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 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.
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.
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.
2. In Vitro Proliferation Assay
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 "Clean" and contains sterilized supplies; Area 2 is labeled "Dirty" and contains used materials.
4. Bioluminescence Imaging of GL261 Tumor Growth
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.
Figure 1. Luciferase activity in GL261.luc cells. 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. Please click here to view a larger version of this figure.
Figure 2. Luciferase expression does not affect GL261 cell proliferation. 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 ± 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± SEM) from quadruplicate samples on each day. Please click here to view a larger version of this figure.
Figure 3. Luciferase expression dose not affect on in vivo tumor growth and animal survival. (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). Please click here to view a larger version of this figure.
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-β 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.
The authors have nothing to disclose.
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.
D-Luciferin, Potassium Salt | Gold Biotechonology | LUCK-100 | Store away from light |
Living Image Software | Caliper Life Sciences | Contact for Quote | none |
Xenogen Lumina | Caliper Life Sciences | Contact for Quote | none |
24-well; Standard tissue culture; flat-bottom | Falcon | 353047 | |
CellTiter 96® AQueous One Solution Cell Proliferation Assay | Promega | G3582 | none |
Synergy 2 Multi-Mode Reader | BioTek | Contact for Quote | none |
96 well Assay Plate, Black Plate, Clear bottom with Lid, Tissue culture Tretaed | Coster | 3603 | none |
Ketaset | Pfizer | NDC 0856-2013-01 | Control substance |
Xylazine | Sigma-Aldrich | 23076-35-9 | none |
28G Needle (with syringe) | Fisher | 22-004-270 | AKA Insulin Syringe |
2% Chlorhexidine | Fisher | NC9756995 | AKA “Nolvasan” |
3% Hydrogen Peroxide | Fisher | H312P-4 | Store away from light |
Ophthalmic Ointment | Cardinal Health | 1272830 | AKA “Akwa Tears” |
25G 1 1/2 needle | BD Becton Dickinson | 305127 | none |
Disposable Scalpels | Feather | 2975 | No. 21 |
Gauze | Fisher | 22028563 | Autoclave before use |
Heating Pad | Dunlap | HP950 | none |
Skin Stapler, Staples, Remover | Stoelting | 59020 | none |
PDI Alchol Prep Pads | Fisher | 23-501-711 | none |
Reflex 7mm Wound Clips 100 pack | Brain Tree Scientific, INC | 203 1000 | Autoclave before use |
Reflex 7mm Wound Clip Applier | Brain Tree Scientific, INC | 204 1000 | Autoclave before use |
Reflex 7mm Wound Clip Remover | Brain Tree Scientific, INC | 205 1000 | none |
Single Ended Round Tip Swab with Wood handle | Qosmedix | 10107 | Autoclave before use |
Hanks' Balanced Salt Solution without Ca2+ and Mg2+ (HBSS) | Gibco | 14170-112 | none |
Hamilton Syringe | Hamilton | 80300 | none |
FuGENE 6 Transfection Reagent | Promega | E2961 | none |
Bone Wax | Harvard Apparatus | 599864 | none |