High-risk blastemal Wilms tumor can be modeled by 3D spheroid cultures in vitro

Jenny Wegert,Lisa Zauter,Rhoikos Furtwängler,Christoph Otto,Christian Vokuhl,Sabrina Bausenwein,Karen Ernestus,Norbert Graf,Manfred Gessler,Silke Appenzeller

doi:10.1038/s41388-019-1027-8

Abstract

In vitro models represent a critical tool in cancer research to study tumor biology and to evaluate new treatment options. Unfortunately, there are no effective preclinical models available that represent Wilms tumor (WT) — the most common pediatric renal tumor. Especially the high-risk blastemal WT subtype is not represented by the few primary cell lines established until now. Here, we describe a new 3D approach for in vitro cultivation of blastemal WT cells, where primary cultures grown in suspension as spheroids could be propagated long-term. Besides blastemal cultures, we could generate spheroids representing epithelial and stromal WT. Spheroid cultures were analyzed by immunohistochemistry in comparison to corresponding tumor sections and were further characterized by RNA sequencing. Histological appearance of spheroids resembled the original tumor and they expressed marker genes characteristic of early renal development and blastemal WT elements. The cultures were amenable to genetic manipulation and they formed xenograft tumors, which resemble the primary human tumor. This collection of WT spheroids that carry different genetic drivers forms a long-sought tool for drug testing and in vitro modeling.

Highlights

Studying in particular the high-risk blastemal Wilms tumor (WT) subtype has been hampered by the lack of any WT blastemal cell cultures
That propagation of blastemal WT cells is possible, if cells are grown as 3D spheroids and in media containing ROCK inhibitor (Y-27632) to avoid anoikis
Even under long-term cultivation blastemal spheroids retain expression of genes typically seen in condensing mesenchyme and nephron progenitors (e.g., SIX2, SALL1, EYA1, CITED1, or PAX2), suggesting that blastemal cells are trapped in early renal development and retain this undifferentiated status

Summary

Introduction

Wilms tumor (WT) or nephroblastoma is the most common pediatric renal tumor that is diagnosed at a median age of 3.5 years [1]. Overall survival is at 90%, but strongly dependent on histology and stage [2]. While stromal- or epithelial-type, triphasic, and regressive tumors are classified as intermediate risk, the blastemal type and tumors with diffuse anaplasia represent the high-risk group [3]. WT as an embryonal tumor is thought to arise from renal precursor cells, which explains the histological heterogeneity reminiscent of embryonal kidney development. While blastemal cells are similar to condensed metanephric mesenchyme, the epithelial cells represent early tubular structures. Stromal cells show greater diversity from fibroblast-like appearance to skeletal muscle or even cartilage differentiation. Analysis of WT provides insight into cancer biology as well as into normal kidney development

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Conclusion