We would like to acknowledge Dr. Joshua B. Rubin for the generous gift of the plasmids for the lentivirus system as well as Mahil Rao for helpful suggestion on the preparation of the GL261-luc cells.
We thank Students Supporting Brain Tumor Research (SSBTR), The Barrow Neurological Foundation and The Wallace Foundation for their generous support.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee of St. Joseph's Hospital and Medical Center.

1. Cell Culture
<ol>
 <li>The GL26 cell line was obtained from the Division of Cancer Treatment and Diagnosis (DCTD) National Cancer Institute (NCI), Frederick, MD. To facilitate a quantitative measurement of tumor growth rate GL261 cells were made bioluminescent using the Lentiphos HT System (Clontech Laboratories, Inc., Mountain View, CA) with the Lenti-X HT Packaging Mix (Clontech Laboratories, Inc.) and the FUW-GL plasmid (a generous gift from the laboratory of J.B. Rubin, MD, PhD). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% tetracycline-free Fetal Calf Serum (FCS; Clontech Laboratories, Inc.). GL261 cells were also stably transfected with the gene encoding luc2 using the pGL4.51[luc2/CMV/Neo] vector (Promega Corp, Madison, WI) and FuGENE 6 Transfection Reagent (Roche Applied Science, Indianapolis, IN) following conditions specified by the manufacturer. The luc2 gene is a codon optimized version of firefly luciferase that provides a significantly higher light output than the standard luc gene. Stable transfectants were selected and maintained in DMEM media containing 10%FCS and 100 &mu;g/ml Geneticin (G418, Invitrogen Corp., Carlsbad, CA).</li>
 <li>Prior to implantation the cultured cells are harvested by trypsinzation, washed once in DMEM without serum and resuspended in DMEM without serum at a concentration of 1 x 107 cells/ml.</li>
</ol>
2. Surgery Setup 2
<ol>
 <li>A sterile environment is maintained throughout the surgery, including all surgical instruments, supplies, gloves, drapes, etc..</li>
 <li>Ten week old C57BL/6-cBrd/cBrd/Cr (albino C57BL/6) mice are purchased from the National Cancer Institute at Frederick Animal Production Program and used at an average weight of 20 grams (NCI, Frederick, MD).</li>
 <li>Animals are anesthetized by intraperitoneal injection of xylazine (5 mg/kg), ketamine (50 mg/kg) and buprenorphine (0.05 mg/kg). Toe pinch is done to ensure that the animal is adequately anesthetized before the surgery is begun. Movement (even if slight) of any part of the animal is an indication of a reduction in the level of anesthesia. Animal is immediately given an additional 3.3 mg/kg xylazine and 26.6 mg/kg ketamine. Body temperature is maintained using a lamp and sterile dressings covering the body.</li>
 <li>Once the animal is properly anesthetized, they are placed on the cushioned bed of the stereotactic headframe (Model 900 Small Animal Stereotaxic, David Kopf Instruments)[Figure 1].</li>
 <li>A small amount (1/4 inch) of AKWA Tears Opthalmic Lubricant Ointment is applied over the corneas.</li>
 <li>The animal is secured in the stereotactic frame by opening the animal's mouth (by placing your index finger and thumb around the animal's jaw) and sliding the top front teeth into the indentation in the stereotactic frame. The clamp is tightened to secure the animal's head into the headset making sure that the head is aligned and the eyes are centered, making sure however not to exert undue force on the animal's head.</li>
 <li>The O2 hose is taped down near the animal's nostrils. Oxygen flow rate is 0.5 l/min.</li>
 <li>The lower back and tail is taped down to the stereotactic bed being careful not to compromise respirations.</li>
 <li>The surgical incision site is shaved. Povidine-Iodine Swab Sticks are used to wash the area between the eyes going back to the area between the ears with iodine, making sure the iodine does not drip into the animal's eyes.</li>
 <li>The cells are prepared for implantation just prior to the surgery and are periodically mixed to ensure they don't settle.</li>
 <li>A 10 &mu;l, 50 mm World Precision Instrument (WPI), Sarasota, FL, syringe with 26 gauge beveled needle is loaded with the cell inoculum. If the cells seem to be clumped together it may be necessary to reload the syringe.</li>
 <li>The 10 &mu;l syringe is then placed into the UMP3-1 UltraMicroPump micro injector (WPI, Sarasota, FL).</li>
</ol>
3. Intracranial Implantation 2
<ol>
 <li>The skin incision is made using a size 15 scalpel blade and toothed forceps. A 10-15 mm incision is made longitudinally from between the animal's eyes, moving towards the animal's ears exposing the bregma (junction of the sagittal and coronal sutures at the top of the skull). Be sure to properly identify the bregma for it can be easily confused with the sinus area which is distal to the bregma [Figure 2].</li>
 <li>After the animal is sedated it receives an intra-incisional injection of 0.25% (2.5 mg/ml) bupivacaine.</li>
 <li>A burrhole is made 0.1 mm posterior to bregma and 2.3 mm to right of the midline by slowly twisting a 16 gauge 1&frac12; inch needle by hand, applying little pressure to the needle while twisting until the skull is penetrated and the brain is exposed.</li>
 <li>The syringe needle is moved down into position using the microdrive stereotactic syringe holder until it just touches the brain's surface. From this position, the needle is advanced into the brain to a depth of 3 mm and kept in place for 3 minutes.</li>
 <li>The needle is withdrawn .4 mm to a total depth of 2.6 mm below the surface of the brain, creating a small pocket where the cells are to be infused. It is optional at this point to take an X-ray image of the needle in the animal to ensure proper placement and depth.</li>
 <li>The cell suspension is infused over 3 minutes using the micro injector set to a volume of 2000 nL (2 &mu;L) with an infusion rate of 667 nL/minute.</li>
 <li>The needle is left in place for 2 minutes to prevent leakage from the site of infusion.</li>
 <li>The needle is slowly withdrawn completely.</li>
 <li>The burrhole is filled with bone wax using a Penfield dissector.</li>
 <li>The incision is sutured using a 4-0 (1.5 Metric) vicryl suture making sure no large gaps are left in the skin. Vicryl is a suture material that dissolves, and therefore sutures do not need to be removed when the incision is healed.</li>
 <li>After surgery the animals are placed in a cage under a heating lamp that is set at the height required to warm the bottom surface of the cage to 30&deg;C. When the animals are fully awake (as judged by normal movement in the cage) they are returned to group housing. Oral ibuprofen is added to their drinking water for 5 days post-operatively. 100 mg of children's ibuprofen (100mg/5ml) is added to a standard 473 ml rodent water bottle. Animals are observed daily and imaged and weighed every 3 days. Two weeks post-implantation observation is increased to twice per day. Animals are euthanized when they show signs of declining health which includes hunched posture, reduced mobility and visible body weight loss (&ge;20%). These symptoms are a published response to the tumor and they reproducibly appear approximately 1 day prior to death due to the tumor.</li>
</ol>
4. In vivo Bioluminescence Imaging 3
<ol>
 <li>Start the [Living Image] software.</li>
 <li>The IVIS Imaging System is Initialized by clicking the [Initialize IVIS System] button on the bottom right side of the control panel.</li>
 <li>Select the [Luminescent] Imaging Mode on the top left side of the control panel.</li>
 <li>To determine the optimal time of imaging after luciferin injection a kinetic study is necessary [Figure 3]. This description is for firefly luciferase;
 <ol class="lalpha">
 <li>Inject 10&mu;l/g body weight of D-luciferin firefly (15mg/ml in PBS; Caliper Life Sciences Catalog XR-1001 or a similar product from another vendor) into the animal, as described below.</li>
 <li>Wait 3 minutes, and then anesthetize the mouse by placing it into the gas anesthesia chamber (2% Isoflurane gas in O2).</li>
 <li>Turn off the gas anesthesia to the chamber and open the anesthesia valve and the vacuum to the IVIS manifold. Immediately place the sedated animal on the temperature controlled imaging platform, making sure the mouse's nostril is properly placed in the gas anesthesia manifold. The first image should be taken approximately 5 minutes after the luciferin injection. Up to 5 animals can be imaged at one time in the IVIS Spectrum instrument. If less than 5 animals are to be imaged, it is possible to plug the unused manifold(s) to conserve on isoflurane gas.</li>
 <li>Continue to take images every 3 minutes by creating a sequence for up to an hour to generate a kinetic curve for luciferin expression.
 <ol>
 <li>Click on the [Sequence Setup] button in the control panel.</li>
 <li>The sequence editor appears.</li>
 <li>In the control panel, specify the settings for the first bioluminescent image in the sequence.
 <ol class="lroman">
 <li>We recommend starting with Medium Binning.</li>
 <li>We also recommend Auto-Exposure to determine the optimal exposure time.</li>
 <li>Select the [Delay] button in the sequence editor and specify a delay time of 3 minutes between each acquisition.</li>
 </ol>
 </li>
 <li>Click [Add] in the sequence editor. Acquisition parameters are then added to the table.</li>
 <li>Repeat step 4 for each image in the sequence.</li>
 </ol>
 </li>
 <li>Once the curve is established, the optimal imaging time can be determined by plotting the signal strength (intensity) versus time. Image animals at the time of highest in vivo photon count to get the strongest and most accurate signal.</li>
 </ol>
 </li>
 <li>Early experiments used intraperitoneal (i.p.) injection of luciferin, however, i.p. injections occasionally resulted in intermittent results showing little to no bioluminescence in the tumor. We hypothesized that the occasional random lack of signal was due to the delivery of the luciferin to the bowel or other internal organs. We therefore began to use subcutaneous (s.c.) luciferin injections and saw greater imaging reproducibility [Figure 3].</li>
 <li>Twenty minutes after s.c. injection, the animals are anesthetized by placing them in a chamber with 2% Isoflurane gas in O2 until they are unresponsive.</li>
 <li>The anesthetized animal(s) are moved to the imaging chamber. Ophthalmic ointment should be used for the kinetic study because of the length of imaging. It is not needed for the other imaging procedures because they are short in duration.</li>
 <li>The image is acquired at medium binning with a 5 minute exposure time. The auto acquisition option may also be used.</li>
 <li>If the signal is saturated and/or faint at medium binning, binning or exposure time can be adjusted.</li>
 <li>The program files and subsequent image comments are saved in the user's computer directory.</li>
</ol>
5. 3D Imaging 3
<ol>
 <li>Click on the [Imaging Wizard] tab in the [Sequence Setup] window of the control panel.</li>
 <li>Select the [Bioluminescence] imaging mode on the Imaging Wizard start screen and click [Next].</li>
 <li>In the "Bioluminescence - DLIT" window of the imaging wizard, select the [Firefly] reporter probe. The emission / excitation of the selected firefly source spectrum will appear with the six corresponding filter selections to be acquired.</li>
 <li>The last screen will appear with default choices that include Auto exposure acquisition parameters and a of Field of View C. Default settings work very well; however, these settings can be modified if necessary.</li>
 <li>Click [Next] and the sequence editor window will be populated with the sequence of six spectral regions at 20 nm wide filters (560 nm, 580 nm, 600nm, 620 nm, 640 nm, and 660nm). Press [Acquire Sequence]. The first filter (560 nm) will include a structured light pattern using a laser galvanometer to establish surface topography.</li>
 <li>Select the [Surface Topography] tab in the Tool Palette. Surface smoothing can be applied to account for any sharp angles created during the reconstruction process. The default low smoothing is recommended.</li>
 <li>Click [Create] and the tomography analysis box will appear. Draw a crop box that includes the entire animal and then click [Next].</li>
 <li>The threshold tool will appear as a purple mask over the selected region. The mask should automatically be set to match the photograph of the animal. If necessary, adjust the threshold of the mask to more appropriately fit the outline of the animal.</li>
 <li>Click [Finish] and the reconstructed mesh will appear. The reconstruction can then be saved in the results tab.</li>
 <li>Following the creation of the animal's surface topography, proceed to the [DLIT 3D Reconstruction] drop down on the Tool Palette.</li>
 <li>Under the [Analyze] tab select all six wavelengths to perform the reconstruction. Deselect images that yielded saturated pixels or counts below 600. Leave settings in the [Parameters] tab as default.</li>
 <li>Under the [Properties] tab, "Muscle" should be listed as the default choice for Tissue Properties, and "Firefly" should be listed as the source spectrum.</li>
 <li>Click [Reconstruct] under the Analyze tab, and the 3D reconstruction of the animal's surface and the corresponding reconstruction of the signal source should appear [Figure 4].</li>
 <li>To determine signal location and intensity, select the Voxels button on the 3D Tools tab of the Tool Palette.
 <ol>
 <li>Draw a square around all displayed voxels and the total flux measurements is shown in the bottom of the volume tab.</li>
 <li>Click on [Center of Mass] to identify the signal location of the selected voxels. Coronal, sagittal and transaxial slices of the animal will appear and using the [Measurement Cursor Display] the distance from the animal's surface to the voxel center can be measured.</li>
 </ol>
 </li>
 <li>Please refer to the Living Image Software User's Manual for further information on co-registration of organ atlases and other advanced features.</li>
</ol>
6. Data Analysis 3
<ol>
 <li>After the image is acquired and saved, access the program file by clicking the [Browse] button and selecting the file.</li>
 <li>The image information can be found under [View] &rarr; [Image Information].</li>
 <li>The intensity of the signal can be quantified by selecting the Region Of Interest (ROI) [ROI Tools] button. Make sure the image is analyzed under the [Photon] mode by selecting "Photon" on the drop-down list on the top left corner of the image control panel.</li>
 <li>Select the [Measurement ROI] button from the "Type" drop-down list. Select the ROI shape of interest; options include Circle, Square, and or Grid. Cover all areas of intensity on the acquired image.</li>
 <li>The ROI position is set by dragging the ROI shape selection to the region containing the bioluminescent signal.</li>
 <li>The signal intensity of the ROI is computed by clicking the [Measure] button. The ROI label displays the intensity. ROI's can be managed and saved using the Living Image software.</li>
</ol>
7. Representative Results:
Successful cell implantation is evident when implanted cells are detectable using the IVIS spectrum on the day of surgery. Both GL261-luc and GL261-luc2 cells are detectable, however, the luc2 gene will provide a higher level of bioluminescence [Figure 5]. Images taken soon after implantation may have non-specific signaling on the animal's paws and nose which should be disregarded as background. Signal located at the site of implantation is real and the signal will increase with time [Figure 6]. The decline in signal intensity on day 6 is reproducible and most likely due to loss of tumor take by some of the implanted cells. Quantitative measurements of tumor burden are reproducible in that they should rise steadily until the animal ultimately succumbs to the disease. However, growth curves will largely depend on the state of implanted cells, and or unavoidable minor variability in the implantation procedure. Our laboratory has elected to image implanted animals every three days.
<img alt="Figure 1" src="/files/ftp_upload/3403/3403fig1.jpg" /> 
Figure 1. After the mouse is properly anesthetized, it is placed in the stereotactic frame. The mouse head is secured using the mouth clamp.
<img alt="Figure 2" src="/files/ftp_upload/3403/3403fig2.jpg" /> 
Figure 2. After the skin is opened the main anatomical landmarks are identified including include the bregma, the coronal and sagittal sutures. The burrhole is made 2.3mm to the right of the bregma by slowly twisting a 16 gauge 1&frac12; inch needle with a small amount of pressure until the skull is penetrated and the brain is exposed.
<img alt="Figure 3" src="/files/ftp_upload/3403/3403fig3.jpg" /> 

Figure 3. A kinetic comparison of subcutaneous (<img src="/files/ftp_upload/3403/3403fig3_1.jpg" alt="Figure3.1" />) versus intraperitoneal (<img src="/files/ftp_upload/3403/3403fig3_2.jpg" alt="Figure3.2" />) luciferin injection was performed to demonstrate the utility of a subcutaneous luciferin injection and to identify the optimal time to image following luciferin administration. Three minutes after the luciferin was injected the mouse was sedated, placed in the IVIS Spectrum instrument and imaged every 3 minutes for up to an hour and every 6 minutes after that to generate a kinetic curve of bioluminescence. This demonstrated that a subcutaneous route of luciferin administration was superior to an intraperitoneal injection in our hands, and that the optimal time to image the animals was approximately 25 minutes following the luciferin injection when GL261-luc cells were used.
<img alt="Figure 4" src="/files/ftp_upload/3403/3403fig4.jpg" /> 
Figure 4. Multiple views of a 3-Dimensional reconstruction of the intracranial implantation of GL261-luc2 cells co-registered with the mouse skeleton and brain.
<img alt="Figure 5" src="/files/ftp_upload/3403/3403fig5.jpg" /> 
Figure 5. Photon counts obtained from tumors resulting from GL261-luc cells vs GL261-luc2 cells. Results are an average of 5 animals.
<img alt="Figure 6" src="/files/ftp_upload/3403/3403fig6.jpg" /> 
Figure 6. Graph of GL261-luc tumor cell growth in an albino C57BL/6 mouse. Bioluminescence was measured every 3 days and plotted as in vivo photon count versus days post-implantation. Photographs show bioluminescence at various time points. Coloration is an indication of bioluminescence (pixel intensity) which is relative to tumor cell number (color bar is shown to the right). After the animal succumbed to the disease the brain was dissected and luciferin was added topically to obtain the ex vivo image shown in the figure inset.

<ol><li>Candolfi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracranial+glioblastoma+models+in+preclinical+neuro-oncology%3A+neuropathological+characterization+and+tumor+progression%5BTitle%5D)+AND+%22J+Neurooncol%22%5BJournal%5D)">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.</a> J Neurooncol. 85, 133-148 (2007).</li>
<li>Stafford, P., Abdelwahab, M. G., do, K. im, Preul, Y., Rho, M. C., M, J., Scheck, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ketogenic+diet+reverses+gene+expression+patterns+and+redudes+reactive+oxygen+species+levels+when+used+as+an+adjuvant+therapy+for+glioma%5BTitle%5D)+AND+%22Nutr+Metab%22%5BJournal%5D)">The ketogenic diet reverses gene expression patterns and redudes reactive oxygen species levels when used as an adjuvant therapy for glioma.</a> Nutr Metab. 7, (2010).</li>
<li>Caliper Life Sciences, Inc. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Living+Image+Software+Version+4.0.+VivoVision+Systems%22">Living Image Software Version 4.0. VivoVision Systems</a>. , (2010).</li>
<li>Paxinos, G. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPaxinos%2C+G+%22The+Mouse+Brain+in+Stereotaxic+Coordinates%22">The Mouse Brain in Stereotaxic Coordinates</a>. , 2nd Ed, Academic Press. (2001).</li>
<li>Jouanneau, E., Poujol, D., Gulia, S., Le, M. I., Blay, J. Y., Belin, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+cells+are+essential+for+priming+but+inefficient+for+boosting+antitumour+immune+response+in+an+orthotopic+murine+glioma+model%5BTitle%5D)+AND+%22Cancer+Immunol+Immunother%22%5BJournal%5D)">Dendritic cells are essential for priming but inefficient for boosting antitumour immune response in an orthotopic murine glioma model.</a> Cancer Immunol Immunother. 55, 254-267 (2006).</li>
</ol>

Free Access to this article is sponsored by Caliper Life Sciences.

The cell inoculum is infused at a depth of 2.6 mm from the surface of the brain after creating a 0.4 mm pocket. To ensure proper placement and depth of needle an X-ray can be taken using a C-Arm or similar X-ray image intensifying device; however, this is optional. Complications of the surgery may arise if the animal is not properly sedated, at which point the animal may move during cell infusion. This can cause leakage of cell mix or bleeding from needle track. Leakage of cells causes ectopic growth of tumor cells. It is also important not to puncture the ventricle which can be done if the burrhole is made medial to the 2.3 mm described in the protocol 4. Proper placement of the needle and cell spread was tested by infusing a mouse with 2 &micro;l methylene blue dye and dissecting the brain tissue to check the location of the infused dye.
In this protocol we have used the IVIS Spectrum in vivo imaging system and the Living Image software (v 4.0) designed for use with this instrument. (Caliper Life Sciences). Any comparable in vivo imaging system and image analysis tools can be used to obtain similar results. These systems provide some advantages over traditional magnetic resonance imaging (MRI) to follow the growth of an experimental intracranial tumor. The most obvious is the relative costs of the 2 instruments &ndash; animal MRI machines are far more expensive and typically require the services of a skilled MRI technician. In vivo imaging such as what was described here can be done by the end user. The bioluminescence data is quantitative, while quantitation of MRI data is time-consuming and somewhat inexact. Furthermore, MRI images show edema and inflammation in addition to tumor cells and it can be difficult to separate tumor from treatment effect. For these reasons obtaining accurate volumetric measurements of growing tumor can be a challenge. Bioluminescence requires ATP, therefore only living tumor cells contribute to the tumor size data. Despite this, there are some advantages to MRI if a machine is available. Cells do not have to be labeled with a bioluminescent marker to be visualized by MRI. The ability to visualize peri-tumoral edema may be an advantage for some experimental protocols. A strength of both technologies is that using one does not preclude the use of the other, so data can be obtained on the growth of the tumor as well as the presence of peri-tumoral edema and inflammation from the same animal when both technologies are available to the researcher.

<table><tbody><tr><td>GL261-luc2 Bioware Ultra</td><td>Caliper Life Sciences</td><td>GL261-luc2</td><td> </td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Invitrogen</td><td>10313039</td><td> </td></tr><tr><td>Geneticin (G418)</td><td>GIBCO, by Life Technologies</td><td>11811-023</td><td> </td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Invitrogen</td><td>26140079</td><td> </td></tr><tr><td>Phospate Buffered Saline (PBS)</td><td>Invitrogen</td><td>70011044</td><td> </td></tr><tr><td>C57BL/6-cBrd/cBrd/Cr Mice</td><td>National Cancer Institute</td><td></td><td> </td></tr><tr><td>AKWA Tears Lubricant Opthalmic Ointment</td><td>Akorn Inc</td><td>17478-062-35</td><td> </td></tr><tr><td>Ketaset (ketamine hydrochloride)</td><td>Wyeth Animal Health</td><td>11570775</td><td> </td></tr><tr><td>Sedazine (xylazine hydrochloride)</td><td>Wyeth Animal Health</td><td>10031894</td><td> </td></tr><tr><td>Small Animal Stereotaxic Instrument</td><td>Kopf Instruments</td><td>900</td><td> </td></tr><tr><td>UltraMicroPump with SYS-Micro4 Controller</td><td>World Precision Instruments, Inc.</td><td>UMP3-1</td><td>If not available, it is possible to infuse manually</td></tr><tr><td>10&amp;mu;l syringe with 26 gauge beveled needle</td><td>World Precision Instruments, Inc.</td><td>SGE010RNS</td><td> </td></tr><tr><td>Adison Forceps</td><td>World Precision Instruments, Inc.</td><td>500092</td><td> </td></tr><tr><td>Penfield Dissector</td><td>Codman</td><td>65-1015</td><td> </td></tr><tr><td>16g 1&amp;frac12; Precision Glide Needle</td><td>BD Biosciences</td><td>305198</td><td> </td></tr><tr><td>Surgical Blade Handle</td><td>BD Biosciences</td><td>371030</td><td> </td></tr><tr><td>Size 15 Blade</td><td>BD Biosciences</td><td>371315</td><td> </td></tr><tr><td>4-0 Vicryl Suture</td><td>Ethicon Inc.</td><td>VCP496G</td><td> </td></tr><tr><td>Bone Wax</td><td>Medline Industries</td><td>DYNJBW25</td><td> </td></tr><tr><td>Povidine-Iodine Swab Sticks</td><td>Medline Industries</td><td>MD93901</td><td> </td></tr><tr><td>D-Luciferin Potassium Salt</td><td>Caliper Life Sciences</td><td>122796</td><td> </td></tr><tr><td>Forane (Isoflurane)</td><td>Baxter Internationl Inc.</td><td>1001936060</td><td> </td></tr><tr><td>OPMI Pentero Microscope</td><td>Carl Zeiss, Inc.</td><td></td><td>Any surgical microscope will suffice</td></tr><tr><td>Xenogen IVIS Spectrum with optional anesthesia system</td><td>Caliper Life Sciences</td><td></td><td> </td></tr></tbody></table>

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.

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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.

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 ...

intracranial-implantation-with-subsequent-3d-vivo-bioluminescent

Nanyang Technological University

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20.5K Views.  Barrow Neurological Institute of St. Joseph’s Hospital and Medical Center. 中文

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

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

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 ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

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

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

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

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

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 ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

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 ...

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 ...

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

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

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