Mice and Housing Conditions All in vivo experiments were approved by the Stanford University Institutional Animal Care and Use Committee and performed in accordance with institutional guidelines. Thy1::ChR2 mice (line 20; The Jackson Laboratory, Bar Harbor, ME) were first intercrossed with NSG mice (NOD-SCID-IL2R gamma chain-deficient, The Jackson Laboratory, Bar Harbor, ME) to produce the Thy1::ChR2;NSG genotype. All experiments were performed either on animals heterozygous for Thy1::ChR2 or on WT control NSG littermates. Animals were housed according to standard guidelines with free access to food and water in a 12 hr light/dark cycle.

Cell Culture For all human tissue studies, informed consent was obtained and Institutional Review Board (IRB) approval was granted. For all cultures, authenticity is verified and monitored using short tandem repeat (STR) DNA fingerprinting every three months. Pediatric cortical high-grade glioma culture was generated as follows: Tissue from a pediatric cortical high-grade glioma (WHO grade IV) tumor was obtained at the time of initial biopsy from a 15-year-old male patient under sterile conditions. The tissue was dissociated mechanically, followed by gentle enzymatic dissociation with TrypLE (5 min at 37°C; Life Technologies, Carlsbad, CA) and then passed through a 100-μm filter. The flow-through was collected and cultured in a T75cm2 flask. The filter was inverted and the tissue was flushed and cultured into a separate flask. A defined, serum-free medium designated “Tumor Stem Media (TSM)” was used throughout, consisting of Neurobasal(-A) (Invitrogen, Carlsbad, CA), B27(-A) (Invitrogen, Carlsbad, CA), human-bFGF (20 ng/mL) (Shenandoah Biotech, Warwick, PA), human-EGF (20 ng/mL) (Shenandoah, Biotech, Warwick, PA), human PDGF-AA (10 ng/mL) and PDGF-BB (10 ng/mL) (Shenandoah, Biotech, Warwick, PA) and heparin (2 ng/mL) (Stem Cell Technologies, Vancouver, BC, Canada). When neurospheres were visible in the primary culture it was passed through a 40-μm filter to remove debris and single cells such as red blood cells; the matter that did not pass through the filter, containing the larger than 40-μm neurospheres, was recovered and subsequently dissociated using TrypLE. A second filtration was then performed using 40-μm filters, the single cells in the flow-through were centrifuged at 300 g for 5 min, and the pellet was resuspended in TSM and recultured, generating secondary neurospheres. This cell line was designated SU-pcGBM2 (Stanford University, pediatric cortical glioblastoma line 2), grown in nonadherent neurosphere culture in the above medium, and passaged every one to two weeks. Caretti et al., 2014 Caretti V.

Sewing A.C.

Lagerweij T.

Schellen P.

Bugiani M.

Jansen M.H.

van Vuurden D.G.

Navis A.C.

Horsman I.

Vandertop W.P.

et al. Human pontine glioma cells can induce murine tumors. Monje et al., 2011 Monje M.

Mitra S.S.

Freret M.E.

Raveh T.B.

Kim J.

Masek M.

Attema J.L.

Li G.

Haddix T.

Edwards M.S.

et al. Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma. Diffuse intrinsic pontine glioma (DIPG) tumor neurosphere cultures SU-DIPGIV and SU-DIPGVI were generated as previously described () from early post-mortem tissue donations and grown as tumor neurospheres in defined, serum-free TSM medium as above. SU-DIPGXIII was generated similarly, but using the mechanical dissociation protocol described above. JHH-DIPGI cells, a kind gift from Dr. Eric Raabe at Johns Hopkins Hospital, were generated similarly to the Stanford cultures as neurospheres from post-mortem tissue and are grown under the same media conditions. Monje et al., 2011 Monje M.

Mitra S.S.

Freret M.E.

Raveh T.B.

Kim J.

Masek M.

Attema J.L.

Li G.

Haddix T.

Edwards M.S.

et al. Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma. The adult high-grade gliomas (WHO grade IV) used were obtained at the time of biopsy and cultured as described previously for DIPG tissue () but using the mechanical dissociation protocol described above and without PDGF-AA or BB supplementation. The oligodendroglioma culture was obtained at the time of biopsy. The tumor specimen was chopped into fine pieces with a sterile razor blade, and dissociated enzymatically by incubating in Liberase DH (Roche Life Science, Indianapolis, IN) on a rotator at 37°C for 30 min. Tissue was then passed through a 10 ml serological pipette 8 times, followed by 8 passages through a 1 ml pipette tip. The resulting cell suspension was strained through a 100-μm cell strainer to remove large debris, and the flow-through was pelleted and myelin removed by centrifugation in 30% sucrose solution at 800 g. Red blood cells in the resulting pellet were removed by treatment with ACK lysis solution (Life Technologies, Carlsbad, CA), and the remaining cells were plated in culture flasks pre-coated with Matrigel (BD Biosciences, San Jose, CA) in TSM media.

Orthotopic Xenografting A single-cell suspension from cultured SU-pcGBM2 neurospheres at passage 19-22 was prepared in sterile PBS immediately prior to the xenograft procedure. Animals at P34-36 were anesthetized with 1%–4% isoflurane and placed in a stereotactic apparatus. The cranium was exposed via midline incision under aseptic conditions. 600,000 SU-pcGBM2 cells in 3 μl sterile PBS were stereotactically implanted in the premotor cortex (M2) of the right hemisphere through a 31-gauge burr hole, using a digital pump at infusion rate of 0.4 μL/min and 31-gauge Hamilton syringe. Stereotactic coordinates used were as follows: 0.5 mm lateral to midline, 1.0 mm anterior to bregma, –1.75 mm deep to cranial surface. At the completion of infusion, 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 to minimize backflow of the injected cell suspension.

Fiber Optic Placement and In Vivo Optogenetic Stimulation Gibson et al., 2014 Gibson E.M.

Purger D.

Mount C.W.

Goldstein A.K.

Lin G.L.

Wood L.S.

Inema I.

Miller S.E.

Bieri G.

Zuchero J.B.

et al. Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain. 2 light density at the tip of the patch cord; with the optical ferrule placed just below the pial surface this would deliver ∼3 mW/cm2 approximately midway through the cortex to reach the layer V apical dendrites ( Yizhar et al., 2011 Yizhar O.

Fenno L.E.

Davidson T.J.

Mogri M.

Deisseroth K. Optogenetics in neural systems. Gibson et al., 2014 Gibson E.M.

Purger D.

Mount C.W.

Goldstein A.K.

Lin G.L.

Wood L.S.

Inema I.

Miller S.E.

Bieri G.

Zuchero J.B.

et al. Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain. th) stimulation session. Fiber optic placement was performed as previously described () a minimum of 7 days prior to optogenetic stimulation. Animals were anesthetized with 1%–4% isoflurane and placed in a stereotactic apparatus. The cranium was exposed using a midline incision under aseptic conditions. A fiber optic ferrule (Doric Lenses, Quebec, Canada) was placed at the premotor cortex (M2) of the right hemisphere using the following coordinates: 0.5 mm lateral to midline, 1.0 mm anterior to bregma, −0.6 mm deep to cranial surface in the right hemisphere. At 10-11 weeks post-xenograft (allowing a minimum of 7 days of recovery following ferrule placement procedure), all animals were connected to a 100-mW 473-nm DPSS laser system with a mono fiber patch cord, which freely permits wakeful behavior of the animal. Pulses of light with ∼10 mW measured output at tip of the patch cord were administered at a frequency of 20 Hz for periods of 30 s, followed by 90 s recovery periods, for a total session duration of 30 min for single-session stimulation paradigms or 10 min per day for 7 consecutive days for the repetitive stimulation paradigm. This power represents ∼30 mW/cmlight density at the tip of the patch cord; with the optical ferrule placed just below the pial surface this would deliver ∼3 mW/cmapproximately midway through the cortex to reach the layer V apical dendrites (). This was the minimum light required to reliably elicit complex motor behavioral output. This paradigm is modified slightly from that previously described (), with a 90 s recovery period rather than 120 s and higher light density required to elicit the complex motor behavior in tumor-bearing NSG mice rather than non-tumor-bearing mice with a CD1/B6 mixed background. When tumor-bearing Thy1::ChR2;NSG animals are stimulated in parallel with non-tumor bearing Thy1::ChR2 animals on a mixed CD1/B6 background, the tumor-bearing NSG background mice consistently required higher light power to elicit the expected motor behavior (unidirectional ambulation). During periods of light administration, all Thy1::ChR2;NSG animals responded with unidirectional ambulation to the left for the duration of light exposure ( Movie S1 ), confirming proper ferrule placement over right M2 and effective neuronal stimulation. All WT;NSG animals demonstrated no change in behavioral output in response to light stimulation. Following stimulation in the single-session paradigm, an intraperitoneal injection of 5-ethynyl-2′-deoxyuridine (EdU; 40 mg/kg; Invitrogen, Carlsbad, CA) was administered to the animal. Mice were sacrificed 24 hr after administration of EdU in the single-session paradigm experiment. For the repetitive stimulation experiment, mice were sacrificed 48 hr following the final (7) stimulation session.

Perfusion and Immunohistochemistry Animals were anesthetized with intraperitoneal Avertin (tribromoethanol), then transcardially perfused with 20 ml of PBS. Brains were fixed in 4% paraformaldehyde overnight at 4°C, then transferred to 30% sucrose for cryoprotection. Brains were embedded in Tissue-Tek O.C.T. (Sakura, Torrance, CA) and sectioned in the coronal plane at 40 μm using a sliding microtome (Microm HM450; Thermo Scientific, Waltham, MA). For immunohistochemistry, a 1 in 6 series of 40-μm coronal sections was stained using the Click-iT EdU cell proliferation kit and protocol (Life Technologies, Carlsbad, CA) to expose EdU labeling, then 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 (1:100; Millipore, Billerica, MA), rabbit anti-Ki67 (1:500; Abcam, Cambridge, MA), rat anti-MBP (1:200; Abcam, Cambridge, MA) and rabbit anti-cleaved caspase-3 (1:200; Cell Signaling, Danvers, MA) were diluted in 1% blocking solution (1% normal donkey serum in 0.3% Triton X-100 in TBS) and incubated overnight at 4°C. Sections were then rinsed three times in 1X TBS and incubated in secondary antibody solution (Alexa 488 goat anti-mouse IgG, 1:500 (Life Technologies, Carlsbad, CA); Alexa 488 donkey anti-rabbit IgG, 1:500 (Life Technologies, Carlsbad, CA); Alexa 594 donkey anti-mouse IgG, 1:500 (Life Technologies, Carlsbad, CA); Alexa 647 donkey anti-rabbit IgG, 1:500 (Life Technologies, Carlsbad, CA); Alexa 594 donkey anti-rat IgG, 1:1000 (Life Technologies, Carlsbad, CA)) in 1% blocking solution at 4°C overnight. The next day, sections were rinsed 3 times in TBS and mounted with ProLong Gold mounting medium with DAPI (Life Technologies, Carlsbad, CA).

Confocal Imaging and Quantification Analysis of Cell Proliferation and Cell Death Paxinos and Franklin, 2008 Paxinos G.

Franklin K.B.J. The Mouse Brain in Stereotaxic Coordinates. 6 μm3; six fields within each of three coronal sections were selected for a total quantified volume of 18.432 × 106 μm3 per animal. These selected premotor cortex and corpus callosum areas lie within the active premotor circuit, but are deep to the path of tumor cell injection by our stereotactic coordinates, and thus avoid the principal areas of inflammatory change involving tissue more proximal to the injection site. Within each field, all human nuclear antigen (HNA)-positive tumor cells were quantified to determine tumor burden within the areas quantified. HNA-positive tumor cells were then assessed for double-labeling with either EdU or Ki67 (cell proliferation), or with cleaved caspase-3 (cell death). To calculate proliferation index (the percentage of proliferating tumor cells for each animal), the total number of HNA-positive cells co-labeled with EdU across all areas quantified was divided by the total number of human nuclei-positive cells counted across all areas quantified. This was repeated for human nuclei-positive cells assessed for double-labeling with Ki67 (proliferation index) or for cleaved caspase-3 (cell death). For cleaved caspase-3 staining, ischemic mouse brain tissue was used as a positive staining control (brain placed in PBS after removal at 37°C for five hours prior to fixation with 4% paraformaldehyde overnight, then transferred to 30% sucrose for cryoprotection). Cell quantification was performed by live counting at 400x magnification using a Zeiss LSM700 scanning confocal microscope and Zen 2011 imaging software (Carl Zeiss Inc., Pleasanton, CA). For the single-session stimulation paradigm, the area for quantification within the active circuit was selected as follows: of a 1 in 6 series of 40-μm coronal sections, 3 consecutive sections were selected at approximately 1.1–0.86 mm anterior to bregma (Figures 22, 23, 24 in); using our stereotactic coordinates for tumor xenograft, these sections are expected to include the tissue most proximal to the site of tumor cell implantation in the coronal plane. For each of the three consecutive sections, the cingulum bundle was first identified as an anatomic landmark, and a 160x160-μm field area for quantification ( Figure S1 C, Field 1) was selected immediately superficial to this landmark within cortical layer 6b of M2. A second field (Field 2) was selected immediately deep to this landmark in the corpus callosum. Two additional quantification fields (3,4) were selected so as to lie within cortical layer 6b/6a, immediately superficial to the topmost edge of Field 1, and juxtaposed side-by-side about the Field 1 mediolateral midpoint. Similarly, two additional quantification fields (5,6) were selected so as to lie within the corpus callosum, immediately deep to the bottommost edge of Field 2, and juxtaposed side-by-side about the Field 2 mediolateral midpoint (see schematic Figure S1 C). As each field was live-counted through the entire slice thickness of 40 μm, the total volume quantified per field was 1.024 × 10μm; six fields within each of three coronal sections were selected for a total quantified volume of 18.432 × 10μmper animal. These selected premotor cortex and corpus callosum areas lie within the active premotor circuit, but are deep to the path of tumor cell injection by our stereotactic coordinates, and thus avoid the principal areas of inflammatory change involving tissue more proximal to the injection site. Within each field, all human nuclear antigen (HNA)-positive tumor cells were quantified to determine tumor burden within the areas quantified. HNA-positive tumor cells were then assessed for double-labeling with either EdU or Ki67 (cell proliferation), or with cleaved caspase-3 (cell death). To calculate proliferation index (the percentage of proliferating tumor cells for each animal), the total number of HNA-positive cells co-labeled with EdU across all areas quantified was divided by the total number of human nuclei-positive cells counted across all areas quantified. This was repeated for human nuclei-positive cells assessed for double-labeling with Ki67 (proliferation index) or for cleaved caspase-3 (cell death). For cleaved caspase-3 staining, ischemic mouse brain tissue was used as a positive staining control (brain placed in PBS after removal at 37°C for five hours prior to fixation with 4% paraformaldehyde overnight, then transferred to 30% sucrose for cryoprotection). 6 μm3 per animal ( Using the same selected coronal tissue sections of the 1 in 6 series, the area of quantification outside the active circuit was selected as follows: for each of the three consecutive sections per animal, the cingulum bundle was first identified as an anatomic landmark, and a 160x160-μm field area for quantification was selected in the prefrontal cortex adjacent to the longitudinal fissure and medial to the cingulum bundle. Two additional fields were selected side-by-side moving laterally in the prefrontal cortex but outside the active circuit, for a total quantified volume of 9.216 × 10μmper animal ( Figure S1 D). Analysis of Tumor Cell Burden Animals were included that had well-matched xenografts with respect to location of injection tract in the rostro-caudal dimension and with respect to cortical depth. The target location was midway through the cortical depth and just outside of the rostro-caudal center of premotor area M2 but within ∼240 μm of the premotor area M2 midpoint in the rostro-caudal dimension; in this way, the xenografted cells diffusely infiltrate the area of optogenetic stimulation in M2 but xenograft needle injury and optical ferrule placement are not induced in the same coronal plane. For the repetitive stimulation experiment, two litters of mice born one day apart were xenografted two days apart at ∼P35 using two separate flasks of SU-pcGBM2 cells (at passage 19 and passage 20, respectively). The two litters had optical-neural interfaces placed as above at 9 weeks following xenotransplantation and were stimulated at 10 weeks following xenotransplantation in parallel. Because these two litters were xenografted on different days with cells from different flasks, the data were normalized to the mean tumor cell density of the identically manipulated WT control animals from each litter. Tumor cell burden was analyzed in the superior ∼1/3 of the corpus callosum, 100 μm deep to the cortical-corpus callosum border, the region containing the most dense projections from both M2 and M1 motor cortices (Allen Brain Atlas). Quantification of HNA-positive cell density was performed by a blinded investigator as follows: three consecutive sections of a 1 in 2 series of 40-μm coronal sections were selected for closest proximity to the site of fiber optic placement. For each section, a maximum-intensity projection image of a z-stack of 6 slices 6 μm apart through the thickness of the section was obtained at 100x magnification using a Zeiss LSM700 scanning confocal microscope and Zen 2011 imaging software (Carl Zeiss, Pleasanton, CA). HNA-positive tumor cells were then counted within five 100 μm x 100 μm boxed counting areas aligned with the cortical-corpus callosum border and centered about the apex of the cingulum bundle in the maximum intensity projection images using Adobe Photoshop software (Adobe, San Jose, CA).

Electrophysiology in Slices Coronal brain sections containing the motor cortex were cut from Thy1::ChR2 mice. Following isoflurane anesthesia, animals were decapitated and the brain rapidly removed and placed in ice-cold, oxygenated artificial CSF (aCSF) containing (in mM): 118 NaCl, 2.5 KCl, 2.5 NaHCO3, 10 glucose, 1.3 MgCl2, 2.5 CaCl2, and 1.2 NaH2PO4. Slices were cut in 300 μm sections using a vibratome (Leica VT 1200S, Buffalo Grove, IL), and incubated at 32°C for 30 min before being allowed to equilibrate at room temperature for at least a further 30 min. During recording, slices were perfused with heated aCSF (32 ± 2°C). For whole cell current clamp recordings from Layer V/VI motor cortical pyramidal cells, recording pipettes (3-5 MΩ) fabricated from borosilicate glass were filled with a solution containing (in mM): 135 KMeSO4, 8 NaCl, 10 HEPES, 2 Mg2ATP, 0.3 Na3GTP, 0.1 spermine, 7 phosphocreatine, and 0.3 EGTA. GABAA antagonist picrotoxin (100 μM) and AMPA receptor antagonist NBQX (10 μM) were added to the aCSF during all the experiments. 25-ms light pulses were delivered at 20 Hz for 15 min to evoke ChR2-mediated action potentials. All recordings were made using MultiClamp 700B (Molecular Devices, Sunnyvale, CA), filtered at 10 kHz and digitized at 20 kHz using an ITC-16 board (Instru-Tech, Port Washington, NY) and acquired using Axograph X software (Berkeley, CA).

Verification of Slice Health All slices evaluated pre- and post-stimulation as in Figure S2 were fixed in formalin, paraffin-embedded, and stained with hematoxylin and eosin by standard protocol. Histological evaluation of cortical slice health was performed by a board-certified neuropathologist (H.V.).

Generation of Conditioned Media from Acute Stimulation Thy1::ChR2 or WT mice between the age of 4-7 weeks were briefly exposed to CO 2 and immediately decapitated. Extracted brains were placed in oxygenated high-sucrose solution and sliced in 350-μm sections. Slices were then placed in buffering solution (aCSF) and allowed to recover for at least one hr (as above). After recovery, slices were then moved into fresh aCSF in a 24-well plate and stimulated using a blue-light LED from a microscope objective. The stimulation paradigm mirrored in vivo experiments, using 20-Hz pulses of blue light for 30 s on, 90 s off over a period of 30 min. Surrounding medium was then collected for immediate use or frozen at −80°C for future experiments.

Generation of Conditioned Media from 4 Hr Incubation As above, WT slices were sectioned on the vibratome and recovered in aCSF solution. Slices were then placed in 24-well plates in oxygenated aCSF in the presence or absence of 1μM tetrodotoxin (TTX; Tocris Biosciences, Minneapolis, MN) for 4 hr duration. After the 4 hr time period, surrounding media was collected.

EdU Incorporation Assay 8-well chamber slides were coated with poly-L-lysine. Cells were then seeded at 40,000 cells per well and exposed to either blue light-exposed or unexposed aCSF or conditioned media from stimulated or unstimulated Thy1::ChR2 slices or WT slices, or various recombinant proteins (concentrations and conditioning methods vary by assay). 10 μM EdU was added to each well. Cells were fixed after 24 hr using 4% paraformaldehyde in PBS and stained using the Click-iT EdU kit and protocol (Invitrogen, Carlsbad, CA). Proliferation index was then determined by quantifying the fraction of EdU labeled cells/DAPI labeled cells using confocal microscopy at 200x magnification, as above.

CellTiter-Glo Assay To assess overall cell number, 5,000 cells per well of SU-pcGBM2, SU-DIPGIV, SU-DIPGXIII, SU-DIPGVI, SU-GBM034, SU-GBM035, SU-GBM047, SU-GBM052, or SU-AO2 were seeded in minimal growth media in a 96-well plate with either CM from optogenetically stimulated Thy1::ChR2 slices, CM from blue light-exposed WT slices, or aCSF. After 72 hr, CellTiter-Glo reagent (Promega, Madison, WI) was added at a 1:1 ratio. Luminescence was measured after 10 min incubation at room temperature to stabilize signal.

Annexin V Apoptosis Assay SU-pcGBM2 cells were cultured in minimal growth media together with 50nM NLGN3 recombinant protein (dissolved in PBS) or PBS only as a vehicle control in duplicate for 24 hr. Cells were harvested and FACS analyses of apoptosis were performed using Annexin V-FITC Apoptosis Detection Kit II (556570, BD Biosciences, San Jose, CA) according to the manufacturer’s instruction with slight modifications. DAPI was used in combination with Annexin V-FITC to stain the apoptotic cell population. The stained cells were analyzed using a BD Fortessa FACS machine (BD Biosciences, San Jose, CA). The data were analyzed using FlowJo software (FlowJo, LLC).

Biochemical Assays Fractionation experiments were performed using Amicon ultracentrifugal filters with either 10KDa or 100KDa cutoff membranes (Millipore, Billerica, MA). Conditioned medium from either blue light-exposed WT or optogenetically stimulated Thy1::ChR2 slices was spun through filters at 12,000 rpm for 30 min. All proteins were resuspended in equal volumes of aCSF. Protein denaturation was achieved by boiling the conditioned media from blue light-exposed WT or stimulated Thy1::ChR2 slices for 7 min at 100°C. Nucleic acid degradation was achieved by treatment of the conditioned media with RNase and DNase at a concentration of 2 μg/mL and incubated for 1 hr before being added to the cells. All experiments were performed in triplicate.

Two-Dimensional Gel Electrophoresis Two-dimensional gel electrophoresis (2-D DIGE) and subsequent Protein ID were performed by Applied Biomics, Inc (Hayward, CA). Preparation of Samples and CyDye Labeling Protein sample buffer was exchanged into 2-D cell lysis buffer (30 mM Tris-HCl, pH 8.8, containing 7 M urea, 2 M thiourea and 4% CHAPS). Protein concentration was measured using Bio-Rad protein assay method (Hercules, CA). For each sample, 30 μg of protein was mixed with 1.0 μl of diluted CyDye, and kept in the dark on ice for 30 min. The labeling reaction was stopped by adding 1.0 μl of 10 mM Lysine to each sample, and incubating in the dark on ice for an additional 15 min. The labeled samples were then mixed together. The 2X 2-D Sample buffer (8 M urea, 4% CHAPS, 20 mg/ml DTT, 2% pharmalytes and trace amount of bromophenol blue), 100 μl destreak solution and Rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mg/mL DTT, 1% pharmalytes and trace amount of bromophenol blue) were added to the labeling mix to make the total volume of 250 μl for the 13-cm IPG strip. IEF and SDS-PAGE After loading the labeled samples, IEF (pH 3-10) was run following the protocol provided by GE Healthcare. Next, the IPG strips were incubated in the freshly made equilibration buffer-1 (50 mM Tris-HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, trace amount of bromophenol blue and 10 mg/mL DTT) for 15 min with gentle shaking. Then the strips were rinsed in the freshly made equilibration buffer-2 (50 mM Tris-HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, trace amount of bromophenol blue and 45 mg/mL Iodoacetamide) for 10 min with gentle shaking. Next, the IPG strips were rinsed in the SDS-gel running buffer before transferring into 12% SDS-gels. The SDS-gels were run at 15°C until the dye front ran out of the gels. Image Scan and Data Analysis Gel images were scanned immediately following the SDS-PAGE using Typhoon TRIO (GE Healthcare, Waukesha, WI). The scanned images were then analyzed by Image Quant software (version 6.0, GE Healthcare, Waukesha, WI), followed by quantitation analysis using DeCyder software (version 6.5, GE Healthcare, Waukesha, WI). The fold change of the protein expression levels was obtained from in-gel DeCyder analysis.

Protein Identification by Mass Spectrometry Spot Picking and Trypsin Digestion The spots of interest were picked up by Ettan Spot Picker (Amersham BioSciences, Piscataway, NJ) based on the in-gel analysis and spot picking design by DeCyder software. The gel spots were washed a few times, then digested in-gel with modified porcine trypsin protease (Trypsin Gold, Promega, Madison, WI). The digested tryptic peptides were desalted by Zip-tip C18 (Millipore, Billerica, MA). Peptides were eluted from the Zip-tip with 0.5 μl of matrix solution (α-cyano-4-hydroxycinnamic acid 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid, 25 mM ammonium bicarbonate) and spotted on the AB SCIEX MALDI plate (Opti-TOF 384 Well Insert, AB SCIEX, Framingham, MA). Mass Spectrometry MALDI-TOF MS and TOF/TOF tandem MS/MS were performed on an AB SCIEX TOF/TOF 5800 System (AB SCIEX, Framingham, MA). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each of the 10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions). Database Search Both the resulting peptide mass and the associated fragmentation spectra were submitted to GPS Explorer workstation equipped with MASCOT search engine (Matrix Science, Boston, CA) to search the Swiss-Prot database. Searches were performed without constraining protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine residues, and with one missed cleavage also allowed in the search parameters. Candidates with either protein score C.I.% or Ion C.I.% greater than 95 were considered significant.

Spectral Counting and Tandem Mass Tags Proteomic Analyses Proteomics Materials Ammonium bicarbonate, dithiothreitol (DTT), and iodoacetamide were purchased from Sigma (St. Louis, MO). Sequencing grade modified porcine trypsin was purchased from Promega (Madison, WI). Formic acid, HPLC grade acetonitrile, HPLC grade water and Amino reactive TMT reagents (126 to 131) were purchased from ThermoFisher Scientific (Waltham, MA). BCA protein assay kit was purchased from Pierce (Waltham, MA). C18 Magic bead size 5 μm, pore size 300 Å was purchased from Michrom BioResources (Auburn, CA). Symmetry 300 C18 5 μm NanoEase trap column was purchased from Waters (Milford, MA). Proteomic Sample Preparation Faca et al., 2007 Faca V.

Pitteri S.J.

Newcomb L.

Glukhova V.

Phanstiel D.

Krasnoselsky A.

Zhang Q.

Struthers J.

Wang H.

Eng J.

et al. Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes. Kani et al., 2012 Kani K.

Sordella R.

Mallick P. Investigation of acquired resistance to EGFR-targeted therapies in lung cancer using cDNA microarrays. 4 HCO 3 buffer (pH 8.0) with 10 mM DTT, 0.1% PPS detergent (Agilent Technologies, Santa Clara, CA), for 2 hr at 56°C. The reaction was cooled to room temperature and the sample was alkylated with 50 mM iodoacetamide for 60 min at room temperature in the dark. The resulting mixtures were diluted 6-fold with 50 mM NH 4 HCO 3 (pH 8.0), and then trypsin was added at a trypsin-to-protein ratio of 1:50 (w/w). The reaction was incubated for 18 hr at 37°C. The digestion was interrupted and acidified by addition of 5 μl of 10% formic acid solution. For TMT analysis, Amino reactive TMT reagents (126 to 131, 0.8 mg; Thermo Scientific, Waltham, MA) were dissolved in 41 μl acetonitrile, and 10 μl of the solution was added to 100 μg of peptides. After incubating for 1 hr at room temperature (22°C), the reaction was quenched by adding 8 μl of 5% w/v hydroxylamine for 15 min. Following labeling, the sample was combined in equal ratios. The peptide-containing samples were then dried using a speed vacuum concentrator. The samples were reconstituted with 10–20 μl of 0.1% (vol/vol) formic acid in water. Protein digestion and identification by LC-MS/MS was performed as described previously (). Briefly, sample concentration was estimated by Pierce BCA protein assay (Thermo Scientific, Waltham, MA). Next, in-solution digestion was performed with lyophilized samples that were resuspended, denatured and reduced in 50 mM NHHCObuffer (pH 8.0) with 10 mM DTT, 0.1% PPS detergent (Agilent Technologies, Santa Clara, CA), for 2 hr at 56°C. The reaction was cooled to room temperature and the sample was alkylated with 50 mM iodoacetamide for 60 min at room temperature in the dark. The resulting mixtures were diluted 6-fold with 50 mM NHHCO(pH 8.0), and then trypsin was added at a trypsin-to-protein ratio of 1:50 (w/w). The reaction was incubated for 18 hr at 37°C. The digestion was interrupted and acidified by addition of 5 μl of 10% formic acid solution. For TMT analysis, Amino reactive TMT reagents (126 to 131, 0.8 mg; Thermo Scientific, Waltham, MA) were dissolved in 41 μl acetonitrile, and 10 μl of the solution was added to 100 μg of peptides. After incubating for 1 hr at room temperature (22°C), the reaction was quenched by adding 8 μl of 5% w/v hydroxylamine for 15 min. Following labeling, the sample was combined in equal ratios. The peptide-containing samples were then dried using a speed vacuum concentrator. The samples were reconstituted with 10–20 μl of 0.1% (vol/vol) formic acid in water.

Mass Spectrometric Data Acquisition Nano-LC-MS/MS was performed using an Eksigent nanoLC 2D system (Dublin, CA) interfaced with a LTQ-Velos-Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA) which is coupled with a CaptiveSpray source (Michrom BioResources, Auburn, CA). The composition of solvent A was 0.1% (v/v) of formic acid in water and solvent B consisted of 0.1% (v/v) of formic acid in HPLC-grade acetonitrile. 10 μl of the digested samples were injected using a CTC autosampler (Leap Technologies, Carrboro, NC) onto a Symmetry 300 C18 5 μm NanoEase trap column. Samples were loaded onto the trap column at a flow rate of 5 μL/min, for 20 min using 0.1% formic acid in water. The peptides were eluted from the trap column and subsequently separated on an IntegraFrit capillary analytical column (150 mm × 75 μm i.d.) packed in-house with Magic C18, using 90 min linear gradient (3%–40% solvent B) at a flow rate of 600 nL/min. The mass spectrometer was operated in a data-dependent MS/MS mode. A single full MS scan, collected in the Orbitrap in profile mode over the mass range of 400–2000 m/z, was accompanied by 10 MS/MS scans, collected in centroid mode in the LTQ, of the 10 most intense peaks. Dynamic exclusion parameters included: repeat count = 1, repeat duration = 30, exclusion list size = 400, exclusion duration = 30 s, and dynamic exclusion width = 1.5 (high and low by mass). For TMT analysis, HCD fragmentation in the Orbitrap was performed.

Western Blot Analysis Briefly, as presented in Figure 5 B and 5D, cells were lysed after 1 hr exposure to either 0 nM, 5 nM, 10 nM, or 50 nM recombinant NLGN3 (Origene Technologies, Rockville, MD), using RIPA buffer and protease inhibitors. In Figure 6 I, cells were exposed to 50 nM NLGN3 for 12 hr, and then allowed to recover in fresh media for 24 hr. After thorough washing, cells were lysed as above. For all experiments, lysates were incubated on ice for 10 min and then centrifuged for 10 min at 4°C. Protein concentration in the lysate was determined using a Bradford assay. Samples were then normalized to protein concentration, mixed with Laemmli loading buffer (1:4), boiled for 5 min, and loaded onto BioRad Mini-Protean TGX precast gels. Protein was transferred to PVDF membranes and blocked with 5% bovine serum albumin (BSA) in TBST for one hr. Primary antibodies were diluted in 1% BSA/TBST and incubated with the membrane overnight. Antibodies against AKT, phospho-AKT (Ser473), DYKDDDDK Tag, p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), 4E-BP1, phospho-4E-BP1 (Thr37/46), PI3K p110a, and mTOR were purchased from Cell Signaling (Danvers, MA) and used at a concentration of 1:1000. Anti-Neuroligin-3 (NovusBio, Littleton, CO) was used at a concentration of 1:300. Anti- β-tubulin (Abcam, Cambridge, MA) was used at 1:5000. Secondary anti-rabbit conjugated to HRP (BioRad, Hercules, CA) was then added for one hour (1:1000). Proteins were visualized using Clarity ECL Western Substrate (BioRad, Hercules, CA) and quantified using ImageJ.

qPCR Analysis 500,000 SU-pcGBM2 or SU-DIPGXIII cells were exposed to either aCSF, 50 nM NLGN3 (Origene Technologies, Rockville), 100 nM BKM120 (SelleckChem, Houston, TX), 100 nM RAD001 (SelleckChem, Houston, TX), 50 nM EGF (Shenandoah, Warwick, RI), or a combination of the above treatments. RNA was extracted using the TRIzol Reagent (Life Technologies, Carlsbad, CA) at either 1 hr ( Figure 5 A) or 12 hr ( Figure 6 A-H, S5 D-E, S6 ) after treatment. For qPCR analysis, cDNA was prepared using iScript cDNA Synthesis Kit (BioRad, Hercules, CA). RT-PCR was performed on Eppendorf Mastercyler Realplex2 using Universal SYBR Green Supermix (BioRad, Hercules, CA). Differential expression was determined using the delta CT method. Primers used were as follows: FOS forward: 5′ CCTAACCGCCACGATGATGT 3′; FOS reverse: 5′ TCTGCGGGTGAGTGGTAGTA 3′; NLGN3 forward: 5′ GGGAGTCCCCTTTCTGAAGC 3′; NLGN3 reverse: 5′ CCTTCATGGCCACACTGACT 3′; ACTB forward: 5′ TGAAGTGTGACGTGGACATC 3′; ACTB reverse: 5′ GGAGGAGCAATGATCTTGAT 3′.

RNA Sequencing RNA sequencing was performed by Elim Biopharm (Hayward, CA). Total RNA was treated with RiboZero (EpiCentre, Madison, WI) to remove rRNA. The resulting RNA was subject to cDNA synthesis with standard protocol for the first and second strands of cDNA synthesis. Illumina library was prepared from the ds cDNA according to Illumina’s standard NGS library preparation method. The libraries were quantified and Q.C.’ed by Qubit, Bioanalyzer, and qPCR. The libraries were then sequenced on Illumina HiSeq2500 (Madison, WI) with 50 bp paired-end read run, generating 179.62M reads. For data analysis, TopHat was used for mapping to the reference genome (hg19(UCSC)), and Cufflinks was used for differential expression analysis.

Pharmacologic Inhibition SU-pcGBM2 or SU-DIPGXIII cells were treated with 100 nM BKM120 (SelleckChem, Houston, TX) or 100 nM RAD001 (SelleckChem, Houston, TX) dissolved in DMSO. All experiments using inhibitor treatments used vehicle DMSO treatment as control.

shRNA-Expressing Lentivirus Preparation and Infection shRNA expressing lentiviral constructs against human PIK3CA and mTOR from the RNAi consortium human collection were purchased from Sigma (St. Louis, MO). Lentiviral expressing constructs were co-transfected with packaging plasmids (pDelta 8.92 + VSV-G) into 293T cells to generate lentiviral particles. Lentiviral particles were then concentrated by the polyethylene glycol precipitation method. The precipitated lentiviruses were resuspended in PBS and aliquoted for −80°C storage. For lentiviral infection, SU-pcGBM2 cells were incubated with shRNA expressing lentivirus; 48 hr post-infection, puromycin (0.5 ug/ml) was added to select virally infected cells for further experiments.

Glutamate Assay Glutamate concentration in the conditioned media was determined using the reagents and protocols in the Glutamate Assay Kit purchased from Sigma (St. Louis, MO).

Recombinant Proteins Used GRP78 (Abcam, Cambridge, MA), BDNF, Brevican, Apolipoprotein E (R&D Systems, Minneapolis, MN), NLGN3 (Origene, Rockville, CA), NRXN1β (R&D Systems, Minneapolis, MN).