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Diffuse midline gliomas driven by lysine27-to-methionine mutations in histone 3 (H3-K27M DMGs) are among the most fatal brain tumors. Molecular studies including single cell RNA-sequencing (scRNA-seq) of pediatric and predominantly pontine H3-K27M DMGs have shown that the H3-K27M oncohistone keeps glioma cells locked in a stem-like oligodendrocyte precursor cell (OPC) state that is capable of self-renewal and tumor-initiation. However, a comprehensive dissection of the cellular architecture of H3-K27M DMGs across different midline regions and age groups is required to better understand the cell-intrinsic and contextual regulation of H3-K27M DMG cell identities. In particular, the more recently described group of adult H3-K27M DMGs remains understudied. Here, we have collected and characterized 45 H3-K27M mutant patient tumors, spanning pontine (n=26), thalamic (n=17), and spinal (n=2) locations. Median age at surgery was 12 (2-68) years, encompassing 21 early childhood (0-10 years), 12 adolescent (11-20 years), and 12 adult (≥ 21 years) tumors. The majority of samples were obtained pre-treatment (n=28), as opposed to post-treatment or at autopsy (n=17). We profiled all 45 tumors by single cell/single nucleus RNA-seq and selected tumors were further characterized by the single cell assay for transposase-accessible chromatin (scATAC-seq). Our integrated analyses highlight the predominance of transcriptionally and epigenetically defined OPC-like tumor cells as the main cell population of H3-K27M DMGs across all age groups and locations. We further identify distinct age- and location-specific OPC-like cell subpopulations. Comparison of pediatric and adult tumors further demonstrates a significant increase of mesenchymal cell states in adult H3-K27M DMGs, which we link to differences in glioma-associated immune cell compartments between age groups. Together, this study sheds light on the effects of age- and region-dependent microenvironments in shaping cellular identities in H3-K27M DMGs.

Tumor cell extensions called tumor microtubes (TMs) in glioma resemble neurites during neurodevelopment and connect glioma cells to a network that has considerable relevance for tumor progression and therapy resistance. The determination of interconnectivity in individual tumors has been challenging and the impact of tumor cell connectivity on patient survival remained unresolved so far. Here, a connectivity signature from single-cell RNA-sequenced (scRNA-Seq) xenografted primary glioblastoma (GB) cells was established and clinically validated. Thirty-four of 40 connectivity genes were related to neurogenesis, neural tube development or glioma progression, including the TM-network-relevant GAP43 gene. Astrocytic-like and mesenchymal-like GB cells had the highest connectivity signature scores in scRNA-Seq data of patient-derived xenografts and patient samples. In 230 human GBs, high connectivity correlated with the mesenchymal expression subtype, TP53 wildtype, and with dismal patient survival. CHI3L1 was identified as a robust molecular marker of connectivity. Thus, the connectivity signature allows novel insights into brain tumor biology, provides a proof-of-principle that tumor cell connectivity is relevant for patients’ prognosis, and serves as a robust biomarker that can be used for future clinical trials.Statement of significanceIntegration of GB cells into functional networks drives tumor progression and resistance. Here, we established and validated a novel connectivity gene expression signature of single GB cells and whole tumors that can be easily applied to clinical and preclinical samples. It is shown that connectivity is determining prognosis combining molecular, functional and clinical insights into the disease.

Histone 3 lysine27-to-methionine mutant diffuse midline gliomas (H3-K27M DMGs) are among the most lethal brain tumors. Their putative cellular hierarchy has been shown to be driven by self-renewing stem-like cells arrested in an oligodendrocyte precursor-like (OPC-like) state, of which few cells are able to differentiate towards more mature astrocyte (AC)-like and oligodendrocyte (OC)-like cells. However, the spatial organization underlying this tumor cell architecture and its microenvironmental interactions in intact H3-K27M DMG tissues remain unknown. Here, we profiled the single cell transcriptomes of 45 patient H3-K27M DMGs and derived cell population-specific marker gene combinations to characterize the single cell spatial organization of 16 tumors using targeted in situ sequencing. We thereby resolved different malignant and non-malignant cell populations including cycling, OPC-like, AC-like, OC-like, mesenchymal tumor cells, and non-malignant oligodendrocytes, astrocytes, neurons, myeloid cells, T cells, and vascular cells directly in situ. Global neighborhood analyses indicate a higher tendency of cycling OPC-like cells, vascular cells, and neurons to localize within a more restricted homogeneous compartment, whereas AC-like cells, non-malignant astrocytes and myeloid cells tend to intermingle with different cell populations in a more diffuse manner. Among malignant cells, we observed cycling OPC-like and OC-like cells to co-localize within a niche-like structure that is surrounded by more differentiated AC-like cells. We further validated this stem-like niche at the protein level using multiplexed immunofluorescence via the CODEX system. Finally, we characterized relationships between malignant and non-malignant cells, consistently identifying preferred neighborhoods of mesenchymal tumor cells with vascular and myeloid cells. Together, this study resolves the spatial architecture of H3-K27M DMG malignant and non-malignant cells at single cell resolution and identifies a local niche of the oligodendroglial lineage containing the OPC-like cancer stem-like cells, thus providing novel insights into the cancer stem-like compartment in H3-K27M DMGs and suggesting potential avenues for its perturbation.

BACKGROUND: Medulloblastoma with extensive nodularity (MBEN) represents a rare type of cerebellar tumors of infancy comprising two histologically distinct components that differ in cell differentiation and mitotic activity. Whereas some children suffering from MBEN experience disease recurrence, MBEN can also spontaneously differentiate and discontinue to grow. The underlying mechanisms of this variable biological behavior may offer insight into how embryonal tumors develop. METHODS: Fresh frozen and FFPE tumor tissue from nine MBEN-patients was subjected to multi-omics characterization including bulk sequencing, microdissection followed by RNA sequencing, single nucleus RNA-sequencing using the 10X Genomics- and SMART Seq. V2-protocols and spatial transcriptomics via RNAscope. RESULTS: All cases were molecularly classified as Sonic Hedgehog (SHH)-MB, and harbored somatic mutations within the SHH-pathway. After quality control, a total of ~30.000 cells were subjected to downstream analysis. Several non-malignant cell types, such as glial cells, were identified. In accordance with previous studies, we found only sparse immune infiltration. Unsupervised clustering identified cell clusters that differed in differentiation state and represented a continuum from embryonal-like cells with SHH-upregulation over intermediate cell states, to neuronal-like, postmitotic cells. Mapping to a single nucleus sequencing atlas of cerebellar development indicated that tumor cells reflected various stages of normally developing cerebellar granular precursors. Interestingly, one cluster of malignant cells with tumor-specific copy number alterations showed both transcriptomic features of astrocytes and embryonal cells. Using spatial transcriptomics, we were able to correlate different clusters of MBEN cells with distinct histologic MBEN compartments, with astrocyte-like tumor cells being located in the internodular compartment and in close proximity to mitotically active cancer cells. CONCLUSION: MBEN is formed by a continuum of malignant cell differentiation along the granular precursor lineage, with a subset of cells developing into cells that may represent tumor astrocytes. This differentiation process is reflected in the bicompartmental structure of MBEN.

Oncogenic fusion events involving c-MET have been observed in up to 12% of pediatric high-grade glioma (pHGG). MET inhibitors have displayed potent initial responses in MET rearranged tumors but acquired resistance to single agent modalities invariably occurs. To identify new treatment options against these tumors, we established two novel orthotopic mouse models including an immunocompetent, murine allograft and an intracranial patient-derived xenograft (PDX), both harboring distinct MET fusions. We analyzed the pharmacokinetic and pharmacodynamic profiles of two MET inhibitors, crizotinib and capmatinib, and examined their efficacy against tumor cell cultures derived from the aforementioned models. Capmatinib outperformed crizotinib in terms of specificity, potency and brain availability, resulting in a highly differential cellular response compared to crizotinib treatment. We evaluated the efficacy of both compounds in combination with radiotherapy (RT) and found that radiation further potentiated the inhibitory effect of capmatinib on tumor cell growth. We then utilized both models to assess the combinatorial effect of capmatinib and radiation on intracranial tumors in vivo and found that the combination therapy significantly increased overall survival in both cohorts. In the PDX model, the combination, relative to either intervention alone, induced a remarkable decrease of tumor burden, which persisted throughout the observation period in all treated animals. RNA-sequencing of capmatinib-treated tumors and tumor cell cultures revealed impaired expression of DNA repair genes. Further, we showed that capmatinib enhanced radiation-induced DNA damage, as demonstrated by increased γ-H2AX foci in treated cells, providing mechanistic insight for the cooperative effects of the combined treatment. Our results validate capmatinib as an effective inhibitor of MET in pHGG and demonstrate the outstanding efficacy of capmatinib and radiation against MET-driven pHGG in two complementary preclinical models, informing future clinical trials.

Recent advances allowing the genomic analysis of individual cells from a bulk population have provided intriguing new insights into areas such as developmental processes and tumor heterogeneity. Most approaches to date, however, rely on the availability of fresh surgical specimens, thereby dramatically reducing the ability to profile particularly rare tissue types. Pediatric central nervous system tumors – the leading cause of childhood cancer deaths – represent one such example, where often only frozen rather than native material is available. Due to an increasing need for advanced techniques to understand the heterogeneity of these tumors, we optimized a method to isolate intact nuclei from long-term frozen pediatric glioma tissues. We performed a technical comparison between different single nucleus RNA-sequencing (snRNA-seq) systems using a patient-derived xenograft model as a test sample. Further, we applied the established nucleus isolation method to analyze frozen primary tissue from two pediatric central nervous system tumors – one pilocytic astrocytoma and one glioblastoma – allowing the identification of distinct tumor cell populations and infiltrating microglia. The results show that our fast, simple and low-cost nuclear isolation protocol provides intact nuclei, which can be used in both droplet-based 3’ transcriptome amplification (10X Genomics) and plate-based whole transcriptome amplification (Fluidigm C1) single-cell sequencing platforms, thereby dramatically increasing the potential for application of such methods to rare entities.

Medulloblastoma (MB) is a highly malignant pediatric brain tumor originating from the cerebellum and brainstem. Identification of molecular subgroups forming this heterogeneous tumor entity was initially achieved from transcriptome characterization and further strengthened using DNA methylation profiling. While subgroup classification improved clinical diagnosis and treatment options, the lack of knowledge of the cell-of-origin for some of the subgroups hinders further treatment improvements. In addition identification of the precise cells of origin for each subgroup could help to understand tumor cell biology. Single cell sequencing is the optimal way to solve this task; recently, there were attempts to uncover putative MB cell-of-origin by using such information obtained from mouse embryonic cerebellum. However, such a comparative strategy can miss important results due to the differences between mouse and human. To solve this issue, we performed global single nucleus sequencing on human cerebellum pre- and postnatal materials across several developmental time points and generated transcriptome profiles from ~200k single cells. We identified known cell types forming the human cerebellum and performed detailed comparison of normal cells to RNA-seq bulk data from MB brain tumors across all subgroups. By selecting an optimal analysis strategy, we verified granule neuron precursors as cells of origin for the SHH MB subgroup. Additionally, we also found other cell types in conjunction with the remaining MB subgroups, suggesting new potential targets for investigation. Notably, this strategy can be further applied to the examination of other brain tumors and has perspectives in medical application.

Atypical teratoid/rhabdoid tumors (ATRT) represents a genomically homogeneous disease characterized by loss of SMARCB1 protein in the vast majority of cases. In recent years, it has become clear that these tumors display a high degree of intertumoral heterogeneity with three molecularly distinct subgroups. However, the degree of intratumoral heterogeneity and the information on cellular subpopulations currently remains largely an unchartered territory. To explore the transcriptomic composition of ATRTs, we performed single nuclei RNA sequencing for 16 ATRTs representing all three molecular subgroups (5 ATRT-TYR, 7 ATRT-SHH, 4 ATRT-MYC). By performing tSNE cluster analyses of all the single cell data (~50.000 cells have been sequenced), we were able to gain unprecedented insights into the phenotypic composition of ATRTs and unravelled substantial differences between the three subgroups. Integrating transcriptomic information from non-neoplastic brain cells and the data derived from single nuclei sequencing, we found an OPC like gene signature in ATRT-SHH. In contrast, ATRT-TYR subpopulations overexpressed more markers of neuronal stem cells suggesting a larger fraction of undifferentiated cells in this subgroup. We also identified a subpopulation of cells with a clear overexpression of cell cycle associated genes (CDK4, CDKN3), predominantly present in ATRT-MYC samples, a finding which may harbour important consequences for a targeted therapy with e.g. CDK inhibitors. In summary, our analyses reveal different cellular compartments in ATRT and provide important insights into the cellular differentiation of the three ATRT-subgroups. Further analyses to achieve a specific mapping of ATRT to its physiological cell of origin are currently being pursued.