿瘤免疫增加疗效。方法体外使用细胞计数试剂盒8(CCK8)检测小鼠Lewis肺癌细胞系(LLC)对AMG510的敏感性,并对LLC进行RAS基因测序。使用C57BL/6小鼠构建移植瘤模型,随机分为4组【对照组、放疗(RT)组、AMG510组和AMG510+RT组】,分别接受AMG510和(或)放疗的治疗方式,观察抑制肿瘤生长的情况。通过流式细胞仪及免疫组化的方法检测肿瘤浸润T淋巴细胞的情况。对治疗后的肿瘤进行基因表达测序,使用热图及信号通路富集等方法分析免疫相关基因表达变化。组间比较采用单因素方差分析或非参数检验中的中位数检验。结果LLC细胞同时具有KRASG12C和NRASQ61H位点突变。CCK8结果显示,AMG510单药对LLC生长抑制至63%。体内实验结果显示,AMG510单药控制肿瘤生长作用有限,但联合放疗后肿瘤生长体积受到明显控制,优于任何单一治疗(均P<0.05)。流式细胞术结果显示,AMG510十RT组的肿瘤浸润CD3^(+)T、CD4^(+)T和CD8^(+)T细胞的比例为3.29%(2.81%,6.44%)、1.04%(0.72%,2.43%)和2.16%(0.93%,4.19%),相较于其他3组浸润增加,均P<0.05;免疫组化结果显示趋势与流式细胞术一致。免疫组化结果显示,AMG510^(+)RT组与对照组和RT组相比,能够增加干扰素(IFN)-^(+)T细胞的浸润(均P=0.009),但调节性T细胞(Treg)及巨噬细胞浸润数量4组间差异无统计学意义,x^(2)值分别为2.22和0.50,P值分别为0.528和0.918。基因测序分析显示,AMG510联合放疗对比对照组差异基因数据最多,包括399个上调和46个下调的基因,免疫相关基因表达上调。AMG510联合放疗及AMG510单药均能够增强趋化因子信号通路及T细胞受体信号通路。结论AMG510与放疗联合能够显著抑制KRASG12C突变肺癌生长,提高T细胞相关的肿瘤免疫,具有一定转化医学意义。

Objective To explore whether the combination of AMG510 and radiotherapy can enhance the therapeutic effect by enhancing anti-tumor immunity.Methods The sensitivity of the mouse Lewis lung cancer cell line(LLC)to AMG510 was detected using cell count kit 8(CCK8)in vitro,and the RAS gene sequencing was performed.Constructed a transplant tumor model using C57BL/6 mice and randomly divided them into 4 groups[control group,radiotherapy(RT)group,AMG510 group,and AMG510+RT group].They received treatment with AMG510 and/or radiotherapy respectively,and observed the inhibition of tumor growth.Detected the infiltration of T lymphocytes in tumors using flow cytometry and immunohistochemistry methods.Performed gene expression sequencing on the treated tumor and analyzed changes in immune related gene expression using methods such as heatmaps and signal pathway enrichment.The median test in one-way ANOVA or non parametric tests was used for inter group comparison.Results LLC cells had both KRAS G12C and NRAS Q61H mutations,and CCK8 showed that AMG510 alone inhibited the growth of LLC by 63%.In vivo experiments showed that AMG510 alone had limited effect on tumor growth,but the tumor growth volume was significantly controlled after combined radiotherapy,which was significantly better than any single therapy(all P<O.05).Flow cytometry results showed that the proportion of tumor infiltrated CD3^(+)T,CD4^(+)T and CD8^(+)T cells in the AMG510 combined with radiotherapy group was 3.29%(2.81%,6.44%),1.04%(0.72%,2.43%)and 2.16%(0.93%,4.19%),which was significantly increased compared with the other three groups(all P<o.05),and the above results were consistent with the immunohistochemical results.In addition,immunohistochemical results showed that AMG510 combined with radiotherapy could increase the infiltration of interferon(IFN)-γ^(+)T cells compared with control group and radiotherapy group(both P=0.009),but there was no statistical significance in the number of regulatory T cells(Treg)and macrophage infiltration between the four groups,the x values were 2.22 and 0.50,respectively,and the P values were 0.528 and 0.918,respectively.Gene sequencing analysis showed that AMG510 combined with radiotherapy compared with the control group had the most differential gene data,including 399 up-regulated and 46 down-regulated genes,and the expression of immune-related genes was up-regulated.AMG510 combined with radiotherapy and AMG510 enhanced chemokine and T cell receptor signaling pathways.Conclusion The combination of AMG510 and radiotherapy can significantly inhibit the growth of KRAS G12C mutant lung cancer and improve the anti-tumor immune function of T cells,which has translational medicine significance.