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

Abstract

Background: Upper tract urothelial carcinoma (UTUC) and bladder urothelial carcinoma (BLCA) both originate from uroepithelial tissue, sharing remarkably similar clinical manifestations and therapeutic modalities. However, emerging evidence suggests that identical treatment regimens may lead to less favorable outcomes in UTUC compared to BLCA. Therefore, it is imperative to explore molecular processes of UTUC and identify biological differences between UTUC and BLCA.

Methods: In this study, we performed a comprehensive analysis using single-cell RNA sequencing (scRNA-seq) on three UTUC cases and four normal ureteral tissues. These data were combined with publicly available datasets from previous BLCA studies and RNA sequencing (RNA-seq) data for both cancer types. This pooled analysis allowed us to delineate the transcriptional differences among distinct cell subsets within the microenvironment, thus identifying critical factors contributing to UTUC progression and phenotypic differences between UTUC and BLCA.

Results: scRNA-seq analysis revealed seemingly similar but transcriptionally distinct cellular identities within the UTUC and BLCA ecosystems. Notably, we observed striking differences in acquired immunological landscapes and varied cellular functional phenotypes between these two cancers. In addition, we uncovered the immunomodulatory functions of vein endothelial cells (ECs) in UTUC, and intercellular network analysis demonstrated that fibroblasts play important roles in the microenvironment. Further intersection analysis showed that MARCKS promote UTUC progression, and immunohistochemistry (IHC) staining revealed that the diverse expression patterns of MARCKS in UTUC, BLCA and normal ureter tissues.

Conclusion: This study expands our multidimensional understanding of the similarities and distinctions between UTUC and BLCA. Our findings lay the foundation for further investigations to develop diagnostic and therapeutic targets for UTUC.

Keywords: cellular heterogeneity; interaction network; single-cell RNA sequencing; tumor microenvironment; upper tract urothelial carcinoma.

Figure 1

Workflow of the sample preparation process, sequencing and bioinformatic analysis of in-house UTUC and retrieval of publicly available BLCA scRNA-seq datasets.

Figure 2

scRNA-seq and bulk RNA-seq profiling of UTUC. (A) UMAP displaying ten major cell clusters identified in the in-house UTUC scRNA-seq dataset. (B) Marker genes and relative proportions of sample origins for the ten major cell clusters. (C) Relative distribution of the major cell clusters. (D) Barplots showing the counts of DEGs between the cancer and health samples of each cell cluster in the UTUC scRNA-seq dataset. The pie plots at the top of the bar show the KEGG pathway enriched enrichment for each group of DEGs. (E) Relative distribution of GEO UTUC cohort. (F) Barplots showing the counts of DEGs between the UTUC and healthy cases of GEO UTUC cohort. The pie plots at the top of the bar show the KEGG pathway enrichment for each group of DEGs.

Figure 3

scRNA-seq and bulk RNA-seq profiling of BLCA. (A) UMAP displaying ten major cell clusters identified in the publicly available BLCA scRNA-seq dataset. (B) Marker genes and relative proportions of sample origins for the ten major cell clusters. (C) Relative distribution of the major cell clusters. (D) Barplots showing the counts of DEGs between the cancer and health samples of each cell cluster in the BLCA scRNA-seq dataset. The pie plots at the top of the bar show the KEGG pathway enrichment for each group of DEGs. (E) Relative distribution of TCGA BLCA cohort. (F) Barplots showing the counts of DEGs between the BLCA and healthy cases of TCGA BLCA cohort. The pie plots at the top of the bar show the KEGG pathway enrichment for each group of DEGs.

Figure 4

Identification and comparison of T cell subpopulations between the UTUC and BLCA. (A) UMAP showing the sample origins of T cell subpopulations. (B) The relative expression of marker genes in the T cell subpopulations. (C) Relative constituent ratio of each T cell subpopulation. (D) Relative distribution of six T cell subpopulations. (E) Bubble plot indicating the specific DEGs of CD8⁺ T cells in the UTUC relative to BLCA and normal ureter. (F) GO enrichment analysis of the upregulated DEGs of CD8⁺ T cells in UTUC relative to BLCA and normal ureter. (G) Bubble plot indicating the specific DEGs of Tregs in the UTUC and BLCA. (H) GO enrichment analysis of the downregulated DEGs of Tregs in UTUC relative to BLCA and normal ureter.

Figure 5

Identification and comparison of EC subpopulations in the UTUC and BLCA (A) UMAP showing the sample origins of EC subpopulations. (B) The relative expression of marker genes for EC subpopulations. (C) Relative constituent ratio of each EC subpopulation. (D) Relative distribution of five EC subpopulations. (E) Pseudotime trajectory for EC subpopulations. (F) Bubble plot indicating the specific DEGs of vein ECs in the UTUC compared to BLCA and normal ureter. (G) GO enrichment analysis for the downregulated vein ECs in UTUC compared to BLCA and normal ureter.

Figure 6

Identification and comparison of fibroblasts subpopulations in the UTUC and BLCA. (A) UMAP showing the sample origins of fibroblasts subpopulations. (B) The relative expression of marker genes for fibroblasts subpopulations. (C) Relative constituent ratio of each fibroblast subpopulation. (D) Relative distribution of three fibroblast subpopulations. (E) Bubble plot indicating the specific DEGs of inflammatory fibroblasts in the UTUC compared to BLCA and normal ureter. (F) GO enrichment analysis for the upregulated inflammatory fibroblasts in UTUC relative to BLCA and normal ureter.

Figure 7

Heterogeneity and differentiation trajectory of tumor cells in UTUC. (A) UMAP displaying the four tumor cell subtypes in the UTUC. (B) Differentiation trajectory of the tumor cell lineages in the UTUC assessed by Monocle3. (C) Differentiation trajectory of the tumor cell lineages in the UTUC assessed by RNA velocity. (D) Four tumor cell subtypes in the UTUC and corresponding enriched pathways of their top 30 expressed gene.

Figure 8

Intercellular network constructions of the UTUC and BLCA ecosystems. (A) Total interaction strengths in the UTUC ecosystems compared with corresponding normal tissue. (B) Circle plots showing the numbers of interactions in each cell subpopulation in UTUC. (C) Circle plots showing the numbers of interactions in each cell subpopulation in normal ureter tissues. (D) Total interaction strengths in the BLCA ecosystems compared with corresponding normal tissue (E) Circle plots showing the numbers of interactions in each cell subpopulation in BLCA. (F) Circle plots showing the numbers of interactions in each cell subpopulation in normal bladder tissues.

Figure 9

Identification of the key genes in the tumor cells of UTUC. (A) Identification of DEGs in tumor cells between BLCA and UTUC (B) KEGG pathway enrichment analysis of the upregulated DEGs in the tumor cells of UTUC. (C) KEGG pathway enrichment analysis of the upregulated DEGs in the tumor cells of BLCA. (D) Upset plot showing the intersection analysis between the groups compared (E) Venn plot showing upregulated DEGs in the tumor cells and RNA-seq data for UTUC.

Figure 10

Identification of the key genes in the fibroblasts of UTUC. (A) Identification of DEGs in fibroblasts between BLCA and UTUC. (B) KEGG pathway enrichment analysis of the upregulated DEGs in the fibroblasts of UTUC. (C) KEGG pathway enrichment analysis of the upregulated DEGs in the fibroblasts of BLCA. (D) Upset plot showing the intersection analysis between the groups compared. (E) Venn plot showing upregulated DEGs in the tumor cells and fibroblasts for UTUC.

Figure 11

MARCKS is a prominent driver of UTUC progression. (A) Expression levels of MARCKS in tumor cell subpopulations of UTUC. (B) Expression levels of MARCKS during the differentiation of tumor cell subpopulations of UTUC. (C) IHC showing upregulated MARCKS in the UTUC tissues relative to BLCA and normal ureter.