抗PD-1单抗联合顺铂的新辅助治疗策略对NSCLC人源化小鼠模型肿瘤复发与转移的影响

Neoadjuvant treatment strategy by anti-PD-1 immunotherapy combined with cisplatin inhibits recurrence and metastasis of NSCLC in a humanized mouse model

目的利用人源化小鼠模型探讨抗PD-1单抗联合顺铂的新辅助治疗策略对人非小细胞肺癌(nonsmall-cell lung cancer,NSCLC)术后复发与转移的影响。方法选择重度联合免疫缺陷小鼠NCG,通过序贯移植人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)和人非小细胞肺癌细胞的方法,构建免疫系统-肿瘤双人源化NSCLC小鼠移植模型;评估不同新辅助治疗策略的疗效,包括顺铂治疗、抗PD-1单抗(帕博利珠单抗)治疗以及联合治疗;通过小动物活体成像观察各治疗组小鼠术后肿瘤复发与转移情况;通过免疫组化分析肿瘤组织中Ki67的表达,观察肿瘤细胞增殖情况;利用免疫荧光分析肿瘤免疫微环境的变化。结果成功构建了免疫系统-肿瘤双人源化NSCLC小鼠模型,重建小鼠外周血、各脏器以及肿瘤组织中均有人免疫细胞的浸润;活体成像结果显示新辅助抗PD-1单抗治疗能抑制肿瘤的复发及转移,联合治疗组效果最为显著;免疫组化结果显示联合治疗组中小鼠肿瘤组织Ki67表达明显降低;免疫荧光结果表明帕博利珠单抗治疗可增加肿瘤组织中人CD45^(+)免疫细胞、CD8^(+)免疫细胞的浸润,联合治疗后CD45^(+)免疫细胞、CD8^(+)T细胞的浸润更为明显;此外,颗粒酶B(granzyme B,GzmB)在CD8^(+)T细胞杀伤肿瘤中发挥重要作用,免疫组织化学染色结果显示帕博利珠单抗治疗可增加肿瘤组织中granzyme B的浸润,联合治疗后granzyme B阳性细胞的浸润更为明显。结论新辅助帕博利珠单抗联合顺铂有效提高了人源化小鼠免疫系统杀伤NSCLC的能力,显著抑制了NSCLC的复发和转移。

Objective To investigate the effect of a neoadjuvant treatment strategy of an anti-PD-1 monoclonal antibody combined with cisplatin on postoperative recurrence and metastasis of non-small cell lung cancer(NSCLC)in a humanized mouse model.Methods Human peripheral blood mononuclear cells and human NSCLC cells were sequentially transplanted into the severe combined immunodeficiency mouse(NOD/ShiLtJGpt-Prkdc^(em26Cd52)Il2rg^(em26Cd22)/Gpt,NCG)to establish an immune system-tumor double humanized NSCLC model.We elevated the treatment effect of various neoadjuvant strategies in the double humanized NSCLC model,including cisplatin,anti-PD-1(pembrolizumab),and combined treatments.Tumor recurrence and metastasis were observed by optical imaging.Ki67 expression in tumor tissue was analyzed by immunohistochemistry to assess tumor cell proliferation.Changes in the tumor immune microenvironment were detected by immunofluorescence.Results The double humanized NSCLC model was successfully established.Immune cells had infiltrated into peripheral blood,organs,and tumors.Optical imaging showed that neoadjuvant anti-PD-1 treatment inhibited tumor recurrence and metastasis,and the effect of combined treatment was more obvious.Moreover,combination treatment significantly decreased Ki67 expression in tumor tissues.Large numbers of CD8^(+)and CD45^(+)cells in tumors were observed after anti-PD-1 treatment,and many more CD8^(+)and CD45^(+)T cells were found in the combined therapy group.Granzyme B plays an important role in tumor killing by CD8^(+)T cells.Immunohistochemical staining showed that anti-PD-1 treatment increased granzyme B in tumor tissues,which was more obvious after combined treatment.Conclusions Neoadjuvant pembrolizumab combined with cisplatin effectively improves the anti-tumor effect in humanized mice and significantly inhibits tumor recurrence and metastasis.