We are particularly indebted to patients at Vanderbilt University Medical Center who provided invaluable research material for the Molecular Neurosurgical Tissue Bank. We thank those who established and maintain the Tissue Bank, Reid C. Thompson MD (principal investigator), Cherryl Kinnard RN (research nurse) and Larry A. Pierce MS (manager). Histological services were performed, in part, by the Vanderbilt University Medical Center (VUMC) Translational Pathology Shared Resource (supported by award 5P30 CA068485 to the Vanderbilt-Ingram Cancer Center). This work was supported by grants to MKC from the NINDS (1R21NS070139), the Burroughs Wellcome Fund and VMC development funds. MKC is supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development through grant 1 I01 BX000744-01. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.

1. Preparation of Tumor Cell Suspension
Note: Appropriate institutional approvals for the use of patient material and animals are required to establish and maintain primary orthotopic glioma xenografts. At Vanderbilt University Medical Center, resected tumor material that is in excess of that required for diagnostic purposes is collected with patient consent for a research tissue repository. Specimens are labeled with a randomized 5-digit REDcap database number and all patient-specific identifiers are removed. The REDcap database contains deidentified clinical data for each specimen in the tissue repository that includes gender, age (in years), race, survival status, pathology, cancer treatments, year of resection, and time to progression. To maintain sterility and optimize success of the primary xenograft method, all of the reagents, steam-autoclaved surgical instruments, and the surgical site should be assembled or prepared in advance.
Note: Glioma specimens often contain large amounts of myelin and debris. Depending on specimen size, it may be necessary to use more than 4 discontinuous gradient tubes for adequate removal of myelin and debris.
Note: The process is completed when there is no, or little, clearly visible undissociated tissue remaining. Avoid the introduction of bubbles during the trituration process.
Note: Cell viability following dissociation is typically &#62;90%. Lower viabilities may reflect many variables including suboptimal tissue handling or transport time from the operating room, cryopreservation method, or papain dissociation technique. Lower cellular viabilities may still be adequate, however, as long as a sufficient number of viable may be transplanted in the appropriate volume. For each mouse, 50,000-200,000 viable cells are implanted in volume of 2 &#181;l. Due to the beveled tip of the syringe needle, an additional 5 &#181;l of cell suspension should be included in the final volume to ensure that adequate material can be drawn into the syringe for each injection. For example, to inject 2 &#181;l/mouse for 5 mice the final volume should be 15 &#181;l (15 &#181;l = (5 x 2 &#181;l) + 5 &#181;l).
<ol>
	<li>Place freshly resected, deidentified patient material in a sterile, capped container on ice for transport to the laboratory. Process the specimen directly for transplantation or cryopreserve the specimen in liquid nitrogen (see below) for storage in liquid nitrogen and later use.</li>
	<li>Prepare the papain dissociation solutions (papain solution, wash/protease inhibitor solution and discontinuous gradient solution) in a sterile hood with the kit components and instructions supplied by the manufacturer. Label sterile 15 ml polystyrene conical tubes and distribute the solutions as follows:
		<ul>
			<li>Tube 1: 5 ml of papain solution</li>
			<li>Tube 2: 3 ml of wash/protease inhibitor solution</li>
			<li>Tubes 3-6: 5 ml each of discontinuous gradient solution</li>
		</ul>
	</li>
	<li>Place the glioma specimen in tube 1 with 5 ml of papain solution and triturate with a 5 ml pipette over a 10-20 min time period.</li>
	<li>Pipette the material up and down 10 times and then incubate the material for 2-3 min at room temperature before initiating the next cycle of trituration.</li>
	<li>Position the tip of the 5 ml pipette closer to the bottom of the conical tube with each cycle of trituration such that the tip is touching the bottom of the tube for the last 2 cycles.</li>
	<li>Centrifuge at 300 x g for 5 min at room temperature. Remove the supernatant and pipette the pellet up and down 10x in 3 ml of the wash/protease inhibitor solution.</li>
	<li>Carefully layer an equal volume of the suspension over each of the 5 ml discontinuous gradient solutions (tubes 3-6), with a pipettor set at the &#8220;gravity&#8221; dispense speed.</li>
	<li>Place the tubes in a centrifuge equipped with a swinging-bucket rotor, with the acceleration speed reduced to the lowest setting and the brake turned off. Centrifuge at 76 x g for 12 min at room temperature. With the brake turned off, centrifugation is generally completed after 20 min.</li>
	<li>Remove the supernatant, resuspend and combine each pellet in 5 ml of sterile balanced salt solution and place the cells on ice.</li>
	<li>Remove a portion (10-100 &#181;l) of the cell suspension and determine the density of viable cells by trypan blue exclusion with a hemocytometer.</li>
	<li>Centrifuge at 300 x g for 5 min. Remove the supernatant and resuspend the cell pellet at a density of 25,000-125,000 viable cells per &#181;l.</li>
	<li>Transfer an adequate volume of the cell suspension to a 200 &#956;l microcentrifuge tube (PCR tube) and place on ice.</li>
</ol>
2. Preparation of Surgical Site and Instruments
Note: Sterile surgical gloves are worn during the procedure. Depending upon requirements of each institution, surgical gowns, caps and face guards may be required.
<ol>
	<li>Prepare a disinfected benchtop for rodent surgery with separated adjoining areas for animal preparation, operating field and animal recovery.</li>
	<li>Sterilize surgical instruments in a steam autoclave. Subsequently, use a hot bead sterilizer to flash-sterilize instruments when transitioning from one animal subject to the next.</li>
</ol>
3. Induction, Anesthesia, and Analgesia
Note: Surgeries exceeding 15 min from the time the mice is anesthetized require a contact heat source. To prevent hypothermia, the mice are provided with a heat source (circulating hot water blanket) during the pre- and post-operative periods. 
<ol>
	<li>Prepare ketamine/xylazine cocktail for induction anesthesia such that each mouse receives ketamine 100 mg/kg and xylazine 10 mg/kg by injecting 10 &#181;l of the cocktail per gram of body weight.</li>
	<li>Table 1. Anesthesia cocktail.
		<table>
			<tbody>
				<tr>
					<td rowspan="2"></td>
					<td rowspan="2">Stock concentration</td>
					<td colspan="3">Volume to prepare for </td>
				</tr>
				<tr>
					<td>one mouse</td>
					<td>five mice</td>
					<td>ten mice</td>
				</tr>
				<tr>
					<td>Ketamine</td>
					<td>100 mg/ml</td>
					<td>25 &#181;l</td>
					<td>125 &#181;l</td>
					<td>250 &#181;l</td>
				</tr>
				<tr>
					<td>Xylazine</td>
					<td>100 mg/ml</td>
					<td>2.5 &#181;l</td>
					<td>12.5 &#181;l</td>
					<td>25 &#181;l</td>
				</tr>
				<tr>
					<td>Isotonic saline</td>
					<td></td>
					<td>223 &#181;l</td>
					<td>1,113 &#181;l</td>
					<td>2,225 &#181;l</td>
				</tr>
				<tr>
					<td colspan="2">Total</td>
					<td>250 ul</td>
					<td>1,250 &#181;l</td>
					<td>2,500 &#181;l</td>
				</tr>
			</tbody>
		</table>
	</li>
	<li>Prepare ketoprofen solution for analgesia by diluting the stock solution (10 mg/ml) 1:20 in sterile, pyogen-free water such that each mouse receives a dose of 5 mg/kg by injecting 10 &#181;l of the solution per gram of body weight.</li>
	<li>Weigh and record presurgical weight. Individual mouse weights vary, but generally range from 18-22 g.</li>
	<li>Inject 10 &#181;l of the ketamine/xylazine cocktail per gram of mouse body weight into the intraperitoneal space with an insulin syringe. For example, inject 200 &#181;l for a 20 g mouse.</li>
	<li>Determine whether mouse is fully anesthetized by toe pinch of the hind leg. It generally takes 3-5 min for the mouse to no longer withdraw from the pinch.</li>
	<li>Inject 10 &#181;l of the ketoprofen solution per gram of mouse body weight subcutaneously in the loose skin of the flank with an insulin syringe. For example, inject 200 &#181;l for a 20 g mouse.</li>
	<li>Shave hair over the skull with clippers, scrub the exposed scalp with povidone-iodine using a cotton tip applicator and then wipe an alcohol pad. Repeat these steps, povidone-iodine scrub and alcohol wipe, two more times.</li>
	<li>Apply ophthalmic ointment to the eyes of the mouse.</li>
	<li>Make a 1 cm midline incision extending from behind the ears to just in front of the eyes with a sterile scalpel blade.</li>
	<li>Place the mouse under a dissecting scope and expose the skull to identify the suture lines. Using circular motions with a drill, center a burr hole 2.5 mm lateral to the bregma and 1 mm posterior to the coronal suture.</li>
</ol>
4. Intracranial Transplantation
Note: Injection volumes exceeding 2.5 &#181;l may be lethal to mice.
<ol>
	<li>Position the mouse on a stereotactic frame by hooking the upper incisors over the incisor bar and applying the nose clamp so that the skull is firmly held in a neutral position.</li>
	<li>Draw 5-10 &#181;l of the cells in a microcentrifuge tube up and down several times into a 10 &#181;l Hamilton syringe clamped in the probe holder of the stereotactic manipulator to mix the suspension and eliminate bubbles. Depress the plunger so that the syringe is loaded with 2 &#181;l (the adequate volume to inject one animal).</li>
	<li>Pull the lateral edge of the skin incision to expose the burr hole and, with the micromanipulator, introduce the needle into the burr hole so that the beveled portion is just below the skull surface.
		<ol>
			<li>Advance the needle 3 mm and then withdraw the needle 0.5 mm to create a space for injection.</li>
			<li>Advance the plunger slowly over 30 sec and then allow the needle to remain in the brain for an additional 2 min. Withdraw the needle gradually by ascending 0.5 mm and waiting for an additional 30 sec before removing the needle from the brain.</li>
		</ol>
	</li>
	<li>Remove the mouse from the stereotactic frame and close the wound with an autoclip.</li>
	<li>Place the mouse on a warming pad to maintain body temperature and monitor continuously the respiration pattern and color of mucous membranes and exposed tissues (soles of feet).</li>
	<li>Place the mouse in a sterile microisolator cage once it recovers fully from anesthesia (sternal and ambulatory), and return it to the animal housing facility.</li>
</ol>
5. Post-implantation Animal Care and Observation
Note: The most reliable indication of tumor engraftment and infiltrative growth is gradual, sustained weight loss accompanied by development of hunched posture and rough hair coat. On rare occasions, neurological deficits are noted that include ataxia, paresis, and seizures. Orthotopic primary xenografts are highly invasive and develop over a long period of time. Mice typically manifest symptoms of tumor growth 3-6 months after transplantation.
<ol>
	<li>Observe mice with orthotopic xenografts daily for food and water intake, behavior, grooming, and signs of infection at the incision site.</li>
	<li>Administer ketoprofen (5 mg/kg subcutaneously, 1-2x a day) if pain or distress is observed postoperatively.</li>
	<li>Remove autoclips 8-10 days after surgery.</li>
	<li>Weigh mice weekly.</li>
	<li>Monitor for signs and symptoms of tumor engraftment.</li>
</ol>
6. Tumor Harvesting
Note: By this method, the first coronal slice (#1) contains the posterior aspect of the olfactory bulbs and the anterior aspect of the frontal lobes. Engrafted tumor is most abundant in the right hemisphere of the subsequent 3 coronal slices (slice #2, #3, and #4) and the injection sight is routinely contained in coronal slice #3. Depending upon experimental needs, the material may be used for tumor passage, frozen tissue sections, formalin fixed paraffin embedded tissue sections, flow cytometry of dissociated tissue, cell culture or lysates for DNA, RNA, or protein analysis. Portions of the tumor may be cryopreserved for future needs with cell viability ranging from of 80-95%.
<ol>
	<li>Euthanize mice by prolonged exposure to carbon dioxide followed by cervical dislocation.</li>
	<li>Decapitate euthanized mice at the base of the brain (foramen magnum level) with a larger pair of surgical scissors, and pull the skin from incision forward to fully expose the skull.</li>
	<li>Remove the brain.
		<ol>
			<li>Insert one tip of pair of fine surgical scissors into the lateral aspect of the foramen magnum to level of the left external auditory meatus to cut the occipital bone along the left side of the skull. Perform the same cut on the right side.</li>
			<li>Cut the occipital bone along a coronal plane from one external auditory meatus to the other and remove the bone to expose the cerebellum.</li>
			<li>Cut the nasal bone plates between the eyes.</li>
			<li>Cut the sagittal suture by cutting posteriorly along the sagittal suture from the nasal bone plates to the bregma. Complete the midline incision along the sagittal suture by cutting anteriorly from the lambda to the bregma.</li>
			<li>Carefully insert the tip of a fine pair of forceps along the sagittal opening on each side to tear any dural adhesion of the skull to the brain and then peel the two skull halves laterally to expose the brain and olfactory bulbs dorsally.</li>
			<li>Slide the forceps between the ventral aspect of the olfactory bulbs and the skull to free any adhesions.</li>
			<li>Tilt the skull back to allow the brain to be retracted so that the optic and other cranial nerves are exposed. Sever the cranial nerves to release the brain into a dish containing sterile PBS.</li>
		</ol>
	</li>
	<li>Transfer the brain with a weighing spatula to a matrix slicer and generate 2 mm coronal slices with razor blades.</li>
	<li>Arrange the coronal slices in a dish containing sterile PBS for further visual inspection and further tissue processing.</li>
</ol>
7. Tumor Cryopreservation
<ol>
	<li>Prepare a stock solution of cryopreservation medium.
		<ol>
			<li>Add 50 ml of proliferation supplement, 5 ml of 100x penicillin and streptomycin solution, and 450 &#181;l of 0.2% heparin to 450 ml of basal medium.</li>
			<li>Store stock solution at 4 &#176;C protected from light.</li>
		</ol>
	</li>
	<li>Prepare a working solution of cryopreservation medium.
		<ol>
			<li>Combine 47.5 ml of cryopreservation medium and 2.5 ml of sterile DMSO in a 50 ml polypropylene conical tube and mix well.</li>
			<li>Aliquot 1.0 ml of working solution to each cryovial and store at 4 &#176;C protected from light.</li>
		</ol>
	</li>
	<li>Place one 2 mm coronal slice of xenografted brain into a cryovial containing cryopreservation medium on ice.</li>
	<li>Tightly cap the vial and place it in a freezing container at -80 &#176;C. Transfer the cryovial the following day to a liquid nitrogen tank for longer storage.</li>
</ol>

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The authors declare that they have no competing financial interests.

Cultured cell lines, xenografts and genetically engineered mice are the most common methods for modeling gliomas, and there are distinct benefits and limitations for each model system3,13,14. Relevant benefits of primary orthotopic glioma xenografts include infiltrative growth that typifies diffuse gliomas and the retention of genetic alterations and important signaling mechanisms that can be exceedingly difficult to maintain in cultured glioma cells. For example, isocitrate dehydrogenase mutations and Sonic hedgehog ligand production can be maintained in primary orthotopic xenografts15,16 but only rarely in cultured glioma cells14,17-19. It is important to consider significant disadvantages of primary orthotopic xenografts, however, depending upon experimental needs. Notwithstanding access to adequate patient material, it is more difficult to measure glioma growth over time in the brain than in a heterotopic site. Additionally, primary orthotopic xenografts grow at a significantly slower rate than heterotopic xenografts. Thus investigators may prefer to transplant cells into the flank of an immunocompromised mouse, where important histological and molecular features may also be retained4.
Two important considerations for establishing primary orthotopic xenografts are the type of immunocompromised mouse recipient and the grade of glioma malignancy. We have had severely limited success establishing primary xenografts in athymic (nu-nu) mice and equally good success with NOD/SCID and NSG (NOD/SCID/Interleukin-2 receptor gamma chain deficient) mice. NSG mice are preferable recipients because of increased engraftment rate of tumorigenic cells and longer lifespan as compared to NOD/SCID mice20,21. With regard to WHO tumor grade, we have observed glioma engraftment of grade III and IV tumors but not grades I and II.
For primary orthotopic glioma xenografts, dissociated tumor cells may be transplanted directly into the brains of immunocompromised mice9 or tumor-initiating cells may be prospectively isolated (for example by selecting CD133-expressing cells) for transplantation22. Either source of cells is suitable for successful tumor engraftment in our experience15,16, though the use of CD133-enriched cells requires more starting material. Whether preparing sorted or unsorted cells for transplantation, it is important to adequately remove myelin and debris from dissociated glioma specimens. Failure to adequately remove myelin and debris markedly hampers the ability to draw the suspension into a Hamilton syringe and transplant a uniform number of cells into each recipient. Following papain dissociation, it is helpful to evaluate a small aliquot using a hemocytometer in order to determine the number of discontinuous gradient tubes that will be required to sufficiently eliminate myelin and debris. Complete removal is not feasible, but one can gain an appreciation for the difficulty quantifying cells in a dissociated specimen prior to myelin removal and the relative ease following removal with discontinuous gradients.
The yield of viable cells from dissociated patient specimens can be highly variable. Optimally, 105 cells are transplanted into each of five recipient mice though we have obtained engraftment with as few as 3,000 cells/animal. Approximately half of the primary malignant glioma specimens (WHO grades III and IV) that we have transplanted have engrafted in 40-100% of the initial recipient mice. Following orthotopic transplantation, recipient mice must be weighed weekly and monitored regularly for signs and symptoms of tumor engraftment and growth. Primary orthotopic xenografts demonstrate diffuse, infiltrative growth and focal neurological symptoms such as hemiparesis or seizures are observed only on rare occasions. The most sensitive indication of tumor engraftment is significant and sustained weight loss that may be accompanied by development of hunched posture, rough hair coat or domed skull. The rate at which mice develop sustained weight loss is highly variable depending upon the patient specimen and can range from 60-350 days. NSG mice typically weigh 18-25 g at transplantation and post-operatively achieve adult weights of 28-33 g. Bedding material should be changed regularly as cage conditions may alter animal weights to a considerable extent. An animal&#39;s weights may fluctuate by as much as a gram from one week to the next, and determining whether this represents tumor engraftment can be difficult. A good indication of tumor engraftment in NSG mice that have achieved adult weights is sustained weight loss of greater than 3 g. Although engrafted mice typically develop gradual weight loss that can be measured over a several week period, occasionally an animal&#39;s health may decline rapidly due to tumor growth, following the development of hydrocephalus for example, or from unrelated causes such as infection.
Following euthanasia, tumor engraftment may not be readily apparent by gross inspection of the brain and histological confirmation is essential. For initial tumor recipients, we process each brain in the same fashion so that one portion is evaluated histologically and other portions are cryopreserved for subsequent tumor passage if engraftment is verified. As discussed above, the brain is placed in a matrix slicer to generate 2 mm coronal slices with each slice representing a defined region of the brain along the anterior to posterior axis. Coronal slice #3, which routinely contains the injection site, is fixed in formalin and each of the remaining slices is placed in a separate, labeled cryovial containing 1 ml of cryopreservation medium. Formalin fixed paraffin embedded (FFPE) slides are stained with hematoxylin and eosin to evaluate the histology and with antibodies against Ki67 and human vimentin to visualize engrafted cells.
Once engraftment is verified, the tumor may be passaged and engraftment rates in secondary recipients are typically higher at 80-100%. We recommend passaging a tumor the first time in a larger number of recipient mice so that adequate cryopreserved material may be generated to maintain a given tumor line at low-passage (2-5) for future studies. Median survival times for a given tumor are generally similar from passage to passage when the same number of tumor cells are transplanted each time.

Malignant gliomas are primary glial tumors of the central nervous system that occur in the brain and occasionally the spinal cord. Gliomas are classified by the World Health Organization (WHO) according to histologic resemblance to astrocytes, oligodendrocytes or ependymal cells and then numerically graded (I to IV) for pathologic features of malignancy. The most common histologic subtypes are astrocytomas, oligodendrogliomas and mixed oligoastrocytomas. Malignant gliomas encompassing WHO grades II to IV are characterized by invasive growth and recalcitrance to current therapies. Each year in the United States, approximately 15,750 individuals are diagnosed with a malignant glioma and an estimated 12,740 patients succumb to this disease. These statistics highlight the particularly lethal nature of malignant gliomas and important need for enhanced therapeutic efficacy.
Cancer models are essential for investigating tumor biology and therapies. Human cancer cell lines represent an important first step for in vitro manipulations and in vivo xenografting studies (secondary xenografts)1. However, standard cancer cell cultures undergo phenotypic and genotypic transformation2-4 that may not be restored in secondary xenografts5. Furthermore, genetic alterations such as isocitrate dehydrogenase (IDH) mutations6, distinct stem cell populations7 and dependency on key signaling pathways8 can be lost in cancer cell cultures. Genomic profiles can be maintained to better extent in cancer sphere culture, though still fail to mirror fully the genotype of primary tumors2,3. Direct orthotopic transplantation negates the influences of in vitro culture, provides a proper microenvironment, and preserves the integrity of tumor-initiating cells9,10. Therefore, primary xenografts represent a powerful and relevant preclinical model for rigorously testing targeted agents to aid in the rational design of future clinical trials5,11,12.

<table><tbody><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>14040-133</td><td></td></tr><tr><td>Papain dissociation system</td><td>Worthington Biochemical Corp.</td><td>LK003150</td><td></td></tr><tr><td>Trypan blue solution 0.4%</td><td>Life Technologies</td><td>15250061</td><td></td></tr><tr><td>Ketamine HCl</td><td>Obtained from institutional pharmacy or local veterinary supply company</td><td></td><td></td></tr><tr><td>Xylazine HCl</td><td></td><td></td><td></td></tr><tr><td>Ketoprofen</td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;Ophthalmic ointment&lt;/p&gt; </td><td></td><td>&lt;p&gt;&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Povidone-iodine&lt;/p&gt; </td><td>Fisher Scientific</td><td>&lt;p&gt;190061617&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Cryopreservation medium and proliferation supplement&lt;/p&gt; </td><td>StemCell Technologies</td><td>&lt;p&gt;05751&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;0.2% Heparin sodium salt in PBS&lt;/p&gt; </td><td>StemCell Technologies</td><td>&lt;p&gt;07980&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Penicillin-streptomycin&lt;/p&gt; </td><td>Life Technologies</td><td>&lt;p&gt;15140-122&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Dimethyl sulfoxide&lt;/p&gt; </td><td>Sigma-Aldrich</td><td>&lt;p&gt;D6250-5X10ML&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;NOD.Cg-&lt;em&gt;Prkdc&lt;sup&gt;scid&lt;/sup&gt; I/2rg&lt;sup&gt;tm1Wjl&lt;/sup&gt;&lt;/em&gt;/SzJ mice&lt;/p&gt; </td><td>The Jackson Laboratory</td><td>&lt;p&gt;005557&lt;/p&gt;</td><td> &lt;p&gt;NSG mice&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-human vimentin antibody&lt;/p&gt; </td><td>Dako</td><td>&lt;p&gt;M7020&lt;/p&gt;</td><td> &lt;p&gt;Use 1:200 to 1:800&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-human IDH1 R132H antibody&lt;/p&gt; </td><td>Dianova</td><td>&lt;p&gt;DIA-H09&lt;/p&gt;</td><td> &lt;p&gt;Use 1:100 to 1:400&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Centrifuge with swinging bucket rotor&lt;/p&gt; </td><td></td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Pipetter with dispensing speed control&lt;/p&gt; </td><td></td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Disposable hemocytometer&lt;/p&gt; </td><td>Fisher Scientific</td><td>&lt;p&gt;22-600-100&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Sterile surgical gloves&lt;/p&gt; </td><td>Fisher Scientific</td><td>&lt;p&gt;11-388128&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Disposable gown&lt;/p&gt; </td><td>Fisher Scientific</td><td>&lt;p&gt;18-567&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Surgical mask&lt;/p&gt; </td><td>Fisher Scientific</td><td>&lt;p&gt;19-120-1256&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Tuberculin syringe&lt;/p&gt; </td><td>BD</td><td>&lt;p&gt;305620&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Alcohol pads&lt;/p&gt; </td><td>Fisher Scientific</td><td>&lt;p&gt;22-246-073&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Portable electronic scale&lt;/p&gt; </td><td>Fisher Scientific</td><td>&lt;p&gt;01-919-33&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Zoom stereomicroscope&lt;/p&gt; </td><td></td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Surgical clipper&lt;/p&gt; </td><td>Stoelting</td><td>&lt;p&gt;51465&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Scalpel handle&lt;/p&gt; </td><td>Fine Science Tools</td><td>&lt;p&gt;10003-12&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Scalpel blades, #10&lt;/p&gt; </td><td></td><td>&lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Stereotaxic instrument&lt;/p&gt; </td><td>Stoelting</td><td>&lt;p&gt;51730&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;High-speed drill&lt;/p&gt; </td><td>Stoelting</td><td>&lt;p&gt;51449&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td>Drill bit, 0.6 mm</td><td>Stoelting</td><td>514552</td><td></td></tr><tr><td> &lt;p&gt;Hamilton syringe&lt;/p&gt; </td><td>Hamilton</td><td>&lt;p&gt;80336&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Autoclip, 9 mm&lt;/p&gt; </td><td>BD</td><td>&lt;p&gt;427630&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Circulating water warming pad&lt;/p&gt; </td><td>Kent Scientific</td><td>&lt;p&gt;TP-700&lt;/p&gt; &lt;p&gt;TP-1215EA&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Hot bead dry sterilizer&lt;/p&gt; </td><td>Kent Scientific</td><td>&lt;p&gt;INS300850&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Surgical scissors&lt;/p&gt; </td><td>Fine Science Tools</td><td>&lt;p&gt;14101-14&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Fine scissors&lt;/p&gt; </td><td>Fine Science Tools</td><td>&lt;p&gt;14094-11&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Spring scissors&lt;/p&gt; </td><td>Fine Science Tools</td><td>&lt;p&gt;15018-10&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Dumont forceps&lt;/p&gt; </td><td>Fine Science Tools</td><td>&lt;p&gt;11251-30&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Semimicro spatulas&lt;/p&gt; </td><td>Fisher Scientific</td><td>&lt;p&gt;14374&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Mouse brain slicer matrix&lt;/p&gt; </td><td>Zivic Instruments</td><td>&lt;p&gt;BSMAS002-1&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Cryogenic storage vials&lt;/p&gt; </td><td>Fisher Scientific</td><td>&lt;p&gt;12-567-501&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr></tbody></table>

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.

Dissociated glioma cells are transplanted directly into the brains of immunocompromised mice to obtain primary orthotopic xenograft lines. Each tumor specimen is assigned a randomized number prior to transplantation, as part of the deidentification process to remove protected health information. We use a 5-digit REDcap database number for this purpose. Figure 1 illustrates the process and nomenclature for establishing a xenograft line from a glioblastoma (GBM 17182) with isocitrate dehydrogenase 1 (IDH1) mutation arginine 132 to histidine (R132H). Following transplantation an &#34;X&#34; is added to the tumor identification number to denote xenografted tumor, followed by a &#34;P&#34; to track the passage number. P0 designates the initial transplantation recipient and P1 designates recipient mice of the first xenograft passage. Typically, 3-5 mice are initially transplanted to establish a line and xenografted tumor is then passaged into 10 recipient mice (P1) to expand the line for future studies.
Following orthotopic transplantation, recipient mice are monitored for signs and symptoms of tumor engraftment and growth. The most sensitive indication of tumor engraftment is significant and sustained weight loss. As mice are transplanted at a young age, post-operative weight gain is expected. Mouse weights may fluctuate in seemingly dramatic fashion from week to week, however sustained loss of greater than 3 g is generally a good indication of tumor engraftment. Typically, most of the mice in a xenograft cohort develop weight loss over a similar period of time ranging from as short as 60 days to as long as 350 days depending on the tumor. Median survival times for a given tumor are generally similar from passage to passage when the same number of tumor cells are transplanted each time. Illustrated in Figure 2 is a cohort of 5 mice transplanted with a GBM (17182XP2) that had been previously engrafted and passaged once before. Four of the five 17182XP2 mice demonstrated significant weight loss 17 weeks after transplantation. The remaining mouse (mouse 3) did not exhibit weight loss and no tumor was detected at post-mortem examination, indicating tumor engraftment in only 80% of the recipients.
When the brain of a symptomatic mouse is harvested, a brain slicer matrix is an extremely valuable tool for tissue processing. The generation of multiple, anatomically defined sections from each brain allows for multiple modes of analysis with some portions and cryopreservation of other portions of the tissue. For example, mutational status of IDH1 may be assessed by Sanger sequencing with material from one portion or immunohistochemistry with another (Figure 3). Tumor engraftment may be readily apparent by visual inspection of the coronal sections, however, histology with immunohistochemistry may be required for some xenografts (Figures 4 and 5). When dissociating a xenograft for serial passage or other studies, it is important start with a small and defined portion of material so that myelin and debris may be adequately removed (Figure 6). The discontinuous gradient of the Papain Dissociation System is best suited for processing small portions of an adult mouse brain. We recommend cutting each coronal section along the midline and using two discontinuous gradients for each half.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50865/50865fig1highres.jpg" src="/files/ftp_upload/50865/50865fig1.jpg" width="500px" /> 
Figure 1. Primary orthotopic xenograft lines are established by transplanting dissociated glioma cells directly into the brains of immunocompromised mice and then expanded by serial transplantation. Heterozygous somatic mutations at arginine 132 of the IDH1 gene occur in more than 70% of diffuse astrocytomas, oligodendrogliomas, oligoastrocytomas and secondary glioblastomas, and less than 7% of primary glioblastomas. GBM 17182 contains the most common IDH1 mutation, arginine 132 to histidine (R132H). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50865/50865fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50865/50865fig2highres.jpg" src="/files/ftp_upload/50865/50865fig2.jpg" width="500px" /> 
Figure 2. Growth curves of serial transplant recipients of GBM 17182XP1 demonstrate significant weight loss 130-150 days after surgery in 4 of 5 GBM 17182XP2 mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50865/50865fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50865/50865fig3highres.jpg" src="/files/ftp_upload/50865/50865fig3.jpg" width="500px" /> 
Figure 3. Heterozygous, somatic IDH1 R132H mutation in the patient tumor (GBM 17182) and primary xenograft (17182XP0) can be detected by Sanger sequencing or antibody staining. Immunohistochemistry with an IDH1 R132H-specific antibody demonstrates mutant glioma cells densely clustered around blood vessels or in more diffusely infiltrated regions of the patient specimen and mouse brain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50865/50865fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/50865/50865fig4highres.jpg" src="/files/ftp_upload/50865/50865fig4.jpg" width="500px" /> 
Figure 4. Primary orthotopic xenografts display highly infiltrative growth that may be difficult to fully appreciate by hematoxylin and eosin (H &#38; E) staining alone. Immunohistochemistry with a human-specific antibody is a particularly useful method for identifying engrafted human cells. Anti-human vimentin staining of an analplastic oligoastrocytoma xenograft (14175XP2) reveals typical features of an infiltrative glioma with a diffuse tumor mass in the injected right hemisphere and invasive growth along myelinated tracts of the corpus callosum (asterisks) and the striatopallidal fibers (black arrowheads) and leptomeningeal dissemination (black arrow). Not appreciated at this magnification (1.25X) is clustering of human tumor cells around mouse neurons (perineuronal satellitosis) in the invaded cortex. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50865/50865fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="6in" fo:src="/files/ftp_upload/50865/50865fig5highres.jpg" src="/files/ftp_upload/50865/50865fig5.jpg" width="500px" /> 
Figure 5. Analysis of analplastic oligoastrocytoma 14175XP2 coronal sections at higher power (40X) in boxed regions A-C reveals IDH mutant human cells around blood vessels (region A), in striatopallidal tracts (&#34;pencil fibers;&#34; region B) and the corpus callosum (region C). As with GBM 17182XP2, IDH1 R132H staining of anaplastic oligoastrocytoma 14175XP2 demonstrates that IDH mutation is maintained in a primary orthotopic xenograft following serial passages in recipient mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50865/50865fig5highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 6" fo:content-width="6in" fo:src="/files/ftp_upload/50865/50865fig6highres.jpg" src="/files/ftp_upload/50865/50865fig6.jpg" width="500px" /> 
Figure 6. Human tumor cells can be distinguished from mouse cells in dissociated xenografts based upon size. To quantify human tumor cells in a dissociated xenograft for serial passage, human cells are identified in a hemocytometer under light microscopy by their large size and dark green luminescence (arrowheads). By flow cytometry, human cells have distinct scatter properties from mouse material and can be gated for further analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50865/50865fig6highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation at JoVE.com

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.

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

primary-orthotopic-glioma-xenografts-recapitulate-infiltrative-growth

Nanyang Technological University

Research

JoVE Journal

Medicine

8.0K Views.  Vanderbilt University Medical Center. 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.

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

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Neuroscience

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

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

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

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

Cancer Research

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 ...

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

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 ...

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.

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 ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 &#176;C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P &lt; 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

This study introduces an automated method to generate patient-derived glioblastoma 3-dimensional organoids utilizing a tissue chopper. The method provides a suitable and effective approach to obtain such organoids for therapeutical testing.

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing ...

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 ...

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 ...

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

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Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

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

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

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

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

Translational Orthotopic Models of Glioblastoma Multiforme

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

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

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma