This work was supported by National Institutes of Health/National Institute of Neurological Disorders &#38; Stroke (NIH/NINDS) Grants R37-NS094804, R01-NS074387, R21-NS091555 to M.G.C.; NIH/NINDS Grants R01-NS076991, R01-NS082311, and R01-NS096756 to P.R.L.; NIH/NINDS R01-EB022563; the Department of Neurosurgery; Leah&#39;s Happy Hearts, and Chad Though Foundation to M.G.C. and P.R.L. RNA Biomedicine Grant F046166 to M.G.C. F.M.M. is supported by an F31 NIH/NINDS-F31NS103500. R.I.Z.-V. is supported by the NIH/ NIGMS grant 5T34GM007821-37.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.
1. Generation of brain tumor NS and culturing conditions.
<ol>
	<li>Prepare Neural Stem Cell Medium (NSC) with Dulbecco&#39;s Modified Eagle Medium/F12B27 supplement (1x), N2 supplement (1x), and an antimicrobial reagent. Supplement NSC medium on the day of use with human recombinant endothelial growth factor (EGF) and basic-fibroblast growth factor (FGF) at a final concentration of 20 ng/mL each.</li>
	<li>Induce the brain tumor in mouse using the Sleeping Beauty transposon system in which plasmids encoding oncogenes or shRNA targeting tumor suppressors is injected into the lateral ventricles of neonates to generate spontaneous brain tumors9. Select a mouse with a large tumor, which is confirmed with bioluminescence imaging. 
	NOTE: More detailed information about the Sleeping Beauty Transposon System and plasmid constructs used can be found in Calinescu et al.9.
	<ol>
		<li>To image the mice, inject 100 &#181;L of 30 mg/mL luciferin solution intraperitoneally using a 26 gauge needle.</li>
		<li>5 min after injection of luciferin, anesthetize the animal using an anesthesia chamber with oxygen/isoflurane flow set to 2% isoflurane.</li>
		<li>When the animals are anesthetized (usually 2-3 min), place the animals in the bioluminescence chamber with oxygen isoflurane set to 2% isoflurane. If the animals will be under anesthesia for longer than 5 min, use an ophthalmic ointment to prevent dryness while under anesthesia.</li>
		<li>Image the mice using an in vivo optical imaging system with the following parameters: automatic exposure, large binning, and aperture f=1. The parameter to consider it a large tumor is a bioluminescence &#62;106 photons/s/cm2/sr.</li>
	</ol>
	</li>
	<li>Euthanize the mouse with an overdose of isoflurane inhalation anesthetic in a closed chamber, check by toe pinch that the animal is anesthetized, and decapitate the mouse.</li>
	<li>Extract the brain and dissect it as described previously in Calinescu et al.9.</li>
	<li>Place the brain in a 10 cm Petri dish. If the tumor induced expresses a fluorescent protein, use a fluorescent dissecting microscope set to the appropriate fluorescent channel and 0.3X magnification, otherwise identify tumor mass by the differences in tissue color and texture. Use scalpel and forceps to separate the tumor tissue from the normal brain.</li>
	<li>Mince the tumor into small pieces in the Petri dish. Place the dissected tumor in a 1.5 mL tube with 300 &#181;L of the NSC medium.</li>
	<li>Use a disposable plastic pellet pestle that fits to the walls of a conical microcentrifuge tube to gently homogenize the tumor. Add 1 mL of cell detachment medium and incubate the sample for 5 min at 37 &#176;C. 
	NOTE: The use of a pestle may kill some cells. If the reader wants to maximize the cell viability, the use of a pestle may be omitted.</li>
	<li>Pass the cell suspension from step 1.7 through a 70 &#181;m cell strainer and wash the strainer with 25 mL of NSC medium.</li>
	<li>Centrifuge the suspension at 300 x g for 5 min at room temperature, decant the supernatant and re-suspend the pellet into 6 mL of NSC medium.</li>
	<li>Plate the tumor cell suspension from step 1.9 into a T-25 culture flask and culture it at 37 &#176;C in a tissue culture incubator with an atmosphere of 95% air and 5% CO2. Usually after 3 days, there will be a mixture of some dead cells, adhered cells and some cells forming NS which float in the medium.</li>
	<li>Transfer the cell suspension into a 15 mL conical tube, collect only the cells that sink to the bottom using a micropipette and re-plate them onto a T-75 tissue culture flask.</li>
	<li>Maintain the cells at relatively high density (1-3 X 106 cells in a T-25). Passage the cells when they are confluent. 
	NOTE: Confluency is determined by the spheres size. Black centers of large spheres mean that there are dead cells in the center and indicate that the cells should be passaged immediately.</li>
	<li>Add the growth factors (EGF and basic-FGF; 20 ng/mL each) every three days. Change the medium when the cells are not confluent and the medium turns light orange.</li>
	<li>To change the medium, collect the old medium and centrifuge it at 300 x g at room temperature for 5 min and re-suspend the cell pellet in NSC medium supplemented with EGF and FGF at the concentration specified in step 1.1. 
	NOTE: If the spheres are at mid-to high confluency, but not ready to be passaged (sphere size is small with a diameter &#60; 100 &#181;m), then the pellet can be split into two flasks.</li>
	<li>To passage the cells, centrifuge the cells at 300 x g for 5 min at room temperature, decant the supernatant and incubate the cells in cell detachment medium for 5 min at 37 &#176;C.
	<ol>
		<li>Add 5 mL of balanced salt solution to dilute detachment medium and centrifuge it at 300 x g for 5 min at room temperature.</li>
		<li>Decant the supernatant, resuspend the cell pellet in 18 mL of NSC medium and divide the suspension equally into three T-25 flasks.</li>
	</ol>
	</li>
	<li>To freeze the aliquots of cells, dissociate the cells as in step 1.15, and freeze the cells in 1 mL aliquots of 90% fetal bovine serum with 10% dimethyl sulfoxide. Slowly cool down the cells by placing the cells on ice for 10 min, then keeping the cells -20 &#176;C for 30 min, and -80 &#176;C for 1 day. The next day, transfer the cells to a liquid nitrogen storage container.</li>
</ol>
2. Native ChIP
<ol>
	<li>Chromatin preparation
	<ol>
		<li>After making stock of derived NS, culture NS in a T-75 in 15 mL of NSC medium at 37 &#176;C in a tissue culture incubator with an atmosphere of 95% air and 5% CO2 until NS become confluent. One confluent T-75 plate will yield 3-5 X 106 cells.</li>
		<li>When the cells become confluent, centrifuge NS at 300 x g for 5 min, decant the supernatant and add 1 mL of cell dissociation medium. Incubate the cells at 37 &#176;C for 5 min. Add 5 mL of medium and count the cells using a hemocytometer10. 
		NOTE: 1 x 106 cells are needed per immunoprecipitation (IP) reaction. Note that a pre-immune serum or IgG control must also be included11.</li>
		<li>Centrifuge the NS in one 1.5 mL microcentrifuge tube at 300 x g for 5 min, decant the supernatant and re-suspend NS pellet with 1 mL of balanced salt solution and transfer the suspension to low protein binding 1.5 mL microcentrifuge tubes.</li>
		<li>Centrifuge NS at 300 x g for 5 min, decant the supernatant and re-suspend the cell pellet in 95 &#181;L of digestion buffer (50 mM Tris-HCl, pH 8.0, 1 mM CaCl2, 0.2% polyethylene glycol octylphenyl ether) per 1 x 106 cells supplemented with protease inhibitor cocktail at a high concentration (1:100). Immediately pipet the cells up and down to prevent clumping and avoid creating any bubbles.</li>
		<li>Resuspend MNase in 50% glycerol to make 1 mg/mL solution (1.15 units/&#181;L, stored at -20 &#176;C) that will be referred to as &#34;1x&#34; stock. Make a &#34;0.1x&#34; stock by doing a second 1:9 dilution with 50% glycerol (e.g., 900 &#181;L of &#34;1x&#34; MNase and 100 &#181;L of 50% glycerol) and store it at -20 &#176;C.</li>
		<li>Mix 5 &#181;L of &#34;0.1x&#34; MNase and 145 &#181;L of digestion buffer. Keep the enzyme on ice all the times. Add 5 &#181;L of diluted MNase in digestion buffer per 1 x 106 cells. Flick the tube to mix and place the tube in 37 &#176;C block for exactly 12 min. Process the samples one at a time to ensure an accurate incubation time of 12 min.</li>
		<li>Add 10 &#181;L of 10x MNase Stop Buffer (110 mM Tris-HCl, pH 8.0, 55 mM EDTA) per 1 x 106 cells. Samples must be kept on ice from this point onwards.</li>
		<li>Add 110 &#181;L &#34;2x RIPA buffer&#34; (280 mM NaCl, 1.8% polyethylene glycol octylphenyl ether, 0.2% sodium dodecyl sulfate (SDS), 0.2% Na-deoxycholate, 5 mM ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA); stored at 4 &#176;C) supplemented with protease inhibitor cocktail at high concentration (1:100) per 1 x 106 cells. Flick the tube to mix.</li>
		<li>Centrifuge the tubes for 15 min in a microfuge at 4 &#176;C and 1700 x g. Transfer the supernatant to a new regular 1.5 mL microcentrifuge tubes (low protein binding microcentrifuge tubes are no longer necessary) and store them on ice. Discard the pellet.</li>
	</ol>
	</li>
	<li>Immunoprecipitation (IP)
	<ol>
		<li>Mix Protein A and Protein G magnetic beads in a 1:1 ratio. For every IP, prepare 25 &#181;L of beads.</li>
		<li>Wash the beads by adding 1 mL of RIPA buffer (10 mM Tris pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% polyethylene glycol octylphenyl ether, 0.1% SDS, 0.1% Na-deoxycholate; stored at 4 &#176;C) supplemented with protease inhibitors at low concentration (1:1000).</li>
		<li>Use a magnet to allow beads to separate from the washing solution and decant the washing solution (approximately 1 min). Perform the wash step twice.</li>
		<li>Re-suspend the beads to the original volume using RIPA buffer supplemented with protease inhibitors (1:1000).</li>
		<li>If necessary, dilute the chromatin using RIPA buffer supplemented with protease inhibitors (1:1000) so that each IP has a volume of 100-200 &#181;L. Reserve 10% of the volume used per IP of chromatin for the input. 
		NOTE: For example, if 200 &#181;L are used per IP, 20 &#181;L of diluted chromatin should be reserved for the input. For a total of four IPs, total chromatin volume should be diluted to 820 &#181;L.</li>
		<li>Prepare the input. Add 100 &#181;L of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) supplemented with proteinase K (0.5 mg/mL) to the input and incubate the input at 55 &#176;C for 1 h. 
		NOTE: Steps 2.2.6-2.2.7 can be performed along with steps 2.3.4 and 2.4.1.</li>
		<li>Purify the input using a PCR purification kit following the manufacturer&#39;s instructions and elute in 50 &#181;L of elution buffer provided in kit.</li>
		<li>Measure the concentration of the input using a microvolume spectrophotometer and record the results in a notebook. Blank with elution buffer from kit. 
		NOTE: In mouse neurospheres, we used approximately 6 &#181;g of DNA for each IP.</li>
		<li>Run input on a 1% gel or load 1 &#181;L on a DNA Bioanalyzer using standard settings. Store the remainder for use as input in downstream applications.</li>
		<li>Add 10 &#181;L of washed protein A &#38; G magnetic beads for every IP and incubate IP for 1 h at 4 &#176;C. 
		NOTE: For example, add 30 &#181;L of protein A &#38; G magnetic beads for three IPs.</li>
		<li>Place the samples on a magnet and allow beads to separate. Divide chromatin by transferring the previously determined amount into a new 1.5 mL tube.</li>
		<li>Add the antibody and wrap the caps with plastic paraffin film to avoid evaporation. Incubate the samples overnight at 4 &#176;C with rotation at 20 rpm. 
		NOTE: Concentration should be determined empirically following manufacturer recommendations.</li>
		<li>The next day, spin the tubes using a mini centrifuge at room temperature, 2000 x g, for 10 s. Then add 10 &#181;L of beads to each IP and incubate IP for 3 h at 4 &#176;C with rotation at 20 rpm.</li>
	</ol>
	</li>
	<li>IP Washes and Protein Digestion
	<ol>
		<li>Add 150 &#181;L of RIPA buffer supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 &#176;C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow the magnetic beads to separate. Use a pipet to remove the supernatant. Repeat this tep five washes.</li>
		<li>Add 150 &#181;L of LiCl buffer (250 mM LiCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5 % Na-deoxycholate; store at 4 &#176;C) supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 &#176;C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove supernatant.</li>
		<li>Add 150 &#181;L of cold TE (no protease inhibitors) and incubate the sample at 4 &#176;C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove the supernatant.</li>
		<li>After the final washing step, re-suspend the sample in 100 &#181;L of TE buffer supplemented with proteinase K (0.5 mg/mL) and incubate the sample at 55 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>DNA Purification and ChIP Quantitative Real-Time PCR (qPCR)
	<ol>
		<li>Purify immunoprecipitated DNA using a PCR purification kit. After addition of DNA binding buffer from the kit to each sample, place the tube on a magnet and wait until magnetic beads aggregate, then transfer the supernatant to the cleanup column. Follow the manufacturer&#39;s instructions and elute in 50 &#181;L of elution buffer provided in kit.</li>
		<li>To check if the IP is successful, perform a qPCR. Ideally, primers should be designed for positive and negative control regions. 
		NOTE: For example, for an IP performed with H3K4me3 and H3K27me3, the following samples should be run on qPCR: H3K4me3, H3K27me3, and IgG ChIP DNA, input DNA, and a no template control (NTC) using primers for regions to be enriched with H3K4me3 (positive control) and regions to be enriched with H3K4me3 (negative control); likewise, for H3K27me3.</li>
		<li>Follow standard protocols to perform qPCR12. Briefly, perform a qPCR using SYBR green master mix, 0.5 &#181;L of 10 &#181;M working concentration of each forward and reverse primer, and 2 &#181;L of ChIP or input DNA. Read plates using a Real Time PCR system. Primer sequences are provided in Table 2. The Gapdh primer sequences from Hwang et al. were used13.</li>
		<li>Analyze ChIP data relative to the input, i.e., percent input method (% IP)14. Calculate the percent input using the following formulas: 
		Adjusted input = mean ct(input)-log2(DF) 
		where DF is the dilution factor of the starting input and 
		DF = total volume of IP/ volume of input 
		% IP = 100&#215;2^(Adjusted input - Ct (IP)) 
		where Ct is the threshold factor.</li>
	</ol>
	</li>
</ol>

<ol>
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The authors have nothing to disclose.

The protocol presented here will enable the user to perform native ChIP on NS derived from genetically engineered brain tumors. In contrast to cross-linking ChIP, this protocol is limited for the study of proteins that associate tightly with DNA6. The number of cells used can be modified as necessary and the protocol can be scaled up. We used 1 X 106 cells per IP, however, native ChIP may also be performed with as little as 4 x 104 cells5. In this prot...

Epigenetic events are frequent in gliomas and likely play an important role in tumor pathogenesis. Indeed, in pediatric high-grade glioma, mutations in genes encoding histone variants H3.3 and H3.1 occur frequently1. The mutations affect histone modifications and have major epigenetic consequences2,3. In the adolescent to adult spectrum, recurrent mutations in isocitrate dehydrogenase gene 1/2 (IDH1/2), a mutation that inhibits &#945;-KG dependent histone and DNA de-methylasaes, and genetic alterations in other chromatin regulators such as ATRX and DAXX occur4. Therefore, it is of critical importance to study how mutations that affect epigenetic regulators alter chromatin structure and regulatory histone modifications, which, in turn, have a dramatic impact of the tumor cells&#39; transcriptome.
Chromatin immunoprecipitation (ChIP) is a powerful tool used to evaluate the impact of epigenetic modifications in the genome5,6,7. In native ChIP, chromatin is digested with micrococcal nuclease (MNase), immunoprecipitated using an antibody raised against the protein of interest, and then DNA is purified from the immunoprecipitated chromatin complex6. Cells are not fixed during the procedure so this technique is only applicable for the study of proteins that interact tightly with DNA6. The absence of cross-linking aids antibody specificity since antibodies are usually raised against unfixed peptides or proteins7. In addition, since there is no cross-linking step, this reduces the chances of fixing transient protein-DNA interactions that are non-specific and not regulatory7,8. ChIP can be used to identify the enrichment of histone modifications in a specific genomic region. Here, we detail a protocol for performing native ChIP in neurospheres (NS) generated from genetically engineered mouse models of glioma.

<table><tbody><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent</td><td>G2946-90004</td><td>bioanalyzer</td></tr><tr><td>AccuSpin Micro 17R, refrigerated</td><td>Fisher Scientific</td><td>13-100-676</td><td></td></tr><tr><td>C57BL/6</td><td>Taconic</td><td>B6-f</td><td>C57BL/6 mouse</td></tr><tr><td>Calcium Chloride</td><td>Aldrich</td><td>22350-6</td><td>buffer reagent</td></tr><tr><td>D-Luciferin, Potassium Salt</td><td>Goldbio</td><td>LuckK-1g</td><td></td></tr><tr><td>DiaMag1.5 magnetic rack</td><td>Diagenode</td><td>B04000003</td><td>for magnetic bead washes</td></tr><tr><td>DiaMag Rotator EU</td><td>Diagenode</td><td>B05000001</td><td>rotator</td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>11330-057</td><td>NSC component</td></tr><tr><td>Dynabeads Protein A</td><td>Thermo Fisher Scientific</td><td>10001D</td><td>protein A magnetic beads</td></tr><tr><td>Dynabeads Protein G</td><td>Thermo Fisher Scientific</td><td>10003D</td><td>protein G magnetic beads</td></tr><tr><td>EGF</td><td>PeproTech</td><td>AF-100-15</td><td>prepare 20 &amp;mu;g/mL stock in 0.1% BSA and aliquot.</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>E-4884</td><td>buffer reagent</td></tr><tr><td>ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&#039;,N&#039;-tetraacetic acid) (EGTA)</td><td>Sigma</td><td>E-4378</td><td>buffer reagent</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>Applied Biosystems</td><td>4385612</td><td>qPCR reagent</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10437028</td><td>for freezing cells</td></tr><tr><td>FGF</td><td>PeproTech</td><td>100-18b</td><td>Prepare 20 &amp;mu;g/mL stock in 0.1% BSA and aliquot.</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11008-13</td><td>for dissection of tumor</td></tr><tr><td>Glycerol, MB Grade</td><td>EMD- Millipore</td><td>356352</td><td></td></tr><tr><td>H3K4me3</td><td>Abcam</td><td>Ab8580</td><td></td></tr><tr><td>H3K27me3</td><td>Millipore</td><td>07-449</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175-103</td><td>balanced salt solution</td></tr><tr><td>Fluriso</td><td>VETone</td><td>501017</td><td>inhalation anesthetic</td></tr><tr><td>Ivis Spectrum</td><td>Perkin-Elmer</td><td>124262</td><td>in vivo optical imaging system</td></tr><tr><td>Hyqtase</td><td>HyClon</td><td>SV3003001</td><td>cell detachment media</td></tr><tr><td>Lithium Chloride</td><td>Sigma</td><td>L8895</td><td>buffer reagent</td></tr><tr><td>Low binding microtubes</td><td>Corning Costar</td><td>CLS3207</td><td>low protein binding microcentrifuge tube</td></tr><tr><td>Microcentrifuge tube</td><td>Fisher</td><td>21-402-903</td><td>regular microcentrifuge tube</td></tr><tr><td>Micrococcal Nuclease</td><td>Thermo Fisher Scientific, Affymetrix</td><td>70196Y</td><td>each batch may differ; purchase sufficient amount for experiments and aliquot.</td></tr><tr><td>N2</td><td>Gibco</td><td>17502-048</td><td>NSC component</td></tr><tr><td>Normal Rabbit IgG</td><td>Millipore</td><td>12-372</td><td></td></tr><tr><td>Normocin</td><td>Invivogen</td><td>NOL-36-063</td><td>anti-microbial agent, use at 0.1 mg/mL.</td></tr><tr><td>NP-40 (Igepal CA-630)</td><td>Sigma</td><td>18896-50ML</td><td>buffer reagent</td></tr><tr><td>Kimble Kontes Pellet Pestle</td><td>Fisher Scientific</td><td>K749515-0000</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>P8340</td><td>aliquot and store at -20 &amp;deg;C.</td></tr><tr><td>Protinase K</td><td>Sigma-Aldrich</td><td>P2308</td><td>make 10 mg/mL stock in water; aliquot and store at -20 &amp;deg;C.</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td>DNA purification kit</td></tr><tr><td>Scalpel</td><td>Fine Science Tools</td><td>10007-16</td><td>for dissection of tumor</td></tr><tr><td>Sodium Chloride</td><td>VWR</td><td>0241-5KG</td><td>buffer reagent</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>D670-25G</td><td>buffer reagent</td></tr><tr><td>Sodium Dodecyl sulfate (SDS)</td><td>Sigma</td><td>L-4390</td><td>buffer reagent</td></tr><tr><td>Tris Base</td><td>Thermo Fisher Scientific</td><td>Bp152-1</td><td>buffer reagent</td></tr><tr><td>Triton X-100</td><td>Thermo Fisher Scientific</td><td>BP 151-500</td><td>polyethylene glycol octylphenyl ether</td></tr><tr><td>Standard Mini Centrifuge</td><td>Fisherbrand</td><td>12-006-901</td><td>standard mini centrifuge</td></tr><tr><td>SZX16 microscope</td><td>Olympus</td><td>SZX16</td><td>flourescent dissecting microscope</td></tr><tr><td>ViiA 7 Real-Time PCR System with Fast 96-Well Block</td><td>Applied Biosystems</td><td>4453535</td><td></td></tr><tr><td>Nanodrop One</td><td>Thermo-Fisher Scientific</td><td>ND-ONEC-W</td><td></td></tr></tbody></table>

native chromatin immunoprecipitation using murine brain tumor neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

A schematic representation of tumor NS generated from a brain tumor where brain tumor cells are Katushka positive is presented in Figure 1. Figure 2 is a schematic representation of the entire ChIP technique. Figure 3 shows the representative results of chromatin from brain tumor NS digested with MNase for 12 min, yielding a majority of mono, di-, and tri- nucleosomes. Following ChIP, a qPCR may be p...

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Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

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JoVE Journal

Cancer Research

7.6K Views.  University of Michigan Medical School. Epigenetische mechanismen worden regelmatig gewijzigd in glioma. Chromatine immunoprecipitation kan worden gebruikt om de gevolgen van genetische wijzigingen in glioma die voortvloeien uit wijzigingen in histone modificaties tot regeling van de chromatine structuur en gene transcriptie. Dit protocol beschrijft een native chromatine immunoprecipitation op lymfkliertest hersenen tumor neurospheres.

Video: Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Biology

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Neuroscience

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Medicine

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of neuro-oncology. One of the most widely used techniques is the orthotopic intracranial injection, a method that allows investigating of tumor growth, invasion, and dissemination within a controlled setting. This technique involves transplanting tumor cells from a specific patient region into the corresponding anatomical site in an animal. By doing so, these orthotopic brain tumor models offer a unique advantage, as they more accurately replicate cancer's biological behavior and its interactions with the brain environment seen in human patients. This makes them especially valuable for preclinical therapeutic testing, where a close resemblance to the clinical scenario is essential for evaluating potential treatments. This protocol shares experiences in developing patient-derived xenograft (PDX) models for pediatric brain tumors, including diffuse midline glioma (DMG), glioblastoma (GBM), medulloblastoma, and ependymoma. This method delineates the procedure for conducting intracranial stereotaxic injections in mice, ensuring the correct targeting of the injection site within the brain. Additionally, we describe the post-procedural monitoring system employed to detect signs of successful tumor engraftment. Following tumor injection, a rigorous monitoring system is implemented to observe the animals for any signs of neurological impairment, behavioral changes, and/or weight loss, which are common indicators of tumor progression. This system allows for timely intervention and provides critical data regarding the tumor's growth dynamics. By refining these models and protocols, we aim to enhance the reliability and translational potential of preclinical studies, contributing to the development of more effective treatments for pediatric CNS tumors.

The development of orthotopic pediatric brain tumor models requires meticulous precision, using a stereotaxic device to implant cancer cells precisely. The methodology presented here outlines the steps involved in preparing brain tumor cells, performing intracranial injections, and implementing a post-operative monitoring system to assess brain tumor engraftment.

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Immunology and Infection

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections

<a href='https://app.jove.com/v/58931/evaluation-biomarkers-glioma-immunohistochemistry-on-paraffin'>Source: N&#250;&#241;ez, F. J., et al. Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures.&#160;J. Vis. Exp. (2019).</a>This video demonstrates an immunohistochemical technique for labeling tumor biomarkers in paraffin-embedded brain tumor neurospheres. The neurosphere sections undergo paraffin removal and rehydration, followed by treatment to expose biomarker sites. Primary and secondary antibodies are then applied to label these biomarkers. The secondary antibodies are bound by avidin-conjugated peroxidase enzymes, which oxidize chromogenic substrates to indicate biomarker locations. Finally, nuclear counterstaining and slide preparation are completed for microscopic analysis.

Source: N&#250;&#241;ez, F. J., et al. Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere ...

JoVE Encyclopedia of Experiments

Neuropathology

Tumors

Native Chromatin Immunoprecipitation of Neurosphere Cells

<a href='https://app.jove.com/v/57016/native-chromatin-immunoprecipitation-using-murine-brain-tumor'>Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).</a>This video demonstrates the isolation of protein-DNA complexes from neurosphere cells derived from a mouse brain tumor using native chromatin immunoprecipitation. It details the steps for chromatin preparation, immunoprecipitation, washing, and protein digestion to release DNA for downstream analysis.

Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).This video ...

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

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