安罗替尼联合抗PD-1抗体重塑小鼠结肠癌模型免疫微环境的机制研究

Mechanisms of anlotinib combined with anti-PD1 antibody for remodeling the immune microenvironment of a colon cancer mouse model

目的探讨抗血管生成小分子药物安罗替尼联合抗PD-1抗体对小鼠结肠癌的抑制效应,并探索其可能的重塑肿瘤免疫微环境机制。方法构建肠癌细胞CT26荷瘤BALB/c小鼠模型,随机分为对照组、安罗替尼组、抗PD-1抗体组和安罗替尼/抗PD-1抗体联合组,每组6只。期间每2 d用游标卡尺测量肿瘤体积,在实验结束(d14)后对各组移植瘤称重,并使用流式细胞仪检测肿瘤组织中免疫浸润细胞如CD4^(+)T细胞、CD8^(+)T细胞、单核细胞型髓源性抑制细胞(M-MDSC)、粒细胞型髓源性抑制细胞(G-MDSC)以及M2型肿瘤相关巨噬细胞(M2型TAM)数量变化。采用ELISA方法检测小鼠血清中血管内皮生长因子(VEGF)、干扰素γ(IFN-γ)、白细胞介素17(IL-17)和IL-10的水平。结果与对照组、安罗替尼组及抗PD-1抗体组相比,联合组小鼠移植瘤体积及重量下降(P<0.05),细胞因子VEGF、IL-10水平下降(P<0.05),IL-17水平下降(P<0.01),IFN-γ水平升高(P<0.05)。在免疫浸润细胞数量方面,与对照组相比,各治疗组M-MDSC数量均下降,但无统计学差异(P>0.05);联合组中M2型TAM数量较对照组和抗PD1抗体组下降(P<0.05)。与对照组、安罗替尼组及抗PD-1抗体组相比,联合组小鼠CD8^(+)T细胞数量增加(P<0.05);CD4^(+)T细胞数量较其他组略有下降,但仅与安罗替尼组有统计学差异(P<0.05)。结论安罗替尼联合抗PD-1抗体可调节细胞因子VEGF、IFN-γ、IL-10和IL-17水平,影响肿瘤微环境中免疫浸润细胞数量,重塑肿瘤免疫微环境,抑制小鼠结肠癌移植瘤的生长。

Objective To investigate the inhibitory effect of anlotinib combined with anti-PD1 antibody on a colon cancer mouse model,and to explore its possible mechanismfor remodeling the immune system and tumor microenvironment.Methods A BALB/c mouse model was established with colon cancer cells CT26,and the mice were divided randomly into four groups:the control group,the anlotinib group,anti-PD1 antibody group and anlotinib combined with anti-PD1 antibody group,with 6 mice in each.During the experiment,tumor volumes were measured every 2 days using a vernier caliper.After the experiment(on day 14),the weight of the tumors of mice in each group was measured.Flow cytometry was used to detect changes in the number of immune infiltrating cells in tumor tissues,including CD4^(+)T cells,CD8^(+)T cells,monocytic myeloid-derived suppressor cells(M-MDSCs),granulocytic myeloid-derived suppressor cells(G-MDSCs),tumor-associated macrophages(M2-TAM).Furthermore,ELISA was employed to detect the levels of vascular endothelial growth factor(VEGF),interferon-γ(IFN-γ),interleukin-17(IL-17),and IL-10 in the serum of mice.Results Compared with the control group,the other three groups showed a decrease in the volume and weight of transplanted tumors in mice(P<0.05),as well as decreased levels of cytokines VEGF,IL-10(P<0.05),and IL-17(P<0.01).Additionally,there was an increase in the level of IFN-γ(P<0.05).In terms of the number of immune infiltrating cells,the number of M-MDSCs decreased in each treatment group compared to the control group,but without statistically significant difference(P>0.05).In the combined group,the number of M2-type TAMs decreased compared to the control group and the anti-PD-1 antibody group(P<0.05).Furthermore,flow cytometry results indicated that compared to the control group,the other three groups showed an increase in the number of CD8^(+)T cells in mice(P<0.05).The number of CD4^(+)T cells decreased slightly compared to the other groups,but the statistically significant difference was only observed when compared to the anlotinib group(P<0.05).Conclusion The combination of anlotinib and anti-PD1 antibody may regulate the levels of cytokines VEGF,IFN-γ,IL-10,and IL-17,thereby influencing the number of immunosuppressive cells in the tumor microenvironment.The tumor microenvironment and immunity can also be improved,thus significantly inhibiting the growth of mouse colonic transplant tumors.