Prognostic Significance of Lineage Diversity in Bladder Cancer Revealed by Single-Cell Sequencing

Abstract

Bladder cancer is the most common malignant tumor of the urinary system. We investigated the clinical implications of cell lineages in bladder cancer by integrating single-cell and bulk transcriptome data. By investigating the single-cell transcriptional profiles of 12,424 cells from normal bladder, eleven cell types and five types of epithelial sub-population were identified. Based on the signature of cell types identified in single-cell profiles, deconvolution analysis was employed to estimate cell types and epithelial lineages in the bulk RNA sequencing bladder cancer cohort. Cancer subtypes with clinical implications were further identified based on the heterogeneity of the epithelial lineage across patients. This study suggests that the EMT-like subtype is robustly correlated with poor prognosis and the umbrella subtype is a positive factor for the patient survival. Our research has a high potential for accurate prognostic and therapeutic stratification of bladder cancer.

Keywords: bladder cancer; cell lineages; deconvolution; intratumoral heterogeneity; prognosis; single-cell RNA sequencing.

FIGURE 1

A cell census of human bladder cells. (A) Uniform manifold approximation and projection (UMAP) showing the dimensionality reduction of single-cell transcriptomes from the three samples. The cells are colored by their patient sources (left), clusters (middle), or cell types (right). (B) Heat map showing the marker genes of each cluster of all bladder cell types, plot the top 10 markers (or all markers if less than 10) for each cluster. (C) Bubble plot showing expression of specific marker genes across different cell types.

FIGURE 2

A cell census of epithelial cells within the human bladder. (A) UMAP plot representation of 7529 epithelial cells and clusters are colored and distinctively labeled. (B) Heat map showing the marker genes of sub-clusters of epithelial cells, plot the top five markers for each cluster. (C) Violin plots showing the average fold change of the top50 DEGs in the five sub-clusters. The average is indicated by dots in each cluster.

FIGURE 3

Characteristics of EMT-like cells (A) Overlaps of the upregulated DEGs of five subtypes on hallmark gene sets. The pathway names are labeled on the left and color-coded by cell types. (B) Bubble plot showing expression of traditional classic EMT marker genes across different epithelial types. (C) Heatmap shows large-scale CNVs for individual cells (rows) from epithelial cells, inferred based on the average expression of 100 genes surrounding each chromosomal position (columns). Red: amplifications; blue: deletions. (D) Histogram shows the distribution of CNV scores (average expression of epithelial marker genes) of various epithelial cell types based on CNV classification. The average is indicated by lines in each cell type.

FIGURE 4

Repertoire of the phenotypic heterogeneity of BCa and functional enrichment analysis. (A) Stacked bar plot shows the deconvolution result of 412 tumors from the TCGA bladder cancer study. The colors of the bars denote five epithelial cell states as shown in the legend. The y-axis represents the proportion of each state in a given bulk tumor sample. On the x-axis, each column represents one tumor case. The annotation bar on the right denotes the subtypes of bulk tumors that are defined by the dominant cell state within each tumor. (B) Overlaps of the DEGs of EMT-like subtypes and umbrella subtypes on hallmark gene sets, KEGG gene sets, and GO biological process. Only the pathways (rows) that were differentially expressed across different tumor cell lineages are shown. The pathway names are labeled on the left and color-coded by gene sets. The red underlines highlight the pathways that highly associated with patient prognostic significance.

FIGURE 5

Diversity in tumor cell lineage compositions links to ITH in cancer staging. (A) The stacked bar plot visualizes the result of differences in the average expression levels of the four epithelial cell lines in 412 TCGA bladder cancer patients with different cancer staging. The y-axis denotes the proportion of the four cell states (colors) across different stages of TCGA tumor samples (rows). We divided T1–2 into T_low, T3–4 into T_high, and the N stage into N0 and above. (B,C) The violin plots for the differences of bladder cancer epithelial cell (B) and fibroblasts and smooth muscle cells (C) lineages in cancer staging. The average is indicated by lines in each stage, colored according to the stages. The p values were calculated by the rank-sum test. All statistically significant p values were marked in the figures, and the degree of the difference in stages of the lineages we are focusing on was identified by ***.

FIGURE 6

Correlation between the tumor cell lineage and patient survival. (A) Kaplan–Meier curves show the effect of the five subtypes scores on survival in TCGA. Each short vertical line indicates a censoring event. (B) Kaplan–Meier curves show the effect of the umbrella cells and EMT-like cell scores on survival in TCGA. Patients are dichotomized into high/low by the median of the umbrella cells and EMT-like cell scores in each dataset. (C) Kaplan–Meier curves show the effect of the umbrella cell scores on survival in MSKCC as in (B).

FIGURE 7

The EMT-like subtype is correlated with high-grade tumors. (A) UMAP plot showing unbiased clustering analysis of the predicted cell types on eight tumor samples. Each dot of the UMAP plot represents a single cell. Cells are color-coded for their associated cell types. (B) Boxplot showing the percentage of the EMT-like subtype in high-grade tumor cells was higher than that in low-grade tumor cells. The average is indicated by lines in each subtype.