Presentation Authors: Astgik Petrosyan*, Matthew Thornton, Brendan Grubbs, Roger De Filippo, Stefano Da Sacco, Laura Perin, Los Angeles, CA
Introduction: Wilms' tumor (WT) accounts for 95% of renal malignancies in children and is characterized by postnatal uncontrolled proliferation of nephron progenitors (NP) without generation of functional nephrons. However, little is known about WT formation, its microenvironment or the involvement of NP in tumor progression. Since the extracellular matrix (ECM) is known to play a crucial role during morphogenesis and in tissue homeostasis, we first characterized the ECM composition within the NP niche and WT blastema. Moreover, using our validated Smartflares technique we isolated NP expressing SIX2 and CITED1 (the master genes regulating nephrogenesis) from WT samples and human fetal kidneys (hFK) and compared them by RNA-seq. We also performed in vivo and in vitro experiments to study the regulation of self-renewal vs. differentiation of NP.
Methods: Immunofluorescence (IF) for collagens, laminin and fibronectin were performed to characterize WT and hFK ECM in the NP niche. WT (#3) and hFK (#3) samples were digested to single cell suspension, incubated with Smartflare-probe and SIX2+CITED1+ cells were immediately sorted and processed for RNA-seq. Using a nephrogenic specific media, we established conditions for long-term culture of NP cells and study in vitro mechanism of self-renewal vs differentiation of NP from both WT and hFK. We also transplanted NP in vivo to study tumorigenesis.
Results: IF staining confirmed marked similarities in ECM protein composition between the undifferentiated NP within the hFK and WT. hFK-NP and WT-NP showed limited expression of collagen types 1a1, 3a1, 4a12 and FBN but expressed collagen types 18, 16, 21, and laminina5. We verified the expression of SIX2+CITED1+ cells in WT samples within the blastema that accounted for ~ 9% of the total population compared to only ~0.2% of the total hFK. RNA-Seq data confirmed the nephrogenic signature of hFK-NP and WT-NP but highlighted differences in expression of pluripotency and self-renewal related genes like OCT4, FOXO1, SALL, NANOG along with a lower expression of B-catenin, TCF, and other growth factors known to promote differentiation (BMPs, FGFs). We show that WT-NP like hFK-NP can also be extracted and cultured for multiple passages in vitro and when injected in vivo form tumors similar to the primary tumor isolated from the patient.
Conclusions: This work represents the first characterization of SIX2+CITED1+ cells from WT and suggests the importance of matrix-cell interaction in development and tumor formation. We confirmed similarities and differences in hFK-NP biology vs. WT-NP in regards to ECM deposition and developmental and pluripotency gene expressions. These studies can help increase our knowledge of human nephrogenesis and the development of new strategies aimed at halting tumor progression. Since WT arises from undifferentiated NP retained after birth, identification of factors affecting nephron maintenance, such as self-renewal/differentiation balance, has the potential for advancing our understanding of WT initiation and progression.
Source of Funding: GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics