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.
GL261 murine gliomaceller, når de implanteres intrakranielt i syngene immunokompetente C57BL / 6-mus, tilbyder flere fordele i forhold til humane gliom xenograft dyremodeller. Mange af xenotransplanterede tumorer vokse som indkapslede læsioner, der ikke nøjagtigt rekapitulere invasiv sygdom hos mennesker. I modsætning hertil GL261 tumor viser ikke kun invasion i tilstødende hjernen, men også neovaskularisering, mitotiske tal, og dyb nekrose 7. Vigtigst når man studerer tumorimmunologi eller immunterapeutiske strategier 15, 16, C57BL / 6 mus bevarer et intakt immunsystem 8. Tidligere undersøgelser har vist robust ekspression af det immunosuppressive cytokin TGF-β og intrakranielle tumorer indeholder immunosuppressive T-regulatoriske celler svarende til human glioblastoma 9, 10.
Den enkle her beskrevne teknik muliggør stabil ekspression af luciferase fra GL261 celler. Vi har for nylig rapporteret SimiLAR in vitro og in vivo væksten af celler GL261.luc sammenlignet med GL261 celler, foruden lignende tumor histologiske karakteristika og immuncelle infiltrere 17. GL261.luc celler stabilt udtrykker luciferase, som katalyserer oxidationen af substratet luciferin konvertere kemisk energi til fotoner og således detekterbar lys. Luciferin kan administreres sikkert til dyr og krydser blodhjernebarrieren efter intraperitoneal eller intravenøs injektion. I små forsøgsdyr såsom mus, kan bioluminescens detekteres eksternt på en ikke-invasiv måde 11. Derfor kan tumorvækst serielt vurderes uden behov for aflivning af dyr eller dyre MRI eller CT-billeddannelse.
De kritiske trin i protokollen indebærer, ikke kun den lentivirale transduktion, men også kontrol af stabil ekspression af luciferase in vitro inden påbegyndelsen af in vivo forsøg. Tab af transduktion kan vandskravenere gentagelse lentivirus-infektion. Indførelse af et antibiotisk resistensgen i ekspressionsvektoren kan også anvendes til at selektere for luciferaseekspression. Omhyggelig teknik er nødvendig for intrakraniel implantation til reproducerbart et sammenhængende antal celler i en præcis anatomisk stedet for at muliggøre sammenligning af forskellige behandlingsgrupper. En væsentlig forskel mellem den nuværende undersøgelse og tidligere undersøgelser af luciferase udtrykker GL261 celler er anvendelsen af en fri hånd teknik til implantering celler sammenlignet med anvendelsen af et stereotaktisk ramme 18. Vi har tidligere demonstreret konsistente implantation resultater med den frie hånd teknik 19. Fordelen ved den frie hånd teknik er evnen til at udføre high throughput analyser af forskellige behandlingsmidler. I vores hænder, kan vi implantere cirka 60 dyr i 1 time.
Efter mastering teknikken, de fremtidige anvendelser af teknikken er ubegrænsede. Som GL261 demonstration indATES forhøjede mitose og vækst hurtige tumor ligner humant glioblastom, kan antiproliferative behandlinger evalueres. Ligeledes kan anti-invasive og antiangiogene terapier anvendes som GL261 tumorer er invasiv og angiogen. Der bør udvises forsigtighed ved vurderingen af effekten af kemoterapeutika rettet mod menneskelige mål. Sammenlignet med tidligere undersøgelser med humane glioblastom xenotransplantater udtrykker luciferase 20, ville dette blive betragtet som en begrænsning af vores model. Desuden kan virkningen af immunterapeutiske strategier for tumorvækst blive undersøgt i GL261 xenotransplanteret 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 |