EPEN-06. Single cell resolution spatial transcriptomic analysis of PFA ependymoma reveals appearance of a proliferative UEC-A progenitor subpopulation at recurrence

Ependymoma is incurable in approximately 50 percent of childhood posterior fossa subgroup A (PFA). Characterization of the cellular heterogeneity within the PFA tumor microenvironment (TME) is crucial to understanding and preventing disease progression. The underlying cellular components of the PFA TME have been revealed by single-cell RNAseq (scRNAseq), and low resolution Visium spatial transcriptomics (ST) was able to partially validate and histologically chart these subpopulations. We used the newly developed Xenium single cell resolution ST platform to more definitively define the single cell histology of PFA. A custom Xenium panel of 480 genes was designed based on PFA subpopulation marker genes previously defined by scRNAseq and Visium. Xenium probes were hybridized to a variety of PFA patient samples from presentation and recurrence using the Xenium Analyzer. Through integration of transcript and morphological markers, we were able to distinguish the main PFA TME subpopulations that had previously been identified. Importantly, the single cell resolution of Xenium enabled identification of cell types that showed scattered intermixing with more abundant cell types that had not been detectable by lower resolution Visium ST. The newly visualized cell types include a putative progenitor population previously identified by single cell and ST, termed undifferentiated ependymoma cell -A (UEC-A). The abundance of UEC-A as measured by Xenium was significantly higher than had been estimated by Visium, and Xenium provided greater granularity of UEC-A, which was shown to include a subpopulation with high cell-cycle activity. UEC-A subpopulations were intermixed in neuroepithelial ependymally-differentiated zones but absent from areas that had undergone mesenchymal transition. Comparison of the cellular architecture of matched presentation and recurrence samples revealed a significant increase in the abundance of proliferating UEC-A at recurrence. Xenium ST therefore provides a sensitive method to identify high-risk PFA subpopulations and track the impact of experimental treatments targeting these cells.