肿瘤细胞和肿瘤微环境（TME）的异质性与尿路上皮癌（UC）患者的临床结果和治疗反应显著相关。综合评估恶性细胞和TME之间的细胞多样性和相互作用，可以阐明UC进展的机制并指导新疗法的开发。本研究旨在扩展我们对UC内肿瘤异质性和免疫抑制TME的了解，并为开发新的UC疗法提供基础支持。研究对象包括在2020年7月至2020年10月间在我们医院接受根治手术的7名UC患者。我们在7个肿瘤和6个匹配的邻近正常组织中进行了单细胞RNA测序（scRNA-seq）分析，并将结果与两个公共scRNA-seq数据库集成。对单个细胞的功能特性和细胞间相互作用进行了表征，并使用多重免疫荧光染色、流式细胞术和大量转录组数据集对结果进行了验证。通过双边检验的R软件包进行了所有统计分析，合适地使用了威尔科克森秩和检验、对数秩检验、单因素方差分析（ANOVA）检验和皮尔逊相关分析。 无监督的t分布随机近邻嵌入聚类分析在尿路上皮组织中确定了十个主要细胞子簇。其中，注意到了七个上皮亚型，恶性上皮细胞具有增加的细胞增殖和降低的免疫原性。CD8+T细胞亚群在UC组织中表现出增强的细胞毒性活性以及增加的疲劳程度标志，CD4+T调节细胞的招募在肿瘤组织中也增加了。对于髓样细胞，浸润的中性粒细胞、极化的M2型巨噬细胞和LAMP3+树突状细胞的协调重编程有助于UC组织中的免疫抑制TME。肿瘤组织表现出由KDR+内皮细胞和RGS5+/ACTA2+平滑肌细胞介导的增强血管生成。通过解卷积分析，我们确定了多个细胞亚型可能影响UC患者的PD-L1免疫疗法反应。 我们的scRNA-seq分析证明了UC组织内的异质性，并描述了其促肿瘤和免疫抑制的微环境，从而为发现新的治疗靶点提供了可能。 版权所有©2023中国医师协会，由Wolters Kluwer，Inc.根据CC-BY-NC-ND许可证生产。

Heterogeneity of tumor cells and the tumor microenvironment (TME) is significantly associated with clinical outcomes and treatment responses in patients with urothelial carcinoma (UC). Comprehensive profiling of the cellular diversity and interactions between malignant cells and TME may clarify the mechanisms underlying UC progression and guide the development of novel therapies. This study aimed to extend our understanding of intra-tumoral heterogeneity and the immunosuppressive TME in UC and provide basic support for the development of novel UC therapies.Seven patients with UC were included who underwent curative surgery at our hospital between July 2020 and October 2020. We performed single-cell RNA sequencing (scRNA-seq) analysis in seven tumors with six matched adjacent normal tissues and integrated the results with two public scRNA-seq datasets. The functional properties and intercellular interactions between single cells were characterized, and the results were validated using multiplex immunofluorescence staining, flow cytometry, and bulk transcriptomic datasets. All statistical analyses were performed using the R package with two-sided tests. Wilcoxon-rank test, log-rank test, one-way analysis of variance (ANOVA) test, and Pearson correlation analysis were used properly.Unsupervised t-distributed stochastic neighbor embedding clustering analysis identified ten main cellular subclusters in urothelial tissues. Of them, seven urothelial subtypes were noted, and malignant urothelial cells were characterized with enhanced cellular proliferation and reduced immunogenicity. CD8+ T cell subclusters exhibited enhanced cellular cytotoxicity activities along with increased exhaustion signature in UC tissues, and the recruitment of CD4+ T regulatory cells was also increased in tumor tissues. Regarding myeloid cells, coordinated reprogramming of infiltrated neutrophils, M2-type polarized macrophages, and LAMP3+ dendritic cells contribute to immunosuppressive TME in UC tissues. Tumor tissues demonstrated enhanced angiogenesis mediated by KDR+ endothelial cells and RGS5+/ACTA2+ pericytes. Through deconvolution analysis, we identified multiple cellular subtypes may influence the programmed death-ligand 1 (PD-L1) immunotherapy response in patients with UC.Our scRNA-seq analysis clarified intra-tumoral heterogeneity and delineated the pro-tumoral and immunosuppressive microenvironment in UC tissues, which may provide novel therapeutic targets.Copyright © 2023 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.