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Childhood Ependymoma Single-Cell Sequencing Study Reveals New Cellular Subpopulations

NEW YORK – To further expand on the understanding of and identify possible therapeutic targets for childhood ependymoma, a team of researchers from the University of Colorado Denver has studied the cellular diversity of this type of brain tumor using single-cell transcriptome sequencing.

Ependymoma remains fatal in most children, is incurable, and has not seen new therapies in several decades. In previous studies, scientists have mostly analyzed bulk tumor samples.

As described in a publication in Cell Reports on Tuesday, the researchers found a potential progenitor subpopulation of cells after analyzing more than 11,000 individual neoplastic cells using single-cell RNA-seq along with histology, deconvolution, and in vitro functional analysis. The findings came from 26 childhood ependymoma patient samples, which were classified as either posterior fossa (PF) or supratentorial (ST) ependymoma.

"The recent development of single-cell technology provided us with an unbiased method to chart the different neoplastic and non-neoplastic cell types within this and other pediatric brain tumors," said Andrew Donson, a researcher at the University of Colorado Denver and Children's Hospital Colorado, and the senior author of the study, in an email.

The team analyzed 19 patient samples classified as posterior fossa group A (PFA) and five samples classified as supratentorial RELA, as well as one each from subgroups PFB and YAP, generating 10X Genomics Chromium single-cell transcriptome data for about 18,500 cells.

They then applied Harmony alignment to their dataset and identified cell clusters. Some of these had gene expression profiles consistent with subpopulations of non-neoplastic cells, such as myleoid cells, lymphocytes, and oligodendrocytes, while others had profiles consistent with neoplastic ependymoma cells. Inferred copy-number variation (CNV) analysis (inferCNV) was used in addition to confirm neoplastic versus non-neoplastic cells. Using deconvoluted subpopulation fractions and immunohistochemistry scores in the clinically annotated sample cohort, the researchers examined the biology of PFA subpopulations further.

"Within most samples, we observed a divergent lineage trajectory, where progenitors develop into more differentiated subpopulations that are driven by either hypoxic or developmental stimuli," said Donson. "Moreover, the ratio of hypoxia- to developmental-driven differentiation was shown to underlie assignment of samples to different ependymoma molecular subgroups based on bulk tumor profiling. This demonstrates how our findings and the accompanying ependymoma single-cell browser will help researchers to more accurately interpret ependymoma biological data."

For histological characterization of PFA, the researchers focused on PFA subpopulations named CEC, TEC, UEC-1, and MEC, which appeared to have distinct biological roles based on their transcriptome profiles. In comparison to ST tumor samples, PFA tumors seemed to conceal multiple conserved and well-defined subpopulations corresponding to normal ependymal functional states, neurodevelopmental states, and stress-response mechanisms, according to the study.

The CEC and TEC subpopulations of PFA may have a favorable clinical outcome because of their normal ependymal phenotypes of cilia function and cellular transport.

The study also identified the UEC-1 and MEC subpopulations, which were associated with a more aggressive clinical course. These represent novel candidate drivers of relapse in PFA and could be targets for future therapeutics, the authors wrote. Further functional and preclinical studies are required, though, to demonstrate the progenitor-cell characteristics and therapeutic resistance properties of these subpopulations, they added.

Remarkably, single-cell RNA sequencing also identified the conserved subpopulations of TEC and UEC-1 that have distinct and clinically relevant properties and were not found in prior bulk-tumor transcriptomic analyses.

"This surprising finding further underscores the power of single-cell analyses in oncology research and the critical need to address cancer cell heterogeneity in our goal to provide effective therapy for this devastating childhood tumor," the authors wrote.

Donson said that the findings of the study have started many new research directions for the laboratory and have led to awards and grants, including two from the National Institutes of Health and one from the Department of Defense. 

"We have been awarded two NIH R01s to more thoroughly characterize the biology of these novel neoplastic subpopulations," he said. "We also hope to utilize these findings to refine treatment strategies for ependymoma, and have been awarded a DoD grant that explores the impact of ependymoma experimental therapeutics that specifically target the more aggressive progenitor- and hypoxia-driven subpopulations."