瑞戈非尼通过CSF1-R信号通路调节肿瘤相关巨噬细胞增强PD-1抗体在结直肠癌中疗效的研究  

Regorafenib enhances anti-tumor activity of PD-1 antibody in colorectal cancer by regulation of tumor-associated macrophages through CSF1-R signaling in vivo

作　　者：苏燕燕[1] 杨翀[1] 张宗祥[1] 叶宇[1] 应佳可 SU Yan-yan;YANG chong;ZHANG zong-xiang;YE yu;YING jia-ke(Department of General Surgery,Hangzhou Red Cross Hospital,Hangzhou 310005,China)

机构地区：[1]杭州市红十字会医院普外科,杭州310005

出　　处：《浙江中西医结合杂志》2023年第10期909-914,共6页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省医药卫生科技计划项目(No.2020KY221)。

摘　　要：目的探讨瑞戈非尼(Reg)通过集落刺激因子(CSF1-R)信号通路调节肿瘤相关巨噬细胞(TAMs)增强PD-1抗体(PD-1 antibody,anti-PD-1)在结直肠癌中的疗效,并分析其机制。方法接种结肠癌细胞CT26于24只BALB/c小鼠,建立结直肠癌移植瘤模型,模型小鼠采用随机数字表法分为CT26组、CT26+Reg组、CT26+anti-PD-1组和CT26+Reg+anti-PD-1组4个组,每组6只,进行药物治疗后观察荷瘤小鼠移植瘤的生长情况。取肿瘤组织,通过Western Blot和免疫组化检测瘤内磷酸化CSF1-R(p-CSF1-R)和CSF1-R表达,流式细胞术检测肿瘤单细胞悬液中TAMs并区分M1、M2表型。提取小鼠骨髓来源巨噬细胞(BMDMs),分别诱导成M1、M2型巨噬细胞,并用Reg处理细胞,qRT-PCR检测相关基因表达水平,Western blot检测巨噬细胞表面CSF1-R蛋白表达。结果与CT26组比较,药物处理后肿瘤体积[(1174.33±56.84)mm3、(1221.00±42.42)mm3、(949.50±15.92)mm3比(1451.00±43.10)mm3]和重量[(2.32±0.08)g、(2.40±0.10)g、(1.73±0.08)g比(3.04±0.06)g]均显著减小,与单独使用Reg或anti-PD-1比较,Reg和anti-PD-1联合用药物使移植瘤的体积[(949.50±15.92)mm3比(1174.33±56.84)mm3、(1221.00±42.42)mm3]和重量[(1.73±0.08)g比(2.32±0.08)g、(2.40±0.10)g]进一步减小;Western blot和免疫组化结果显示,Reg处理后,瘤内p-CSF1-R表达显著下调(P<0.05);流式结果显示Reg处理后,M2型巨噬细胞比例显著下降[(13.67±1.80)%比(35.33±2.21)%、(21.33±2.36)%、(28.67±1.89)%,(P<0.05)],M1型巨噬细胞比例显著增加[(19.00±2.00)%比(5.50±0.96)%、(12.50±1.71)%、(8.00±0.82)%,(P<0.05)];qRT-PCR结果也表明,Reg处理能增加BMDMs中M1表型比例[IL-23:(1.98±0.08)比(1.00±0.05);IL-12:(3.10±0.12)比(1.00±0.10);CD86:(2.56±0.13)比(1.00±0.10)],并下调M2表型比例[Arg1:(0.57±0.04)比(1.00±0.09);CD206,(0.73±0.08)比(1.00±0.15);CD163:(0.81±0.07)比(1.00±0.20)],同时Western blot结果显示,Reg处理能显著下调M2型巨噬细胞表面p-CSF1-R蛋白的表达水平(PObjective To assess the effect of Regorafenib(Reg)on enhancement of PD-1 antibody(anti-PD-1)anti-tumor activity in vivo and then explore the underlying molecular events.Methods Colon cancer cell line CT26 was inoculated into BALB/c mice to establish colorectal cancer cell xenografts in vivo and then randomly divided into control(without treatment),CT26+Reg,CT26+anti-PD-1,and CT26+Reg+anti-PD-1 groups.Expression of colony stimulating factor 1 receptor(CSF1-R)and phosphorylated CSF1-R(p-CSF1-R)in tumor cell xenografts was analyzed using Western blot and immunohistochemistry and numbers of tumor-associated macrophages(TAMs)and M1 and M2 phenotypes were assayed in tumor cell xenograft single cell suspension using flow cytometry.Mouse bone marrow-derived macrophages(BMDMs)were isolated and induced to differentiate into M1 and M2 type macrophages in vitro and then treated with Reg.The cells were then subjected to qRT-PCR analysis of various gene mRNA and Western blot analysis of CSF1-R,respectively.Results Compared with CT26 controls,these treatments significantly reduced the tumor cell xenograft volumes(1174.33±56.84,1221.00±42.42,and 949.50±15.92 vs.1451.00±43.10 mm3;P<0.05)and weight(2.32±0.08,2.40±0.10,and 1.73±0.08 vs.3.04±0.06 g;P<0.05)and the combination of Reg with anti-PD-1 further reduced xenograft volumes(949.50±15.92 vs.1174.33±56.84 and 1221.00±42.42 mm3,P<0.05)and weight(1.73±0.08 vs.2.32±0.08 and 2.40±0.10 g,P<0.05)in these mice compared with single drug treatment.At a protein level,Reg treatment significantly downregulated expression of p-CSF1-R,while these treatments reduced the proportion of M2 macrophages(35.33±2.21,21.33±2.36,and 28.67±1.89 vs.13.67±1.80%,respectively;P<0.05)but they significantly induced the proportion of M1 macrophages(5.50±0.96,12.50±1.71,and 8.00±0.82 vs.19.00±2.00%,respectively;P<0.05).Moreover,the Reg treatment also increased level of Interleukins(IL)-23 mRNA(1.98±0.08 vs.1.00±0.05),IL-12 mRNA(3.10±0.12 vs.1.00±0.10),and CD86 mRNA(2.56±0.13 vs.1.00±

关 键 词：小鼠 结直肠癌 瑞戈非尼 CSF1-R信号通路 肿瘤相关巨噬细胞 

分 类 号：R735.34[医药卫生—肿瘤]