We would like to thank Rajwant Kaur for technical assistance. We would like to thank Maxwell Tom for assistance with lentivirus preparation. This work was supported by the Reza and Georgianna Khatib Endowed Chair in Skull Base Tumor Surgery at UCSF, and the Michael J. Marchese Professor and Chair at Northwestern University.

All procedures described below have been reviewed and approved by the Institutional Animal Use and Care Committee at Northwestern University and have been performed under sterilized conditions.
1. Modification of GL261 cells with Firefly Luciferase Expressing Reporter
NOTE: HIV-1-based lentiviral vectors express firefly luciferase (Fluc) under the control of the spleen focus-forming virus (SFFV) promoter. The optical reporter gene has been cloned into the vector plasmid pHRSIN-CSGW-dlNotI.
<ol>
	<li>Maintain 293-T cells in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and GL261 cells in RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 &#176;C in a humidified atmosphere containing 95% air and 5% carbon dioxide (CO2).</li>
	<li>When the 293-T cell culture reaches 80% confluency in a T-75 flask, wash the cells once with phosphate buffer saline (PBS) without Ca2+ and Mg2+, incubate the cells with 3 ml of trypsin (0.05%) at 37 &#176;C for 5 min, triturate with a 5 ml pipette by holding the flask slightly slanted and collect all the cells from the bottom of flask. Transfer the 3 ml of trypsinized cell suspension into a 15 ml conical tube, and add 7 ml of flesh RPMI medium (a total of 10 ml of the cell suspension).</li>
	<li>Mix the cell suspension well with a 5 ml pipette. Take 100 &#956;l of the cell suspension from the tube and count the number of the cells using hemocytometer. To do so, mix 100 &#956;l of the cell suspension with 300 &#956;l of trypan blue solution and insert 2 &#956;l of the mixture into a hemocytometer. Count the number of the cells without blue staining from four separate views under a microscope. Average the cell number from each view, as this number represent 104 cells/ml.</li>
	<li>Meanwhile, centrifuge the tube containing the cell suspension at 300 x g for 7 min. Aspirate the media and resuspend the cell pellets with fresh RPMI medium to make a GL261 cell suspension at density of 1.0 x 106/ml. Seed 2.5 x 106 GL261 cells with 2.5 ml of RPMI media into a T-175 flask with 27.5 ml DMEM (day 1).</li>
	<li>After 24 hr, replace the medium with 20 ml fresh DMEM (day 2).</li>
	<li>To produce the lentiviral vector, mix 54 &#956;l transfection reagent with 2 ml serum free DMEM for 5 min. Add 3 &#956;g of gag-pol, 3 &#956;g of vesicular stomatitis virus G envelope (VSV-G), and 4.5 &#956;g of Fluc (pSIN-Fluc) into the DMEM with transfection reagent, and incubate for 20 min at RT. Drop the entire DNA mixture into 293-T flask (T-175) and incubate at 37&#176;C in a humidified chamber containing 95% air and 5% CO2 for 48 hr (day 2).</li>
	<li>Add 10 ml of fresh DMEM at 24 hr after transfection (total volume of 293-T cell culture medium should be approximately 30 ml) (day 3).</li>
	<li>To split GL261 cells into a 24-well plate, wash the cells once with PBS without Ca2+ and Mg2+, incubate the cells with 5 ml of trypsin (0.05%) at 37 &#176;C for 5 min, triturate with a 5 ml pipette by holding the flask slightly slanted, collect all the cells from the bottom of flask, and count cells via hemocytometer as in step 1.2. Meanwhile, centrifuge the tube at 300 x g for 7 min. Aspirate the media and resuspend the cell pellets with fresh RPMI medium to make a GL261 cell suspension at density of 2.5 x 104/ml. Seed 2.5 x 104 GL261 cells with 1 ml of RPMI media in a 24-well plate (day 3).</li>
	<li>Collect 30 ml of the lentiviral supernatant with 10 ml pipette from the 293-T flask (T-175) at 48 hr following transfection and transfer the supernatant into 50 ml conical tube. Aspirate the 30 ml lentiviral supernatant via a 50 ml syringe, through a 0.22 &#956;m syringe filter, and aliquot the viral supernatant into 1 ml aliquots (day 4).</li>
	<li>Replace the 1 ml of RPMI medium in GL261 cell plate (24-well) with 1 ml of the lentiviral supernatant and incubate at 37 &#176;C in a humidified chamber (day 4).</li>
	<li>After 48 hr of lentiviral infection, replace the medium with 1 ml of fresh RPMI (day 6).</li>
	<li>After 24 hr, repeat the lentiviral infection of the GL261 cells (day 7).</li>
	<li>After the 2nd 48 hr of lentiviral infection, replace the medium with 1 ml of fresh RPMI (day 9).</li>
	<li>Continue to grow the luciferase modified GL261 cells (to be referred to as GL261.luc cells) and expand the cell culture to a T-75 flask.</li>
	<li>When the GL261.luc cell culture reaches 70% confluency in a T-75 flask, harvest the cells by trypsinization and count by hemocytometer as in step 1.2. Plate the GL261.luc cell into a 24-well plate at graduating densities (1.0 x 106, 5.0 x 105, 2.5 x 105, 1.0 x 105, 5.0 x 104, 2.5 x 104 cells/well) and incubate at 37&#176;C in a humidified chamber containing 95% air and 5% CO2 for 3 hr.</li>
	<li>Measure cellular luciferase activity relative to luciferase modified U-87 MG (U87.luc) cells using bioluminescence imaging (Figure 1).
	<ol>
		<li>Start the image software (click icon on the desktop) and initialize the imaging system (click &#34;Initialize&#34; button). Initialization will begin automatically and may take several min for the system check-up and the camera to cool down to -90 &#176;C.</li>
		<li>Add 25 &#956;l of luciferin (D-Luciferin potassium salt, 150 mg/kg) into each well.</li>
		<li>Incubate the cells with luciferin for 1 min at RT and image the plate for 10 sec. To set up the imaging system, select &#34;Luminescent&#34;, &#34;10 sec&#34; exposure time, and &#34;Medium&#34; binning. Place the 24-well plate on the imaging station and click the &#34;Acquire&#34; button to start image.</li>
		<li>After the image is successfully acquired, you will see an image window and tool palette on the screen. Click &#34;ROI (regions of interest) Tools&#34; and select 6 x 4 from the slit icon. Create 6 x 4 slits to cover the area of signal on the image of the 24-well plate and click the measurements tab (pencil icon). Observe a window with a table of ROI measurements as unit of photons per second per steradian per square cm (photons/sec/sr/cm2).</li>
		<li>Use U87.luc cells, previously modified with the same lentivirus used here for GL261 modification11, as a control for assessing luciferase activity in transduced GL261.luc cells.</li>
	</ol>
	</li>
</ol>
2. In Vitro Proliferation Assay
<ol>
	<li>When the GL261 and GL261.luc cell cultures reach 70% confluency in a T-75 flask, harvest the both cell lines by trypsinization and count as in step 1.2, and plate cells into a 96-well plate at a density of 1500 cells in 80 &#956;l of RPMI medium per well using a multichannel pipette to minimize time and pipetting error. Seed each cell line into 20 wells total. To limit the evaporation in the wells populated by cells, fill empty wells with 100 &#956;l of fresh RPMI medium.</li>
	<li>Assess the cellular proliferation using a colorimetric cell proliferation assay. Here we use the MTS cell proliferation assay. Using a multi-channel pipette, add 20 &#956;l of MTS reagent into each well of the 96-well plate that contains cells.</li>
	<li>Incubate the plate at 37&#176;C in a humidified chamber containing 95% air and 5% CO2 for 3 hr. Measure absorbance at 490 nm using a microplate reader. This is repeated at days 1, 2, 4, and 7, after plating. To normalize absorbance values, the values of each day are divided by the corresponding readings obtained at day 1 (Figure 2).</li>
</ol>
3. Tumor Cell Implantation
Note: Autoclave all equipment. Prepare surgical area by spraying a disinfectant (2% chlorhexidine solution) and cover with absorbent drapes. In order to maintain sterile conditions, wear sterile surgical gloves during surgery, and divide the surgical area into two areas by color tape. Area 1 is labeled &#34;Clean&#34; and contains sterilized supplies; Area 2 is labeled &#34;Dirty&#34; and contains used materials.
<ol>
	<li>Prior to animal surgery, prepare a cell suspension for intracranial injection. Harvest cells from a 70% confluent T-75 flask by trypsinization and count cells via hemocytometer as in step 1.2, and make a cell suspension at a density of 1.0 x 105 cells/&#181;l in Hanks&#39; Balanced Salt Solution without Ca2+ and Mg2+ (HBSS).</li>
	<li>Anesthetize mice with intraperitoneal injection of 200 &#956;l mixture containing 100 mg/kg of ketamine and 10 mg/kg of xylazine in 0.9% saline. To confirm proper anesthetization, pinch the toe&#160;of the mouse. If the mouse is completely under anesthesia, the mouse should not respond to the stimulus.</li>
	<li>Administer 0.1 mg/kg buprenorphine via subcutaneous injection to relieve the post-operative pain.</li>
	<li>Shave the top of the head of the animals using clippers and clean with a cotton-tip applicator dipped in 2% chlorhexidine solution. Apply eye ointment to maintain adequate lubrication during the surgery.</li>
	<li>Make a sagittal incision approximately 1 cm long over the parieto-occipital bone using a sterile scalpel. To visualize the bregma, wipe the surface of the skull with a cotton-tip applicator soaked in a 3% hydrogen peroxide solution.</li>
	<li>Drill the skull with a sterile 25-G needle to make a small hole 3 mm to the right of the bregma and just behind the coronal suture.</li>
	<li>To ensure that intracranial injection site is to a depth of 3 mm from the skull, cut 3 mm off the pointed end of a 20 &#956;l pipette tip using scissors and insert a syringe into the pipette tip.</li>
	<li>Insert the syringe perpendicularly to the hole previously created, and slowly inject the tumor cell suspension containing 3.0 x 105 cells in 3 &#181;l HBSS, over a 1 min period. Leave the needle in place for another minute, and then slowly pull out the needle.</li>
	<li>Swab the skull with cotton-tip applicator dipped in 3% hydrogen peroxide and the 2% chlorhexidine solution. Dry the surface of skull with the cotton-tip applicator. Cut out the sterile bone wax approximately 3mm3 in size and attach the bone wax on the end side of the cotton-tip applicator. Apply the bone wax to cover the hole with the cotton-tip applicator.</li>
	<li>Draw the each side of the scalp together over the skull using forceps, and staple to close the wound. Clean the wound with a cotton-tip dipped in 2% chlorhexidine solution.</li>
	<li>Monitor all mice post-operatively until they become ambulant and retain normal activity. Typically, recovery time is approximately 30 min. Do not return the mice to the cage with other animals until they have fully recovered from anesthesia.</li>
</ol>
4. Bioluminescence Imaging of GL261 Tumor Growth
<ol>
	<li>Spray a generous amount of disinfectant (0.05% dimethyl ammonium chloride solution) on a clean paper towel. Use the disinfectant-soaked paper towel to thoroughly wipe the black sheet, nose cones and divider inside the imaging chamber where the animals will be in contact with the device. Thoroughly wipe all surfaces with a clean, dry paper towel. Repeat this once more and wait 5 min.</li>
	<li>Initialize the imaging station as in step 1.14.1.</li>
	<li>Anesthetize the mouse with a 300 &#956;l mixture containing 200 &#956;l of Ketamine/Xylazine and 100 &#956;l of luciferin (D-Luciferin potassium salt, 150 mg/kg) by intraperitoneal injection.</li>
	<li>Wait 10 min after injection and confirm if the mice are completely under anesthesia by pinching the tail. To set up the imaging system, select &#34;Luminescent&#34;, &#34;Auto&#34; exposure time, and &#34;Medium&#34; binning. Place the mice on the imaging station and click the &#34;Acquire&#34; button to start image. Ensure that the time after injection is consistent with each imaging session.</li>
	<li>After the image is successfully acquired, an image window and tool palette should appear. Click &#34;ROI Tools&#34; and select &#34;auto&#34; from the circle icon. To define ROIs, encircle the area of signal on the image, and then take measurements of the ROI as unit of photons/sec/sr/cm2.</li>
	<li>Take the mice out of the imaging chamber and monitor the mice until they become ambulant and retain normal activity. Typically recovery time is approximately 15 min. Do not return the mice to the cage with other animals until they have fully recovered from anesthesia.</li>
	<li>Monitor tumor growth by bioluminescence imaging twice a week until animals present the following symptoms: weight loss (loss of more than 20% relative to the weight prior to implantation), inappetance (complete anorexia for 24 hr), and lack of sustained purposeful response to gentle stimuli (weak attempt to get up), at which time they should be euthanized. Euthanize animals with these symptoms using a CO2 chamber followed by cervical dislocation.
	<ol>
		<li>Place mice into the euthanasia chamber (do not overcrowded the chamber) and inflow compressed CO2 for 5-6 min. Observe that all animals are unconsciousness and not breathing after approximately 5 min.</li>
		<li>Take the mice out of the chamber. Ensure that the heart is not beating by feeling the chest between your thumb and forefinger and check that there is no blink reflex.</li>
		<li>Restrain the mouse in a prone position on a flat surface and grasp the back of the neck at the base of skull with one hand and base of the tail with your thumb and first finger of the other hand.</li>
		<li>Restrain the head and quickly pull the tail backward. Ensure that skull is separated from the first cervical vertebra using your thumb and first finger.</li>
	</ol>
	</li>
	<li>Normalize luminescence values for each day against corresponding readings obtained at the first day of bioluminescence imaging as in step 2.3 (Figure 3A).</li>
	<li>For statistical analysis, generate survival curves using the Kaplan-Meier estimator12 and compare the differences between survival curves using a log-rank test13.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+luciferase+expression+does+not+alter+immunologic+or+in+vivo+growth+properties+of+GL261+murine+glioma+cells.">Stable luciferase expression does not alter immunologic or in vivo growth properties of GL261 murine glioma cells.</a> J Transl Med. 12, 345-34 (2014).</li><li>Abdelwahab, M. G., Sankar, T., Preul, M. C., Scheck, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+implantation+with+subsequent+3D+in+vivo+bioluminescent+imaging+of+murine+gliomas.">Intracranial implantation with subsequent 3D in vivo bioluminescent imaging of murine gliomas.</a> J Vis Exp. (57), e3403 (2011).</li><li>Ozawa, T., James, C. 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There are no conflicts of interest to disclose.

GL261 murine glioma cells, when implanted intracranially into syngeneic immunocompetent C57BL/6 mice, offer several advantages compared to human glioma xenograft animal models. Many of xenografted tumors grow as encapsulated lesions that do not accurately recapitulate the invasive human disease. In contrast, GL261 tumor not only demonstrates invasion into adjacent brain, but also neovascularization, mitotic figures, and profound necrosis7. Most importantly when studying tumor immunology or immunotherapeutic strategies15, 16, the C57BL/6 mouse retains an intact immune system8. Previous studies have shown robust expression of the immunosuppressive cytokine TGF-&#946; and intracranial tumors contain immunosuppressive T-regulatory cells similar to human glioblastoma9, 10.
The simple technique described here allows for stable expression of luciferase by GL261 cells. We have recently reported similar in vitro and in vivo growth of GL261.luc cells compared to GL261 cells, in addition to similar tumor histologic characteristics and immune cell infiltrate17. GL261.luc cells stably express luciferase which catalyzes the oxidation of the substrate luciferin converting chemical energy to photons and therefore detectable light. Luciferin can be safely administered to animals and crosses the blood brain barrier after intraperitoneal or intravenous injection. In small research animals such as mice, the bioluminescence can be detected externally in a non-invasive manner11. Therefore, tumor growth can be serially assessed without the need for animal sacrifice or costly MRI or CT imaging.
The critical steps in the protocol involve, not only the lentiviral transduction, but also verification of stable expression of luciferase in vitro before beginning any in vivo experiments. Loss of transduction may require repeat lentivirus infection. Introduction of an antibiotic resistance gene into the expression vector could also be used to select for luciferase expression. Meticulous technique is required for intracranial implantation to reproducibly place a consistent number of cells in a precise anatomic location to allow comparison of different treatment groups. A major difference between the current study and previous studies of luciferase expressing GL261 cells is the use of a free hand technique for implanting cells compared to the use of a stereotactic frame18. We have previously demonstrated consistent implantation results with the free hand technique19. The advantage of the free hand technique is the ability to perform high throughput analyses of different treatment agents. In our hands, we can implant approximately 60 animals in 1 hr.
After mastering the technique, the future applications of the technique are limitless. As GL261 demonstrates elevated mitoses and rapid tumor growth similar to human glioblastoma, anti-proliferative treatments can be evaluated. Similarly, anti-invasive and anti-angiogenic therapies can be used as the GL261 tumors are invasive and angiogenic. Caution should be used when assessing the effect of chemotherapeutic agents aimed at human targets. Compared to previous studies utilizing human glioblastoma xenografts expressing luciferase20, this would be considered a limitation of our model. Also, the effect of immunotherapeutic strategies of tumor growth can be studied in the GL261 xenografted model.

Malignant glioma is the most common and most lethal human brain tumor. Even when treated with maximal surgical resection, radiotherapy, and chemotherapy, median survival remains 15-20 months at major brain tumor referral centers1, 2, 3. The most aggressive form of malignant glioma, glioblastoma, is characterized by mitotic figures, neovascularization, invasion into adjacent brain, and pseudopalisading necrosis. Orthotopic brain tumor xenografts using human brain tumor cells have served an essential function in neuro-oncology research and facilitated pre-clinical translation for testing of new agents, prior to entry into human clinical trials. Whereas most of human xenograft models recapitulate many features of the human disease, a fundamental limitation with all human-based xenograft model systems is the use of immunodeficient animals4. 
The absence of an intact host response clearly modifies tumor growth both in terms of tumor morphology, and efficiency of engraftment. While certain assumptions are acceptable in the interpretation of results from xenografted models, achieving relevant conclusions in the area of therapeutic assessment is a challenge. Certain fundamental biologic features are likely to be similar in immunodeficient and immunocompetent animals, but others such as tumor associated inflammation, tissue invasion, response to injury are probably very different. In contrast, GL261 is a chemically induced murine glioma cell line that accurately recapitulates glioma when implanted into the brains of immunocompetent syngeneic C57BL/6 mice5. Similar to the human disease, intracranial tumor growth rapidly and reproducibly leads to neurologic symptoms and animal demise6-10. 
Subcutaneous tumor growth can be directly measured. Intracranial tumor growth can only be measured with animal sacrifice or with costly imaging studies. Stable expression of the enzyme luciferase allows non-invasive and cost-effective intracranial bioluminescence imaging11. Bioluminescence of luciferase transfected GL261 cells correlates well with intracranial tumor growth. We have recently directly compared GL261 to luciferase modified GL261 cells and demonstrated no difference in in vitro growth and invasion, immunologic cytokine profile, in vivo survival of intracranial tumor bearing C57BL/6 mice, or immune cell infiltrate. This technique is applicable to glioblastoma preclinical studies which involve non-invasive monitoring of tumor growth.

<table><tbody><tr><td>D-Luciferin, Potassium Salt</td><td>Gold Biotechonology</td><td>LUCK-100</td><td>Store away from light</td></tr><tr><td>Living Image Software</td><td>Caliper Life Sciences</td><td>Contact for Quote</td><td>none</td></tr><tr><td>Xenogen Lumina</td><td>Caliper Life Sciences</td><td>Contact for Quote</td><td>none</td></tr><tr><td>24-well; Standard tissue culture; flat-bottom</td><td>Falcon</td><td>353047</td><td></td></tr><tr><td>CellTiter 96 AQueous One Solution Cell Proliferation Assay&amp;nbsp;</td><td>Promega</td><td>G3582</td><td>none</td></tr><tr><td>Synergy 2 Multi-Mode Reader</td><td>BioTek</td><td>Contact for Quote</td><td>none</td></tr><tr><td>96 well Assay Plate, Black Plate, Clear bottom with Lid, Tissue culture Tretaed</td><td>Coster</td><td>3603</td><td>none</td></tr><tr><td>Ketaset</td><td>Pfizer</td><td>NDC 0856-2013-01</td><td>Control substance</td></tr><tr><td>Xylazine</td><td>Sigma-Aldrich</td><td>23076-35-9</td><td>none</td></tr><tr><td>28G Needle (with syringe)&amp;nbsp;</td><td>Fisher&amp;nbsp;&amp;nbsp;</td><td>22-004-270</td><td>AKA Insulin Syringe</td></tr><tr><td>2% Chlorhexidine</td><td>Fisher</td><td>NC9756995</td><td>AKA &amp;ldquo;Nolvasan&amp;rdquo;</td></tr><tr><td>3% Hydrogen Peroxide</td><td>Fisher</td><td>H312P-4</td><td>Store away from light</td></tr><tr><td>Ophthalmic Ointment</td><td>Cardinal Health</td><td>1272830</td><td>AKA &amp;ldquo;Akwa Tears&amp;rdquo;</td></tr><tr><td>25G 1 1/2 needle</td><td>BD Becton Dickinson</td><td>305127</td><td>none</td></tr><tr><td>Disposable Scalpels</td><td>Feather</td><td>2975</td><td>No. 21</td></tr><tr><td>Gauze</td><td>Fisher</td><td>22028563</td><td>Autoclave before use</td></tr><tr><td>Heating Pad</td><td>Dunlap</td><td>HP950</td><td>none</td></tr><tr><td>Skin Stapler, Staples, Remover</td><td>Stoelting</td><td>59020</td><td>none</td></tr><tr><td>PDI Alchol Prep Pads</td><td>Fisher</td><td>23-501-711</td><td>none</td></tr><tr><td>Reflex 7 mm Wound Clips 100 pack</td><td>Brain Tree Scientific, INC</td><td>203 1000</td><td>Autoclave before use</td></tr><tr><td>Reflex 7 mm Wound Clip Applier</td><td>Brain Tree Scientific, INC</td><td>204 1000</td><td>Autoclave before use</td></tr><tr><td>Reflex 7 mm Wound Clip Remover</td><td>Brain Tree Scientific, INC</td><td>205 1000</td><td>none</td></tr><tr><td>Single Ended Round Tip Swab with Wood handle</td><td>Qosmedix</td><td>10107</td><td>Autoclave before use</td></tr><tr><td>Hanks&#039; Balanced Salt Solution without Ca2+ and Mg2+ (HBSS)</td><td>Gibco</td><td>14170-112</td><td>none</td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>80300</td><td>none</td></tr><tr><td>FuGENE 6 Transfection Reagent</td><td>Promega</td><td>E2961</td><td>none</td></tr><tr><td>Bone Wax</td><td>Harvard Apparatus</td><td>599864</td><td>none</td></tr></tbody></table>

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 cells were infected with luciferase containing lentivirus as described above in steps 1. In vitro bioluminescence imaging demonstrates robust luciferase expression similar to levels in the positive control U87.luc human glioblastoma cell line (Figure 1). As expected, uninfected GL261 cells demonstrate no background luciferase expression. This step is simple yet critical to perform prior to further studies using the infected cell line to confirm stable luciferase expression.
Luciferase expression after intracranial implantation.
Before intracranial implantation, we demonstrated no difference in vitro growth rate of GL261.luc compared to GL261 cells (Figure 2). For intracranial growth and survival analysis, cells were injected into the brains of C57BL/6 mice as per protocol step 3. Tumor growth in mice bearing GL261.luc tumors was serially analyzed for bioluminescence imaging using protocol step 4 and demonstrated detectable tumor growth (Figure 3A). Mice bearing GL261.luc tumors rapidly and consistently became moribund and were euthanized. Importantly, there is no difference in overall survival between GL261.luc and GL261 tumor bearing animals (Figure 3B). In vivo bioluminescence imaging can therefore be used to monitor response to experimental glioblastoma treatments. Rapid reliable tumor growth and short overall survival can yield large amounts of data in a relatively short amount of time.
<img alt="Figure 1" src="/files/ftp_upload/53287/53287fig1.jpg" /> 
Figure 1. Luciferase activity in GL261.luc cells.&#160; U87.luc, GL261.luc and GL261 (negative control) cells were plated at densities of 1.0 x 106, 5.0 x 105, 2.5 x 105, 1.0 x 105, 5.0 x 104, and 2.5 x 104 cells/well from left to right. Luminescence (photon/sec/sr/cm2) was measured by the lumina imaging station. <a href="https://www.jove.com/files/ftp_upload/53287/53287fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53287/53287fig2.jpg" /> 
Figure 2. Luciferase expression does not affect GL261 cell proliferation.&#160; GL261.luc cells do not cause a difference in proliferation as demonstrated by MTS cell proliferation assay. The graph shows fold increase, relative to day 1, as determined by comparing the average absorbance value (mean &#177; SEM) at thespecified time point to the average value at day 1 (unpaired t-test values for comparisons between each cell line: P = 0.7796)14. Error bars represent the average values (mean&#177; SEM) from quadruplicate samples on each day. <a href="https://www.jove.com/files/ftp_upload/53287/53287fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53287/53287fig3.jpg" /> 
Figure 3. Luciferase expression dose not affect on in vivo tumor growth and animal survival. &#160;(A) Bioluminescence monitoring demonstrates progressive growth of intracranial GL261.luc tumor in C57BL/6 mice. (B) Kaplan-Meier survival analysis demonstrates no difference in overall survival for mice implanted intracranially with either GL261 (solid line) or GL261.luc cells (dashed line). <a href="https://www.jove.com/files/ftp_upload/53287/53287fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma at JoVE.com

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

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 ...

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14.7K Views.  University of California San Francisco. 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.

Video: Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

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

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 ...

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 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Immunology and Infection

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 ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

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Chapters in this video

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

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

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

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

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

Bioluminescent Bacterial Imaging In Vivo

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

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

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models