We are grateful to Mr. Alan E. Kulaga for excellent technical assistance and to Ms. Michelle L. Gumprecht for refining the surgical techniques. We thank Dr. Philip L. Martin for pathology analysis and Ms. Lilia Ileva and Dr. Joseph Kalen of the Frederick National Laboratory Small Animal Imaging Program for MRI scans.
This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261201500003I. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

The study protocol described here was approved by the NCI at Frederick Animal Care and Use Committee. NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the &#8220;Guide for Care and Use of Laboratory Animals (National Research Council, 2011; The National Academies Press, Washington D.C.).
1. Preparation of cells for injection
NOTE: Mouse brain tumor primary cells (MBRs) used for this model were originally isolated from tamoxifen induced TRP GEM mice, as described in El Meskini et al.5. Details on the cell preparation can be found in this reference.
<ol>
	<li>Perform the following steps using sterile technique in a biosafety cabinet.</li>
	<li>Culture primary brain tumor cells in vitro until they reach the exponential growth phase.</li>
	<li>Harvest the cells in 0.25% trypsin, and once they have detached, dilute them with growth medium to inactivate the trypsin.</li>
	<li>Pellet the cells by centrifugation at 400 x g and wash with serum-free media. Repeat once prior to counting.</li>
	<li>Count the cells manually or with an automated cell counter. Resuspend the cells in 5% sterile methylcellulose in 1x phosphate buffered saline (PBS) at the desired concentration, based on a final injection volume of 2 &#181;L. The desired cell concentration may vary based on the cell line and brain tumor model. For GBM GEM TRP cells, we inject 2 &#181;L of a 25 x 106 cells/ml solution, or 50,000 cells.</li>
</ol>
2. Mouse strain
<ol>
	<li>Breed or purchase an appropriate mouse strain that matches the strain background of the brain tumor cells. For TRP cells, use 9-week-old B6D2F1/J mice (from a cross between C57BL/6J [B6] females and DBA/2J [D2] males) as tumor cell recipients.</li>
</ol>
3. Setting up the surgical area
NOTE: All surgical steps are conducted using aseptic technique in a clean, sanitized environment. Scrubs and personal protective equipment, including a mask, should be worn by the surgeon. Surgical tools must be heat-sterilized prior to use.
<ol>
	<li>Place surface protectors under the stereotaxic apparatus and on the work surface.</li>
	<li>Attach the vinyl tubing from the anesthesia machine to the IN port of the gas anesthesia platform of the stereotaxic apparatus, and another tube to the OUT port.</li>
	<li>Connect the digital display to a power source and to the apparatus.</li>
	<li>Connect the micropump controller (Figure 1A) to a power source and attach the micropump (Figure 1A) to the manipulator arm of the apparatus (also see the manufacturer instructions).</li>
	<li>Set the micropump to 5.6 nL/s for the 2,000 nL volume injection.</li>
	<li>Turn on the hot bead sterilizer.</li>
	<li>Plug the mouse heating pad into the temperature controller and connect to a power source. Place the plate on the stage platform. Set the plate temperature to 37 &#176;C.</li>
	<li>Backload a 30 G precision syringe, being careful not to introduce bubbles. Insert the plunger and attach the needle. Make sure to grip the needle by the hub only. Depress the plunger until a drop of the methylcellulose cell mixture dispenses through the needle. Clean the needle with a 70% alcohol preparation pad by carefully wiping the sides of the needle, or by placing a sterile gauze pad on the work surface and blotting it. Take care not to bend or break the needle.</li>
	<li>Attach the precision syringe to the micropump and rotate manipulator arm away from the stage, to prevent syringe-needle displacement prior to placing the mouse on the stage.</li>
</ol>
4. Preparing the mouse for surgery
<ol>
	<li>Anesthetize the mouse by placing it in the induction chamber with the isoflurane vaporizer set to 2.5%, or according to institutional guidelines. Turn on the isoflurane flow to the nosecone.</li>
	<li>Transfer the mouse to the stereotaxic apparatus and secure to the nosecone, with the top teeth positioned onto the nosecone support (Figure 1B, 9). Tighten the knob on the nosecone to secure (Figure 1B). Ensure an appropriate level of anesthesia by performing a toe pinch to check for reflex.
	<ol>
		<li>Monitor the ears, feet, and mucous membranes for color (pink) to ensure adequate oxygenation. Also, monitor the respiratory rate (an increase or decrease could indicate the need to adjust isoflurane levels).</li>
	</ol>
	</li>
	<li>Insert ear bars (Figure 1B) into both ears and tighten the knob to secure the head.</li>
	<li>Apply eye ointment to lubricate the eyes while the mouse is under anesthesia.</li>
	<li>Use curved forceps to pluck the hair on the mouse&#39;s head to an area about 150% larger than the planned incision, to ensure adequate aseptic conditions.</li>
	<li>Inject 0.5-1 mg/kg buprenorphine SR analgesia subcutaneously, or use another protocol-approved analgesic.</li>
	<li>Position the mouse rectal probe to monitor internal temperature and prevent hypothermia due to anesthesia. Keep the mouse body temperature between 36.5 and 38.5 &#176;C.</li>
	<li>Sanitize the surgical field using outward circles, alternating between a surgical scrub and ethanol three times.</li>
	<li>Using forceps to pull the skin taut, make an incision of approximately 1 cm with the scalpel blade, starting between the eyes. The bregma should be visible through the incision. 
	NOTE: For proper sterile technique, touch the surgical site only with the tips of sterilized tools, and place tool tips down on a sterile surface only (such as the inside of an autoclaved tool pack).</li>
	<li>Use the wooden end of cotton-tipped applicator to scrape away excess connective tissue, and then the cotton end of another applicator to dry.</li>
</ol>
5. Cell injection
<ol>
	<li>Return the manipulator arm with the syringe attachment over the mouse, tightening the knob to secure. Use the X and Y knobs in the horizontal plane to move the syringe mount over bregma. Lower the needle using the Z knob to confirm the bregma position. Set the digital readout console to zero.</li>
	<li>Use the X and Y knobs and the corresponding digital readout to move the needle to the desired position. For the cortical cortex location, the appropriate coordinates are 3 mm posterior, 2 mm lateral right to the bregma, and 2 mm deep from the dura mater. Use the Z knob to move the needle to surface of skull.</li>
	<li>Puncture a hole in the skull using a 1 mL syringe with an attached 25 G needle. Place the bevel of the needle toward the 30 G precision syringe and needle, and carefully rotate the manipulator arm to the side. Using thumb and finger, roll the needle back and forth slowly with gentle pressure until the needle tip just pierces the skull cap.</li>
	<li>Use a cotton tipped applicator to dab any blood away from the needle hole. Replace the manipulator arm to the appropriate position with the loaded precision syringe needle above the hole. Align the tip of the needle with the hole, using the Z knob to lower the needle down to the brain dura, and set the digital readout console to zero.</li>
	<li>Using the Z knob, lower the needle 1 mm, and then wait 1 min. Repeat until the desired depth is reached (2 mm as indicated). 
	NOTE: The needle is lowered slowly to prevent additional damage to the surrounding brain tissue and back-flow of the cell solution.</li>
	<li>Start the micropump, and then monitor to ensure that the pump stops when 2 &#181;L has been injected. This process should take about 6 min at the indicated speed. Then, wait 1 min prior to moving the needle.</li>
</ol>
6. Removal of the needle and wound closure
<ol>
	<li>Raise the needle 1 mm and then wait 1 min. Repeat until the needle is completely free from the skull.</li>
	<li>If necessary, use a cotton-tipped applicator to dab any blood away from the injection site.</li>
	<li>Using the wooden end of a cotton-tipped applicator, take a small piece of bone wax (~1 mm) and shape it into a cone. Place it into the opening in the skull, pushing the wax into the hole.</li>
	<li>Heat forceps using a bead sterilizer and use them to melt remaining wax on the skull and smooth it.</li>
	<li>Place roughly two drops of bupivacaine (anesthetic) solution into the incision and use forceps to pull the edges of skin together.</li>
	<li>Pull the skin taut and place one or two wound clips to close the skin.</li>
	<li>Place the mouse in a clean recovery cage on a heating pad in a draft-free area, and closely observe the mouse. Allow the mouse to wake up fully from the anesthesia, resuming normal activity, before returning it to regular housing.</li>
	<li>Check on the mouse daily following the surgical procedure and administer pain management, according to institutional guidelines.
	<ol>
		<li>If buprenorphine SR (Step 4.6) is used for analgesia, the sustained-release formula lasts for 72 h. Repeat the injection only if visible pain or discomfort is present after 72 h. When performed correctly, the mice recover well from the intracranial injections and additional analgesia is not needed.</li>
		<li>Remove the staples 7-10 days post-surgery.</li>
	</ol>
	</li>
	<li>Monitor tumor growth by live animal imaging (MRI).
	<ol>
		<li>Euthanize the mice when humane endpoints are reached (i.e., if the animal loses 20% body weight or becomes hypothermic).</li>
		<li>Due to the invasive nature of these brain tumors, observe the mice for neurological symptoms, such as uneven gait, partial paralysis, spinning, or head tilt. Euthanize a mouse if any of these clinical signs of advanced tumor growth are observed.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Tamimi, A. F., Juweid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+Outcome+of+Glioblastoma.">Epidemiology and Outcome of Glioblastoma.</a> Glioblastoma. , (2017).</li><li>Robertson, F. L., Marques-Torrejon, M. A., Morrison, G. M., Pollard, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+and+tools+to+tackle+glioblastoma.">Experimental models and tools to tackle glioblastoma.</a> Disease Models &amp; Mechanisms. 12 (9), (2019).</li><li>Wen, P. Y., Kesari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+gliomas+in+adults.">Malignant gliomas in adults.</a> The New England Journal of Medicine. 359 (5), 492-507 (2008).</li><li>Haddad, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+glioblastoma+for+the+evaluation+of+novel+therapeutic+strategies.">Mouse models of glioblastoma for the evaluation of novel therapeutic strategies.</a> Neuro-Oncology Advances. 3 (1), (2021).</li><li>El Meskini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preclinical+orthotopic+model+for+glioblastoma+recapitulates+key+features+of+human+tumors+and+demonstrates+sensitivity+to+a+combination+of+MEK+and+PI3K+pathway+inhibitors.">A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors.</a> Disease Models &amp; Mechanisms. 8 (1), 45-56 (2015).</li><li>Song, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+etiology+of+high-grade+astrocytomas.">Evolutionary etiology of high-grade astrocytomas.</a> Proceedings of the National Academy of Sciences. 110 (44), 17933-17938 (2013).</li><li>Cancer Genome Atlas Research Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+genomic+characterization+defines+human+glioblastoma+genes+and+core+pathways.">Comprehensive genomic characterization defines human glioblastoma genes and core pathways.</a> Nature. 455 (7216), 1061-1068 (2008).</li><li>Motomura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+analysis-based+proteomic+subclassification+of+newly+diagnosed+glioblastomas.">Immunohistochemical analysis-based proteomic subclassification of newly diagnosed glioblastomas.</a> Cancer Science. 103 (10), 1871-1879 (2012).</li><li>Choyke, P. L., Dwyer, A. J., Knopp, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tumor+imaging+with+dynamic+contrast-enhanced+magnetic+resonance+imaging.">Functional tumor imaging with dynamic contrast-enhanced magnetic resonance imaging.</a> Journal of Magnetic Resonance Imaging. 17 (5), 509-520 (2003).</li><li>Raza, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+necrosis-associated+genes+in+glioblastoma+by+cDNA+microarray+analysis.">Identification of necrosis-associated genes in glioblastoma by cDNA microarray analysis.</a> Clinical Cancer Research. 10, 212-221 (2004).</li><li>Raza, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrosis+and+glioblastoma:+a+friend+or+a+foe?+A+review+and+a+hypothesis.">Necrosis and glioblastoma: a friend or a foe? A review and a hypothesis.</a> Neurosurgery. 51 (1), 2-12 (2002).</li><li>Hambardzumyan, D., Bergers, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma:+defining+tumor+niches.">Glioblastoma: defining tumor niches.</a> Trends in Cancer. 1 (4), 252-265 (2015).</li><li>Kijima, N., Kanemura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma.">Glioblastoma.</a> Mouse Models of Glioblastoma. , (2017).</li><li>Casanova, F., Carney, P. R., Sarntinoranont, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+needle+insertion+speed+on+tissue+injury,+stress,+and+backflow+distribution+for+convection-enhanced+delivery+in+the+rat+brain.">Effect of needle insertion speed on tissue injury, stress, and backflow distribution for convection-enhanced delivery in the rat brain.</a> PloS One. 9 (4), 94919 (2014).</li><li>Jin, F., Jin-Lee, H. J., Johnson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+of+Experimental+Glioblastoma.">Mouse Models of Experimental Glioblastoma.</a> Gliomas. , (2021).</li><li>Zalles, M., Towner, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-Clinical+Models+and+Potential+Novel+Therapies+for+Glioblastomas.">Pre-Clinical Models and Potential Novel Therapies for Glioblastomas.</a> Gliomas. , 1-13 (2021).</li><li>Wierzbicki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+and+therapeutic+monitoring+of+H3K27M-mutant+glioma.">Targeting and therapeutic monitoring of H3K27M-mutant glioma.</a> Current Oncology Reports. 22 (2), 19 (2020).</li></ol>

The authors declare no conflicts of interest.

Preclinical models are essential for the evaluation of new therapeutic targets and novel treatment strategies in GBM. Genetically engineered mouse models for GBM have the advantage of tumor occurrence in the autochthonous site, but often with a long latency and unpredictable tumor growth13. The GEM model tumors exhibit a latency of 4-5 months, and the ideal time window for imaging, recruitment, and treatment is variable among individual mice. The orthotopic model has a well-established and tractab...

Glioblastoma (GBM; grade IV glioma) is the most common and malignant brain tumor, and current therapies are ineffective, resulting&#160;in a median survival of 15 months1. Reliable and accurate preclinical models that represent the complex signaling pathways involved in brain tumor growth and pathogenesis are essential to expedite the progress in evaluating new therapeutic regimens for GBM. Mouse models in which human brain tumor cell lines are implanted subcutaneously in immunocompromised mice do not reflect the native immune environment of brain tumors, nor can they be used to evaluate the ability of therapeutics to cross the blood-brain barrier2. Ideally, preclinical mouse models should also reproduce closely the human GBM histopathology, including the high level of invasiveness into the surrounding parenchyma3. Although genetically engineered mouse (GEM) models develop tumors in the context of an intact immune system, complicated breeding schemes are often required, and tumors may develop slowly and inconsistently4. GEM-derived allograft models are better suited for preclinical therapeutic studies, where large cohorts of tumor-bearing mice are needed in a shorter time frame.
In a previous report, we described an orthotopic GBM mouse model derived directly from GEM tumors. Tumorigenesis in the GEM is initiated by genetic events in cell populations (primarily astrocytes) expressing glial fibrillary acidic protein (GFAP), that result in progression to GBM. These TRP GEMs harbor a TgGZT121 transgene (T), which expresses T121 after exposure to the GFAP-driven Cre recombinase. T121 protein expression results in the suppression of Rb (Rb1, p107, and p103) protein activity. Co-expression of a GFAP-driven Cre transgene (GFAP-CreERT2) targets expression to adult astrocytes after induction with tamoxifen. TRP mice also harbor a Cre-dependent mutant Kras (KrasG12D; R) allele, to represent activation of the receptor tyrosine kinase pathway, and are heterozygous for the loss of Pten (P)5,6. Concurrent gene aberrations in the receptor tyrosine kinase (RTK), PI3K, and RB networks are implicated in 74% of GBM pathogenesis7. Therefore, the primary signaling pathways altered in human GBM are represented by the engineered mutations in TRP mice, in particular GBM tumors, in which shared downstream targets of RTKs are activated5.
The GEM-derived syngeneic orthotopic model was validated as a model that recapitulates features of human brain tumors, including invasiveness and the presence of subtype biomarkers, for use as a platform to evaluate cancer therapeutics targeting aberrant pathways in GBM. Cells were cultured from tumors harvested from TRP brains and re-implanted in the brain of strain-matched mice, using stereotactic equipment for intracranial injection in the cortex. This preclinical orthotopic mouse model developed GBM tumors that were highly cellular, invasive, pleomorphic with a high mitotic rate, and displayed linear foci of necrosis by neoplastic cells and dense vascularization, as observed for human GBM. Tumor volumes and growth were measured by in vivo magnetic resonance imaging (MRI).
In this report, we describe the optimal technique for the intracranial injection of primary GBM cells or cell lines into the wild-type mouse brain, using TRP tumors as an example. The same protocol may be adapted for immunocompromised mice and other GBM cell lines. Crucial tips are given for avoiding common pitfalls, such as suboptimal cell preparation or cell leakage at the injection site, and for using the stereotactic equipment correctly to ensure model reproducibility and reliability. For translational purposes, we validate the model by MRI detection of brain tumor growth in live animals, histological characterization, and present an example of treatment in tumor-bearing mice.

<table><tbody><tr><td>5% methylcellulose in 1X PBS, autoclaved</td><td>Millipore Sigma</td><td>M7027</td><td></td></tr><tr><td>1mL Tuberculin Syringe, slip tip</td><td>BD</td><td>309659</td><td></td></tr><tr><td>6&quot; Cotton Tipped Applicators</td><td>Puritan</td><td>S-18991</td><td></td></tr><tr><td>Adjustable stage platform</td><td>David Kopf Instruments</td><td>Model 901</td><td></td></tr><tr><td>Aerosol Barrier Tips</td><td>Fisher Scientific</td><td>02-707-33</td><td></td></tr><tr><td>Alcohol Prep Pads Sterile, Large - 2.5 x 3 Inch</td><td>PDI</td><td>C69900</td><td></td></tr><tr><td>B6D2&amp;nbsp; mouse strain (C57Bl/6J x DBA/2J)</td><td>Jackson Laboratory</td><td>Jax #10006</td><td></td></tr><tr><td>Bone Wax</td><td>Surgical Specialties</td><td>901</td><td></td></tr><tr><td>Bupivacaine 0.25%</td><td>Henry Schein</td><td>6023287</td><td></td></tr><tr><td>BuprenorphineSR</td><td>ZooPharm</td><td>n/a</td><td></td></tr><tr><td>Clear Vinyl Tubing 1/8ID X 3/16OD</td><td>UDP</td><td>T10004001</td><td></td></tr><tr><td>CVS Lubricant Eye Ointment</td><td>CVS Pharmacy</td><td>247881</td><td></td></tr><tr><td>Disposable Scalpels, #10 blade</td><td>Scalpel Miltex</td><td>16-63810</td><td></td></tr><tr><td>Gas anesthesia machine with oxygen hook-up and anesthesia box</td><td>Somni Scientific</td><td>n/a</td><td>Investigator may use facility&lt;br/&gt; standard equipment</td></tr><tr><td>Gas anesthesia platform for mice</td><td>David Kopf Instruments</td><td>Model 923-B</td><td></td></tr><tr><td>GraphPad Prism</td><td>Graphpad</td><td>Prism&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 9&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; version 9.4.1</td><td></td></tr><tr><td>Hamilton 30 g needle, &amp;frac12; &amp;ldquo;, small hub, point pst 3</td><td>Hamilton</td><td>Special Order</td><td></td></tr><tr><td>Hamilton precision microliter syringe, 1701 RN, no needle 10 &amp;micro;L</td><td>Hamilton</td><td>7653-01</td><td></td></tr><tr><td>Hot bead sterilizer with beads</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Invitrogen Countess 3 Automated Cell Counter</td><td>Fisher Scientific</td><td>AMQAX2000</td><td></td></tr><tr><td>IsoFlurane</td><td>Piramal Critical Care</td><td>29404</td><td></td></tr><tr><td>Isopropyl Alcohol Prep Pads</td><td>PDI</td><td>C69900</td><td></td></tr><tr><td>ITK_SNAP (Version 36.X, 2011-present)</td><td>Penn Image Computing and Science Laboratory (PICSL) at the University of Pennsylvania, and the Scientific Computing and Imaging Institute (SCI) at the University of Utah</td><td></td><td></td></tr><tr><td>KOPF Small Animal Stereotaxic Instrument with digital readout console</td><td>David Kopf Instruments</td><td>Model 940</td><td></td></tr><tr><td>Masterflex Fitting, PVDF, Straight, Hose Barb Reducer, 1/4&quot; ID x 1/8&quot; ID</td><td>Masterflex</td><td>HV-30616-16</td><td></td></tr><tr><td>Mouse Heating Plate</td><td>David Kopf Instruments</td><td>PH HP-4M</td><td></td></tr><tr><td>Mouse Rectal Probe</td><td>David Kopf Instruments</td><td>PH RET-3-ISO</td><td></td></tr><tr><td>Nalgene Super Versi-Dry Surface Protectors</td><td>ThermoFisher Scientific</td><td>74000-00</td><td></td></tr><tr><td>P20 pipette</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>Povidone Iodine Surgical Scrub</td><td>Dynarex</td><td>1415</td><td></td></tr><tr><td>Reflex 9 mm Wound Clip Applicator</td><td>Fine Science Tools</td><td>12031-09</td><td></td></tr><tr><td>Reflex 9 mm Wound Clip Remover</td><td>Fine Science Tools</td><td>12033-00</td><td></td></tr><tr><td>Reflex 9 mm Wound Clips</td><td>Fine Science Tools</td><td>12032-09</td><td></td></tr><tr><td>Semken forceps, curved</td><td>Fine Science Tools</td><td>11009-13</td><td></td></tr><tr><td>Temperature Controller</td><td>David Kopf Instruments</td><td>PH TCAT-2LV</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Tuberculin Syringe with 25g needle, slip tip</td><td>BD</td><td>309626</td><td></td></tr><tr><td>UltraMicroPump 3 with Micro2T Controller</td><td>World Precision Instruments</td><td>Model UMP3T</td><td></td></tr></tbody></table>

translational orthotopic models of glioblastoma multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Mice injected with brain tumor cells should be monitored daily for signs of tumor growth such as seizures, ataxia, or weight loss. Brain tumor growth may also be monitored by MRI scanning at regular intervals. Weekly MRI scans allow the visualization of increasing tumor burden within the brain and tumor volume measurements (Figure 1C). In particular, TRP tumors exhibit aggressive growth, and 3D tumor volumes are measurable by MRI within 2 to 3 weeks post-intracranial injection (with an avera...

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Translational Orthotopic Models of Glioblastoma Multiforme

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

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2.6K Views.  National Cancer Institute. We beschrijven de optimale techniek voor intracraniële injectie van GEM-afgeleide glioblastoomcellen in een immunocompetent muizenbrein, resulterend in tumoren die de belangrijkste kenmerken van menselijk glioblastoom recapituleren. Deze techniek voor orthotopische injectie van GEM-afgeleide TRP-cellen levert zeer reproduceerbare, schaalbare en consistente resultaten op voor tumoren die hun oorsprong vinden in de cortex, maar toch de invasiviteit van GEM-tumoren behouden. Muis glioblastoom h...

Video: Translational Orthotopic Models of Glioblastoma Multiforme

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Medicine

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Neuroscience

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 ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

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 ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

Translational Orthotopic Models of Glioblastoma Multiforme

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