Name: translational orthotopic models of glioblastoma multiforme
Uploaded: 2023-02-17T13:40:00
Description: Watch this Scientific Journal Video about Translational Orthotopic Models of Glioblastoma Multiforme at JoVE.com

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>
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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.

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			<td>5% methylcellulose in 1X PBS, autoclaved</td>
			<td>Millipore Sigma</td>
			<td>M7027</td>
			<td></td>
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		<tr>
			<td>1mL Tuberculin Syringe, slip tip</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
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			<td>6&#34; Cotton Tipped Applicators</td>
			<td>Puritan</td>
			<td>S-18991</td>
			<td></td>
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			<td>Adjustable stage platform</td>
			<td>David Kopf Instruments</td>
			<td>Model 901</td>
			<td></td>
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			<td>Aerosol Barrier Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-33</td>
			<td></td>
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			<td>Alcohol Prep Pads Sterile, Large - 2.5 x 3 Inch</td>
			<td>PDI</td>
			<td>C69900</td>
			<td></td>
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		<tr>
			<td>B6D2&#160; mouse strain (C57Bl/6J x DBA/2J)</td>
			<td>Jackson Laboratory</td>
			<td>Jax #10006</td>
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			<td>Bone Wax</td>
			<td>Surgical Specialties</td>
			<td>901</td>
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			<td>Bupivacaine 0.25%</td>
			<td>Henry Schein</td>
			<td>6023287</td>
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			<td>BuprenorphineSR</td>
			<td>ZooPharm</td>
			<td>n/a</td>
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			<td>Clear Vinyl Tubing 1/8ID X 3/16OD</td>
			<td>UDP</td>
			<td>T10004001</td>
			<td></td>
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			<td>CVS Lubricant Eye Ointment</td>
			<td>CVS Pharmacy</td>
			<td>247881</td>
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			<td>Disposable Scalpels, #10 blade</td>
			<td>Scalpel Miltex</td>
			<td>16-63810</td>
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			<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>
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		<tr>
			<td>Gas anesthesia platform for mice</td>
			<td>David Kopf Instruments</td>
			<td>Model 923-B</td>
			<td></td>
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			<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>
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			<td>Hamilton 30 g needle, &#189; &#8220;, small hub, point pst 3</td>
			<td>Hamilton</td>
			<td>Special Order</td>
			<td></td>
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			<td>Hamilton precision microliter syringe, 1701 RN, no needle 10 &#181;L</td>
			<td>Hamilton</td>
			<td>7653-01</td>
			<td></td>
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			<td>Hot bead sterilizer with beads</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
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			<td>Invitrogen Countess 3 Automated Cell Counter</td>
			<td>Fisher Scientific</td>
			<td>AMQAX2000</td>
			<td></td>
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			<td>IsoFlurane</td>
			<td>Piramal Critical Care</td>
			<td>29404</td>
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			<td>Isopropyl Alcohol Prep Pads</td>
			<td>PDI</td>
			<td>C69900</td>
			<td></td>
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			<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>
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		<tr>
			<td>KOPF Small Animal Stereotaxic Instrument with digital readout console</td>
			<td>David Kopf Instruments</td>
			<td>Model 940</td>
			<td></td>
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			<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>
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			<td>Mouse Heating Plate</td>
			<td>David Kopf Instruments</td>
			<td>PH HP-4M</td>
			<td></td>
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			<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>
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		<tr>
			<td>P20 pipette</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
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			<td>Povidone Iodine Surgical Scrub</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
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		<tr>
			<td>Reflex 9 mm Wound Clip Applicator</td>
			<td>Fine Science Tools</td>
			<td>12031-09</td>
			<td></td>
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		<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>
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		<tr>
			<td>Temperature Controller</td>
			<td>David Kopf Instruments</td>
			<td>PH TCAT-2LV</td>
			<td></td>
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			<td>Trypsin-EDTA (0.25%)</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
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			<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>
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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 ...

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