PD-1抗体增强奥沙利铂体内外抗结肠癌的效果

PD-1 antibody enhanced anti-tumor efficacy of oxaliplatin against colon cancer in vitro and in vivo

目的:探讨奥沙利铂(oxaliplatin, OXA)联合PD-1抗体对结肠癌的抗肿瘤作用。方法:选用结肠癌细胞系HCT-116和HT-29,用流式细胞术检测细胞中PD-L1的表达。采用T细胞共培养的方法检测OXA预处理HCT-116细胞联合PD-1抗体作用后细胞因子分泌及CD4/CD8亚型变化。建立BALB/c小鼠的结肠癌细胞CT26移植瘤模型,用OXA联合PD-1抗体治疗,评估其抗肿瘤活性;同时采用CD8抗体清除小鼠CD8^+T细胞,评估CD8^+T细胞在OXA抗肿瘤中的作用。结果:OXA能够显著上调结肠癌细胞表面PD-L1的表达。OXA预处理后的结肠癌HCT-116细胞和T细胞共培养后,与单纯培养的T细胞相比,能降低其培养上清中IL-2、IFN-γ和TNF的水平(均P<0.05)及体系中CD4^+记忆性T细胞和CD8^+T_(EMRA)比率(P<0.05)、增加CD4^+(P>0.05)和CD8^+(P<0.05)初始T细胞比率;联合PD-1抗体后,与OXA预处理的HCT-116和T细胞共培养组相比,T细胞培养上清中IFN-γ和IL-10含量(P<0.05)以及CD8^+T_(CM)和T_(EMRA)比率增加(P>0.05)。体内抑瘤实验表明,OXA联合PD-1抗体可以增强其抗肿瘤活性,抑瘤率显著高于单用OXA组和αPD-1组(58.2%vs 25.6%、29.1%,均P<0.05);清除CD8^+T细胞后,OXA的抗肿瘤活性由68.4%下降到46.2%(P<0.05)。结论:OXA联合PD-1抗体具有联合增效的作用,同时CD8^+T细胞在OXA的抗肿瘤活性中具有重要作用。

Objective： To explore the anti-tumor effects of oxaliplatin （OXA） combined with PD-1 antibody on colon cancer. Methods：Flow cytometry was used to detect the expression of PD-L1 in colon cancer cell lines HCT-116 and HT-29. Co-culture method was used to detect the secretion of cytokines and the changes of CD4/CD8 subsets in T-cells that co-cultured with HCT-116 cells, which were pretreated with OXA in combination with/without PD-1 antibody; The CT26 transplanted tumor model of colon cancer in BALB/c mice was established and treated with the combination of OXA and PD-1 to evaluate their anti-tumor efficacy. Meanwhile, CD8 antibody was used to scavenge CD8＋ T cells in mice, and to evaluate the role of CD8＋ T cells in the anti-tumor effect of OXA in vivo. Results：OXA could significantly increase the expression of PD-L1 on the surface of colon cancer cells. Compared with pure T-cells, the T cells co-cultured with colon cancer HCT-116 cells that pre-treated by OXA, exhibited significantly reduced IL-2, IFN-γ and TNF levels （all P〈0.05） in its culture supernatant and decreased ratio of CD4＋ memory T cell / CD8＋TEMER （P〈0.05）, whereas there was increased cell proportion of the CD4＋ （P〉0.05） and CD8＋ （P〈0.05） na？ve T cells. After co-treated with PD-1 antibody, compared with the single treatment of OXA, IFN-γ and IL-10 content （P〈0.05） in culture supernatant and the subsets of CD8＋ TCM and TEMRA ratio （P〉0.05） were increased. In vivo experiments showed that OXA combined with PD-1 antibody could enhance its anti-tumor activity, the tumor suppression rates were 25.6% （OXA） and 29.1% （αPD-1）, respectively, however, the rate of tumor inhibition was increased to 58.2% when combined （P〈0.05, compared to OXA or αPD-1 group）. After scavenging CD8＋ T cells in mice, the antitumor activity of OXA dropped from 68.4% to 46.2% （P〈0.05）. Conclusion： OXA combined with PD-1 antibody had synergistic anti-tumor effect, and CD8＋ T cells played an important role in the antitumor activity of OXA.