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: 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.

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: 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.

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: 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.

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: 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.

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

Journal: Nature protocols

Article Title: Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications

doi: 10.1038/nprot.2013.141

Figure Lengend Snippet: Troubleshooting guide.

Article Snippet: Solutions from the tissue processor are too old or diluted from previous solutions Replace the solutions from the tissue processor and soak the paraffin block with water to soften the tissue before cutting 22 Wrinkles and/or holes in paraffin sections Microtome blade is not sharp enough Shift the microtome blade a little bit to use a new area, and replace the blade with a new one when necessary 47 DNA fragmentation Excessive homogenization in Step 34 or sample vortexing Do not vortex DNA samples, homogenize gently and do not use mechanical disruption of tissues 47, 55A(xii), 55B(vi,viii) Low DNA and RNA yields Incomplete tissue disruption/homogenization Make sure to process about 5-30 mg of tissue and to completely disrupt it with the RNase-free pestle on dry/wet ice and pass through a QIAshredder column Tissue of choice contains much lower levels of nucleic acids than expected Increase up to 60 mg the amount of tissue to be homogenized; be careful not to clog the AllPrep column because of the fibrous nature of the tissue Low DNA but high RNA yield Overloading the AllPrep column Homogenize less tissue (cells in some tumors are densely packed, resulting in high levels of nucleic acids) or load it into two AllPrep columns 55A(xii), 55B(vi,viii) Low RNA A 260 /A 280 ratio Phenol contamination during organic extraction Centrifuge again for 5 min at maximum speed (16,000 g ) at room temperature to separate the aqueous and organic phases, and do not remove any lower phase RNA samples diluted with RNase-free water If a NanoDrop spectrophotometer is not available, dilute the RNA samples with Tris-HCl (pH 7.5) and measure them in a regular spectrophotometer RNA degradation RNA degradation before sample processing Freeze tissues immediately in liquid nitrogen and store at -80 °C (if samples cannot be frozen immediately, treat tissues with RNA later or Allprotect Tissue Reagent) RNase contamination Ensure the samples are handled in RNase-free conditions and no RNases have been introduced.

Techniques: Staining, Blocking Assay, Homogenization, Spectrophotometry