We are grateful to Dr. Andrew Hollander, Sara Davis, Lee Shuman, Tim Jenkins, and Dr. Xiangsheng Xu for their expert assistance. We acknowledge the support of Dr. Ann Kennedy. B.C.B. was supported on the Radiation Biology Training Grant C5T32CA009677. J.F.D. was supported on the Burroughs Wellcome Career Award for Medical Scientists (1006792). J.L.B. was supported on the SUPERs grant (5 R25 CA140116-03). We would like to acknowledge Dr. Steve Hahn whose encouragement and support has helped make our research possible. We would also like to thank the University of Pennsylvania Nano-Bio Interface Center (NBIC) and Dr. Dennis Discher for encouragement and helpful comments. We acknowledge the Small Animal Imaging Facility (SAIF) at the University of Pennsylvania for usage of their MRI and Optical/Bioluminescence Core Facilities. These techniques were developed as part of projects that were supported by the National Institutes of Health (RC1 CA145075 and K08 NS076548-01).

A. Pre-Operative Tumor Cell Preparation
<ol>
 <li>Transduce U251 glioblastoma multiforme cells with a lentiviral expression vector (pGreenFire, System Biosciences) to stably express the firefly luciferase gene.</li>
 <li>These cells were grown in 10 ml of complete Dulbecco's Modification of Eagle's medium (DMEM), which consists of DMEM supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, and 1% nonessential amino acids in a T75 tissue culture flask incubating at 5% C02 and 37 &deg;C.</li>
 <li>Perform standard cell culture, beginning with washing the cells with phosphate buffered saline (PBS) followed by trypsinizing.</li>
 <li>Quench the trypsin with complete DMEM, transfer the solution to a 50 ml conical flask and determine the cellular concentration using the Coulter Counter.</li>
 <li>Centrifuge the harvested cells in complete DMEM for 3 min at approximately 3,000 revolutions per minute.</li>
 <li>After centrifugation, aspirate off the media leaving only a pellet of cells at the bottom of the 50 ml conical flask.</li>
 <li>Working quickly to prevent the cells from drying out, re-suspend the cells in a volume of fresh complete DMEM to achieve the desired concentration of 50,000 cells/&mu;l and transfer to a 2 ml sterile vial placed on ice.</li>
 <li>Cells on ice should be briefly vortexed on a low setting every ~20 min to prevent cell adhesion. Cells can be safely used for intracranial implantations up to two hours after placement on ice.</li>
</ol>
B. Orthotopic Xenograft Implantation
<ol>
 <li>Weigh nude athymic NCr mouse (nu/nu) to establish a baseline, pre-implantation weight. We usually select mice 6-10 weeks of age with weights between 17 and 24 grams.</li>
 <li>1 hr prior to implantation, pre-medicate the mouse with 5 mg/kg of the non-steroidal anti-inflammatory drug meloxicam via subcutaneous injection as a treatment for post-operative pain and inflammation.</li>
 <li>Anesthetize the animal with an intraperitoneal injection of a ketamine/xylazine mixture at a dose of 140 mg/kg and 10 mg/kg respectively.</li>
 <li>Confirm that the mouse is sufficiently anesthetized by assessing the animal's spontaneous response to a toe pinch. If the animal responds to the toe pinch, delay the procedure until the anesthesia can take effect or consider injecting additional anesthesia (&lt;25% of the original amount).</li>
 <li>Prepare the anesthetized mouse for positioning in the Stoelting Digital Just for Mouse Stereotactic platform (Stoelting Inc.) by hooking the mouse's incisor teeth in the bite bar of the snout restrainer and tightening the nose clamp over the snout while ensuring that the mouse's head is on a level plane.</li>
 <li>Transfer the restrained mouse to the Stoelting stereotactic platform, adjusting the positioning of the animal so that the tips of the ear bars are at the caudal end of the ear canal. Once the animal's cranial-to-caudal positioning has been adjusted, secure the bite bar to the stereotactic frame. The mouse should be resting on a firm plastic heating plate (Harvard Apparatus) secured with tape to the Stoelting platform to provide feedback-controlled temperature regulation during the surgery.</li>
 <li>Adjust the height of the ear bars as necessary and then advance the ear bars into the caudal portion of the ear canal, securing them such that the mouse's head is in a level plane and immobilized on finger touch. Monitor carefully for signs of respiratory distress after placement of the ear bars. Loosen and re-position if the animal is in distress.</li>
 <li>Insert the lubricated tip of a rectal temperature probe connected to the TCAT-2DF Temperature Controller (Harvard Apparatus) to monitor the animal's temperature and to provide feedback control to the heating plate positioned underneath the mouse to maintain the animal's body temperature at 36 &deg;C during the procedure.</li>
 <li>Apply ophthalmic ointment to the eyes to prevent drying.</li>
 <li>Apply betadine solution to the top of the mouse's head to disinfect the incision site, taking care to avoid the eyes.</li>
 <li>Perform a toe pinch to confirm the mouse is unconscious, delivering a small amount of additional ketamine/xylazine (&lt;25% of the initial dose) if necessary.</li>
 <li>Clean the 0.45 mm burr drill bit (Stoelting) attached to the drill (Foredom Microdrill) using an alcohol pad and then sterilize the drill bit in a glass bead sterilizer (Germinator 500, CellPoint Scientific) for 15 sec, immersing only the drill bit into the sterilizer beads.</li>
 <li>Using a sterile scalpel, make a 0.75 cm incision longitudinally in the mid-scalp extending from the level of the eyes caudally. Confirm visualization of the bregma.</li>
 <li>Attach the drill holder to the Stoelting platform and place a drill (Foredom Microdrill) with a 0.45 mm burr drill bit (Stoelting) in the drill holder, securing it in position.</li>
 <li>Place the drill bit exactly over the bregma and then zero-out the x, y, z coordinates of the digital stereotactic display. Move the tip of the drill to a position 2 mm posterior and 1.5 mm lateral to the bregma in the right cerebral hemisphere and drill into the animal's skull with the Foredom drill, piercing only the bone. A sterile cotton swab can be used to gently retract the edges of the incision to facilitate visualization of the skull.</li>
 <li>Remove the drill and the drill holder from the stereotactic platform.</li>
 <li>Attach the Nanomite injector syringe pump (Harvard Apparatus) to the stereotactic platform.</li>
 <li>Remove the cells from ice and either gently vortex the vial containing the tumor cells with brief pulses or gently flick the vial to re-suspend the cells. Draw up 7 &mu;l of the cell suspension through a 30 gauge 1" long flat bevel needle attached to a 10 &mu;l syringe (Hamilton Syringe). Avoid large air bubbles in the syringe. Ensure there are no air bubbles in the initial 6 &mu;l of fluid which will be the total volume injected into the mouse. Small air bubbles in the remaining 1 &mu;l are of no concern. Avoid repeated draw-ups of the cell suspension if possible to minimize damage to the cells.</li>
 <li>Position the loaded syringe into the syringe injector. Using an alcohol pad, remove any of the cell suspension fluid that appears at the tip of the needle to prevent contamination of the incision site with cancer cells which may result in tumor growing in the extracranial space.</li>
 <li>Set the injector pump to deliver 6.0 &mu;l at a rate of 0.5 &mu;l/min. The 1 &mu;l of cell suspension remaining in the syringe after implantation ensures that the air bubbles that frequently collect in the syringe are not injected into the animal. Injection of air bubbles could result in a fatal air embolism.</li>
 <li>Advance the syringe needle into the burr hole maintaining the needle perpendicular (90 degrees) to the skull. Once the needle has traversed the skull, zero out the coordinates on the stereotactic digital display and then slowly advance the tip of the needle over a period of 4 min until it reaches a depth of 2.5 mm. The needle passes through the cortex and portions of the posterior hippocampus. The x,y,z coordinates for stereotactic implantation were chosen to generate a laterally positioned tumor within the cerebral hemisphere while avoiding injury to critical brain structures such as the thalamus and proximity to the ventricles thus reducing the likelihood of seeding the cerebrospinal fluid with tumor cells that may give rise to undesirable spinal tumors. A posterior location relative to the bregma was selected to reduce the likelihood of a large, locally advanced tumor ulcerating into the orbit. Pause with the needle at depth for 2 min and then initiate implantation of cells.</li>
 <li>During implantation, dry the skull repeatedly with a microsurgical sponge spear to remove any tumor-containing fluid that may have refluxed out of the burr hole during implantation. Avoid disrupting the needle with the microsurgical sponge. Removing this fluid should reduce the likelihood of tumor growing in the extracranial space.</li>
 <li>After injection is complete, leave the needle in the brain for approximately 2 min and then slowly withdraw the needle over a period of 3-4 min.</li>
 <li>Loosen the ear bars and snout restrainer and remove the mouse from the stereotactic apparatus.</li>
 <li>Close the burr hole in the skull with sterile bone wax deposited by rubbing wax back and forth across the burr hole from the wooden end of a sterile cotton-tipped applicator. Continue applying bone wax until the hole is completely sealed and the bone wax sealing the burr hole is flush with the adjacent skull.</li>
 <li>Re-approximate the edges of the incision with sterile cotton swabs and apply veterinary tissue glue to seal the wound, taking care to avoid glue exposure to the animal's eyes.</li>
 <li>Finally, place the mouse on a heating pad set to 37 &deg;C until the animal recovers consciousness. Transfer the animal back to its original cage once the mouse is alert and responsive.</li>
</ol>
C. Bioluminescent Imaging (BLI) to Monitor Tumor Growth and Response to Therapy
Brief instructions follow for bioluminescent imaging.
<ol>
 <li>Anesthetize mice previously implanted with tumor in a chamber with 2% isoflurane and oxygen.</li>
 <li>While the mice are anesthetized, inject them either subcutaneously or intraperitoneally with 60 &mu;l of D-luciferin potassium salt diluted in PBS to a concentration of 50 mg/ml.</li>
 <li>Turn on the flow of anesthesia to the nose cones in the bioluminescent imaging scanner and quickly transfer the mice to the scanner placing their snouts in the nose cones.</li>
 <li>Position black dividers between the mice to limit the bleeding of bioluminescent signal from a tumor with high signal intensity to an adjacent tumor with much lower signal.</li>
 <li>Using the Living Image software, take frequent serial exposures for a total duration of up to 30 min after the time of injection of D-luciferin. We favor setting the Exposure time to "Auto" to limit the likelihood of an under or over-exposed image. No single exposure should be &gt;5 min.</li>
 <li>Perform repeat bioluminescent imaging at appropriate intervals. Serial weekly imaging is a reasonable option for many longitudinal experiments testing response to treatment.</li>
 <li>After acquisition of images, use the Living Image software program to analyze tumors by drawing a region of interest (ROI) around each tumor in each image acquired during the bioluminescent imaging session. Apply a second, smaller region of interest to the lower flank of each mouse in each image as a background region of interest to correct the bioluminescent signal of the tumor based on the degree of background bioluminescent signal intensity. We prefer using the "Radiance" setting rather than "Counts" as the output values for bioluminescent signal.</li>
 <li>Export the ROI results into Excel and determine the maximum background-adjusted bioluminescent signal for each mouse.</li>
 <li>Repeat bioluminescent imaging as indicated for serial monitoring of tumor growth. In our experiments, we perform weekly BLI. We prefer determining the maximal BLI signal for a given imaging session by taking frequent exposures over 5 - 30 min after injection of D-luciferin.</li>
</ol>
D. Representative Results
This stereotactic implantation technique is associated with a successful tumor-take rate of 90-100% and with low perioperative mortality that is usually less than 5%. The risk of unintended side effects is also low with this technique, including such complications as seeding of the spinal cord from tumor cells implanted into the ventricles, or extracranial tumor growth from either seeding of the incision with tumor cells or inadequate closure of the burr hole allowing intracranial tumor to expand through the opening in the skull.
Ex vivo analysis of tumor xenografts demonstrated expected areas of hypoxia, increased VEGF expression, and necrosis. Fluorescent microscopy for green fluorescent protein (GFP) stably expressed by our GBM cell line revealed the infiltrative nature of these xenografts.
Figure 1 shows the results of a typical successful stereotactic implantation of GBM cells into the brain of a mouse. This is a T2-weighted brain MRI scan of a mouse brain performed with a 9.4 Tesla magnet 21 days after implantation with the technique described here. Figure 1 reveals a single focus of tumor in the right hemisphere (contoured in pink) measuring 19 mm3 that localizes to the precise coordinates of the implantation site.
Figure 2 shows the results of bioluminescent imaging using the techniques described here for a group of 10 mice with stereotactically implanted tumors who were evenly stratified based on maximum bioluminescent signal intensity to receive either cranial irradiation (4 Gy x 4 daily fractions) or no treatment at all. In this experiment, bioluminescent imaging shows that radiation therapy inhibits proliferation of the implanted tumors, resulting in no increase in the detected bioluminescent signal, whereas the signal substantially increases in mock-irradiated control tumors, due to unchecked proliferation of the cancer cells.
Figure 1 (Video). Coronal MRI sections of a mouse brain containing a U251 glioblastoma multiforme tumor with contouring of the tumor volume (in pink). The scan was performed using a spin echo T2 weighted protocol on a 9.4 Tesla scanner. <a href="https://www.jove.com/files/ftp_upload/4089/4089movie1.m4v" target="_blank"> Click here to view movie</a>.
<img alt="Figure 2" src="/files/ftp_upload/4089/4089fig2.jpg" /> 
Figure 2. 10 mice with stereotactically implanted glioblastoma multiforme tumors were treated with either external beam radiation therapy to 16 Gy in 4 fractions or no treatment. Mice were imaged with bioluminescent imaging prior to treatment and weekly after the start of treatment. The graph presents relative bioluminescence calculated as median fold change where fold change is defined as the ratio of current maximal BLI value to pre-treatment maximal BLI value.

<ol>
	<li>Stupp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> N. Engl. J. Med. 352, 987-996 (2005).</li><li>Jacobs, V. L., Valdes, P. A., Hickey, W. F., De Leo, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+review+of+in+vivo+GBM+rodent+models:+emphasis+on+the+CNS-1+tumour+model.">Current review of in vivo GBM rodent models: emphasis on the CNS-1 tumour model.</a> ASN Neuro. 3, e00063 (2011).</li><li>Shelton, L. M. <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+pre-clinical+in+vivo+mouse+model+for+malignant+brain+tumor+growth+and+invasion.">A novel pre-clinical in vivo mouse model for malignant brain tumor growth and invasion.</a> J. Neurooncol. 99, 165-176 (2010).</li><li>Brehar, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+xenograft+glioblastoma+implants+in+nude+mice+brain.">The development of xenograft glioblastoma implants in nude mice brain.</a> J. Med. Life. 1, 275-286 (2008).</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), e1986 (2010).</li><li>Radaelli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistopathological+and+neuroimaging+characterization+of+murine+orthotopic+xenograft+models+of+glioblastoma+multiforme+recapitulating+the+most+salient+features+of+human+disease.">Immunohistopathological and neuroimaging characterization of murine orthotopic xenograft models of glioblastoma multiforme recapitulating the most salient features of human disease.</a> Histol. Histopathol. 24, 879-891 (2009).</li><li>Baumann, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+efficacy+of+drug-loaded+nanocarriers+against+brain+tumors+by+targeted+radiation+therapy.">Enhancing the efficacy of drug-loaded nanocarriers against brain tumors by targeted radiation therapy.</a> , (2012).</li><li>Baumann, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+method+for+reproducible+and+accurate+image-guided+stereotactic+cranial+irradiation+of+brain+tumors+using+the+Small+Animal+Radiation+Research+Platform+(SARRP).">An integrated method for reproducible and accurate image-guided stereotactic cranial irradiation of brain tumors using the Small Animal Radiation Research Platform (SARRP).</a> Transl. Oncol. , (2012).</li><li>Park, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroPET/CT+imaging+of+an+orthotopic+model+of+human+glioblastoma+multiforme+and+evaluation+of+pulsed+low-dose+irradiation.">MicroPET/CT imaging of an orthotopic model of human glioblastoma multiforme and evaluation of pulsed low-dose irradiation.</a> Int. J. Radiat. Oncol. Biol. Phys. 80, 885-892 (2011).</li><li>Szentirmai, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+bioluminescence+imaging+of+luciferase+expressing+intracranial+U87+xenografts:+correlation+with+magnetic+resonance+imaging+determined+tumor+volume+and+longitudinal+use+in+assessing+tumor+growth+and+antiangiogenic+treatment+effect.">Noninvasive bioluminescence imaging of luciferase expressing intracranial U87 xenografts: correlation with magnetic resonance imaging determined tumor volume and longitudinal use in assessing tumor growth and antiangiogenic treatment effect.</a> Neurosurgery. 58, 365-372 (2006).</li><li>Dinca, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+monitoring+of+intracranial+glioblastoma+xenograft:+response+to+primary+and+salvage+temozolomide+therapy.">Bioluminescence monitoring of intracranial glioblastoma xenograft: response to primary and salvage temozolomide therapy.</a> J. Neurosurg. 107, 610-616 (2007).</li></ol>

No conflicts of interest declared.

The method of stereotactic implantation of cancer cells in mice described in this paper reproducibly generates tumors that reasonably recapitulate the infiltrative and rapid-growth pattern of clinical glioblastoma multiforme 2, 6-8. This technique is especially well-suited to experiments stratifying mice evenly to different treatment groups where reproducible tumors of comparable size and biological properties and in specific anatomic locations are desirable. Stereotactic implantation of tumor cells using the ...

<table border="1">
 <tbody>
 <tr>
 <td>Description</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Digital Just for Mouse Stereotaxic Instrument</td>
 <td>Stoelting</td>
 <td>51730D</td>
 <td>Stereotactic platform for mouse implantation</td>
 </tr>
 <tr>
 <td>Ketamine/xylazine</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Injectable anesthesia</td>
 </tr>
 <tr>
 <td>Puralube Vet Ointment (ophthalmic)</td>
 <td>Amazon.com</td>
 <td>&nbsp;</td>
 <td>To prevent drying of the mouse's eyes</td>
 </tr>
 <tr>
 <td>drill holder for the stereotactic platform</td>
 <td>Stoelting</td>
 <td>51681</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micromotor Electric Drill</td>
 <td>Stoelting</td>
 <td>51449</td>
 <td>For drilling through the skull</td>
 </tr>
 <tr>
 <td>.45 mm carbide drill bit</td>
 <td>Stoelting</td>
 <td>514551</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile cotton swabs</td>
 <td>Fisher Scientific</td>
 <td>23-400-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass bead dry sterilizer (Germinator 500)</td>
 <td>Braintree Scientific</td>
 <td>GER-5287</td>
 <td>To sterilize metal surgical instruments</td>
 </tr>
 <tr>
 <td>Mouse rectal probe</td>
 <td>Braintree Scientific</td>
 <td>RET-3-ISO</td>
 <td>Compatible with the temperature controller</td>
 </tr>
 <tr>
 <td>Temperature Controller (TCAT-2DF)</td>
 <td>Harvard Apparatus</td>
 <td>727561</td>
 <td>Temperature controller to maintain animal's temperature during surgery</td>
 </tr>
 <tr>
 <td>Small heating plate</td>
 <td>Harvard Apparatus</td>
 <td>727617</td>
 <td>For use with temperature controller to warm mouse during surgery. The heating plate fits under the mouse on the stereotaxic platform.</td>
 </tr>
 <tr>
 <td>Disposable Scalpels</td>
 <td>BD Bard-Parker</td>
 <td>2015-11</td>
 <td>#10 scalpel</td>
 </tr>
 <tr>
 <td>10 microliter syringe</td>
 <td>Hamilton</td>
 <td>7635-01</td>
 <td>For injection of tumor cells</td>
 </tr>
 <tr>
 <td>30 gauge needles, 1" long, with flat point</td>
 <td>Hamilton</td>
 <td>Various</td>
 <td>Must be compatible with the 10 &mu;l syringe</td>
 </tr>
 <tr>
 <td>Nanomite Programmable Syringe Pump</td>
 <td>Harvard Apparatus</td>
 <td>704507</td>
 <td>Digital motorized syringe injector for stereotaxic device</td>
 </tr>
 <tr>
 <td>Cellulose sterile surgical spear sponges</td>
 <td>Ultracell</td>
 <td>40410</td>
 <td>To dry the surgical field</td>
 </tr>
 <tr>
 <td>Bone wax</td>
 <td>Ethicon</td>
 <td>W31</td>
 <td>To seal the burr hole</td>
 </tr>
 <tr>
 <td>Tissumend II synthetic absorbable tissue adhesive</td>
 <td>Veterinary Products Laboratories</td>
 <td>3002931</td>
 <td>To seal the incision</td>
 </tr>
 <tr>
 <td>Hot water pump with warming pad</td>
 <td>Gaymar</td>
 <td>TP-650</td>
 <td>Warms mice in post-operative period</td>
 </tr>
 <tr>
 <td>IVIS Lumina II</td>
 <td>Caliper Life Science</td>
 <td>&nbsp;</td>
 <td>Bioluminescent imager</td>
 </tr>
 <tr>
 <td>D-Luciferin potassium salt</td>
 <td>Gold Biotechnology</td>
 <td>LUCK-1</td>
 <td>Luciferin for bioluminescent imaging</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme at JoVE.com

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.

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 ...

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25.4K Views.  University of Pennsylvania. 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.

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...