超声血流增强效应联合程序性细胞死亡蛋白配体1单抗改善实体肿瘤免疫微环境

Tumor perfusion enhancement effect combined with programmed cell death-Ligand 1 antibody improves the immune microenvironment of solid tumors

目的探讨低强度超声激励微泡空化(USMC)产生的血流增强效应联合PD-L1单抗对实体肿瘤免疫微环境的改善作用。方法将MC38结肠癌荷瘤小鼠随机分为四组:对照组(26只, 无任何治疗)、USMC组(27只, USMC治疗)、程序性细胞死亡蛋白配体1抗体(anti-PD-L1)组(27只, anti-PD-L1治疗)和USMC+anti-PD-L1组(27只, USMC联合anti-PD-L1治疗)。USMC治疗使用VINNO70超声诊疗一体机。采用超声造影评价肿瘤血流增强效应;通过绘制肿瘤生长曲线及记录小鼠生存期, 评价抗肿瘤疗效;流式细胞术分析肿瘤内CD8^(+)T细胞数量及功能、CD4^(+)T细胞分化情况、骨髓来源的抑制性细胞(MDSC)比例及肿瘤相关巨噬细胞(TAM)的极化表型分布;酶联免疫吸附测定(ELISA)方法检测肿瘤内趋化因子CXCL9、CXCL10及HIF-1α含量;免疫荧光分析肿瘤组织中血管内皮生长因子(VEGF)的表达。结果 USMC治疗后超声造影分析肿瘤峰值强度(PI)、曲线下面积(AUC)较治疗前增加(均P<0.05)。USMC联合anti-PD-L1治疗明显抑制了肿瘤进展。USMC+anti-PD-L1组治疗后肿瘤微环境中CD8^(+)T细胞量及其Ki67的表达、γ干扰素(IFN-γ)与颗粒酶B(Grzm-B)的分泌量高于其他3组, 辅助性T细胞(Th1)比例增加, 调节性T细胞(Tregs)、MDSC比例减少, TAM表型向M1型极化(均P<0.05)。ELISA分析显示, 联合治疗还提高了肿瘤中CXCL9、CXCL10的浓度, 降低了HIF-1α的含量(均P<0.05);肿瘤组织VEGF的荧光表达量也显著低于对照组(P<0.05)。结论超声血流增强效应联合PD-L1单抗能改善实体肿瘤免疫微环境, USMC产生的血流增强效应可作为一种增效PD-L1抗体肿瘤免疫治疗的超声新方法。

Objective To investigate the tumor perfusion enhancement induced by low intensity ultrasound stimulated microbubble cavitation(USMC)combined with programmed cell death-Ligand 1(PD-L1)antibody on improving the immune microenvironment of solid tumors.Methods Tumor-bearing mice were divided into 4 groups:Control(n=26)group,USMC(n=27)group,anti-PD-L1(n=27)group and USMC+anti-PD-L1(n=27)group.USMC treatment was performed with a VINNO 70 ultrasound theranostics system.Tumor perfusion was evaluated by contrast-enhanced ultrasound(CEUS).The anti-tumor efficacy was assessed by the tumor growth curve and the survival time of mice.The number and function of CD8^(+)T cells,the differentiation of CD4^(+)T cells,the proportion of MDSC and the phenotype distribution of TAM in tumors were analyzed by flow cytometry.The content of CXCL9,CXCL10 and HIF-1αin tumor were detected by ELISA.The expression of VEGF in tumor tissues was analyzed by immunofluorescence.Results CEUS showed that the values of PI and AUC of tumors were significantly increased after USMC compared with before USMC(all P<0.05).USMC combined with anti-PD-L1 therapy did suppress the tumor progression.FCM showed the number,the expression of proliferation antigen Ki67,the secretion of IFN-γand Granzyme B of CD8^(+)T cells in tumors were higher in combined group than those in other three groups after therapy(all P<0.05).Meantime,the proportion of Th1 was rose while Tregs and MDSC were declined and the polarization of TAM was toward M1 type by combined therapy.ELISA analysis showed that the combined therapy also increased the concentration of CXCL9,CXCL10 and decreased the content of HIF-1αin tumors(all P<0.05).Meanwhile,the immunofluorescence expression of VEGF was significantly lower in combined group than that in the control group after treatment(P<0.05).Conclusions Tumor perfusion enhancement by USMC combined with PD-L1 antibody therapy could improve tumor immune microenvironment and USMC might be a novel effective method for potentiating PD-L1 antibody immunotherapy.