Single-cell transcriptomic analysis of tumor heterogeneity and intercellular networks in human urothelial carcinoma

Abstract

Background: 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. Methods: 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 test, and Pearson correlation analysis were used properly. Results: 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. Conclusion: 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.

Investigating cellular similarities and differences between upper tract urothelial carcinoma and bladder urothelial carcinoma using single-cell sequencing

“…Myeloid cells are the predominant cell type in the TME and contribute significantly to the immunosuppressive microenvironment of UC ( 13 ). Therefore, we further characterized myeloid cell subtypes in the UTUC and BLCA TME, as displayed in Supplementary Figure 2A , which showed the sample origins.…”

“…Similarly, Shao et al. clarified intra-tumoral heterogeneity and delineated the pro-tumoral and immunosuppressive local microenvironment in UC using scRNA-seq, further supporting the development of therapeutic strategies ( 13 ). More recently, Li and their research team conducted scRNA-seq on UTUC tissues, and explored the tumor ecosystem of this disease.…”

Recent contributions of single-cell and spatial profiling to the understanding of bladder cancer

“…scRNA-seq has demonstrated high heterogeneity at the transcriptomic level in tumor cells [ 3 ], for example, in the existence of multiple malignant cell subpopulations ( n = 14 [ 4 ] or n = 7 [ 5 ]), that exceed numbers of the current molecular bulk profiling classes. Schallenberg et al [ 6 ] used tissue microarrays from tumor center and tumor periphery and observed more than one molecular subtype in 24.6% ( n = 47/191) of the samples.…”

“…Comparison of immune cells in malignant and adjacent normal bladder tissue by scRNA-seq yielded insights into the immunosuppressive features of the tumor microenvironment (TME). These immunosuppressive features were characterized by, besides downregulation of the cancer cell-intrinsic antigen-processing machinery, the presence of exhausted CD8 + cells, M2 macrophages, and LAMP3 + dendritic cells [ 5 , 22 ▪▪ ]. Also, TME composition was shown to be profoundly distinct in different stages of bladder cancer [ 23 ▪ ].…”

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“…For instance, Fong and colleagues used scRNA-seq and T cell receptor (TCR) sequencing to uncover distinct cytotoxic CD4 + T cell subtypes within the BLCA TME, highlighting the pivotal role of the unique gene signature of cytotoxic CD4 + T cell in predicting the effectiveness of immunotherapy (12). Similarly, Shao et al clarified intra-tumoral h e t e r o g e n e i t y a n d d e l i n e a t e d t h e p r o -t u m o r a l a n d immunosuppressive local microenvironment in UC using scRNAseq, further supporting the development of therapeutic strategies (13). More recently, Li and their research team conducted scRNAseq on UTUC tissues, and explored the tumor ecosystem of this disease.…”