Astgik Petrosyan, PhD, Valentina Villani, PhD, Paola Aguiari, PhD, Matthew E. Thornton, BS, Brendan Grubbs, MD, Roger De Filippo, MD, Stefano Da Sacco, PhD, Laura Perin, PhD.
Children's Hospital Los Angeles, Los Angeles, CA, USA.
BACKGROUND: Wilms tumor (WT) accounts for 95% of renal pediatric malignancies and is characterized by uncontrolled proliferation of nephron progenitors (NP) without generation of functional nephrons. Due to the inability of isolating these human NP, little is known about WT initiation and growth. In this study, we isolated NP expressing SIX2 and CITED1 (the master genes regulating nephrogenesis) from WT samples and from human fetal kidneys (hFK) and performed in vivo and in vitro experiments to study the regulation of self-renewal vs differentiation of NP.
METHODS: WT and hFK samples were histologically analyzed and were digested to single cell suspension, incubated with Smartflare-probe and SIX2+CITED1+ cells were immediately sorted and processed for RNA-seq, single-cell RNA-seq and for other analysis. Xenografts of WT-NP and FK-NP were generated and tumor formation was assessed. Using a nephrogenic specific media, we established conditions for long-term culture condition of NPs and studies of mechanisms that regulate self-renewal vs differentiation were performed.
RESULTS: Histology confirmed a different pattern of expression for SIX2 and CITED1 across WT with different prognosis and stages compared to hFK. Our RNA-seq analysis confirmed overexpression of tumorigenic genes in WT-NP compared to WT-NP. When transplanted in vivo WT-NP demonstrated marked capacity of tumorigenesis, which in some instances induced metastasis, while hFK-NP did not. Single-cell RNA-seq after xenotransplantation of WT-NP defined precise cancer-associated cellular identities compared to WT-NP. In vitro experiments confirmed that modulation of integrin signaling leads to blockade of self-renewal in NP by decreasing CITED1 expression, activating b-catenin and inducing specification by stimulating the activation of LEF1.
CONCLUSIONS: This work evidenced that SIX2+CITED+ cells in WT represent a population of cancer stem cells with tumorigenic ability. Our characterization also highlights differences in self-renewal potential between favorable and unfavorable WT samples, with b-catenin playing a key role in regulating this biological process. These studies can help to increase our knowledge of human nephrogenesis and the development of new strategies aimed at halting tumor progression.
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