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Medulloblastoma with extensive nodularity (MBEN) are cerebellar tumors with two histologically distinct compartments and varying disease course. In some children MBEN progresses, while others show spontaneous differentiation into more benign tumors. However, the mechanisms that control the tug-of-war between proliferation and differentiation are not well understood. Here, we dissected this process with a multi-modal single cell transcriptome analysis. We found that the internodular MBEN compartment comprised proliferating early cerebellar granular neuronal precursors (CGNP)-like tumor cells as well as stromal, vascular, and immune cells. In contrast, the nodular compartment consisted of postmitotic, neuronally differentiated MBEN cells. Both compartments were connected through an intermediate cell stage of actively migrating CGNPs. Furthermore, astrocyte-like tumor cells were identified that had branched off the main CGNP developmental trajectory. Cells with an astroglial phenotype were found in close proximity to migrating, late CGNP-like and postmitotic neuronally differentiated cells. Our study reveals how the spatial tissue organization is linked to the developmental trajectory of proliferating tumor cells through a migrating precursor stage into differentiated tumor cells with a more benign phenotype. We anticipate that our framework for integrating single nucleus RNA-sequencing and spatial transcriptomics will help to uncover intercompartmental interactions also in other cancers with varying histology.

Ion mobility spectrometry (IMS) represents a considerable asset for analytics of complex samples as it allows for rapid mass spectrometric separation of compounds. IMS is even more useful for the separation of isobaric compounds when classical separation methods such as liquid chromatography or electrophoresis cannot be used, e.g., during matrix-assisted laser desorption/ionization (MALDI) analyses of biological surfaces. In the present study, we proved the usefulness of IMS for pharmacological applications of MALDI analyses on tissue sections. To illustrate our proof-of-concept, we used the anthelmintic drug mebendazole (MBZ) as a model. Using this exemplary drug, we demonstrated the possibility of using ion mobility to discriminate a drug in tissues from the biological background that masked its signal at low concentrations. In this proof-of-concept, the IMS mode together with the use of a profiling approach for sample preparation enabled quantification of the model drug MBZ from tissue sections in the concentration range 5 to 5,000 ng/g and with a limit of detection of 1 ng/g of tissue, within 2 h. This study highlights the importance of IMS as a separation method for on-surface quantification of drugs in tissue sections.

Background Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive pediatric brain tumors that are characterized by a recurrent mutation (K27M) within the histone H3 encoding genes H3F3A and HIST1H3A/B/C. These mutations have been shown to induce a global reduction in the repressive histone modification H3K27me3, which together with widespread changes in DNA methylation patterns results in an extensive transcriptional reprogramming hampering the identification of single therapeutic targets based on a molecular rationale. Methods We applied a large-scale gene knockdown approach using a pooled short hairpin (sh)RNA library in combination with next-generation sequencing in order to identify DIPG-specific vulnerabilities. The therapeutic potential of specific inhibitors of candidate targets was validated in a secondary drug screen. Results We identified fibroblast growth factor receptor (FGFR) signaling and the serine/threonine protein phosphatase 2A (PP2A) as top depleted hits in patient-derived DIPG cell cultures and validated their lethal potential by FGF ligand depletion and genetic knockdown of the PP2A structural subunit PPP2R1A. Further, pharmacological inhibition of FGFR and PP2A signaling through ponatinib and LB-100 treatment, respectively, exhibited strong tumor-specific anti-proliferative and apoptotic activity in cultured DIPG cells. Conclusions Our findings suggest FGFR and PP2A signaling as potential new therapeutic targets for the treatment of DIPGs.

BACKGROUND: A major challenge in the treatment of brain tumors is the limited penetration of many drugs through the blood-brain barrier (BBB). BBB characteristics include endothelial cells connected by tight junction proteins (e.g. Claudin5) and expression of efflux transporters (e.g.P-gp) with the physiological role to limit the accumulation of potentially toxic substances in brain tissue. While BBB permeability shows anatomical region-specific characteristics and changes with age, brain tumors can also impact its integrity. To achieve therapeutically relevant drug concentrations in the tumor, characterization of the pathophysiological BBB is fundamental when developing (pre)clinical trials. Our study seeks to increase our understanding of the BBB composition in various molecular groups of ependymoma. RESULTS: Transcriptional BBB characteristics were assessed for primary ependymoma (n=440), cell lines (n=3), mouse models (n=22), and healthy brain controls (n=200). T-distributed stochastic neighbor embedding (tSNE)-based clustering analyses based on most relevant tight junction and efflux transporter gene sets revealed distinct molecular ependymoma group-specific expression patterns. While patient-derived xenografts models (PDX, n=20) showed high similarity with patient tumor samples, in utero electroporation-based (IUE, n=2) mouse models did not fully recapitulate these BBB characteristics. Supratentorial ependymoma with ZFTA fusions revealed a higher transcriptional expression level of important tight junctions (e.g.Claudin5) compared to other groups and normal brain which was confirmed at protein level in respective PDX models by using both western blot and ultra-high content imaging. CONCLUSION: Analyses of important BBB markers revealed significant differences between molecular groups of ependymoma, which may partly explain drug resistance of ependymoma with ZFTA fusion caused by a low BBB permeability. BBB characteristics of corresponding models suggest that IUE and PDX models representing ZFTA fusion-driven tumors cannot equally be used within preclinical drug trials. Findings will be further validated in preclinical studies while molecular BBB characterization will be expanded to other brain tumors.

Medulloblastoma is a malignant childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH) subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs), and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional corepressor BCOR are recurrent and enriched in SHH medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a genetically engineered mouse model to delete exons 9/10 of Bcor (BcorΔE9–10) in GNPs during development. This mutation leads to reduced expression of C-terminally truncated BCOR (BCORΔE9–10). While BcorΔE9–10 alone did not promote tumorigenesis or affect GNP differentiation, BcorΔE9–10 combined with loss of the SHH receptor gene Ptch1 resulted in fully penetrant medulloblastomas. In Ptch1+/−;BcorΔE9–10 tumors, the growth factor gene Igf2 was aberrantly up-regulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/− GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/−;BcorΔE9–10 tumors. Overall, our data suggests that BCOR–PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.

Ependymal tumors (EPNs) account for ~10% of all pediatric brain tumors. Supratentorial EPN characterized by RELA fusions (ST-EPN-RELA) and posterior fossa EPN group A (PF-EPN-A) form the two most frequent molecular groups, both of which are associated with poor prognosis and for which only limited therapeutic options are available. Since pediatric EPNs have a relatively low mutational burden, identification and characterization of tumor-associated pathways and molecular processes is of critical importance to inform potential therapeutic targets. Previous transcriptional studies implicated aberrant vesicular pathways in ST-EPN-RELA, prompting further investigation into their putative role in EPN pathogenesis. To this aim, we isolated extracellular vesicles (EVs) of ST-EPN-RELA patient derived cell lines and performed protein mass spectrometry. The specific ST-EPN-RELA EV protein content resembles the parental cells as well as primary tumors. Promising candidates to be transferred by ST-EPN-RELA EVs but not control EVs were associated with unfolded protein response and endoplasmic reticulum stress. When uptaken by recipient cells of the tumor microenvironment, brain endothelial cells or microglia, ST-EPN-RELA EVs induced proliferation and had a chemoattractant effect towards the tumor. ST-EPN-RELA EVs stimulated angiogenesis of brain endothelial cells potentially by the transfer of ER stress proteins. Uptake of ST-EPN-RELA EVs by microglia changed their activation status indicating a tumor promoting function through EV transfer. Therefore, we hypothesize that vesicular pathways play an important role in the pathogenesis of pediatric ST-EPN-RELAs and that an improved understanding may promote new therapeutic opportunities.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive childhood tumor of the brainstem with currently no curative treatment available. The vast majority of DIPGs carry a histone H3 mutation leading to a lysine 27-to-methionine exchange (H3K27M). We engineered human induced pluripotent stem cells (iPSCs) to carry an inducible H3.3-K27M allele in the endogenous locus and studied the effects of the mutation in different disease-relevant neural cell types. H3.3-K27M upregulated bivalent promoter-associated developmental genes, producing diverse outcomes in different cell types. While being fatal for iPSCs, H3.3-K27M increased proliferation in neural stem cells (NSCs) and to a lesser extent in oligodendrocyte progenitor cells (OPCs). Only NSCs gave rise to tumors upon induction of H3.3-K27M and TP53 inactivation in an orthotopic xenograft model recapitulating human DIPGs. In NSCs, H3.3-K27M leads to maintained expression of stemness and proliferative genes and a premature activation of OPC programs that together may cause tumor initiation.