Integrated Single-Nucleus and Spatial Transcriptomics Elucidate Heterogeneity and Hypoxia-Driven Organization of Supratentorial Ependymoma

Supratentorial ependymomas (ST-EPN) are often life-threatening brain tumors that are characterized by significant molecular heterogeneity. Hypoxia contributes to this heterogeneity, but the extent and nature of this relationship remain unclear. This poses challenges for effective treatment strategies, demanding a more holistic and detailed understanding of the underlying biology. In this study, we employed single-nucleus (n = 63) and spatial (n = 30) transcriptomics to delineate the cellular and spatial landscape of ST-EPN. Characterizing the transcriptional consensus programs of this entity revealed two previously undescribed programs that are associated with the remodeling of the extracellular matrix and the ZFTA fusion identity. These programs were related to a highly unfavorable outcome and were exclusive to ZFTA fusion-positive tumors. Developing a set of spatial scoring algorithms that are sensitive to mixed spot transcriptomes enabled the identification of the role of hypoxia as a potential driver of spatial organization. Together with recurrent spatial associations inferred between programs, this informed a generalized model of the higher-order architecture. The functionally and spatially defined zones of this model displayed distinct immune presence, with the highest seen in the hypoxic zone. These results provide insights into the molecular heterogeneity and spatial organization of ST-EPNs, which may aid in the development of targeted future treatments. SIGNIFICANCE: The characterization of transcriptional and spatial heterogeneity in supratentorial ependymoma provides a resource that can inform investigations into potential targets for therapeutic interventions aimed at tumor cells within distinct microenvironments.