Gene expression studies of WT1 mutant Wilms tumor cell lines in the frame work of published kidney development data reveals their early kidney stem cell origin

Abstract

In order to get a better insight into the timing of WT1 mutant Wilms tumor development, we compared the gene expression profiles of nine established WT1 mutant Wilms tumor cell lines with published data from different kidney cell types during development. Publications describing genes expressed in nephrogenic precursor cells, ureteric bud cells, more mature nephrogenic epithelial cells and interstitial cell types were used. These studies uncovered that the WT1 mutant Wilms tumor cells lines express genes from the earliest nephrogenic progenitor cells, as well as from more differentiated nephron cells with the highest expression from the stromal/interstitial compartment. The expression of genes from all cell compartments points to an early developmental origin of the tumor in a common stem cell. Although variability of the expression of specific genes was evident between the cell lines the overall expression pattern was very similar. This is likely dependent on their different genetic backgrounds with distinct WT1 mutations and the absence/presence of mutant CTNNB1.

Fig 1. Cluster dendrogram of all WT1 mutant Wilms cell lines and a WT1 wild type cell line, CLS1, and their genotypes.

Fig 2. Analysis of genes from NPC and IPC enriched genes and from the different clusters expressed in Wilms cells [32].

A) Percent of genes from the Lindström NPC enriched (green) and IPC enriched (red) compartments expressed in Wilms cell lines (>1000). B) The percentage of genes expressed in each individual cluster is shown. Cluster 1, immune cells, was not analyzed. The color coding shows which clusters are part of the NP (green) or IP lineages (red). The differentiating NP cells, cluster 5 and 6 are darker green. Two clusters of cycling cells are turquois and developing vasculature cluster is yellow. C) The exclusively expressed genes in the 4 NPC subclusters and the percent of these expressed in common in the Wilms cell lines are shown, cut off >1000.

Fig 3. Go Biological process analysis of genes expressed in common in NPC (cluster4, [32]) and Wilms cells and those that are exclusively expressed in normal NPC.

A) The table lists the top 10 enriched biological processes and the genes expressed in common in both. B) The table lists the top enriched terms that are exclusive for normal huNPC.

Fig 4. Go Biological process analysis of genes expressed in common in IPC (clusters 2, 9, 10, 11, 12, [32]) and Wilms cells and those that are exclusively expressed in normal IPC.

A) The top 10 enriched Biological processes of genes expressed in common in both. B) The top 10 enriched biological terms that are exclusive for normal huIPC.

Fig 5. Common expression of genes in Lindström clusters and Wilms cell lines.

First genes that are specific, i.e., only expressed in one cluster were extracted. This was done separately for clusters 4A, 4B, 4C, and 4D from the NPC compartment. Cluster 4 is the parental cluster and is shown in the same circle. The top expressed genes in Wilms cell lines and also expressed in the respective clusters are listed and labeled green if they are unique for this cluster; black and underlined genes are also listed in other compartments. Clusters from the IPC compartment were also analyzed for genes exclusively expressed in the subclusters, shown in the red circle. The top expressed genes in Wilms cell lines also expressed in the respective clusters are listed and labeled red, if they are unique for this cluster; black and underlined genes are also listed in other compartments. The top genes for differentiating nephrons are shown in green and for cycling cells in turquois. Cluster1 (immune cells) is not shown.

Fig 6. Analysis of genes expressed in the Wilms cells from the Hochane marker set candidates.

A) The percentage of genes expressed in each cluster is shown. B) Genes in each cluster from the three linages that are expressed >50 000 are shown. Ribosomal genes were omitted. No genes in the CnT cluster were expressed above 50 000.

Fig 7. Expression of the TOP 4 genes from the marker set candidates in the Wilms cell lines.

In the heat map the expressed genes from the top 4 genes are shown (>200).

Fig 8. Expression of KeyGenes in Wilms cell lines.

Each compartment has a different number of KeyGenes. The expressed genes from each set are shown (>200) and numbers are listed on the right.

Fig 9. GSVA enrichment scores for the Hochane and Lindström clusters in Wilms cells.

Expression values of Wilms tumor samples were subjected to GSVA analysis using gene sets associated with developmental kidney cell types in the single cell sequencing studies [32, 43]. In the hierarchical cluster analysis of the GSVA enrichment scores two main clusters of samples emerged: group1 (Wilms Tumor samples 1, 4, 5, 6 and 10M) had higher enrichment in differentiated and lower enrichment in nephron progenitor cell (NPC)-like cells; group2 (Wilms Tumor samples 2, 3, 8 and 10T) had lower enrichment in differentiated and higher enrichment in NPC-like cells.

Fig 10. Differentially expressed genes in Wilms cells with and without WT1 expression.

A) Heat map of the genes expressed higher in cells without WT1: Wilms1, Wilms10T and Wilms10M (left) and expression level of selected genes with a higher expression in Wilms1, Wilms10T and Wilms10M (right). B) Heat map of genes expressed lower in cells without WT1 (left) and expression of selected genes with a lower expression in Wilms cells without WT1 expression (right).

Fig 11. Analysis of differentially expressed genes in Wilms cells with and without mutant WT1 expression.

A) ToppGene result for the differentially expressed genes. B) The Venn diagram shows that among the 114 differentially expressed genes in cells with and without WT1 (blue circle) 17 are putative SIX1/2 target genes (yellow circle, 1792 putative SIX1/2 target genes identified by O´Brien et al., 2016 [14]. C) A list of the 17 putative target genes with the number of peaks as detected with ChIP seq by O´Brien et al., 2016 and the regulatory score that they have determined [14].

Fig 12. Go BP-term analysis of the WT signature genes.

A) the 10 top terms using EnrichR and B) the 20 top terms using ToppGene.

Fig 13. Comparison of Wilms cell lines with UdRPC and analysis of the Go BP-terms of the common gene set.

A) The Venn diagram shows the overlap between genes expressed in Wilms tumor cells (red) and UdRPC (green) and the exclusively expressed genes. B) the 30 top Go BP-terms of the 1260 genes exclusively expressed in Wilms cells.

Fig 14. Comparison of Wilms cell lines with UdRPC_CHIR and analysis of the Go BP-terms of the Wilms tumor cell line exclusive gene set.

A) The Venn diagram shows the overlap between genes expressed in Wilms cells (red) and UdRPC_CHIR (green) and the exclusively expressed genes. B) the 30 top Go BP-terms of the 1327 genes exclusively expressed in Wilms cells.