Cell clustering and annotations
A total of 9 clusters were identified in scRNA-seq analysis.(Fig. 2A) Using SingleR package and the Human Primary Cell Atlas[26] as reference, all the cells were recognized (Table S1). Interestingly, cells in the same cluster may be different types of cells. Since the specimens were derived from MIBC, the cell clusters 0, 1, 2, 3 and 7 are considered to be cancer cells, while the cell cluster 4 with significantly different expression patterns may be non-cancerous bladder epithelial cells in the tumor. The cells in cluster 5 and 8 were mainly recognized as stem cells and fibroblasts, while the cell cluster 6 were recognized were endothelial cells. In addition, a small number of cells in each cell cluster were recognized as other types of cells. The marker genes (Table S2) could basically identify cell clusters.(Fig. 2B) The top marker genes (ranked by AUC value) in each cell cluster were IGFBP3, UPK1A, KIAA0101, ARL6IP1, RPL27A, DCN, GNG11, CYBA and IGFBP7, respectively.(Fig. 2C-D)
The stromal component-based subtypes of MIBC
In the TCGA MIBC data sets, the abundance of 9 stromal cell subsets in each sample were estimated using ssGSEA method referenced the marker gene sets of each cell cluster. Subsequently, we performed k-means clustering of the MIBC stromal subtypes. Our analysis indicated that three was the optimal and stable clustering number. (Fig. 3A) The 408 MIBC samples were classified into three heterogeneous types. (Fig. 3B) Type 1 and 3, the “stromal-sufficient” type I and II, were characterized by relatively high stromal cells (cluster 5, 6 and 8) and varying in bladder epithelial cell cluster (cluster 0, 1, 2 and 3). In detail, type 1 was relatively with high clusters 0 and 1while with low clusters 2 and 3. It conversed in type 3. Type 2, the “stromal-desert” type, was characterized by relatively low stromal cells (cluster 5, 6 and 8).
The stromal subtype transformation
Survival analyses indicated that the “stromal-desert” type MIBC had significantly better overall survival than the other two subtypes (log-rank P = 0.031 and univariate Cox proportional hazards P = 0.011) (Fig. 4A). However, it was not an independent prognostic factor adjusted by routine clinicopathological characteristics (multivariate Cox proportional hazards P > 0.05, Table 1). We found that the stromal type transformation happened as the disease progressed. MIBCs with advanced disease were with higher stromal cells (cluster 5, 6 and 8) than those with early disease (Fig. 4B).
Table 1 Univariate and multivariate Cox proportional hazards for overall survival
|
Variables
|
Univariate
|
|
Multivariate
|
HR (95%CI)
|
P
|
|
HR (95%CI)
|
P
|
Sex
|
|
|
|
|
|
Male
|
Ref
|
|
|
|
|
Female
|
1.124 (0.810-1.560)
|
0.483
|
|
|
|
Age (years)
|
1.035 (1.018-1.051)
|
<0.001
|
|
1.032 (1.016-1.048)
|
<0.001
|
Stage
|
|
|
|
|
|
I-II
|
Ref
|
|
|
|
|
III-IV
|
2.269 (1.560-3.301)
|
<0.001
|
|
2.045 (1.391-3.006)
|
<0.001
|
Not available
|
1.862 (0.255-13.612)
|
0.540
|
|
1.934(0.263-14.235)
|
0.517
|
Stromal phenotype
|
|
|
|
|
|
Type 1
|
Ref
|
|
|
|
|
Type 2
|
0.622 (0.431-0.897)
|
0.011
|
|
0.789 (0.541-1.153)
|
0.221
|
Type 3
|
0.882 (0.619-1.256)
|
0.486
|
|
0.981 (0.686-1.402)
|
0.914
|
HR: hazard ratio, CI: confidence interval
|
The enrichment scores of oncogenic pathways vary among stromal subtypes in MIBC
Referred to the published signatures of 10 oncogenic pathways, [19] we calculated the enrichment scores among the three stromal subtypes for each samples. Except for the cell cycle, the enrichment scores of 9 oncogenic pathways differ among stromal types in MIBC. (Fig. 5) In general, the stromal-sufficient type I and II MIBC were with higher enrichment scores of oncogenic pathways than stromal-desert type except for the TGF-β pathway. In addition, stromal-sufficient type II MIBC has higher enrichment scores in HIPPO, PI3K, TP53 pathways, while lower enrichment scores in NOTCH, NRF2, RTK/RAS pathways than stromal-sufficient type I (P < 0.05).
The stromal- sufficient subtype MIBC was active in EMT
After calculation of the EMTness for each sample among the three stromal types of MIBC, as could be expected, stromal-sufficient type MIBC was more active in EMT than stromal-desert type. We also found that stromal-sufficient type II MIBC EMTness was higher than stromal-sufficient type II. (Fig. 6A)
Three stromal subtypes of MIBC with various gene mutation characteristics
High TMB levels in tumor lead to an increase in tumor neoantigens may trigger the immune system to attack the tumor.[27] However, our result indicated that no difference in TMB of the three stromal subtypes of MIBC.(Fig. 6B) A total of 423 gene mutation frequency differs between the three stromal types of MIBC.(Table S3) Among them, the high frequency mutant gene (gene with a mutation frequency greater than 10% in any stromal subtypes) are ELF3, KDM6A, KMT2A, MED12, TP53, RB1, and VCAN.(Fig. 6C)
The stromal-desert type of MIBC may have relatively few TILs
The relative abundance of immune cells among the three stromal subtypes of MIBC were estimated referred to the published studies,[24, 25] interestingly, compared to the two types of stromal-sufficient MIBC, stromal-desert MIBC also fewer TILs except for resting CD4 memory T cells, activated memory T cells and memory B cells. The stromal-sufficient type II MIBC have fewer resting CD4 memory T cells and more activated CD4 memory T cells. The stromal-sufficient type I MIBC have more memory B cells. (Fig. 7A) The landscape of the TILs in the three stromal subtype MIBC was displayed as a heatmap. (Fig. 7B)
Validation of MIBC stromal component-based clustering
As it was in the TCGA MIBC data set, the abundance of the 9 cluster cell subsets in each sample in GSE87304 were estimated using ssGSEA method. We scaled each sample and used Nbclust package to performed kmeans clustering and determined the optimal number of stable stromal subtypes. It was also three was identified as the optimal number for clustering. (Fig. 8A) The “stromal-sufficient” type I and II have more stromal cells (cluster 5, 6 and 8) while the “stromal-desert” type have lower stromal cells (cluster 5, 6 and 8).(Fig. 8B) In addition, the stromal component-based clustering may be independent to a molecular classifications which divides MIBC into four types[7] (Luminal, Infiltrated-luminal, Basal and Claudin-low) (Pearson's Chi-squared test P = 0.849)