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</s...

<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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34; 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&#160; 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 
			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&#160;&#160;&#160;&#160;&#160; 9&#160;&#160;&#160;&#160;&#160; version 9.4.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton 30 g needle, &#189; &#8220;, 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 &#181;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&#34; ID x 1/8&#34; 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...

Watch this Scientific Journal Video about Translational Orthotopic Models of Glioblastoma Multiforme at JoVE.com

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 ...

We describe the optimal technique for intracranial injection of GEM-derived glioblastoma cells into an immunocompetent mouse brain resulting in tumors that recapitulate key features of human glioblastoma. This technique for orthotopic injection of GEM-derived TRP cells yields highly reproducible, scalable and consistent results for tumors that originate in the cortex, yet maintain the invasiveness of GEM tumors. Mouse glioblastoma brain tumors display aggressive growth and invade the surrounding brain parenchyma.Therefore, it can be used to evaluate therapeutics that may suppress tumor infiltration and migration in patients. This protocol can be customized for other brain tumor models, viruses, therapeutics or adapted for injection into different brain regions with the adjustments of stereotaxic coordinates. Demonstrating the procedure will be Devon Atkinson, a research associate from my laboratory.To begin, place surface protectors under the stereotaxic apparatus and on the work surface. Connect the vinyl tube from the anesthesia machine to the in-port of the gas anesthesia platform of the stereotaxic apparatus, and another tube to its out-port. Connect the digital display to a power source and the apparatus.Next, attach the micro pump to the manipulator arm of the apparatus after connecting the micro pump controller to the power source. Set the micro pump to 5.6 nanoliters per second. Connect the mouse heating pad to the temperature controller and to a power source.Back-load a 30-gauge precision syringe with methyl methylcellulose cell mixture by avoiding bubbles. After inserting the plunger, attach the needle, and depress the plunger until the loaded cell mixture drops through the needle. Clean the needle with a 70%alcohol preparation pad.Once the precision syringe is attached to the micro pump, rotate the manipulator arm away from the stage before placing the mouse to prevent syringe needle displacement. Transfer the anesthetized mouse to the stereotaxic instrument and secure it to the nose cone by placing the upper teeth on the nose cone support. After tightening the nose cone with a knob, pinch a toe to check reflexes to ensure proper anesthesia.Then insert ear bars and tighten the knob to secure the head. Apply eye ointment to lubricate the eyes under anesthetized conditions. Inject buprenorphine SR analgesic subcutaneously, and place a rectal mouse probe to monitor internal temperature.Sterilize the surgical field using outward circles, alternating between a surgical scrub and ethanol three times. Pull the taut skin with forceps and use a scalpel blade to make an approximately 1-centimeter-long incision between the eyes. For cell injection, return the manipulator arm with the syringe attached to the mouse.After tightening the knobs, move the syringe using the X and Y knobs in the horizontal plane and mount it over the bregma. Lower the needle, using the Z knob, to confirm the bregma position, and set the digital readout console to zero. Then move the needle to the desired position, as described in the manuscript, using X and Y knobs and the corresponding digital readout, and use the Z knob to move the needle to the surface of the skull.Puncture a hole in the skull using a 1-milliliter syringe with an attached 25-gauge needle. Carefully rotate the manipulator arm to the side, while placing the bevel of the needle toward the 30-gauge precision syringe. Once the manipulator arm with the loaded precision syringe needle is positioned over the hole, align the needle tip with the hole, and use the Z knob to lower the needle into the dura of the brain.After setting the digital readout console to zero, lower the needle 1 millimeter with the Z knob, and wait for one minute. Once the 2 millimeter depth is reached, stop the micro pump, and ensure that the pump stops when 2 microliters of cell suspension has been injected. To remove the needle from the skull, raise it one millimeter, and wait one minute.For wound closure, using the wooden end of a cotton-tipped applicator, take a piece of bone wax and shape it into a cone. After placing the wax into the opening in the skull, push it into the hole. Use the bead sterilized forceps to melt the remaining wax on the skull.Once it becomes smooth, add two drops of anesthetic bupivacaine solution into the incision, and pull the edges of the skin together, using forceps. After pulling the skin taut, using one or two wound clips, close the skin, place the mouse in a clean recovery cage on a heating pad in a draft-free area, and closely observe the mouse. Weekly MRI scans showed increasing tumor burden within the brain, and tumor volume measurements.The MRI tumor volume measurement revealed a lack of tumor growth suppression even after radiation treatment, and the survival of treated mice was not increased. The GBM tumors in syngeneic models appeared to be grown aggressively and breached the skull by the terminal endpoint. If extracranial growth is observed on imaging after cell implantation, this indicates cell leakage during the injection process, and more care should be taken during the withdrawal of the injection needle.Histopathology of orthotopic TRP GBM tumors confirmed the presence of grade 4 astrocytoma, including recapitulation of the distinctive features in tumors such as pseudopalisading and necrosis. Be precise when setting the zero point at bregma to ensure correct cell injection coordinates. Lowering and raising the needle in the brain must be slow to prevent trauma and backflow.After the injection of tumor cells, weekly MRI scans may be used to determine baseline tumor size prior to starting treatment and to visualize changing tumor burden within the brain.

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Cancer Research

2.1K 次观看.  National Cancer Institute. 我们描述了将GEM衍生的胶质母细胞瘤细胞颅内注射到免疫功能正常的小鼠大脑中的最佳技术，从而产生概括了人胶质母细胞瘤关键特征的肿瘤。这种用于原位注射GEM衍生的TRP细胞的技术对起源于皮层的肿瘤产生了高度可重复，可扩展和一致的结果，同时保持了GEM肿瘤的侵袭性。小鼠胶质母细胞瘤脑肿瘤表现出侵袭性生长并侵入周围的脑实质。因此，它可用于评估可能抑?...