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Single cell genomic profiling of renal cancer stem cells

Final Report Summary - SINGLECELLGENOMICS (Single cell genomic profiling of renal cancer stem cells)

Characterization of the cellular heterogeneity developing kidneys and pediatric kidney tumors:
Kidney development occurs during the embryonic stage from week 5 to week 36 of gestation in humans [1] [2][3]. Cells from the cap mesenchyme – a transient kidney progenitor state – undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron. Faults in this process can lead to Wilms’ tumor, a pediatric malignancy of the kidney. Wilms’ tumor (WT), also known as nephroblastoma, is the most common pediatric tumor of the kidney with 75% of cases presenting before the age of 5 years [4]. WT is unique in terms of histology, typically harboring three main elements in varying proportions: blastema, epithelia, and stroma. These components recapitulate normal stages of nephrogenic differentiation. As such, Wilms’ tumors are a model to study the link between normal development and tumorigenesis [1][5].
In the duration of this grant we studied the cellular heterogeneity of the fetal developing kidney and Wilms’ tumors using “bulk” and single-cell RNA sequencing.
In the first grand period we used single cell qPCR and identified 3 major cell subpopulations representing distinct transcriptional states that could be related to the major cell types participating in kidney development:
• A Cap-mesenchyme (CM) population, overexpressing the genes SIX2, WT1, OSR1, CITED1, EYA1, SIX1, and CDH11
• A second mesenchymal subpopulation overexpressing the genes SERPINE1, ZEB1, and CDH11 that we identified as the uninduced metanephric mesenchymal/stromal cells of the nephrogenic zone.
• Early nephric epithelial cells over expressing EPCAM, CDH1, CDH6, PAX2, KRT18, and KRT19.
We also identified cells in an intermediate Mesenchymal to Epithelial Transition (MET) state.
In blastemal Wilms’ tumor xenografts we found only two cell subpopulations: a CM-like subpopulation and an un-induced mesenchyme-like population. This indicates that Wilms’ tumors mimic normal fetal developing kidneys, but lack the full heterogeneity.
In the second period we used FACS to enrich for the different subpopulations and then performed RNA sequencing in order to characterize this cellular heterogeneity more deeply. We found that blastemal Wilms’ tumors are similar to the CM-like populations, representing early stages of kidney development in both gene expression and alternative splicing. Moreover, our data indicates that ESRP1/2, RBFOX2, and QKI are putative splicing regulators that affect MET during kidney development, and possibly, in the development of Wilms’ tumors.
1. Hohenstein P, Pritchard-Jones K, Charlton J. The yin and yang of kidney development and Wilms’ tumors. Genes Dev. 2015;29: 467–482. doi:10.1101/gad.256396.114
2. Little M, Georgas K, Pennisi D, Wilkinson L. Kidney Development: Two Tales of Tubulogenesis. In: Thornhill BA, Chevalier RL, editors. Current Topics in Developmental Biology. 2010. pp. 193–229. doi:10.1016/S0070-2153(10)90005-7
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5. Huff V. Wilms’ tumours: about tumour suppressor genes, an oncogene and a chameleon gene. Nat Rev Cancer. Nature Publishing Group; 2011;11: 111–121. doi:10.1038/nrc3002