吡非尼酮联合PD-L1抑制剂抑制小鼠异位膀胱肿瘤的生长

Efficacy of combined treatment with pirfenidone and PD-L1 inhibitor in mice bearing ectopic bladder cancer xenograft

目的通过小鼠肿瘤模型观察吡非尼酮(PFD)联合程序性死亡受体-配体1(PD-L1)抑制剂对膀胱癌的疗效及对免疫微环境的调控作用。方法构建C57BL/6小鼠异位膀胱肿瘤模型共40只,根据不同处理随机分为4组(10只/组):对照组、PD-L1抑制剂组、PFD组、联合治疗组。对照组:建立肿瘤模型正常饮食;PD-L1抑制剂组:建模后腹腔每3 d按12.5 mg/kg注射PD-L1抑制剂;PFD组:建模后每天按500 mg/kg口服PFD;联合治疗组:建模后PFD及PD-L1抑制剂按上述剂量联合应用。对比各组小鼠生存率和肿瘤生长速度,药物干预21 d后留取肿瘤组织及小鼠血清,免疫组化检测肿瘤组织中CD3、CD8、CD45、E-cadherin及N-cadherin的表达;免疫荧光观察骨髓来源抑制细胞(MDSCs)表达;生化分析小鼠血液中谷丙转氨酶(ALT)、谷草转氨酶(AST)、血尿素氮(BUN)、肌酐(CRE)及乳酸脱氢酶(LDH-L)的水平。结果与对照组相比,PD-L1抑制剂组和PFD组小鼠肿瘤相对生长速率及21 d肿瘤体积均减小(P<0.05),联合治疗组小鼠更加显著(P<0.05)。免疫组化及免疫荧光结果显示,PD-L1抑制剂组与PFD组小鼠肿瘤组织E-cadherin表达增加,N-cadherin表达降低(P<0.05)。CD3+T细胞、CD8+T细胞、CD45+T细胞数量较对照组增加,而Ly-6G+CD11b+MDSCs细胞减少,联合组变化更加明显(P<0.05)。生化分析结果显示,各组小鼠血清ALT、AST、BUN、CRE及LDH-L水平无明显差异(P>0.05)。结论吡非尼酮联合PD-L1抑制剂可显著抑制膀胱癌的进展,其效应可能是通过调节肿瘤微环境,抑制肿瘤细胞的上皮间质转化来实现的。

Objective To assess the efficacy of pirfenidone combined with PD-L1 inhibitor for treatment of bladder cancer in a mouse model and its effect on tumor immune microenvironment modulation.Methods Forty C57BL/6 mouse models bearing ectopic human bladder cancer xenografts were randomized into control group,PD-L1 inhibitor group,pirfenidone group and combined treatment group(n=10).After successful modeling,PD-L1 inhibitor treatment was administered via intraperitoneal injection at 12.5 mg/kg every 3 days,and oral pirfenidone(500 mg/kg)was given on a daily basis.The survival rate of the mice and tumor growth rate were compared among the 4 groups.The expressions of CD3,CD8,CD45,E-cadherin and N-cadherin in the tumor tissues were detected with immunohistochemistry after the 21-day treatment,and bone marrow-derived suppressor cells(MDSCs)were observed with immunofluorescence staining;serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),urea nitrogen(BUN),creatinine(CRE)and lactate dehydrogenase(LDH-L)were analyzed using an automated biochemical analyzer.Results Treatment with PD-L1 inhibitor and pirfenidone alone both significantly decreased tumor growth rate and tumor volume at 21 days(P<0.05),but the combined treatment produced an obviously stronger inhibitory effect(P<0.05).PD-L1 inhibitor and pirfenidone alone significantly increased E-cadherin expression and decreased N-cadherin expression in the tumor tissue(P<0.05).The two treatments both significantly increased the percentage of CD3+,CD8 and CD45+T cells and decreased the percentage of Ly-6G+CD11b+MDSCs in the tumor tissue,and these changes were more obvious in the combined treatment group(P<0.05).No significant differences were found in serum ALT,AST,BUN,CRE or LDH-L levels among the 4 groups(P>0.05).Conclusion Combined treatment with pirfenidone and PD-L1 inhibitor significantly inhibits the progression of bladder cancer in mice possibly by regulating tumor immune microenvironment and inhibiting epithelial-mesenchymal transition of the tumor cells.