Single-nucleus RNA sequencing reveals transcriptional heterogeneity in the blastema of favorable-histology Wilms tumor
Mike Adam, Keri A. Drake, Naomi Pode-Shakked, Katherine VandenHeuvel, S. Steven Potter, James Geller
Mike Adam, Keri A. Drake, Naomi Pode-Shakked, Katherine VandenHeuvel, S. Steven Potter, James Geller
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Research Article Development Nephrology Oncology

Single-nucleus RNA sequencing reveals transcriptional heterogeneity in the blastema of favorable-histology Wilms tumor

Abstract

While Wilms tumors commonly arise from renal precursor cells and maintain features of the developing kidney, recent studies have demonstrated substantial genetic, histologic, and molecular heterogeneity. To further investigate tumor variability as well as unifying features in tumor biology, we performed single-nucleus RNA sequencing (snRNA-seq) on treatment-naive, favorable-histology Wilms tumors utilizing a reference atlas established from tumor-adjacent kidney samples and fetal kidney. Transcriptional profiles of blastemal, stromal, and epithelial components were correlated with tumor histology and demonstrated developmental-lineage plasticity, with PAX2 and PAX8 expression normally restricted to the nephron lineage of the fetal kidney found to be expressed in tumor stroma, as well as the stromal marker POSTN identified in tumor blastema. Further analyses of the blastema show shared transcriptional features with the differentiation trajectory of “uninduced” to “early differentiating” fetal nephron progenitor cells as well as aberrant expression of stromal signatures. A number of pathways from fetal nephron progenitors were maintained in the blastema, including regulation of stem cell maintenance and axonogenesis, whereas other pathways appear enriched in specific tumor samples, demonstrating the ability of snRNA-seq to identify both unifying transcriptional signatures and uncover distinct molecular targets in signaling pathways and/or biological drivers of Wilms tumorigenesis.

Authors

Mike Adam, Keri A. Drake, Naomi Pode-Shakked, Katherine VandenHeuvel, S. Steven Potter, James Geller

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Figure 4

Trajectory analysis of Wilms tumor blastema shows intra- and intertumor heterogeneity.

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Trajectory analysis of Wilms tumor blastema shows intra- and intertumor ...
(A and B) To evaluate how tumor blastema resembles the differentiation trajectory of the nephron lineage, we further analyzed human fetal kidney snRNA-seq samples to identify subclusters of nephron lineage along the differentiation trajectory from self-renewing NPCs to differentiated cells. Unsupervised cell clustering identified 10 clusters (A) based on differential transcriptional profiles with selected genes highlighted in this heatmap (B), with this analyses identifying 3 subclusters of uninduced NPCs (i.e., 8, 3, and 0), 2 subclusters of induced NPCs (i.e., 2 and 5), and precursor populations for early podocytes (i.e., 1 and 6), proximal tubules (i.e., 4), parietal cells (i.e., 7), distal tubules (i.e., 9), and connecting tubules (i.e., 10). (C) Integration of tumor blastema from each of the 9 samples shows that the blastema from the majority of tumors clusters with cells along the spectrum of normal fetal kidney differentiation trajectory, with the exception of T4 that clusters exclusively podocyte precursors. (D–F) To further evaluate this, we first pooled the blastema from all 9 samples and analyzed the differentiation trajectory of noncycling versus cycling cells. As a control, fetal kidney cells were analyzed via the same methodology, with interstitial cells clustering separately from the nephron lineage. However, tumor blastema shows a mix of differential trajectories, with expression of pretubule, podocyte, and interstitial genes among the differentiation trajectories (D). Additionally, the cycling cells show enriched expression of NPCs genes (i.e., EYA1) and decreased expression of tubular markers (i.e., HNF1B and LIMCH1) along with both the cycling and noncycling tumor blastema showing misexpression of interstitial genes (i.e., COL6A3 and EBF1) and PAX3, which is normally not expressed in the human fetal kidney (E). Differential trajectories were then evaluated for the noncycling and cycling blastema from each individual tumor (F). Color coding shows representation along the differentiation trajectory, with orange representing NPC signature, green representing an epithelial signature, and blue representing a mixed signature.