Dr. Keating is funded by DOD grant CA100335 and is a St. Baldrick&#8217;s Foundation Scholar.

All described procedures were reviewed and approved by our institutional animal care and use committee.
1. Plan the Experiment
<ol>
	<li>Choose cells to be injected. Cells from a variety of sources are candidates for injection: adherent cell culture lines, genetically modified clones, neurosphere cells, primary cultures, or disaggregated tumors. The type of model desired will define the most appropriate site of injection.
	<ol>
		<li>Determine cell number for injection. The number of cells required to form tumors varies with cell line and must be determined empirically; tumor growth rate depends heavily on cell type, cell number and tissue culture passage number. Cell numbers ranging from 1 x 104 to 2 x 105 or more per injection have produced tumors of varying growth rates.</li>
		<li>Limit the volume for injection. The cells should be suspended in the smallest volume of serum free media or phosphate buffered saline (PBS) that allows for smooth, easy passage through a 26 G&#160;needle. The smaller the volume actually delivered into the brain, the more precise and defined the resulting tumor; optimal results are obtained by injecting volumes ranging from 3 to 6 &#181;l. Plan to prepare a final total volume of at least 50 &#181;l of suspended cells, regardless of the number of injections planned, to allow accurate measurement, mixing and sampling.</li>
	</ol>
	</li>
	<li>Choose appropriate mice for the model. Murine models of human tumors typically utilize young immune-compromised mice to avoid immune mediated graft rejection. It is not necessary for the work to take place in a biological safety cabinet if appropriate precautions are taken, including decontamination, disinfection, and sterilization of materials and equipment. The work shown here has been done in athymic nude mice, male and female, between 6 and 12 weeks of age. Mice that weigh less than 20 g can be difficult to position on a stereotaxic frame and may be more susceptible to hypothermia.</li>
</ol>
2. Assemble the Equipment
<ol>
	<li>Obtain and decontaminate a mouse stereotaxic frame, alignment console, and mouse gas anesthesia head holder, microsyringe pump, heat pad, anesthesia machine, fiber optic work lamp, and variable speed rotary drill. Clean and decontaminate all equipment.</li>
	<li>Program the injection parameters (including injection volume, flow rate, rate units, syringe type) and other variables necessary for the operation of the syringe pump. Optimize settings for each particular model. 
	NOTE: In these injections the settings were 3,000 nl samples at a rate of 400 nl/min (rate units &#8220;M&#8221;) using a 25 &#181;l syringe (Device Type &#8220;E&#8221;) in the Non-Grouped (&#8220;N&#8221;) mode.</li>
	<li>Clean a precision microsyringe with 26 G&#160;needle with several rinses of sterile deionized water (diH2O) and 70% ethanol (EtOH), doing a final rinse with diH2O. Some but not all models are designed to be autoclaved. Ensure that the plunger movement is smooth and free.</li>
	<li>Obtain anesthesia delivery equipment. Isoflurane is the preferred anesthetic as it is easy to regulate the depth and duration of anesthesia. Ensure that the stereotaxic unit is equipped with a mouse anesthesia gas attachment and that the necessary tubing and connectors are in place to deliver the gas mixture from the anesthesia machine to the mouse.</li>
	<li>Autoclave surgical tools, including fine tip scissors, two forceps/hemostats (with teeth) for suturing, medium sized scissors, medium- and fine-tipped tweezers, and at least two 1 mm dental drill bits. Keep instruments clean during the procedure with 70% EtOH, disinfectant, or a bead sterilizer.</li>
	<li>Obtain isoflurane, analgesic, ophthalmic ointment/lubricant, antibiotic ointment, saline, 30% hydrogen peroxide (H2O2), antiseptic, and bone wax (if desired) from a veterinary or medical supply. Prepare sterile diH2O and 70% EtOH. Disposables such as gauze pads, sterile dressings, sutures, ethanol swabs, cotton tipped swabs, sterile surgical gloves, surgical blades and more are itemized in the materials list. A fine tip permanent marker for marking the skull should be decontaminated and dedicated for surgical use.</li>
</ol>
3. Prepare Cells for Injection
<ol>
	<li>In the experiments shown here, select an immortalized human GBM cell line. Determine the number of cells (2 x 105) and the volume of suspension to be injected (3 &#181;l) empirically; details may vary with cell type. Use a volume of at least 50 &#181;l to facilitate accurate measuring, mixing, and injection.</li>
	<li>Trypsinize cells and resuspend in media or PBS. Perform the following steps just prior to cell injection:
	<ol>
		<li>Remove media from cells by aspiration and rinse with 10 ml PBS per 100 mm plate; remove PBS.</li>
		<li>Add 1 ml trypsin to each 100 mm plate and incubate at RT just long enough to yield a single cell suspension.</li>
		<li>Stop trypsin activity with 5 ml Dulbecco&#8217;s Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) per 100 mm plate.</li>
		<li>Pellet cells by centrifugation at 450 x g and resuspend in ~5 ml serum free DMEM (SF-DMEM) per 100 mm plate.</li>
		<li>Count viable cells using a hemacytometer or an automated cell counter.</li>
		<li>Adjust the live cell density to 2 x 105 cells/3 &#181;l (or as determined by experimentation) by pelleting the cells and resuspending in SF-DMEM.</li>
	</ol>
	</li>
	<li>Keep cells chilled on ice or a chill pack (do not allow cells to freeze). Mix gently by finger flicking.</li>
</ol>
4. Anesthetize and Prepare Mouse for Surgery
<ol>
	<li>Induce anesthesia using ~5% isoflurane (oxygen flow ~2 L/min) or as directed by veterinarian. Induction should take 2 to 3 min. Maintain mouse at an adequate depth of anesthesia with ~3% isoflurane.
	<ol>
		<li>Check depth of anesthesia by toe pinch, adjust isoflurane if indicated and continue to monitor breathing and toe pinch response throughout the procedure. Cover mouse with gauze pads for warmth and monitor mouse regularly throughout the procedure, which may take 45 min to 1 hr, depending on the rate of injection.</li>
	</ol>
	</li>
	<li>Position the mouse in stereotaxic frame by carefully sliding the palate bar over the tongue and into the mouth. Hook the front incisors into the tooth hole. Use ear bars to stabilize the head, with the ear-to-eye surface as near horizontal as possible. Do not force bars into ear canal. It is critical to position the head securely: check side-to-side and up-and-down motion gently with fingertips. Use the adjustable controls of the stereotaxic unit to optimize fit.</li>
	<li>Lubricate the eyes with ophthalmic ointment. Clean the skin between ears and eyes twice with alternating applications of a povidone-iodine antiseptic and 70% EtOH.</li>
	<li>Inject chosen analgesic subcutaneously (SQ) into flank (or as directed).</li>
</ol>
5. Perform Injection
<ol>
	<li>Determine the injection site. The injection site may vary with the size and strain of mouse and the type of cell. Cells injected too close to the ventricles may lead to cranio-spinal spread of disease. Cells injected too shallow may grow out through the needle track. 
	NOTE: In the experiments shown here, the target was defined as the frontal region of the cerebral cortex a site 2.5 mm lateral (right), 1.5 mm anterior, and 3.5 mm ventral with respect to the bregma was chosen (Figure 1), producing a midbrain tumor in mice ranging from 20 to 30 g.</li>
	<li>Make a small incision (~8 mm long) using sterile technique through the skin of the head to expose the bregma and the site of injection. A diagonal cut, from a point between the midline axis and the right eye towards the right ear (Figure 2) allows access to both the bregma and the injection site.</li>
	<li>Retract the skin and use a sterile cotton swab to dry the surface of the skull. Blot the bone with another swab moistened with H2O2 to visualize bregma. The H2O2 will produce oxygen bubbles, leaving thin lines of white foam along the coronal and sagittal sutures that intersect at bregma.</li>
	<li>Lock an empty syringe with needle onto the micropump and maneuver the tip of the needle directly over the bregma; set coordinates on the alignment console to 0.0 mm lateral, 0.0 mm anterior/posterior, and 0.0 mm ventral/dorsal.</li>
	<li>Move the needle to 2.5 mm lateral (right) and 1.5 mm anterior with respect to bregma (or desired location) using the control knobs on the stereotaxic unit. Raise the syringe slightly and mark the exact location on the skull with a dedicated felt-tip pen. If the outer surface of the skull is not on a level plane with bregma (i.e., the dorsal/ventral coordinate no longer reads zero), reset the ventral/dorsal reading to 0.0 to ensure that the depth of the injection is not shifted.</li>
	<li>Drill a small hole into the skull with a hand-held rotary drill outfitted with a sterile dental tip at the appropriate site marked with the pen in step 5.5. Hold the drill at an angle to the skull and very gently touch the tip to the bone. Repeat as needed. Drilling almost but not completely through the bone, leaving the needle to actually penetrate this layer of the skull, results in the cleanest injection.</li>
	<li>Remove bone dust with a cotton swab moistened in PBS or saline. Return the syringe to the pump and lower the needle straight down to the burr hole to confirm location. If the burr hole is not centered with the needle, use the drill to enlarge the opening.</li>
	<li>Gently mix the cell suspension and draw the cells into the syringe using the pump controller. Avoid bubbles and clumps and wipe the needle with an alcohol swab to remove contaminating cells on the exterior surface. If the syringe/needle is clogged or frozen, remove and quickly clean with sterile water and 70% EtOH.</li>
	<li>Lower the needle to the level of the skull surface (0.0 mm ventral/dorsal). Then slowly (over approximately 1 min) lower the needle to pierce through the full thickness of the skull and penetrate the brain to a depth of 4 mm ventrall. Withdraw the needle slowly to 3.5 mm ventral. If the mouse head is held securely in the stereotaxic unit, there should be almost no movement of the skull.</li>
	<li>Confirm that the correct parameters are entered into the pump controller (Section 2.2) and press &#8220;RUN/STOP&#8221; (or as directed by product manual) to autoinject cells. Monitor the mouse and adjust isoflurane if indicated. Watch the syringe and ensure that the plunger is moving in the barrel. Frozen or clogged movement can result in a bent/broken plunger.</li>
	<li>Wait 1 to 2 min with the needle in the brain, then very slowly (over 3 to 4 min) withdraw needle from tissue. Total time to remove the needle after the injection is complete is 5 min. Use a cotton swab to blot the area around the burr hole; leave the edges of the skin open to allow the bone to dry.</li>
	<li>Remove syringe from the pump and quickly rinse 3x&#160;with sterile H2O, 3x&#160;with 70% EtOH, and 3x&#160;with sterile H2O (or as directed by maker). Wipe needle with EtOH and set aside.</li>
	<li>Apply sterile bone wax (equivalent to 1 or 2 &#181;l) to the burr hole and use the wooden end of a sterile cotton swab to tamp the wax onto and into the bone. If the surface is dried adequately, it should stick.</li>
	<li>Spread sterile drape over the mouse and surrounding work area and suture the incision; three stitches should be sufficient. Alternatively, surgical adhesive may be used. Apply a topical antibiotic.</li>
	<li>Reduce isoflurane to 0% and remove the mouse from the stereotaxic unit, being careful to protect teeth. Check ears, mouth and tongue.</li>
	<li>Monitor the mouse closely following injection. Place mouse on a heat pad for 5 to 10 min; then transfer to cage (on heat pad) and observe until mouse wakes and is ambulatory. Do not leave the mouse unattended until it has regained enough consciousness to maintain sternal recumbence. Do not return mouse to the company of other animals until fully recovered. Consider the use of additional analgesic if signs of pain are noticed.</li>
</ol>
6. Finish and Monitor Recovery &#38; Tumor Development
<ol>
	<li>Clean all instruments and equipment using 70% EtOH, a bead sterilizer or disinfectant between mice.</li>
	<li>Monitor the injected mice for two days after the procedure for signs of pain, infection or other complications. Inject analgesic at 24 and 48 hr (or as indicated) after procedure. Remove sutures at 7 to 10 days, if necessary.</li>
	<li>Evaluate the mice for clinical signs of disease, paralysis, decreased activity, weight loss, seizures, or general illness.</li>
	<li>Monitor the tumor development by the appropriate method [magnetic resonance imaging (MRI), In Vivo Imaging Systems (IVIS), etc.].</li>
</ol>

<ol>
	<li>Heyer, J., Kwong, L. N., Lowe, S. W., Chin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-germline+genetically+engineered+mouse+models+for+translational+cancer+research.">Non-germline genetically engineered mouse models for translational cancer research.</a> Nature reviews. Cancer. 10, 470-480 (2010).</li><li>Yamada, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+accurately+inject+tumor+cells+into+the+caudate/putamen+nuclei+of+the+mouse+brain.">A method to accurately inject tumor cells into the caudate/putamen nuclei of the mouse brain.</a> The Tokai journal of experimental and clinical medicine. 29, 167-173 (2004).</li><li>Brooks, A. I., 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=Reproducible+and+efficient+murine+CNS+gene+delivery+using+a+microprocessor-controlled+injector.">Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector.</a> Journal of neuroscience. 80, 137-147 (1998).</li><li>Shankavaram, U. T., 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=Molecular+profiling+indicates+orthotopic+xenograft+of+glioma+cell+lines+simulate+a+subclass+of+human+glioblastoma.">Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma.</a> Journal of cellular and molecular medicine. 16, 545-554 (2012).</li><li>Giannini, C., 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=Patient+tumor+EGFR+and+PDGFRA+gene+amplifications+retained+in+an+invasive+intracranial+xenograft+model+of+glioblastoma+multiforme.">Patient tumor EGFR and PDGFRA gene amplifications retained in an invasive intracranial xenograft model of glioblastoma multiforme.</a> Neuro-oncology. 7, 164-176 (2005).</li><li>Yi, D., Hua, T. X., Lin, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+gene+overexpression+retained+in+an+invasive+xenograft+model+by+solid+orthotopic+transplantation+of+human+glioblastoma+multiforme+into+nude+mice.">EGFR gene overexpression retained in an invasive xenograft model by solid orthotopic transplantation of human glioblastoma multiforme into nude mice.</a> Cancer investigation. 29, 229-239 (2011).</li><li>Carty, N., 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=Intracranial+injection+of+AAV+expressing+NEP+but+not+IDE+reduces+amyloid+pathology+in+APP+PS1+transgenic+mice.">Intracranial injection of AAV expressing NEP but not IDE reduces amyloid pathology in APP+PS1 transgenic mice.</a> PLos ONE. 8, e59626 (2013).</li><li>Thaci, B., 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=Pharmacokinetic+study+of+neural+stem+cell-based+cell+carrier+for+oncolytic+virotherapy:+targeted+delivery+of+the+therapeutic+payload+in+an+orthotopic+brain+tumor+model.">Pharmacokinetic study of neural stem cell-based cell carrier for oncolytic virotherapy: targeted delivery of the therapeutic payload in an orthotopic brain tumor model.</a> Cancer gene therapy. 19, 431-442 (2012).</li><li>Ozawa, T., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+intracranial+brain+tumor+xenografts+with+subsequent+analysis+of+tumor+growth+and+response+to+therapy+using+bioluminescence+imaging.">Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging.</a> J. Vis. Exp. (41), (2010).</li><li>Valadez, J. G., Sarangi, A., Lundberg, C. J., Cooper, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+orthotopic+glioma+xenografts+recapitulate+infiltrative+growth+and+isocitrate+dehydrogenase+I+mutation.">Primary orthotopic glioma xenografts recapitulate infiltrative growth and isocitrate dehydrogenase I mutation.</a> J. Vis. Exp. (83), (2014).</li><li>Baumann, B. C., Dorsey, J. F., Benci, J. L., Joh, D. Y., Kao, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotactic+intracranial+implantation+and+in+vivo+bioluminescent+imaging+of+tumor+xenografts+in+a+mouse+model+system+of+glioblastoma+multiforme.">Stereotactic intracranial implantation and in vivo bioluminescent imaging of tumor xenografts in a mouse model system of glioblastoma multiforme.</a> J. Vis. Exp. (67), (2012).</li><li>Iwami, 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=A+novel+method+of+intracranial+injection+via+the+postglenoid+foramen+for+brain+tumor+mouse+models.">A novel method of intracranial injection via the postglenoid foramen for brain tumor mouse models.</a> Journal of neurosurgery. 116, 630-635 (2012).</li></ol>

The authors have nothing to disclose.

Orthotopic mouse models of human brain cancer can be an excellent tool for assessing the effectiveness of clinical therapies, but care must be taken to optimize the placement of cells in brain tissue. Studies have shown that excessive aliquot volumes, suboptimal injection technique and hasty injection rates can lead to leakiness and the appearance of tumor cells in undesirable locations (ventricles, spinal cord, extradural regions, etc.) and high variation in tumor size2 (personal observations). An an...

In vitro studies of brain tumor cells are invaluable for dissecting molecular mechanisms driving growth, survival, migration, and invasion of cancer cells; cultured cell experiments can define signaling pathways, suggest potential therapeutic targets, and characterize cellular response to drug treatment. But in vitro systems are far too simplistic to predict organismal response to pharmaceuticals; they lack the physiological reactions, immune responses, cell microenvironment, and overall heterogeneity of living animal systems. Genetically engineered models can be invaluable, when available, but molecular differences exist between species and murine cells may not recapitulate events in human processes, resulting in significant discrepancies when comparing animal models to clinical observations1. Mouse xenograft models involving subcutaneous (SQ) injection of human brain tumor cell lines under the skin of the flank are easy to perform and measure; they can be used to address effects of gene modification and drug administration/delivery, metabolism and toxicity. Significant drawbacks, however, limit the utility of SQ models. The microenvironment does not recapitulate that of a naturally occurring brain tumor: the interactions of various cell types and tissues; the local vasculature, and myriad other factors unique to the brain cannot be replicated. To more accurately reproduce the unique milieu of a naturally occurring brain tumor and test the effects of pharmaceutical interventions, a mouse orthotopic model should be utilized. Furthermore, orthotopic techniques may be used as part of a genetically engineered approach in which human primary non-cancerous cells (differentiated or progenitor) are genetically modified and injected into the relevant site of a mouse, with or without human stroma cells, resulting in tumorigenesis similar to that seen in humans1.
This article describes a methodology to precisely and reproducibly create brain tumors in mice. Using this technique, the user can accurately inject a small aliquot of suspended cells into a specified location of the fronto-parieto-temporal region of the mouse cerebral cortex. Mouse mortality is extremely low; in our hands, no mice have died from surgical complications after 185 procedures. Characteristics of the resultant tumor can be compared with that of typical human clinical tumors; for example: rapidity of growth, degree of necrosis, extent of invasion, heterogeneity of cell type, presence of mitotic cells, markers of proliferation and apoptosis, etc. Cell lines or disaggregated human tissue or tumor samples can then be evaluated based on their ability to simulate actual clinical presentation. Pharmaceuticals, selected based on their performance in cell culture, can be tested in the context of a functioning metabolism, circulatory system, and blood-brain barrier as they exist in an animal burdened with a tumor, all in a relevant architectural context. Furthermore, the cells chosen for injection may be genetically modified to investigate the impact of specific knockdowns, deletions, knock-ins, mutations, etc. on tumor growth and survival.
A number of publications document tumors studies using a variety of intracranial techniques. Yamada et al. did a detailed study of the injection of dye and of U87 cells and found that minimizing volume and injection rate produced the best tumor2. Brooks et al. found superior reproducibility and efficiency using a microprocessor-controlled injector rather than a manual method to deliver viral vectors; their conclusions regarding optimal injection parameters are applicable to cell delivery3. Shankavaram et al. showed that glioblastoma multiforme (GBM) cell lines injected orthotopically (using a manual method) into the brain recapitulated the gene expression profile of the clinical tumors more closely than either in vitro or SQ xenografts, supporting the use of intracranial models for preclinical studies4. Giannini et al. injected cells from human surgical specimens that had been sustained in the flanks of nude mice by serial passaging into the brains of additional mice, and showed that this approach preserved patient tumor gene alterations in the model5. Similar results were reported by Yi et al6. Using a stereotaxic setup, carefully defined injection site, and a slow and steady injection rate, they obtained reproducible brain tumors with consistent growth rates and high (100%) engraftment rate. The validity of this technique has therefore been well established; a literature search suggests that the applications of this technique are extensive. Carty et al. used intracranial injections to successfully deliver viral vectors expressing therapeutic genes into the frontal cortex of transgenic model of Alzheimer&#8217;s disease7. Thaci et al. described the use of intracranial injections to deliver therapeutic oncolytic adenovirus in a neural stem cell based carrier into nude mice already carrying orthotopically injected GBM tumors8. Clearly, intracranial injections are a versatile and effective tool for preclinical research. Earlier publications in The Journal of Visualized Experiments describe fundamental approaches9-11, but we take the concept of intracranial tumor injection and orthotopic modeling to a higher level of precision using easy-to-master technology.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Small Animal Stereotaxic Instrument with Digital Display Console</td><td>Kopf</td><td>Model 940</td><td></td></tr><tr><td>Mouse Gas Anesthesia Head Holder</td><td>Kopf</td><td>Model 923-B</td><td></td></tr><tr><td>Mouse Ear Bars</td><td>Kopf</td><td>Medel 922</td><td></td></tr><tr><td>Fiber Optic Illuminator</td><td>Fisher</td><td>12-562-36</td><td></td></tr><tr><td>UltraMicroPump III</td><td>WPI</td><td>UMP3</td><td></td></tr><tr><td>Micro4 microprocessor</td><td>WPI</td><td>UMC4</td><td></td></tr><tr><td>Variable speed hand-held rotary drill</td><td>Dremel</td><td>Model 300</td><td></td></tr><tr><td>Dental drill bit, 1.0 mm</td><td>Spoelting</td><td>514554</td><td></td></tr><tr><td>Adaptor for dental drill bit: 3/32 inch collet</td><td>Dremel</td><td>481</td><td></td></tr><tr><td>Heating pad</td><td></td><td></td><td>for mice</td></tr><tr><td>Isoflurane vaporizer system</td><td></td><td></td><td>for mice</td></tr><tr><td>Medical tubing and connectors</td><td></td><td></td><td>to connect isoflurane vaporizer with stereotaxic frame</td></tr><tr><td colspan="4">&lt;strong&gt;Instruments&lt;/strong&gt;</td></tr><tr><td>Precision 25 &amp;mu;l microsyringe</td><td>Hamilton&amp;nbsp;</td><td>7636-01</td><td>Model 702, without needle</td></tr><tr><td>Microsyringe needles, 26s G</td><td>Hamilton&amp;nbsp;</td><td>7804-04</td><td>RN, 25 mm point style 2</td></tr><tr><td>Fine-tipped scissors (straight, sharp/sharp)</td><td></td><td></td><td></td></tr><tr><td>Medium-sized standard scissors</td><td></td><td></td><td></td></tr><tr><td>Standard serrated forceps</td><td></td><td></td><td></td></tr><tr><td>Serrated hemostats (2)</td><td></td><td></td><td></td></tr><tr><td>Fine-tipped forceps</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Supplies&lt;/strong&gt;</td></tr><tr><td>Sutures 5-0 vicryl P-3 13 mm (Ethicon)</td><td>MWI</td><td>J463G</td><td></td></tr><tr><td>Surgical blades #10, stainless (Feather)</td><td>Fisher</td><td>296#10</td><td></td></tr><tr><td>Isoflurane (Fluriso)&amp;nbsp;</td><td>VetOne&amp;nbsp;</td><td>NDC 13985-528-60</td><td>Item #502017. Liquid inhalation anesthetic. federal law restricts this drug to use by or on the order of a licensed veterinarian.</td></tr><tr><td>Carprofen (Rimadyl Injectable 50 mg/ml)</td><td>Pfizer</td><td>NDC 61106-8507-01</td><td>dilute in saline</td></tr><tr><td>Ophthalmic ointment (artificial tears)</td><td>Rugby</td><td>NDC 0536-6550-91</td><td></td></tr><tr><td>Topical antibiotic (AK-Poly-Bac )</td><td>Akorn</td><td>NDC 17478-238-35</td><td></td></tr><tr><td>Povidone-iodine topical antiseptic, 10% (Betadine)</td><td>Betadine</td><td>NDC 67618-150-04</td><td></td></tr><tr><td>Hydrogen peroxide, 30%</td><td>Fisher&amp;nbsp;</td><td>H325-100</td><td>for visualizing skull landmarks</td></tr><tr><td>Sterile saline</td><td>VetOne&amp;nbsp;&amp;nbsp;</td><td>NDC 13985-807-25</td><td>for diluting solutions, cleaning tissue</td></tr><tr><td>Bone wax</td><td>WPI</td><td>Item #501771</td><td></td></tr><tr><td>Sterile drapes</td><td>McKesson</td><td>25-517</td><td></td></tr><tr><td>Sterile surgical gloves</td><td>McKesson</td><td>(to fit)</td><td></td></tr><tr><td>Sterile gauze pads, 2 x 2</td><td>Fisherbrand&amp;nbsp;</td><td>22028556</td><td></td></tr><tr><td>Sterile gauze pads, 4 x 4</td><td>Fisherbrand&amp;nbsp;</td><td>22-415-469</td><td></td></tr><tr><td>Alcohol prep pads (medium)</td><td>PDI</td><td>B603</td><td></td></tr><tr><td>Sterile cotton-tipped applicators</td><td>Fisherbrand&amp;nbsp;</td><td>23-400-114</td><td></td></tr><tr><td>Sterile 0.5 ml screw cap tube with caps for cells</td><td>USA Scientific</td><td>1405-4700</td><td>for cells</td></tr><tr><td>Individually wrapped sterile dispo pipettes</td><td>Fisher</td><td>BD 357575</td><td>for needle cleaning solutions</td></tr><tr><td>BD insulin syringes with needles&amp;nbsp;</td><td>Fisher</td><td>329461</td><td>for analgesic</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td>for cleaning</td></tr><tr><td>Sterile diH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>for cleaning</td></tr><tr><td>Microfuge tubes for cleaning solutions</td><td></td><td></td><td>for needle cleaning solutions</td></tr><tr><td>Felt tip pen (dedicated)</td><td></td><td></td><td>for marking skull</td></tr></tbody></table>

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.

Reliable intracranial xenografts can be created with this described technique. Identifying the critical structures of the mouse skull (Figure 1) will allow for recognition of the bregma and guide the investigator to a precise and reproducible injection location. In these studies the U251 parental line, U251 cells transfected with luciferase (U251-Luc), or U87 immortalized human GBM tissue culture cells were suspended in 4 to 6 &#181;l of SF-DMEM and injected 2.5 mm lateral (right), 1.5 mm anterior, and 3...

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Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

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 ...

creating-anatomically-accurate-reproducible-intracranial-xenografts

Research

JoVE Journal

Medicine

15.6K Views.  University of Colorado School of Medicine.  De hersenen is een unieke site met eigenschappen die niet goed weergegeven door in vitro of ectopische analyses. Orthotopische muismodellen met reproduceerbare ligging en groei-eigenschappen op betrouwbare wijze kan worden gemaakt met intracraniële injecties met een stereotaxisch fixatie instrument en een lage druk spuit pomp. 

Video: Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

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

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

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 ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

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

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

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

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

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

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

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

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

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 ...

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of neuro-oncology. One of the most widely used techniques is the orthotopic intracranial injection, a method that allows investigating of tumor growth, invasion, and dissemination within a controlled setting. This technique involves transplanting tumor cells from a specific patient region into the corresponding anatomical site in an animal. By doing so, these orthotopic brain tumor models offer a unique advantage, as they more accurately replicate cancer's biological behavior and its interactions with the brain environment seen in human patients. This makes them especially valuable for preclinical therapeutic testing, where a close resemblance to the clinical scenario is essential for evaluating potential treatments. This protocol shares experiences in developing patient-derived xenograft (PDX) models for pediatric brain tumors, including diffuse midline glioma (DMG), glioblastoma (GBM), medulloblastoma, and ependymoma. This method delineates the procedure for conducting intracranial stereotaxic injections in mice, ensuring the correct targeting of the injection site within the brain. Additionally, we describe the post-procedural monitoring system employed to detect signs of successful tumor engraftment. Following tumor injection, a rigorous monitoring system is implemented to observe the animals for any signs of neurological impairment, behavioral changes, and/or weight loss, which are common indicators of tumor progression. This system allows for timely intervention and provides critical data regarding the tumor's growth dynamics. By refining these models and protocols, we aim to enhance the reliability and translational potential of preclinical studies, contributing to the development of more effective treatments for pediatric CNS tumors.

The development of orthotopic pediatric brain tumor models requires meticulous precision, using a stereotaxic device to implant cancer cells precisely. The methodology presented here outlines the steps involved in preparing brain tumor cells, performing intracranial injections, and implementing a post-operative monitoring system to assess brain tumor engraftment.

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

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