Abstract: Objective The aim of this article was to preliminarily identify the components of tumor microenvironment （TME） characterized by high expression of V-domain immunoglobulin suppressor of T-cell activation（VISTA） and/or Programmed cell Death-1 （PD-1）/PD-1 Ligand （PD-L1） utilizing the public databases and our cohort, and test the efficacy of combination therapy of VISTA blockade and PD-1 blockade in mouse subcutaneous urothelial carcinoma of bladder （UCB） model. Methods We utilized TCGA-UCB dataset to screen out differentially expressing genes （DEGs） in tumors with high expression of VISTA and/or PD-1/PD-L1. The differentially enriched pathways were confirmed by GO （Gene Ontology） pathway enrichment analysis. Immune cell composition of the TME with high/low expression of VISTA and/or PD-1/PD-L1 was evaluated by cibersort analysis, which was verified on a protein level using our own cohort. The associations of high expression of VISTA and/or PD-1/PD-L1 with clinicopathological characteristics were accessed in our cohort. We established subcutaneous mouse UCB model and tested the anti-tumor efficacy of VISTA blockade plus PD-1 blockade by monitoring the tumor growth. Results In TCGA-UCB dataset, high expression of VISTA was associated with multiple immunoinhibitory pathways, increased infiltration of M2 macrophages and CD8⁺ T cells and immunosuppressive molecules such as CD163 and MRC1. VISTA was positively associated with multiple immunosuppressive immune checkpoints including PD-1 and PD-L1. Simultaneous high expression of VISTA and PD-1/PD-L1 was associated with M1 macrophage and CD8⁺ T cell infiltration and immunoinhibitory molecules such as CD163 and MRC1. In our own UCB cohort, VISTA was also positively associated with PD-1/PD-L1 expression. Simultaneous high expression of VISTA and PD-1/PD-L1 was associated with higher T stage, histological grade and CD8⁺ T cell infiltration. Combination of VISTA and PD-1 blockade in subcutaneous mouse UCB model showed greater inhibition of tumor growth. Conclusion In this study, we used TCGA-UCB dataset and our own UCB cohort and found that high expression of VISTA was associated with immunosuppressive TME. Simultaneous high expression of VISTA and PD-1/PD-L1 presented different TME features and associated with advanced clinicopathological characteristics of UCB patients. VISTA blockade plus PD-1 blockade exhibited stronger anti-tumor efficacy. These findings provided theoretical support for the application of combination of VISTA and PD-1 blockade in UCB immunotherapy.

Key words: bladder cancer, VISTA, PD-1/PD-L1, immunosuppressive TME, combination therapy

摘要： 目的 应用公共数据库和我院样本初步分析鉴定膀胱癌中高表达T细胞激活抑制物免疫球蛋白可变区结构域（VISTA）与细胞程序性死亡受体-1（PD-1）/PD-1配体（PD-L1）的肿瘤微环境组成特征,并在小鼠皮下膀胱癌模型上测试联合阻断VISTA与PD-1的抗肿瘤效力。方法 利用TCGA膀胱癌数据库,通过差异分析筛选高表达VISTA与PD-1/PD-L1病人对比低表达病人的差异基因,通过GO通路富集分析筛选差异富集的通路;通过cibersort分析高/低表达VISTA与PD-1/PD-L1膀胱癌的免疫浸润特征并利用我院样本进行蛋白水平验证;分析我院膀胱癌病人高/低表达VISTA和PD-1/PD-L1与其临床病理特征的相关性。建立小鼠的皮下膀胱癌模型,联合应用抗VISTA抗体以及抗PD-1抗体,监测肿瘤生长以探究其抗肿瘤效力。结果 TCGA数据库中,VISTA在膀胱癌中高表达与多种免疫抑制性通路相关,与M2型巨噬细胞、CD8⁺ T细胞相关,与CD163、MRC1等免疫抑制分子相关;VISTA还与PD-1、PD-L1等多种负性免疫检查点相关;同时高表达VISTA和PD-1/PD-L1与M1型巨噬细胞、CD8⁺ T细胞相关,与CD163、MRC1等免疫抑制分子相关。我院膀胱癌队列中,VISTA表达与PD-1、PD-L1相关;免疫细胞上同时高表达VISTA和PD-1/PD-L1与更高的T分期、组织学分级以及CD8⁺ T细胞浸润数量相关。小鼠的皮下膀胱癌模型中联合应用抗VISTA抗体以及抗PD-1抗体更能抑制肿瘤生长。结论 本研究结合TCGA膀胱癌数据库和我院膀胱癌队列分析发现,VISTA在膀胱癌中高表达与免疫抑制微环境相关;膀胱癌中同时高表达VISTA和PD-1/PD-L1呈现出不一样的免疫微环境特征且与恶性肿瘤特征相关。联合阻断VISTA以及PD-1显示更加强大的抗肿瘤效力。这些结果为联合阻断VISTA和PD-1/PD-L1免疫疗法在膀胱癌中的应用提供了理论依据。

关键词: 膀胱癌, VISTA, PD-1/PD-L1, 免疫抑制微环境, 联合治疗