Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: a , The Bdnf -TMKI model. CaRE, calcium regulatory element binding site; CRE, cAMP response element; WT, wild type. b , Optogenetic paradigm. M2, mouse premotor frontal cortex; P, postnatal day. c , Representative images of glioma (SU-DIPG-VI) xenografted into wild-type and Bdnf -TMKI cortex following blue-light stimulation of ChR2 + cortical neurons. HNA (grey) marks glioma cells, Ki67 (red) marks proliferating cells. Scale bar, 50 µm. d , Proliferation index (Ki67 + cells/HNA + glioma cells) of xenografted SU-DIPG-VI glioma in wild-type or Bdnf -TMKI mice stimulated optogenetically (ChR2 + cortical neurons) or mock-stimulated (ChR2 − neurons). n = 6 (wild-type ChR2 − ), 4 ( Bdnf -TMKI ChR2 − ), 7 (wild-type ChR2 + ) and 4 ( Bdnf -TMKI ChR2 + ) mice. e , Survival curves of wild-type and Bdnf -TMKI mice bearing SU-DIPG-XIII-P* xenografts. n = 7 (wild type) and 8 ( Bdnf -TMKI mice). f , Survival curves of mice bearing wild-type and NTRK2 -KO orthotopic xenografts (SU-DIPG-VI and SU-pcGBM; n = 7 mice per group). g , Survival curves of SU-DIPG-XIII-P* xenografted mice treated with entrectinib versus vehicle-treated controls. Grey shading indicates drug treatment. h , Representative images (left) and proliferation index (right; EdU + cells/DAPI cells) of wild-type and NTRK2 -KO glioma cultures (SU-DIPG-VI) with or without BDNF treatment ( n = 5 coverslips per group). Scale bar, 100 µm. i , Representative images (left) and proliferation index (right; EdU + cells/Nestin + glioma cells) of wild-type and NTRK2 -KO glioma (SU-DIPG-VI) cultured alone or with neurons ( n = 3 coverslips per group). Scale bar, 50 µm. j , Proliferation index of SU-DIPG-VI wild-type and NTRK2- KO glioma co-culture with neurons (as in representative image in i ), with or without NBQX ( n = 3 coverslips per group; repeated in Extended Data Fig. ). k – m , Experimental scheme ( k ), Representative images ( l ) and quantification of proliferation rate (Ki67 + cells/HNA + glioma cells) of wild-type and NTRK2 -KO glioma xenografts (SU-DIPG-VI) treated with perampanel or vehicle control ( m ). n = 6 (wild type + vehicle), 7 (wild type + perampanel), 5 ( NTRK2 -KO + vehicle) and 6 ( NTRK2- KO + perampanel) mice. Scale bar, 50 µm. Data are mean ± s.e.m. One-way ANOVA with Tukey’s post hoc analysis ( d , h – j , m ); two-tailed log rank analysis ( e – g ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; NS, not significant.

Article Snippet: 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.

Techniques: Binding Assay, Cell Culture, Co-Culture Assay, Control, Two Tailed Test

Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: a , Proliferation index of SU-DIPG-VI WT and NTRK2 KO glioma monoculture (left), or glioma co-culture with neurons (right, as in Fig. ), in the presence and absence of the AMPAR blocker NBQX (10 μM) (quantified as fraction of EdU + /HNA+ co-positive tumor cells assessed by confocal microscopy, n = 3 coverslips/group for glioma monoculture experiments and 6 coverslips/group for neuron-glioma co-culture; experiment replicated in Fig. , WT vehicle vs WT + neurons vehicle P < 0.0001, WT + neurons vehicle vs WT + neurons NBQX P < 0.0001, WT + neurons vs NTRK2 KO + neurons P < 0.0001). b , Representative images of data quantified in a ; wild-type and NTRK2 KO glioma cells (SU-DIPG-VI) co-cultured with neurons in the presence and absence of NBQX (10 μM). Blue denotes HNA positive glioma cells; red denotes EdU (proliferative marker); green denotes MAP2 (neurons). Scale bar = 30 µm. c , Proliferation index of SU-DIPG-VI (red data points) and SU-DIPG-XIII-FL (blue data points) as a monoculture or cocultured with neurons in the presence of a CAMKII inhibitor, KN-93 (10 μM) or vehicle control (quantified as fraction of EdU + /HNA + glioma cells; n = 7 coverslips/group, vehicle vs vehicle + neurons P < 0.0001, vehicle + neurons vs KN-93 + neurons P = 0.0017, vehicle vs KN93 + neurons P = 0.0212). d , Representative images of data quantified in c ; glioma cells (SU-DIPG-VI) in monoculture, or co-cultured with neurons, in the presence and absence of KN-93 (10 μM). Blue denotes HNA positive glioma cells; red denotes EdU (proliferative marker); green denotes MAP2 (neurons). Scale bar = 100 µm. Data are mean ± s.e.m., *P < 0.05, **P < 0.01, ****P < 0.0001, ns = not significant, one-way analysis of variance (ANOVA) with Tukey’s post hoc analysis.

Article Snippet: 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.

Techniques: Co-Culture Assay, Confocal Microscopy, Cell Culture, Marker, Control

Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: a , Representative image of Alexa 568 (red)- filled GFP+ glioma cell following whole-cell patch clamp recording. Co-labelled with GFP (green) and human nuclear antigen (HNA, grey). Scale bars = 10 µm. b , Representative voltage-clamp traces of whole cell patch-clamp electrophysiological recordings in glioma cells. Hippocampal slices were perfused with ACSF containing tetrodotoxin (TTX, 0.5 µM), and response to a local puff (250 msec) application of 1 mM glutamate (black square) was recorded from xenografted glioma cells with sequential application of NMDAR blocker (AP-5, 100 µM), TBOA (200 µM), AMPAR blocker (NBQX, 10 µM). c , Quantification of data in b ( n = 7 glioma cells, 4 mice, P = 0.0165). d , Whole cell patch-clamp electrophysiological recording of glioma cell with ACSF puff, representative voltage clamp trace. e , Representative traces of glutamate-evoked inward currents (black square) in patient-derived glioma xenografted cells before (grey) and after 30-minute perfusion with NLGN3 recombinant protein (100 ng/ml) in ACSF (containing TTX, 0.5 µM) (purple). f , Quantification of data in e ( n = 5 glioma cells, 3 mice). g , Model of calcium imaging of tdTomato nuclear tagged (red nuclei), GCaMP6s-expressing (green calcium transients) glioma cells xenografted into the mouse hippocampal region. h , Quantification of number of xenografted SU-DIPG-XIII-FL or SU-DIPG-VI cells glioma cells demonstrating a calcium transient (as depicted in Fig. and Extended Data Fig. 6j) in response to a glutamate puff (responders, grey, non-responders, white). i , Baseline GCaMP6s intensity in SU-DIPG-VI glioma cells before and 30-min after BDNF exposure, in the absence of glutamate puff ( n = 7 cells, 3 mice). j , GCaMP6s intensity trace of SU-DIPG-VI glioma cells response to glutamate puff before (3 cells, 3 mice: light grey, average: dark grey) and after BDNF perfusion (three cells: light blue, average intensity: dark blue). k , SU-DIPG-VI GCaMP6s cell response to glutamate puff at baseline and after BDNF perfusion (100 ng/ml, 30 min, n = 7 cells, 4 mice, P = 0.0174). l , Duration of calcium transient response to glutamate puff in SU-DIPG-VI hippocampal xenografted cells, before and after perfusion with BDNF (100 ng/ml, 30 min, n = 6 cells, 4 mice, P = 0.0302). m , Representative traces of SU-DIPG-XIII glioma GCaMP6s intensity in the presence of BDNF (100 ng/ml, 30 min). Response to glutamate application (black) recorded with BDNF perfusion (3 cells, 2 mice: light blue, average: dark blue) or with BDNF and NBQX (10 µM, 3 cells: light red, average: red). n , Response of GCaMP6s cells to glutamate puff with BDNF application, in the presence and absence of NBQX ( n = 6 cells, 3 mice, P = 0.0002). Data are mean ± s.e.m., *P < 0.05, ***P < 0.001, ns = not significant, two-tailed paired Student’s t -test for k , l , n , and two-tailed Wilcoxon signed pairs matched rank test for d , f and i .

Article Snippet: 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.

Techniques: Patch Clamp, Derivative Assay, Recombinant, Imaging, Expressing, Two Tailed Test

Journal: Nature

Article Title: Glioma synapses recruit mechanisms of adaptive plasticity

doi: 10.1038/s41586-023-06678-1

Figure Lengend Snippet: a , Schematic depicting AMPAR trafficking downstream of BDNF–TrkB–CAMKII signalling . b , Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI glioma with or without BDNF treatment for 5, 15 and 30 min. c , Quantification of cell surface GluA4 in b ( n = 3 independent biological replicates). d , Western blot analysis of cell surface and total cell protein levels of GluA3 in SU-DIPG-VI glioma with or without BDNF treatment for 30 min. e , Quantification of cell surface GluA3 in d ( n = 3 independent biological replicates). f , Western blot analysis of cell surface and total cell protein levels of GluA4 in SU-DIPG-VI cells treated with NLGN3 for 30 min. g , Quantification of cell surface GluA4 data in f ( n = 3 independent biological replicates). h , Schematic showing GluA2–SEP experiments. i , j , Validation of pHluorin approach. i , Left, representative images of a glioma cell process expressing GluA2(Q)–SEP, PSD95–RFP and whole-cell TagBFP in co-culture with neurons. Right, representative GluA2(Q)–SEP puncta. Scale bars, 5 µm (left) and 1 µm (right). Cells were exposed to pH 7.4 followed by pH 5.5 and then pH 7.4. j , Quantification of fluorescence intensity of GluA2(Q)–SEP puncta before, during and after acidic exposure ( n = 4 puncta from a representative cell). k , Top, representative images of two processes from glioma cells expressing GluA2(Q)–SEP, PSD95–RFP and TAG-BFP2 in co-culture with neurons (scale bar, 5 µm). Middle and bottom, representative images of GluA2(Q)–SEP puncta at 0, 5, 15 and 20 min of BDNF incubation (scale bar=1 µm). l , Fluorescence intensity of co-localized GluA2(Q)–SEP:PSD95–RFP puncta over time with BDNF treatment ( n = 8 puncta, 6 cells). m , Fluorescence intensity of co-localized GluA2(Q)–SEP:PSD95–RFP puncta after 15 min versus basal fluorescence in control (vehicle, n = 4 puncta, 2 cells) or BDNF-treated cells ( n = 8 puncta, 6 cells). Data are mean ± s.e.m. Two-tailed unpaired Student’s t -test ( c , e , g , m ); two-tailed paired Student’s t -test ( j ); two-tailed one-sample t -test ( l ).

Article Snippet: 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.

Techniques: Western Blot, Biomarker Discovery, Expressing, Co-Culture Assay, Fluorescence, Incubation, Control, Two Tailed Test