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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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.

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			<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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

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 ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...