Multidimensional profiling of heterogeneity in supratentorial ependymomas | Nature

Download PDF Subjects CNS cancer Paediatric cancer Abstract Supratentorial ependymomas are aggressive childhood brain cancers that retain features of neurodevelopmental cell types 1 and segregate into molecularly and clinically distinct subgroups 2 , 3 , suggesting different developmental roots. The developmental signatures, as well as microenvironmental factors, underlying aberrant cellular transformation and behaviour across each supratentorial ependymoma subgroup are unclear. Here we integrated single-cell and spatial transcriptomics, as well as in vitro and in vivo live-cell imaging, to define supratentorial ependymoma cell states, spatial organization and dynamic behaviour within the neural microenvironment. We find that individual tumour subgroups have two distinct progenitor-like cell states—neuroepithelial-like and embryonic-like—that are reminiscent of early human brain development and diverge in the extent of their neuronal or ependymal differentiation. We further identify several modes of spatial organization of these tumours, including a high-order architecture that is influenced by mesenchymal and hypoxia signatures, and local neighbourhood structures. Finally, we identify a role for brain-resident cells in shifting supratentorial ependymoma cellular heterogeneity towards neuronal-like cells that co-opt immature neuronal morphology and migratory mechanisms, and a subset of neuroepithelial-like cells that are both proliferative and highly migratory. Collectively, these findings provide a multidimensional framework to integrate transcriptional and phenotypic characterization of tumour heterogeneity in supratentorial ependymoma and its potential clinical implications. Main Brain cancers are the leading cause of cancer-related deaths in children and adolescents, surpassing leukaemia 4 . Deciphering the developmental correlates is pivotal to therapeutic advancement as malignant cells retain molecular and phenotypic properties of their progenitor origins 5 , 6 , 7 . Supratentorial ependymomas (ST-EPNs) are brain tumours that occur in the central nervous system, with varied outcomes and limited available therapies 8 , 9 , 10 . Recent genome-wide DNA methylation profiling studies have classified ST-EPNs into multiple subgroups with distinct fusion genes and patient outcomes 2 , 3 . These include the canonical ST-ZFTA subgroup, characterized by the fusion of NF-κB pathway regulator RELA with the zinc-finger-translocation-associated ZFTA ( ZFTA-RELA ), and the non-canonical ST-ZFTA subgroups ( ZFTA clusters 1 to 4), which have ZFTA-RELA fusions or fusions between ZFTA and other partner genes. Furthermore, the ST-YAP1 subgroup is enriched for fusions of Hippo effector YAP1 . It remains unclear whether these subgroups have distinct cellular origins and composition of malignant cell states, contributing to differences in outcomes and varying resistance to therapy. To address these questions, previous studies have used single-cell (scRNA-seq) and single-nucleus (snRNA-seq) RNA sequencing 1 , 11 to characterize the molecular cell states of ST-EPN tumours based on distinct gene expression profiles. However, these studies did not include the non-canonical ST-ZFTA subgroups, which led to many cell states being sample- or subgroup-specific. These limitations highlight the need for a comprehensive study examining the composition of malignant cell states of tumours across all subgroups of ST-EPNs. In addition to the cell-intrinsic properties, it has become clear how cancer cell diversity is further influenced by their microenvironment, where interactions with adjacent cells and non-genetic factors augment cellular variability and malignant phenotypes 12 . Recent advances in spatial transcriptomics have overcome the loss of spatial information in scRNA-seq analyses, thereby revealing the intricate ecosystem within various tumours 13 , 14 . Moreover, live-cell imaging has proven to be effective for capturing the dynamic nature of tumour cells within their microenvironment while preserving cell viability, morphology and function 15 . Such multilayered analyses linking cell state to microenvironmental influences, dynamic morphology and behaviour of malignant cells have yet to be carried out in ST-EPN tumours. Here we integrate sc/snRNA-seq, spatial transcriptomics, and in vitro and in vivo live-cell imaging to provide a multidimensional framework to characterize tumour heterogeneity in ST-EPNs. Taken together, we identify the extensive tumour heterogeneity in ST-EPNs by shedding light on their developmental cell states, patterns of spatial localization, and cellular morphology and behaviour, thereby opening potential avenues for therapeutic interventions. Transcriptionally distinct ST-EPN subgroups To comprehensively characterize the cellular tumour heterogeneity of ST-EPNs, we compiled a patient cohort of 42 patient tumour samples from 34 patients for single-cell transcriptomics, encompassing ZFTA-RELA ( n = 17), ZFTA