Patient-derived glioma cell models
Table of Contents
Patient-derived glioma cultures were derived as described previously1,58 with informed consent under a protocol approved by the Stanford University Institutional Review Board (IRB). Patient-derived glioma models used include diffuse intrinsic pontine glioma (DIPG): SU-DIPG-VI, SU-DIPG-XIII-FL (derived from tumour in frontal lobe), SU-DIPG-XIII-P (tumour cultured from the pons of the same patient), SU-DIPG-21, SU-DIPG-25; thalamic DMG: QCTB-R059 and paediatric hemispheric glioblastoma: SU-pcGBM2. All cultures are monitored by short tandem repeat (STR) fingerprinting for authenticity throughout the culture period and mycoplasma testing was routinely performed. Characteristics of the glioma models used (patient information, molecular characterization, and other characteristics) have been previously reported1,59,60.
Glioma cultures are grown as neurospheres (unless otherwise stated) in serum-free medium consisting of DMEM (Invitrogen), Neurobasal(-A) (Invitrogen), B27(-A) (Invitrogen), heparin (2 ng ml−1), human-bFGF (20 ng ml−1) (Shenandoah Biotech), human-bEGF (20 ng ml−1) (Shenandoah Biotech), human-PDGF-AA (10 ng ml−1) (Shenandoah Biotech), human-PDGF-BB (10 ng ml−1) (Shenandoah Biotech). The spheres were dissociated using TrypLE (Gibco) for seeding of in vitro experiments.
Neuron–glioma co-culture
For synaptic puncta assays, neurons were isolated from CD1 mice (The Jackson Laboratory) at P0-P1 using he Neural Tissue Dissociation Kit—Postnatal Neurons (Miltenyi), and followed by the Neuron Isolation Kit, Mouse (Miltenyi) per manufacturer’s instructions. After isolation 200,000 neurons were plated onto circular glass coverslips (Electron Microscopy Services) pre-coated with poly-l-lysine (Sigma) and mouse laminin (Thermo Fisher Scientific) as described previously4. Neurons were cultured in BrainPhys neuronal medium containing B27 (Invitrogen), BDNF (10 ng ml−1, Shenandoah Biotech), GDNF (5 ng ml−1, Shenandoah Biotech), TRO19622 (5 µM; Tocris) and β- mercaptoethanol (Gibco). The medium was replenished on days in vitro (DIV) 1 and 3. On DIV 5, fresh medium was added containing 50,000 glioma cells expressing PSD95–RFP for synaptic puncta experiments and incubated for 72 h. The PSD95-RFP-expressing glioma culture (SU-DIPG-VI) was previously described4 and used to knockdown NTRK2 by shRNA. The SU-DIPG-VI wild-type and NTRK2-KO glioma cultures were transduced with the PSD95-RFP-PURO construct (see ‘Cloning constructs’). For EdU proliferation assays, 70,000 wild-type or NTRK2-KO glioma cells were plated and incubated for 48 h, before treatment with EdU (10 μM) with or without the AMPAR blocker NBQX (10 μM, Tocris) and incubated for a further 24 h. Following incubation, the cultures were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and stained for immunofluorescence analysis. For EdU analysis, cells were stained using the Click-iT EdU Cell Proliferation kit (Thermo Fisher Scientific, C10337), before staining with primary antibodies mouse anti-nestin (1:500; Abcam, ab6320) and chicken anti-neurofilament (M + H, 1:1000; Aves Labs, NFM and NFH), or mouse anti-human nuclei clone 235-1 (1:250; Millipore, MAB1281) and rabbit anti-microtubule-associated protein 2 (MAP2; 1:500, EMD Millipore, AB5622), overnight at 4 °C. Following washing, slips were incubated in secondary antibodies, Alexa 488 donkey anti-mouse pig IgG and Alexa 647 donkey anti-chicken IgG, or Alexa 488 donkey anti-mouse IgG and Alexa Fluor 647 donkey anti-rabbit (all 1:500, Jackson Immuno Research) and mounted using ProLong Gold Mounting medium (Life Technologies). Images were collected on a Zeiss LSM800, and proliferation index determined by quantifying percentage EdU-labelled glioma cells over total glioma cells (either nestin or HNA immunopositivity to identify glioma cells).
Mice and housing conditions
All animal 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. Animals were housed according to standard guidelines with unlimited access to water and food, under a 12 h light: 12 h dark cycle, a temperature of 21 °C and 60% humidity. For brain tumour xenograft experiments, the IACUC has a limit on indications of morbidity (as opposed to tumour volume). Under no circumstances did any of the experiments exceed the limits indicated and mice were immediately euthanized if they exhibited signs of neurological morbidity or if they lost 15% or more of their initial body weight.
For the Bdnf-TMKI mice (C57BL/6 J background), knock-in mutations in three calcium regulatory element binding sites in the Bdnf promoter 4: CaRE1, CaRE2 and CaRE3–CRE (gift from M. Greenberg32) were bred to Thy1::ChR2+/− mice (line 18, The Jackson Laboratory, C57BL/6 J background) to produce the Bdnf-TMKI; Thy1::ChR2+/− genotype. These mice were then intercrossed with NSG mice (NOD-SCID-IL2R gamma chain-deficient, The Jackson Laboratory) to produce a Bdnf-TMKI; Thy1::ChR2+/−; NSG genotype to facilitate to facilitate orthotopic xenografting.
Orthotopic xenografting
For all xenograft studies, NSG mice (NOD-SCID-IL2R-gamma chain-deficient, The Jackson Laboratory) were used. Male and female mice were used in cohorts equally. For electrophysiological, immuno-electron microscopy and calcium imaging experiments, a single-cell suspension from patient-derived DIPG cultures SU-DIPG-VI and SU-DIPG-XIII-FL was injected into the hippocampal region. For survival analysis and proliferation immunohistological assays, patient-derived DIPG cultures (SU-DIPG-VI and SU-DIPG-XIII-p*) were injected into the pontine region and for patient-derived H3WT paediatric cortical glioblastoma (SU-pcGBM2), cells were injected into the cortex. For optogenetic stimulation, SU-DIPG-VI cells were stereotactically injected into wild-type and Bdnf-TMKI mouse premotor (M2) frontal cortex; ChR2+ SU-DIPG-XIII-FL cells were similarly stereotactically injected into the M2 cortex. A single-cell suspension of all cultures was prepared in sterile culture medium (see ‘Cell culture’) immediately before surgery. Animals at P28–P35 were anaesthetized with 1–4% isoflurane and placed on stereotactic apparatus. Under sterile conditions, the cranium was exposed via a midline incision and a 31-gauge burr hole made at exact coordinates. For hippocampal injections the coordinates were as follows: 1.5 mm lateral to midline, 1.8 mm posterior to bregma, −1.4 deep to cranial surface. For pontine injections coordinates were: −1mm lateral to midline, 0.8 mm posterior to lambda, −5 deep to cranial surface. For cortical injections coordinates were: 1 mm anterior to bregma, 0.5 mm lateral to midline, 1.4 mm deep (for survival) and 1 mm deep (for optogenetics) to cranial surface. Cells were injected using a 31-gauge Hamilton syringe at an infusion rate of 0.4 µl min−1 with a digital pump. At completion of infusion, the syringe needle was allowed to remain in place for a minimum of 2 min, then manually withdrawn. The wound was closed using 3 M Vetbond (Thermo Fisher Scientific) and treated with Neo-Predef with Tetracaine Powder.
Fibre-optic placement and in vivo optogenetic stimulation
Experiments for the chronic neuronal optogenetic stimulation paradigm of glioma xenografts were performed as previously described1. In brief, a fibre-optic ferrule (Doric Lenses) was placed at M2 of the right hemisphere with the following coordinates: 1.0 mm anterior to bregma, 0.5 mm lateral to midline, −0.7 mm deep to the cranial surface at twelve weeks post glioma xenograft. Following seven days of recovery, all mice were connected to a 473 nm diode-pumped solid-state (DPSS) laser system via a mono fibre patch cord. In awake mice, pulses of light with a power measured at 10 mW were administered at a frequency of 20 Hz for a period of 30 s, followed by 90 s recovery in a repeated cycle for a total of 10 min per day, for 7 consecutive days. The mice were monitored for their unidirectional ambulation response to light stimulation, confirming correct ferrule placement over M2 of the right hemisphere and effective neuronal stimulation. Animals confirmed as ChR2-negative had no response to light stimulation. Mice were sacrificed 24 h following the final stimulation session. Experiments for the chronic optogenetic stimulation paradigm of ChR2+ glioma xenografts were performed as previously described4. In brief, ChR2+ (pLV-ef1-ChR2(H134R)-eYFP WPRE) or control (pLV-ef1-eYFP WPRE) constructs were lentivirally transduced into SU-DIPG-XIII-FL cells. Five weeks after tumour engraftment, a fibre-optic ferrule was placed as described above and previously1,4. After a 7-day recovery, the surgically placed ferrules were connected to a 100-mW 473-nm DPSS laser system and received either 5 ms or 25 ms pulses of light (~5 mW output, 3–30 mW cm−2 light density in the analysed region) at a frequency of 20 Hz over 30 s, followed by 90 s rest for a total of 10 min per day for 5 consecutive days. The mice were euthanized 24 h after the 5th stimulation.
Survival studies
For survival studies, mice were xenografted at 4 to 5 weeks of age with the cultures SU-DIPG-VI (wild type and NTRK2-KO), SU-pcGBM2 (wild type and NTRK2-KO) and SU-DIPG-XIII-P*. After xenografts, mice were continuously monitored for signs of neurological deficits or health decline. For inhibitor treatment, SU-DIPG-XIII-P* was treated with 120 mg kg−1 orally daily of entrectinib (HY-12678, MedChemExpress, 7% DMSO (Sigma), 10% Tween 80 (Sigma) in sterile H2O) for 14 days, starting at 2 weeks post-xenograft. Morbidity criteria were a 15% reduction in weight or severe neurological motor deficits consistent with brain dysfunction (brainstem tumours exhibited circling and barrel roles, cortical tumours displayed seizures and loss of gait). Statistical analyses were performed with Kaplan–Meier survival analysis using log rank testing.
Mouse drug treatment studies
For all drug studies to assess proliferation index of xenografted glioma cells, NSG mice were xenografted as above and blind randomized to a treatment group. Four weeks post-xenograft of SU-DIPG-VI wild-type or NTRK2-KO glioma cells, mice were treated with oral administration of the AMPAR blocker perampanel (5 mg kg−1; Adooq Biosciences; formulated in 10% DMSO, 60% PEG300, 30% water) via oral gavage for three weeks (5 days per week) and controls treated with equivalent volume of vehicle. Similarly, four weeks post-xenograft of SU-DIPG-VI wild-type or NTRK2-KO glioma cells, mice were treated with oral administration with the pan-Trk inhibitor entrectinib (120 mg kg−1; HY-12678, MedChemExpress, 7% DMSO (Sigma), 10% Tween 80 (Sigma) in sterile H2O) for 15 days and controls with equivalent volume of vehicle. For immunohistological analysis of glioma cell proliferation, mice were euthanized on the same day of the last drug dose.
Slice preparation for electrophysiology and calcium imaging experiments
Coronal slices (300 µm thick) containing the hippocampal region were prepared from mice (4–8 weeks after xenografting) in accordance with a protocol approved by Stanford University Institutional Animal Care and Use Committee (IACUC). After rapid decapitation, the brain was removed from the skull and immersed in ice‐cold slicing artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3 and 1.25 NaH2PO4, 3 MgCl2 and 0.1 CaCl2. After cutting, slices were incubated for 30 min in warm (30 °C) oxygenated (95% O2, 5% CO2) recovery ACSF containing (in mM): 100 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 30 sucrose, 2 MgCl2 and 1 CaCl2 before being allowed to equilibrate at room temperature for an additional 30 min.
Cerebral slice conditioned medium
Wild-type or Bdnf-TMKI mice expressing Thy1::Chr2 were used at 4–7 weeks of age. Brief exposure to isoflurane rendered the mice unconscious before immediate decapitation. Extracted brains (cerebrum) were placed in an oxygenated sucrose cutting solution and sliced at 350um as described previously1. The slices were placed in ACSF (see ‘Electrophysiology’) and allowed to recover for 30 min at 37 C and 30 min at room temperature. After recovery the slices were moved to fresh ACSF and stimulated using a blue-light LED using a microscope objective. The optogenetic stimulation paradigm was 20-Hz pulses of blue light for 30 s on, 90 s off over a period of 30 min. The surrounding conditioned medium was collected and used immediately or frozen at −80 °C for future use.
Preparation of cells for in vitro electrophysiology
A single-cell suspension of dissociated glioma cells (see ‘Cell culture’) was plated at a density of 20,000 cells per well of a 24-well plate containing glass coverslips (Electron Microscopy Services) pre-coated with poly-l-lysine (Sigma) and 5 μg ml−1 mouse laminin (Thermo Fisher Scientific). The medium was supplemented with B27(+A) (10 μl ml−1 serum-free medium plus growth factors; Invitrogen). Cells were incubated overnight prior to whole-cell recordings.
Electrophysiology
Slices were transferred to a recording chamber and perfused with oxygenated, warmed (28–30 °C) recording ACSF containing (in mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 1 MgCl2 and 2 CaCl2. Slices were visualized using a microscope equipped with DIC optics (Olympus BX51WI). Recording patch pipettes (2–3 MΩ) were filled with potassium gluconate-based pipette solution containing (in mM): 130 potassium gluconate, 20 KCl, 5 sodium phosphocreatine, 10 HEPES, 4 Mg-ATP, 0.3 GTP, and 50 μM Fluo-4, pH 7.3. Pipette solution additionally contained Alexa 568 (50 μM) to visualize the cell by dye-filling during whole-cell recordings. Glutamate (1 mM; Sigma) in recording ACSF was applied for a period of 250 ms via a puff pipette approximately 100 μm away from the patched cell and controlled by a Picospritzer II (Parker Hannifin). Tetrodotoxin (0.5 µM; Tocris) was perfused with the recording ACSF to prevent neuronal action potential firing in all glutamate puff experiments. Recombinant BDNF human protein (Peprotech, 450-02), or NLGN3 (OriGene Technologies, TP307955), was added to ACSF at 100 ng ml−1 and perfused for 30 min to test changes in response to glutamate puff or evoked stimulation. Other drugs used for electrophysiology were NBQX (10 µM; Tocris), AP-5 (100 µM; Tocris), and TBOA (200 µM; Tocris), KN-93 (10 µM; Tocris), KN-92 (10 µM; Tocris) and perfused for 2 h. 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. Synaptic responses were evoked with a bipolar electrode connected to an Iso-flex stimulus isolator (A.M.P.I.) placed in the strata radiatum. Signals were acquired with a MultiClamp 700B amplifier (Molecular Devices) and digitized at 10 kHz with an InstruTECH LIH 8 + 8 data acquisition device (HEKA).
For in vitro recordings, glioma cells expressing ChR2 were placed in an extracellular Tyrode medium (150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES pH 7.4, and 10 mM glucose). Borosilicate pipettes (Harvard Apparatus, with resistance of 4–6 mΩ) were filled with intracellular medium (140 mM potassium gluconate, 10 mM EGTA, 2 mM MgCl2 and 10 mM HEPES pH 7.2). After break-in, cells were held for at least 5 min before recording to ensure cell health and stability of the recording. Light was delivered with the Lumencor Spectra X Light engine with 470 nm for blue-light delivery, respectively. Light stimulation with 1.0 mW mm−2, 20 Hz light power density at varying light-pulse width (5 ms or 25 ms), and all recordings were performed in triplicate to ensure stable and reproducible data. Recordings were randomized in order across conditions to counterbalance for unknown variables.
Data were recorded and analysed using AxoGraph X (AxoGraph Scientific) and IGOR Pro 8 (Wavemetrics). For representative traces, stimulus artifacts preceding the synaptic currents have been removed for clarity.
Calcium imaging
SU-DIPG-VI and SU-DIPG-XIII-FL were transduced with lentivirus containing the genetically encoded calcium indicator GCaMP6s (pLV-ef1-GCAMP6s-P2A-nls-tdTomato) as described4. Cells were xenograft into the CA1 region of the hippocampus as described above.
Calcium imaging experiments performed on in situ SU-DIPG-XIII-FL xenograft slices were visualized using a microscope equipped with DIC optics (Olympus BX51WI). Excitation light was at 594 (for TdTomato) and 488 (for GCaMP6s) provided by pE-300 ULTRA (CoolLED). The recording software used was FlyCapture2 (Point Grey). Calcium imaging experiments performed on in situ SU-DIPG-VI xenograft slices were visualized using a Prairie Ultima XY upright two-photon microscope equipped with an Olympus LUM Plan FI W/IR-2 40x water immersion objective with Prairie View v5.6 software, as described4. A tunable Ti:Sapphire laser (Spectra Physics Mai Tai DeepSee) provided the excitation light (920 nm) for both tdTomato and GCaMP6s. PMTs were set to 750 V for each channel. These settings resulted in a power of approximately 30 mW at 920 nm. The emission filters wavelength ranges were for PMT1: 607 nm centre wavelength with 45 nm bandpass (full width at half maximum) and PMT: 525 nm with 70 nm bandpass (full width at half maximum). Image recordings were taken at 1 frame per second (1 Hz) over the course of the glutamate stimulation (approx. 1 min). Tumour cells located in the CA1 region of the hippocampus were identified via the expression for nuclear tdTomato. Slices were perfused with oxygenated aCSF, as described above, at a constant temperature of (28–30 °C) and containing (in mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 1 MgCl2 and 2 CaCl2. Tetrodotoxin (0.5 μM) was perfused with the recording ACSF to prevent neuronal action potential firing. Glutamate (1 mM) in recording ACSF was applied via a puff pipette (250 ms) approximately 100 μm away from the tdTomato-expressing cells and cells were stimulated three times, with approximately-2 min intervals, to ensure a reliable response. Glutamate solution contained Alexa 568 (50 μM) to visualize and confirm reliable glutamate puff (for representative images, the tdTomato nuclear signal displayed is prior to ACSF puff for all frames). Recombinant BDNF human protein (Peprotech, 450-02) was added to ACSF at 100 ng ml−1, in addition to tetrodotoxin (0.5 μM) and perfused for 30 min to test changes in response to glutamate puff or evoked stimulation. NBQX (10 µM; Tocris) was added to the ACSF (+tetrodotoxin, +BDNF) perfused for 20 min. Analysis was performed as previously described4. In brief, using imageJ (v.2.1.0/153c), regions of interest (ROIs) of nuclear tdTomato were defined manually for each glutamate responding GCaMP6s-expressing glioma cell. Mean intensity over the image time-course was used to measure the corresponding change in fluorescence and following background subtraction, the intensity values were calculated as ΔF/F, where F is the basal fluorescence of the ROI and ΔF is the change in fluorescence of the ROI at peak response relative to the F value. Duration of response to glutamate was calculated as the length of time that an increase in GCaMP6s signal (ΔF/F) was observed above 0 (F) in seconds.
Biotinylation
Glioma cells (SU-DIPG-VI) were seeded on laminin coated wells of 6-well plates at a density of 500,000 cells per condition. One day after plating, the medium was changed to medium without growth factors to ‘starve’ the cells for three days. Cells were treated with 100 nM BDNF recombinant protein (Peprotech, 450-02, stock 0.25 µg µl−1 in 0.1% BSA in H2O) compared to vehicle (equal volume added of 0.1% BSA in H2O), or 100 nM of NLGN3 (OriGene Technologies, TP307955) compared to vehicle, for specified time periods. To label surface proteins, the cells were washed twice with ice-cold PBS before adding 1 mg ml−1 sulfo-NHS-SS-biotin (Thermo Fisher Scientific) for 10 min at 4 °C with continuous gentle shaking. The reaction was quenched (100 mM glycine, 25 mM Tris-HCL, pH 7.4) for 5 min and then washed in ice-cold PBS three times; all procedures were carried out at 4 °C. The biotinylated cells were then lysed in RIPA lysis buffer (Santa Cruz Biotechnology) supplemented with PMSF, protease inhibitor cocktail and sodium orthovanadate as per manufacturers recommendations. Insoluble material was removed by centrifugation at 10,000g at 4 °C for 10 min and the supernatant was incubated with 50 μl NeutrAvidin agarose resin (Thermo Fisher Scientific) with gentle mixing overnight at 4 °C. Beads were washed with lysis buffer three times and proteins bound to the beads were eluted with NuPage LDS and sample reducing buffer in equal volumes (Life Technologies). Protein lysates were run on 4–20% Tris-Glycine Plus Gels (Novex, Thermo Fisher Scientific) and transferred to PDVF membranes using an iBlot 2 Gel Transfer Device (Thermo Fisher Scientific). Membranes were incubated in 5% BSA in 1× TBS/1% Tween 20 for 1 h. Primary antibodies against GluA4 (Cell Signaling Technology, 8070), GluA3 (Cell Signaling Technology, 4676) and GAPDH (Cell Signaling Technology, 5174) were added at a concentration of 1:1,000 and incubated overnight at 4 °C, before washing and addition of horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, 7074). Chemiluminescent signal was detected using either SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, PI34095) or Clarity Western ECL Substrate (Biorad, 1705061). Quantification was performed using imageJ (v.2.1.0/153c), where an ROI was manually drawn around the lanes, and plotted for the relative density of signal observed for each band (analyze>gels>plot lanes). The cell surface levels were normalized to their respective total protein level. The level of AMPAR at the cell surface was presented as a percentage of biotinylated cell surface GluA4 from the control average.
Western blots
For ligand activation experiments, patient-derived cultures were incubated in medium supplemented with only B27 supplement minus vitamin A for three days. Cells were dissociated using 5 mM EDTA in HBSS and resuspended in medium with B27 supplement for 4 h before incubation with recombinant BDNF protein (100 nM, Peprotech, 450-02) or vehicle (equal volume of 0.1% BSA in H2O). After ligand stimulation for the stated time points the cells were washed in PBS, before lysis using the RIPA Lysis Buffer System containing PMSF, protease inhibitor cocktail and sodium orthovanadate (Santa Cruz Biotechnology). Following quantification using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), equal amounts of total protein were loaded onto for each sample for standard western blot. Protein lysates were run on 4–20% Tris-Glycine Plus Gels (Novex, Thermo Fisher Scientific) and transferred to PDVF membranes using an iBlot 2 Gel Transfer Device (Thermo Fisher Scientific). Membranes were incubated in 5% BSA in 1× TBS/1% Tween 20 for 1 h. Primary antibodies GluA4 (Cell Signaling Technology, 8070), phospho-GluA4 (Ser862; Invitrogen, PA5-36807), TrkB (Cell Signaling Technology, 4606), β-actin (Cell Signaling Technology, 4970), phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204; Cell Signaling Technology, 4370), p44/42 MAPK (ERK1/2, Thr202/Tyr204; Cell Signaling Technology, 9102), phospho-AKT (Ser473; Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 9272), phospho-CAMKII (Thr286; Cell Signaling Technology, 12716), CAMKII (Cell Signaling Technology, 4436), phospho-TrkB (Tyr515; Sigma, SAB4503785) were added at a concentration of 1:1,000 and incubated overnight at 4 °C, before washing and addition of horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, 7074). Chemiluminescent signal was detected using either SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, PI34095) or Clarity Western ECL Substrate (Biorad, 1705061). Quantification was performed using ImageJ (v.2.1.0/153c), where an ROI was manually drawn around the lanes, and plotted for the relative density of signal observed for each band (analyze>gels>plot lanes). The conditions were normalized to loading control.
Synaptic puncta staining and quantification
Fixed neuron–glioma co-culture coverslips were incubated in blocking solution (3% normal donkey serum, 0.3% Triton X-100 in TBS) at room temperature for 1 h. Primary antibodies guinea pig anti-synapsin1/2 (1:500; Synaptic Systems, 106-004), mouse anti-nestin (1:500; Abcam, ab6320), chicken anti-neurofilament (M + H, 1:1,000; Aves Labs, NFM and NFH) or rabbit anti-RFP (1:500; Rockland, 600-401-379) diluted in diluent (1% normal donkey serum in 0.3% Triton X-100 in TBS) and incubated at 4 °C overnight. Following washing, the slides were incubated in secondary antibody (Alexa 594 donkey anti-rabbit IgG; Alexa 405 donkey anti-guinea pig IgG; Alexa 647 donkey anti-mouse IgG and Alexa 488 donkey anti-chicken IgG all used at 1:500 (Jackson Immuno Research)) overnight at 4 °C. Following washing, coverslips were mounted using ProLong Gold Mounting medium (Life Technologies). Images were collected on a Zeiss LSM800 confocal microscope using a 63× oil-immersion objective and post-processed with Airyscan. Co-localization of synaptic puncta images were performed as previously described4 using a custom ImageJ (v.2.1.0/153c) processing script (please refer for extended details). In brief, the quantification determines co-localization of presynaptic synapsin and postsynaptic PDS95–RFP within a defined proximity of 1.5 μm. Background fluorescence is removed using rolling ball background subtraction and peaks detected using imglib2 DogDetection plugin which determines the region of interest for each channel. The percentage of total glioma ROIs that are within 1.5 μm of a neuron ROI is reported. The script was implemented in ImageJ (v.2.1.0/153c).
Cloning constructs
For SEP–GluA2(Q)–TagBFP, Addgene plasmid EFS-Cas9-Puro (#138317) was digested with AgeI, MluI, and EcoRV; the 6 kb lentiviral backbone was isolated via gel extraction. The SEP fragment with Gibson overhangs was amplified from Addgene plasmid pCI-SEP-GluR2(Q) (#24002) with primers: 5′-AACGGGTTTGCCGCCAGAACACAGGACCGGTGCCACCATGCAAAAGATTATGCATATTTC-3′ and 5′-CCCCCTATCTGTATGCTGTTGCTAGCTTTGTATAGTTCATC-3′. Human GRIA2 (GluA2) with Gibson overhangs was amplified from pLV-EF1a-GFP-GRIA24 in two parts to introduce R583Q. GRIA-part 1 was amplified with primers: 5′-CAAAGCTAGCAACAGCATACAGATAGGG-3′ and 5′-TTGGCGAAATATCGCATCCCTGCTGCATAAAGGCACCCAAGGA-3′. GRIA-part 2 was amplified with primers: 5′-TTATGCAGCAGGGATGCGATATTTCGCCAA-3′ and 5′-TCTTCGACATCTCCGGCTTGTTTCAGCAGAGAGAAGTTTGTTGCGCCGGATCCAATTTTAACACTTTCGATGC-3′. TagBFP2 with Gibson overhangs was amplified from Addgene plasmid pLenti6.2-TagBFP (#113724) with primers: 5′-TCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGATGAGCGAGCTGATTAAG-3′ and 5′-TTGTAATCCAGAGGTTGATTGTCGACTTAACGCGTTTAATTAAGCTTGTGCCC-3′. DNA fragments above were stitched together using Gibson Assembly and transformed.
For PSD95-PURO, Addgene plasmid EFS-Cas9-Puro (#138317) was digested with AgeI, BamHI, and EcoRV; the 6.7 kb lentiviral backbone was isolated via gel extraction. PSD95-RFP with Gibson overhangs was amplified from PSD95-RFP4 with primers: 5′-TCGCAACGGGTTTGCCGCCAGAACACAGGTCTAGAGCCACCATGGACTGTCTCTGTATAG-3′ and 5′-TGTTTCAGCAGAGAGAAGTTTGTTGCGCCGGATCCATTAAGTTTGTGCCCCAG. DNA fragments above were stitched together using Gibson Assembly and transformed.
CRISPR deletion and shRNA knockdown
Target sequencing for single guide RNA (sgRNA) was generated using the online predictor at https://cctop.cos.uni-heidelburg.de. The validated sgRNA sequence used for NTRK2 deletion in all cultures was 5′-GTCGCTGCACCAGATCCGAG-3′. The scrambled control sequence used was 5′-GGAGACGTGACCGTCTCT-3′. The custom oligonucleotides were purchased from Elim Biopharmaceuticals. The oligonucleotides were phosphorylated in a reaction with the oligonucleotide (10 μM), 1× T4 DNA ligase buffer (B0202, NEB) and T4 PNK (M020, NEB) with a program 45 min 37 °C, 2 min 30 s at 95 °C, cool 0.1 °C s−1 to 22 °C. The sgRNA was cloned into the Lenti vector (pL-CRISPR.EFS.RFP, Addgene #57819). First the vector was digested in a reaction with Fast Digest Buffer (B64, Thermo Fisher Scientific), BsmBI restriction enzyme (FD0454, Thermo Fisher Scientific), DTT (10 mM) with program 45 min at 37 °C, heat inactivate 10 min at 65 °C. The digested vector backbone was dephosphorylated using Antartica phosphatase (M0289, NEB) in Antarctic phosphatase buffer (B0289, NEB) at 37 °C for 30 min, before purifying after running on a 1% agarose gel. The phosphorylated oligonucleotide duplexes were ligated into the vector backbone in a reaction with T4 DNA ligase buffer and T4 DNA ligase and incubated at room temperature for 1 h. Stabl3 (Invitrogen) cells were transformed with the assembled plasmids and individual colonies picked the next day for propagation and sanger sequencing (ElimBio). Lentiviral particles for were produced following transfection of the lentiviral packaging vectors (pΔ8.9 and VSV-g) and either the NTRK2 CRISPR vector or the control scramble vector into HEK293T cells and collected 48 h later. The viral particles were concentrated using Lenti-X Concentrator (Takara Bio) and resuspended in TSM base and stored at −80 °C for future use. The RFP-positive cells were FACs sorted for purity and returned to culture.
Lentiviral particles for shRNA knockdown of NTRK2 were produced following transfection of the lentiviral packaging vectors (pΔ8.9 and VSV-g) and either the NTRK2 shRNA vector (TRCN0000197207; Sigma) or the control shRNA vector (SHCOO2; Sigma) into HEK293T cells and collected 48 h later. The viral particles were concentrated using Lenti-X Concentrator and resuspended in TSM base and stored at −80 °C for future use. Control or NTRK2 shRNA lentiviral particles were transduced into SU-DIPG-VI cultures and the transduced cells were selected with puromycin (4 μg ml−1) from day 3.
pHluorin live imaging
Glioma cells (SU-DIPG-VI) expressing the SEP-GluA2(Q)-TagBFP and PSD95-RFP-Puro constructs (see ‘Cloning constructs’) were cultured as adherent cells, with mouse neurons, on laminin coated 27 mm glass bottom plates (150682, Thermo Fisher Scientific). ACSF was made at pH 7.4 (see ‘Electrophysiology’) and at pH 5.5 using the membrane impermeable acid MES hydrate (Sigma) to replace NaHCO3 at equimolar concentration. The ACSF was perfused onto the culture dish using a 3D-printed custom-built stage and tubing for manual perfusion of the solution. Images were collected using a Zeiss LSM980 confocal microscope equipped with a plexiglass environmental chamber, heated stage and CO2 module, and post-processed with Airyscan. The cells were kept at 37 °C with 5% CO2 for the duration of the imaging period. SEP puncta (channel setting for Alexa 488) were identified on the glioma cells as co-localized to the PSD95–RFP (channel setting for Alexa 594) puncta signal to the GFP signal from the SEP-GluA2(Q) puncta. In ImageJ (v.2.1.0/153c), an ROI was manually drawn over the PSD95–RFP signal and used to measure mean intensity of the both RFP and the SEP signal, thus blinding the area chosen for the SEP–GluA2(Q) signal. All puncta analysed were identified for the first timepoint and quantified for all the subsequent time points, thus the choice was blind with respect to outcome. The mean fluorescence intensity of SEP–GluA2(Q) were represented as a ratio to the levels of PSD95–RFP, to account for any fluorescence intensity changes that may occur due to photobleaching or z-axis drifting during the imaging time course. For BDNF perfusion experiments, ACSF (pH 7.4) containing 100 nM BDNF (Peprotech, 450-02) was perfused into the chamber. After imaging the signal in response to BDNF, the signal was then quenched with pH 5.5 to confirm the puncta of interest were membrane-bound GluA subunits. The fluorescence intensity was measured using ImageJ (v.2.1.0/153c). For the pH validation experiments, the ratio of SEP–GluA2(Q)/PSD95–RFP fluorescence intensity was reported. For the BDNF perfusion experiments, the intensity values were calculated as ΔF/F, where F is the basal SEP–GluA4(Q)/PSD95–RFP ratio and ΔF is the change in fluorescence of the SEP/PSD95 signal at each subsequent timepoint relative to the F value.
Immuno-electron microscopy
Twelve weeks post xenografting, mice were euthanized by transcardial perfusion with Karnovsky’s fixative: 4% PFA (EMS 15700) in 0.1 M sodium cacodylate (EMS 12300), 2% glutaraldehyde (EMS 16000), p.H 7.4. For all xenograft analysis, transmission electron microscopy was performed in the tumour mass located in the CA1 region of the hippocampus. At room temperature the samples were post fixed in 1% osmium tetroxide (EMS 19100) for 1 h, washed 3 times with ultrafiltered water, before 2-h en bloc staining. The samples were dehydrated in graded ethanol (50%, 75% and 95%) for 15 min each at 4 °C before equilibrating to room temperature and washed in 100% ethanol twice, followed by a 15 min acetonitrile wash. Samples were immersed for 2 h in Embed-812 resin (EMS 14120) with 1:1 ratio of acetonitrile, followed by a 2:1 Embed-812:acetonitrile for 2 h, then in Embed-812 for 2 h. The samples were moved to TAAB capsules with fresh resin and kept at 65 °C overnight. Sections of 40 and 60 nm were cut on an Ultracut S (Leica) and mounted on 100-mes 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, 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 next 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, samples that did not contain any GFP-expressing cells were stained simultaneously to control for any non-specific 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 TEM at 120 kV and images were collected using a Gatan Orius digital camera. Secondary antibody-only controls were used to compare for specific binding and quantification of images was performed by a blinded investigator.
Electron microscopy data analysis
Sections of hippocampal preparations bearing xenografted SU-DIPG-VI cells were imaged as above using TEM imaging. Overall, 280 sections of SU-DIPG-VI wild-type across 7 mice and 253 sections of NTRK2-KO across 7 mice were analysed. Electron microscopy images were captured at 6,000×, with a 15.75 μm2 field of view. Identified synapses were verified by 2 independent, blinded investigators. Glioma cells were counted and confirmed after unequivocal identification of immunogold particle labelling with four or more particles. To confirm clear synaptic structures, the following three criteria needed to be clearly met: (1) presence of synaptic vesicle clusters; (2) visually apparent synaptic cleft; and (3) clear postsynaptic density in the glioma cell. The number of confirmed glioma–neuron synapses identified was divided by the total number of glioma cells identified to provide the percentage of synaptic structures present. Overall, the analyses identified 0–6 glioma processes per section and 0–2 neuron–glioma synaptic structures per section.
Immunohistochemistry
Mice were anaesthetized with intraperitoneal avertin (tribromoethanol), then transcardially perfused with 20 ml of PBS. Brains were fixed in 4% PFA overnight at 4 °C, 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 (AO 860, American Optical).
For immunohistochemistry, coronal or sagittal sections were incubated in blocking solution (3% normal donkey serum, 0.3% Triton X-100 in TBS) at room temperature for 30 min. Mouse anti-human nuclei clone 235-1 (1:200, Millipore), rabbit anti-Ki67 antibody (1:500, Abcam, ab15580) or chicken anti-GFP (1:500, Abcam, ab6320) were diluted in antibody diluent solution (1% normal donkey serum in 0.3% Triton X-100 in TBS) and incubated overnight at 4 °C. Sections were then rinsed once with TBS, before an incubation with DAPI (1 µg ml−1 in TBS, Thermo Fisher Scientific) and then another rinse with TBS. Slices were incubated in secondary antibody solution; Alexa 594 donkey anti-rabbit IgG, Alexa 647 donkey anti-mouse IgG or Alexa 488 donkey anti-chicken all used at 1:500 (Jackson Immuno Research) in antibody diluent at 4 °C overnight. Sections were washed three times with TBS and mounted with ProLong Gold Mounting medium (Life Technologies). Confocal images were acquired on either a Zeiss Airyscan1 800 or Zeiss Airyscan LSM980 using Zen 2011 v8.1. Proliferation index was determined by quantifying the fraction of Ki67-labelled cells/HNA-labelled cells using confocal microscopy at 20x magnification. Quantification of images was performed by a blinded investigator.
EdU incorporation assay
EdU staining was performed on in vitro cell culture slides or on glass coverslips in 24-well plates which were pre-coated with poly-l-lysine (Sigma) and laminin (Thermo Fisher Scientific). Neurosphere culture were dissociated with TrypLE and plated onto coated slides, once the cells had adhered the medium was replaced with growth factor-depleted medium for 72 h. Recombinant proteins BDNF (100 nM, Peprotech, 450-02), NGF (100 nM, Peprotech 450-01), NT-3 (100 nM, Peprotech 450-03) or NT-4 (100 nM, Peprotech, 450-04), inhibitors (500 nM entrectinib HY-12678 and 500 nM larotrectinib HY-12866, both MedChem Express) and vehicle (0.1% BSA and/or DMSO) were added for specified times with 10 μM EdU. After a further 24 h the cells were fixed with 4% PFA in PBS for 20 min and then stained using the Click-iT EdU kit and protocol (Invitrogen) and mounted using Prolong Gold mounting medium with DAPI (Life Technologies). Confocal images were acquired on either a Zeiss Airyscan1 800 or Zeiss Airyscan LSM980 using Zen 2011 v8.1. Proliferation index was determined by quantifying the fraction of EdU-labelled cells divided byDAPI-labelled cells, HNA-labeled cells or nestin-labeled cells using confocal microscopy at 20× magnification. Quantification of images was performed by a blinded investigator.
Bioinformatic analysis
Single-cell expression (in transcripts per million units) and metadata were downloaded from the 3CA Curated Cancer Cell Atlas61 website (https://www.weizmann.ac.il/sites/3CA/) and the analysis was performed using R version 4.1.1. Gene-expression values were divided by 10 and log2-transformed. Genes were considered analysable and included in the analysis (overall 11,518 genes) in case their average log2 expression was greater than 0.25. Cells were separated into malignant and non-malignant populations according to the metadata file downloaded from the 3CA website. Within each tumour sample the malignant cells were scored using the sigScores function (scalop package available at https://github.com/jlaffy/scalop) for the cell-state programmes from Filbin et al.36 (that is, AC-like, OC-like and OPC-like) and for two gene signatures reflecting synaptic transmission (SYN) and tumour microtube structure (TM) that were manually curated. Each malignant cell was assigned with a cell state from Filbin et al.36 if the maximal state score was greater than 0.5. Cells that did not achieve such a score for any of the states were assigned with an ‘unresolved’ state. Lineage and stemness coordinates were computed for each cell as described36 This basic data object was used for generating the panels of Extended Data Fig. 4. NTRK2 detection rate (Extended Data Fig. 4a) was computed for each state by summing the number of cells with NTRK2 expression level greater than zero and dividing by the number of cells assigned to that state. NTRK2 expression level (Extended Data Fig. 4b) was smoothened for the purpose of data visualization by assigning each cell with the average NTRK2 expression of its 10 nearest neighbours (using the k-nearest-neighbours algorithm, FNN package) in the 2-dimensional lineage versus stemness space. Pearson correlation between NTRK2 and analysable genes was computed for each state by centring the matrix across the assignable cells (351 AC-like, 201 OC-like and 720 OPC-like) and computing the Pearson correlation coefficient for each cell state between NTRK2 and the each of the analysable genes (across the cells classified to the particular state). Genes were included in the analysis (Extended Data Fig. 4d) in case the absolute Pearson correlation coefficient was greater than 0.25 in at least one state (overall 519 genes passed this threshold). Gene Ontology enrichment analysis (Extended Data Fig. 4e,f,g) was computed for the positively correlated genes in each state (145, 138 and 97 genes with Pearson correlation coefficient greater than 0.25 for the AC-like, OC-like and OPC-like states, respectively) using the function enrichGo (package clusterProfiler). For scRNAseq processing of individual biopsy samples, RSEM-normalized gene abundances for the Filbin dataset36 were downloaded from the Single Cell Portal (https://singlecell.broadinstitute.org/single_cell, Gene Expression Omnibus (GEO) accession: GSE102130). The data were log-transformed with a pseudocount of one.
For NTRK2 and BDNF expression levels in DIPG patient-derived cell cultures, fragments per kilobase of transcript per million mapped reads (FPKM) data were analysed from datasets kept in-house and are publicly available to download (GEO accession: GSE94259) and (GEO accession: GSE222560). Cultures included were SU-DIPG-IV, SU-DIPG-VI, SU-DIPG-XIII-p, SU-DIPG-XVII, SU-DIPG-XXI, SU-DIPG25 and SU-DIPG-XIII-FL.
For TrkB isoform analysis, our previously published scRNASeq dataset4 of patient-derived orthotopic xenograft models was used. NTRK2 isoform abundances were quantified from FASTQ files using Kallisto62 (v.0.46.1). Reads were pseudoaligned against a reference transcriptome created from the hg38 reference genome using Ensembl hg38 transcript annotations. Estimated counts were library size normalized to counts per million and log-transformed with a pseudocount of one. Cells with libraries containing irregular GC content were identified and removed from analysis, resulting in a total of 321 cells.
For transcriptome analysis of BDNF-treated tumour cells, cultures were grown in serum-free medium without growth factors for three days before addition of 100 nM BDNF recombinant protein (Peprotech, 450-02) and incubated for 16 h before harvesting RNA. RNA was extracted from pelleted cell culture samples using the RNeasy Isolation kit (Qiagen) as per manufacturer’s instructions. Total RNA samples were submitted to Stanford Functional Genomics Facility. RNA integrity was established with Bioanalyzer trace (Agilent). The mRNA was prepared for sequencing using the KAPA Stranded mRNAseq Library prep kit (KK8420), and libraries were indexed with Truseq RNA UD from Illumina (20021454) as per manufacturer’s instructions. The sequencing was performed on the Illumina NextSeq 500.
Reads were mapped to hg19 annotation using Tophat263 (version 2.0.13) and transcript expression was quantified against RefSeq gene annotations using featureCounts64 (v2.0.3). Differential gene expression and log2 fold change calculations were determined using the DESeq2 (v.1.36.0) package in R65. Mitochondrial genes were excluded from analysis. Volcano plot analysis was conducted using the R-based EnhancedVolcano (v1.14.0) package (https://github.com/kevinblighe/EnhancedVolcano). The included volcano plot analysis was filtered for P values <0.4 and log2FC< 1.5 to highlight the top differentially expressed genes.
Statistics and reproducibility
Statistical tests were conducted using Prism v9.1.0 (GraphPad) software unless otherwise indicated. Gaussian distribution was confirmed by the Shapiro–Wilk normality test. For parametric data, unpaired two-tailed Student’s t-test or one-way ANOVA with Tukey’s post hoc tests to examine pairwise differences were used as indicated. Paired two-tailed Student’s t-tests were used in the case of same-cell experiments (as in electrophysiological recordings). For data normalized to a control mean (as in western blot or pHluorin analyses) one-sample t-test were used against the mean of the control (either 0 or 1), with Wilcoxon signed-rank test for non-parametric data. For non-parametric data, a two-sided unpaired Mann–Whitney test was used as indicated, or a one-tailed Wilcoxon matched pairs signed-rank test was used for same-cell 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. On the basis of variance of xenograft growth in control mice, we used at least three mice per genotype to give 80% power to detect effect size of 20% with a significance level of 0.05. All in vitro experiments have been performed in at least three independent coverslips for each experiment and performed in at least two independent experiments. The number of biological replicates (mice for in vivo growth, calcium imaging, electrophysiological recording, optogenetic stimulation and immuno-electron microscopy experiments) is indicated in the figure legends and was three or greater for all experiments. The comparison of wild-type glioma to NTRK2-KO glioma growth was tested in three independent in vivo experiments and in vivo synaptic connectivity in two independent experiments. The effect of entrectinib on glioma growth was tested in two independent in vivo experiments. The increase of glioma synaptic/glutamatergic current strength in response to BDNF application was tested in three independent electrophysiological experiments. All protein experiments assayed by western blot analysis were performed 3 independent times, except for NTRK2 knockdown, which was performed twice, with data shown from one experiment and entrectinib administration, which was performed once. The ChR2+ glioma in vivo optogenetic stimulation experiment was performed in two independent experiments, with data shown from one experiment.
Materials availability
All unique materials such as patient-derived cell cultures are freely available and can be obtained by contacting the corresponding author with a standard materials transfer agreement with Stanford University.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.