Human samples and data
For all human tissue and cell studies, informed consent was obtained, and tissue was used in accordance with protocols approved by the Stanford University Institutional Review Board.
Mice and housing conditions
All in vivo experiments were conducted in accordance with protocols approved by the Stanford University Institutional Animal Care and Use Committee (IACUC) and performed in accordance with institutional guidelines. The IACUC implements regulations from the US Department of Agriculture (USDA), the Public Health Service (PHS) policy, California State Regulations and Stanford University Polices and Guidelines to ensure effective and ethical animal research programmes. Animals were housed according to standard guidelines with free access to food and water in a 12-h light–12-h dark cycle, a temperature of 21 °C and 60% humidity. For brain tumour xenograft experiments, the IACUC does not set a limit on maximal tumour volume but rather on indications of morbidity. In no experiments were these limits exceeded as mice were euthanized if they exhibited signs of neurological morbidity or if they lost 15% or more of their body weight.
Orthotopic xenografting and allografting
For all xenograft studies, NSG mice (NOD-SCID-IL2Rγ chain-deficient; The Jackson Laboratory) were used. Male and female mice were used equally. A single-cell suspension from cultured SU-DIPG-VI–GFP, SU-DIPG-XIII-FL–GFP, SU-DIPG-50–GFP, SU-pcGBM2–GFP or SU-DIPG-XIII-pons patient-derived glioma cells3,47 were prepared in sterile PBS immediately before the xenograft procedure. Animals at postnatal day 28–30 (P28–30) were anaesthetized with 1–4% isoflurane and placed in a stereotactic apparatus. The cranium was exposed via midline incision under aseptic conditions. Approximately 300,000 cells in 3 µl sterile PBS were stereotactically implanted through a 26-gauge burr hole, using a digital pump at an infusion rate of 0.4 µl min−1 and 26-gauge Hamilton syringe. For all electrophysiology and optogenetics experiments, cells were implanted into the CA1 region of the hippocampus (1.5 mm lateral to midline, −1.8 mm posterior to bregma and −1.4 mm deep to the cranial surface). SU-DIPG-XIII-FL–GFP and SU-pcGBM2–GFP for lorazepam treatments were xenografted into the premotor cortex (0.5 mm lateral to midline, 1.0 mm anterior to bregma and −1.75 mm deep to the cranial surface). For pontine xenografting, coordinates were 1.0 mm lateral to midline, −0.8 mm posterior to lambda and −5.0 mm deep to the cranial surface. At the completion of infusion, the syringe needle was allowed to remain in place for a minimum of 2 min, then manually withdrawn at a rate of 0.875 mm min−1 to minimize backflow of the injected cell suspension. The patient-derived glioma cells used were routinely authenticated using short tandem repeat (STR) fingerprinting to verify identity and lack of contamination.
In vivo bioluminescence imaging
For in vivo monitoring of tumour growth, bioluminescence imaging was performed using an in vivo bioluminescent imaging system (Xenogen). Mice were orthotopically xenografted with firefly luciferase-expressing H3K27M-mutant DMG cells (SU-DIPG-VI cell xenografted to the pons)30. For imaging of tumour burden, mice were placed under isofluorane anaesthesia, injected with luciferin substrate and imaged. Animals were imaged at baseline and randomized based on tumour size by an investigator blinded to experimental conditions, so that experimental groups contained an equivalent range of tumour sizes. All total flux values were then normalized to baseline values to determine the fold change of tumour growth.
Mouse lorazepam treatment studies
For histological analyses of tumour proliferation, NSG mice were xenografted as above with SU-DIPG-VI–GFP, SU-DIPG-XIII-FL–GFP, SU-DIPG-50–GFP or SU-pcGBM2–GFP cells and randomized to treatment group by an investigator blinded to experimental conditions. Four-to-six weeks post-xenograft, mice were treated with systemic administration of lorazepam (8 mg kg−1 or 2 mg kg−1 daily; Hospira) via intraperitoneal injection for 4 weeks (5 days per week). Controls were treated with an identical volume of the relevant vehicle.
For analysis of tumour growth, bioluminescence imaging was performed as described above for mice bearing SU-DIPG-VI–GFP-luciferase cells xenografted into the pons before treatment and at 14 days after starting treatment with lorazepam. Tumour burden was assessed as the fold change in change in luminescence from baseline.
For the lorazepam treatment survival study, SU-DIPG-XIII-pons cells were xenografted to the pons as above, and lorazepam was administered (low dose of 2 mg kg−1 intraperitoneal, high dose of 8 mg kg−1 intraperitoneal, or vehicle control) beginning 3 days after xenografting and administered 5 days a week for 28 days. Morbidity criteria used were either reduction of weight by 15% initial weight or severe neurological motor deficits consistent with brainstem dysfunction (that is, hemiplegia or an incessant stereotyped circling behaviour seen with ventral midbrain dysfunction). Kaplan–Meier survival analysis using log-rank testing was performed to determine statistical significance. Of note, mice tolerated both high-dose and low-dose lorazepam regimens, without becoming over-sedated. With the higher dose (8 mg kg−1), mice initially exhibited decreased activity, but appeared to become acclimated to the dose within a couple of days and then behaved normally in their cages. When mice died, which occurred after the 28-day period of lorazepam administration, they exhibited symptoms of increased tumour burden (motor and balance symptoms as typically seen with brainstem tumour disease progression).
Patient-derived and mouse model-derived cell culture
High-grade glioma cultures SU-DIPG-VI, SU-DIPG-XIII-FL, SU-DIPG-50, SU-pcGBM2 and SU-DIPG-XIII-pons were generated as previously described13. In brief, tissue was obtained from high-grade glioma (WHO grade III or IV) tumours at the time of biopsy or from early post-mortem donations in accordance with Institutional Review Board-approved protocols. Tissue was dissociated both mechanically and enzymatically and grown in a defined, serum-free medium designated ‘tumour stem media’, consisting of neurobasal(-A) (Invitrogen), B27(-A) (Invitrogen), human bFGF (20 ng ml−1; Shenandoah), human EGF (20 ng ml−1; Shenandoah), human PDGF-AA (10 ng ml−1) and PDGF-BB (10 ng ml−1; Shenandoah) and heparin (2 μg ml−1; Stem Cell Technologies). For all patient-derived cultures, mycoplasma testing was routinely performed, and STR DNA fingerprinting was performed every 3 months to verify authenticity. The STR fingerprints and clinical characteristics for the patient-derived cultures and xenograft models used have been previously reported48.
Single-cell sequencing analysis
Data curation and preprocessing
We combined publicly available single-cell datasets from ten adult IDH-mutant glioma samples, three adult WT IDH glioma samples, six paediatric H3K27M-mutant DMG samples as well as the single-cell transcriptome of patient-derived SU-DIPG-VI and SU-DIPG-XIII-FL cells15,32. For all analyses of these datasets, R studio 1.4.1106-5 was used. Cell filtering was conducted similarly to the original studies, resulting in the retention of 5,096 IDH-mutant glioma malignant cells, 599 adult WT IDH glioma malignant cells and 2,259 paediatric H3K27M+ DMG malignant cells.
For most analyses, transcripts per million (TPM) entries in the matrix were normalized using the formula \(E={\log }_{2}\left(\frac{{\rm{TPM}}}{10}+1\right)\). The values were divided by 10 because the actual complexity is assumed to be around 100,000 rather than 1 million, as implied by the TPM measure. After normalization, the data were centred by subtracting the mean expression of each gene across all cells.
Before dimensionality reduction, we retained the 7,000 genes with the highest mean expression across all cells in the study. We then computed the UMAP x and y values using the top 100 principal components (Extended Data Fig. 3).
GABAergic synapse-related genes
GABAARα genes included: GABRA1, GABRA2, GABRA3, GABRA4, GABRA5 and GABRA6. GABAARβ genes included: GABRB1, GABRB2 and GABRB3. GABAARγ genes included: GABRG1, GABRG2 and GABRG3.
Total GABAAR genes included: GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6, GABRB1, GABRB2, GABRB3, GABRG1, GABRG2, GABRG3, GABRD, GABRE, GABRP, GABRQ, GABRR1, GABRR2 and GABRR3.
Total GABAergic synapse-related genes included: GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6, GABRB1, GABRB2, GABRB3, GABRG1, GABRG2, GABRG3, GABRD, GABRE, GABRP, GABRQ, GABRR1, GABRR2, GABRR3, GPHN, ARHGEF9, NLGN2, SLC12A2 and SLC12A5.
Violin plots
For each sample, we performed first cell-level normalization, and then centred the gene expression around 0 to allow principal component analysis (PCA) computation. Following the PCA reduction, we clustered the cells using shared nearest neighbour clustering. To examine the various GABAAR signatures of each of the cells in each cluster, we used the function AddModuleScore by the Seurat package, which calculates the average expression levels of the gene set subtracted by the aggregated expression of 100 randomly chosen control gene sets, where the control gene sets are chosen from matching 25 expression bins corresponding to the tested gene set expression. To calculate the P values, we pseudobulked the cells from each sample to generate average gene expression levels per sample using AggregateExpression by the Seurat package. The P values were calculated using Wilcoxon rank-sum test by stat_compare_means by the ggpubr package.
Differentiation versus stemness analysis
In the H3K27M+ DMG dataset15, we computed a ‘stemness score’ for each cell. This score was determined by subtracting the higher of either the OC (oligodendrocyte-like) score or the AC (astrocyte-like) score of a cell from its OPC-like score. We also calculated a ‘differentiation score’ for each cell, defined as the maximum of the OC and AC scores. If the AC score was higher, it was multiplied by −1. In cases in which both AC and OC scores were negative, a value of 0 was assigned, with some added jitter. Centring was performed across all samples, and cells were scored based on the genes highlighted in Fig. 1c.
TagGFP2–GABRG2 cloning and generation of reporter glioma cells
The codon-optimized GABRG2 sequence, with TagGFP2 inserted at the N terminus, was custom synthesized as a gblock (IDT DNA). Subsequently, it was cloned into a lentiviral vector under the Ef1a promoter. The resulting plasmid was transformed into Top10 competent cells (Thermo Fisher) and subjected to sequence verification. Following confirmation of the correct sequence, the plasmid was maxiprepped (Qiagen), yielding a concentration ranging from 1 to 2 μg μl−1. Once cloned, the plasmid was packaged together with helper plasmids (pΔ8,9 and VSV-g) to generate replication-deficient lentivirus from adherent HEK293T cells (Thermo Fisher). One million target SU-DIPG-VI and SU-DIPG-XIII-FL cells were infected with Lenti-X (Takara) concentrated viral supernatant and allowed to recover for at least 2 weeks.
Immuno-electron microscopy
Eight weeks after xenografting, mice were euthanized by transcardial perfusion with Karnovsky’s fixative: 2% glutaraldehyde (EMS 16000) and 4% paraformaldehyde (EMS 15700) in 0.1 M sodium cacodylate (EMS 12300), pH 7.4. Transmission electron microscopy was performed in the xenograft tumour mass within the CA1 region of the hippocampus. The samples were post-fixed in 1% osmium tetroxide (EMS 19100) at room temperature for 1 h, washed three times with ultrafiltered water, and then en bloc stained at room temperature for 2 h. The samples were then dehydrated in graded ethanol (50%, 75% and 95%) for 15 min each at 4 °C. The samples were then allowed to equilibrate to room temperature and rinsed in 100% ethanol twice, followed by acetonitrile for 15 min. The samples were infiltrated with EMbed-812 resin (EMS 14120) mixed 1:1 with acetonitrile for 2 h, followed by EMbed-812 mixed 2:1 with acetonitrile for 2 h, and then in Embed-812 for 2 h. The samples were then placed into TAAB capsules with fresh resin and kept overnight at 65 °C. Sections between 40 nm and 60 nm were taken on an Ultracut S (Leica) and mounted on 100-mesh Ni grids (EMS FCF100-Ni). For immunohistochemistry, microetching was done with 10% periodic acid and eluting of osmium with 10% sodium metaperiodate for 15 min at room temperature on parafilm. Grids were rinsed with water three times, followed by 0.5 M glycine quench, and then incubated in blocking solution (0.5% BSA and 0.5% ovalbumin in PBST) at room temperature for 20 min. Primary rabbit anti-GFP (1:300; MBL International) was diluted in the same blocking solution and incubated overnight at 4 °C. The following day, grids were rinsed in PBS three times, and incubated in secondary antibody (1:10 10-nm gold-conjugated IgG TED Pella15732) for 1 h at room temperature and rinsed with PBST followed by water. For each staining set, secondary-only staining was simultaneously performed to control for any nonspecific binding. Grids were contrast stained for 30 s in 3.5% uranyl acetate in 50% acetone followed by staining in 0.2% lead citrate for 90 s. Samples were imaged using a JEOL JEM-1400 transmission electron microscopy (TEM) at 120 kV and images were collected using a Gatan Orius digital camera.
Sections of hippocampal xenografts of SU-DIPG-VI and SU-DIPG-XIII-FL with GFP-tagged GABRG2 were imaged using TEM imaging. In total, 78 images of SU-DIPG-VI xenografts from 4 mice and 137 images of SU-DIPG-XIII-FL from 3 mice were assessed. Electron microscopy images were taken at ×60,000, ×80,000 or ×12,000. GABAergic neuron-to-glioma synapses were defined as those with unequivocal identification of immunogold particle labelling with three or more particles adjacent to the postsynaptic membrane, in addition to the presence of synaptic vesicle clusters and a visually apparent synaptic cleft.
Neuron-glioma co-culture experiments
Neurons were isolated from the cortices of P0 Sprague-Dawley rat pups of either sex using the Neural Tissue Dissociation Kit–Postnatal Neurons (Miltenyi) and ACK Lysing Buffer (Gibco), followed by the Neuron Isolation Kit, Mouse (Miltenyi) per the manufacturers’ instructions. After isolation, 80,000 neurons were plated onto circular glass coverslips (Electron Microscopy Services) pre-treated for 1 h at 37 °C with poly-l-lysine (Sigma) and then 2 h at 37 °C with 5 µg ml−1 mouse laminin (Thermo Fisher). Neurons were cultured in BrainPhys Neuronal Medium (StemCell Technologies) supplemented with B-27 (Invitrogen), 1× GlutaMAX (Invitrogen), penicillin–streptomycin (Invitrogen), 55 µM 2-mercaptoethanol (Gibco), GDNF (5 ng ml−1; Shenandoah), BDNF (10 ng ml−1; Shenandoah) and TRO19622 (5 µM; Tocris). Half of the medium was replenished on 1 and 3 days in vitro. On 5 days in vitro, half of the medium was replaced in the morning. In the afternoon, 60,000 glioma cells expressing TagGFP2–GABRG2 were plated onto the neurons in half of the conditioned medium. Glioma cells were cultured with neurons for 3 days, and then fixed with 4% paraformaldehyde for 15 min at room temperature and stained for immunofluorescence analysis as described below. The patient-derived glioma cells used were routinely authenticated using STR fingerprinting to verify identity and lack of contamination.
Slice preparation for electrophysiology
Coronal slices (300 µm thick) containing the hippocampal region were prepared from mice (at least 8 weeks after xenografting) in accordance with a protocol approved by Stanford University IACUC. After rapid decapitation, the brain was removed from the skull and immersed in ice‐cold slicing artificial cerebrospinal fluid (ACSF) containing: 125 mM NaCl, 2.5 mM KCl, 25 mM glucose, 25 mM NaHCO3, 1.25 mM NaH2PO4, 3 mM MgCl2 and 0.1 mM CaCl2. After cutting, slices were incubated for 30 min in warm (30 °C) oxygenated (95% O2 and 5% CO2) recovery ACSF containing: 100 mM NaCl, 2.5 mM KCl, 25 mM glucose, 25 mM NaHCO3, 1.25 mM NaH2PO4, 30 mM sucrose, 2 mM MgCl2 and 1 mM CaCl2 before being allowed to equilibrate at room temperature for an additional 30 min.
Electrophysiology
Slices were transferred to a recording chamber and perfused with oxygenated, warmed (28–30 °C) recording ACSF containing: 125 mM NaCl, 2.5 mM KCl, 25 mM glucose, 25 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM MgCl2 and 2 mM CaCl2. NBQX (10 µM) was perfused with the recording ACSF to prevent AMPAR-mediated currents in synaptic response experiments. Tetrodotoxin (0.5 µM) was perfused with the recording ACSF during GABA puff experiments to prevent neuronal action potential firing. Cells were visualized using a microscope equipped with DIC optics (Olympus BX51WI). Recording patch pipettes were filled with CsCl-based pipette solution containing: 150 mM CsCl, 5 mM EGTA, 1 mM MgCl2, 10 mM HEPES, 2 mM ATP and 0.3 mM GTP, pH 7.3. Patch electrodes had resistances of 4–5 MΩ. Pipette solution additionally contained Alexa 568 (50 μM) to visualize the cell through dye filling during whole-cell recordings, and recorded slices were post-fixed and stained to confirm cell type. Gramicidin A (60 μg ml−1) was added to the pipette solution for perforated patch recordings. A perforated patch was considered satisfactory when an access resistance of approximately 30 MΩ was obtained about 30 min after attaining gigaseal resistance. A leak of Alexa 568 dye from the pipette into the cell during perforated patch recordings indicated a damaged membrane, and the data from such recordings were discarded. EGABA was calculated for each individual cell and averaged. In the negative chloride load experiments and in neuronal recordings, recording patch pipettes were filled with CsMe-based pipette solution containing: 135 mM CsMeSO4, 12 mM HEPES, 8 mM NaCl, 0.25 mM EGTA, 2 mM MgCl2, 2 mM ATP, 0.3 mM GTP and 5 mM phosphocreatine, pH 7.3. Glioma cells were voltage clamped at a holding potential of −70 mV. For all whole-cell patch clamp experiments, series resistance was less than 30 MΩ. Synaptic responses were evoked with a bipolar electrode connected to an Iso-flex stimulus isolator (A.M.P.I.) placed 100–200 μm from the patched xenografted cells. A low-intensity stimulation, sufficient to evoke consistent responses but not higher, was used at a frequency of 0.1 Hz. GABA (1 mM) in recording ACSF was applied via a puff pipette, which was placed approximately 100 μm away from the patched cell and controlled by a Picospritzer II (Parker Hannifin Corp.). Optogenetic stimulation of interneurons was achieved with a 598-nm LED using a pE-4000 illumination system (CoolLED). Optogenetic stimulation of interneurons in acute hippocampus slices was performed with 5-ms pulses of blue light, in the absence of inhibitors such as NBQX. Signals were acquired with a MultiClamp 700B amplifier (Molecular Devices) and digitized at 10 kHz with an Axon Digidata 1550B (Molecular Devices) or an InstruTECH LIH 8 + 8 data acquisition device (HEKA). Data were recorded and analysed using pClamp 11 software suite (Molecular Devices), AxoGraph X (AxoGraph Scientific) and/or IGOR Pro 8 (Wavemetrics). For representative traces, stimulus artefacts preceding the synaptic responses have been removed for clarity. Intracellular chloride concentration was calculated using the Nernst equation. Rise time, decay time and synaptic delay were measured from responses to stimulation in SU-DIPG-VI cells in the presence of NBQX. Rise time was calculated as the time between 10% and 90% of the peak response, as measured from the discernible onset of the averaged response for each cell. Decay time was defined as the monoexponential decay time constant for the averaged response for each cell. Synaptic delay was measured by the time from the end of the 0.5-ms stimulus to the visually discernible beginning of the response.
Pharmacological agents
Drugs and toxins used for electrophysiology were picrotoxin (50 µM; Tocris), tetrodotoxin (0.5 µM; Tocris), NBQX (10 µM; Tocris), D-AP5 (100 µM; Tocris), bicuculline (10 μM, Tocris), bumetanide (10 µM or 100 μM; Tocris) and lorazepam (10 μM; Hospira). When used for in vitro slice application, drugs were made up as a stock in distilled water or dimethylsulfoxide (DMSO) and dissolved to their final concentrations in ACSF before exposure to slices. The final concentration of DMSO was less than 1%.
Viral injection and fibre optic placement
Animals were anaesthetized with 1–4% isoflurane and placed in a stereotaxic apparatus. For optogenetic stimulation experiments, 1 µl of AAV8-DLX5/6–ChRmine::mCherry49 (virus titre of 1.19 × 1012; a gift from K. Deisseroth at Stanford University) was unilaterally injected using Hamilton Neurosyringe and Stoelting stereotaxic injector over 5 min. The viral vector was injected into the hippocampal CA1 region in the right hemisphere at coordinates: 1.5 mm lateral to midline, −1.8 mm posterior to bregma and −1.3 mm deep to the cranial surface. Two weeks following the viral injection, SU-DIPG-XIII-FL cells were xenografted as described above. After 7 weeks of tumour engraftment, an optic ferrule was placed above the CA1 of the hippocampus of the right hemisphere at: 1.5 mm lateral to midline, −1.8 mm posterior to bregma and −1.25 mm deep to the cranial surface.
Optogenetic stimulation
Optogenetic stimulations were performed at least 10 weeks after the viral vector delivery, 8 weeks after xenografts and 1 week after optic ferrule implantation. Freely moving animals were connected to a 595-nm fibre-coupled LED laser system with a monofibre patch cord. Optogenetic stimulation was performed with cycles of 595-nm light pulses at 40 Hz frequency, 10-ms light pulse width and a light power output of 10–15 mW from the tip of the optic fibre, which lasted for 30 s, followed by 90 s recovery over a 30-min period. Animals were injected intraperitoneally with 40 mg kg−1 EdU (5-ethynyl-2′-deoxyuridine; E10187, Invitrogen) before the session, and were perfused 24 h after optogenetic stimulation for proliferation assays and 90 min after optogenetic stimulation for Fos expression in interneurons.
Perfusion and immunohistochemistry
Animals were anaesthetized with intraperitoneal avertin (tribromoethanol), then transcardially perfused with 20 ml of PBS. Brains were fixed in 4% paraformaldehyde overnight at 4 °C and then transferred to 30% sucrose for cryoprotection. Brains were then embedded in Tissue-Tek O.C.T. (Sakura) and sectioned in the coronal plane at 40 µm using a sliding microtome (Microm HM450; Thermo Scientific).
For immunohistochemistry, coronal sections were incubated in blocking solution (3% normal donkey serum, 0.3% Triton X-100 in TBS) at room temperature for 1–2 h. Chicken anti-GFP (1:500; Abcam), mouse anti-human nuclei clone 235-1 (1:100; Millipore), rabbit anti-Ki67 (1:500; Abcam), guinea pig anti-VGAT (1:500; Synaptic Systems), mouse anti-gephyrin (1:500; Synaptic Systems), rabbit anti-GAD65 (1:500; Abcam), mouse anti-GAD67 (1:500; Millipore Sigma), rabbit anti-H3K27M (1:500; Millipore) or mouse anti-Fos (1:500; Santa Cruz Biotechnology) were diluted in antibody diluent solution (1% normal donkey serum in 0.3% Triton X-100 in TBS) and incubated 24–36 h at 4 °C. Sections were then rinsed three times in TBS and incubated in secondary antibody solution containing Alexa 488 donkey anti-chicken IgG, Alexa 488 donkey anti-rabbit IgG, Alexa 594 donkey anti-mouse IgG, Alexa 594 donkey anti-rabbit IgG, Alexa 647 donkey anti-mouse IgG, Alexa 647 donkey anti-rabbit IgG and DyLight 405 AffiniPure donkey anti-guinea pig IgG used at 1:500 (Jackson Immuno Research) in antibody diluent at 4 °C for 2 h. Sections were rinsed three times in TBS and mounted with ProLong Gold Mounting medium (Life Technologies).
Human autopsy sample immunohistochemistry for H3K27M antigen was performed on 5-µm sections of FFPE samples in the Stanford University clinical pathology laboratory according to CLIA-certified protocols and counterstained with haemotoxylin.
Immunocytochemistry
For immunocytochemistry, fixed coverslips were incubated in blocking solution (3% normal donkey serum and 0.3% Triton X-100 in TBS) at room temperature for 30 min. Primary antibodies chicken anti-neurofilament (H+M; 1:200; Aves Labs), guinea pig anti-synapsin1/2 (1:500; Synaptic Systems), mouse anti-nestin (1:500; Abcam) and rabbit anti-GFP (1:500; Novus Biologicals) were diluted in the antibody diluent solution (1% normal donkey serum in TBS) and incubated overnight at 4 °C. Coverslips were washed three times with TBS and incubated with secondary antibodies Alexa 594 donkey anti-chicken IgY (1:500; Jackson ImmunoResearch), DyLight 405 donkey anti-guinea pig IgG (1:500; Jackson ImmunoResearch), Alexa 647 donkey anti-mouse IgG (1:1,000; Jackson ImmunoResearch) and Alexa 488 donkey anti-rabbit IgG (1:1,000; Jackson ImmunoResearch) in the antibody diluent solution for 45 min at room temperature. Coverslips were washed three times with TBS and mounted with ProLong Gold Antifade Mountant (Invitrogen).
Confocal imaging
Images were acquired using a ×63 oil immersion objective (synaptic puncta imaging), ×40 oil immersion objective or ×20 air objective of a Zeiss LSM700, Zeiss LSM800 or Zeiss LSM980 scanning confocal microscope and Zen imaging software (v8.1; Carl Zeiss). 3D image reconstructions were processed using IMARIS (v10.0.0) software. Cell quantification within xenografts was performed by an investigator blinded to experimental conditions.
Quantification of cell proliferation
Confocal images were analysed and quantified using ImageJ (v2.1.0/1.53c). For Ki67 analysis, 3 fields for quantification were selected from each of 3 consecutive sections in a 1-in-6 series of 40-μm coronal sections with respect to overall tumour burden. Within each field, all human nuclear antigen (HNA)-positive and GFP-positive tumour cells were quantified to determine tumour burden within the areas quantified. HNA-positive cells were then assessed for co-labelling with Ki67. To calculate the proliferation index (the percentage of proliferating tumour cells for each mouse), the total number of HNA-positive cells co-labelled with Ki67 across all areas quantified was divided by the total number of cells counted across all areas quantified (Ki67+/HNA+).
Unbiased stereology
GABAergic interneurons immunopositive for GAD67 were visualized with a MBF Zeiss Axiocam light microscope. Only those cells for which GAD67 unequivocally marked the soma were counted. HNA was used to mark xenografted patient-derived DMG cells. Cell numbers were determined through unbiased stereology using Stereo Investigator software (MBF Bioscience, version 2023). Frontal cortex regions of interest were defined in sections with a visible corpus callosum by drawing a vertical line from the tip of the cingulum to the brain surface, and a horizontal line from this point towards the midline. The end point of these lines was then connected along the cortical edge to delineate the frontal cortex, including the medial prefrontal cortex and the motor cortex. This method was consistently applied across all sections. Sections were traced at ×2.5 magnification and images were acquired at ×40 from 1 in 6 serial sections throughout the region of interest (3–4 sections per animal). Stereological parameters were determined ensuring that at least 100–300 cells would be counted per animal and the Gunderson m = 1 coefficient of error was less than 0.1. The sampling grid size was set to 250 × 250 μm; the counting frame was set to 100 × 100 μm. Exposure time was kept uniform across all samples imaged within each experiment.
EdU incorporation assay
DMG tumour neurosphere cultures SU-DIPG-VI, SU-DIPG-XIII-FL and SU-DIPG-50 were generated as previously described3,7 from early post-mortem tissue donations and grown as tumour neurospheres in defined, serum-free tumour stem media, consisting of 1:1 mixture of neurobasal(-A) (Invitrogen) and D-MEM/F-12 (Invitrogen), HEPES buffer (Invitrogen), MEM sodium pyruvate (Invitrogen), MEM non-essential amino acids (Invitrogen), GlutaMAX-1 supplement (Invitrogen), B27(-A) (Invitrogen), human bFGF (20 ng ml−1; Shenandoah), human EGF (20 ng ml−1; Shenandoah), human PDGF-AA (10 ng ml−1; Shenandoah), PDGF-BB (10 ng ml−1; Shenandoah) and heparin (2 ng ml−1; Stem Cell Technologies).
One hundred thousand glioma cells were plated onto circular glass coverslips (Electron Microscopy Services) pre-treated for 1 h at 37 °C with poly-l-lysine (Sigma) and then 1 h at 37 °C with 10 µg ml−1 natural mouse laminin (Thermo Fisher). Dimethyl sulfoxide (Sigma-Aldrich) or drugs at the concentrations indicated (dissolved in dimethyl sulfoxide) were added to the coverslips. EdU (10 μM) was added to each coverslip. Cells were fixed after 24 h using 4% paraformaldehyde in PBS and stained using the Click-iT EdU kit and protocol (Invitrogen). The proliferation index was then determined by quantifying the fraction of EdU-labelled cells to DAPI-labelled cells using confocal microscopy.
Statistical analyses
Statistical tests were conducted using Prism (v9.1.0; GraphPad) software. Gaussian distribution was confirmed by the Shapiro–Wilk normality test. For parametric data, unpaired two-tailed Student’s t-tests or one-way ANOVA with Dunnett’s post-hoc test to examine pairwise differences and/or test for linear contrast were used as indicated in figure legends. Paired two-tailed Student’s t-tests or repeated measures one-way ANOVA with Dunnett’s post-hoc analysis were used in electrophysiological experiments within the same cell. Simple linear regression analysis was used to determine the x intercept in the current–voltage relationship experiments. Two-tailed log-rank analyses were used to analyse statistical significance of Kaplan–Meier survival curves. Statistical test results are reported in the figure legends and in Supplementary Table 1. At least three mice for in vivo experiments and at least three independent coverslips for in vitro experiments were used per test group to attain 80% power to detect an effect size of 20% at a significance level of 0.05. Statistical analyses of retrospective patient data are described above.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
