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 gliomceller når implantert intracranially inn i syngeniske immunkompetente C57BL / 6-mus, gir flere fordeler sammenlignet med humane glioma xenotransplantat dyremodeller. Mange av xenopodet svulster vokse som innkapslede lesjoner som ikke nøyaktig rekapitulere invasiv sykdom hos mennesker. I motsetning til dette, GL261 tumor viser ikke bare invasjonen i tilstøtende hjernen, men også neovaskularisering, mitotiske figurer, og dyp nekrose 7. Viktigst når studere tumor immunologi eller immunterapeutiske strategier 15, 16, C57BL / 6 mus beholder et intakt immunsystem 8. Tidligere studier har vist sterk ekspresjon av det immunosuppressive cytokinet TGF-β og intrakranielle tumorer inneholder immunosuppressive T-regulatoriske celler som ligner på human glioblastoma 9, 10.
Den enkle teknikken beskrevet her gir mulighet for stabil uttrykk for luciferase av GL261 celler. Vi har nylig rapportert similar in vitro og in vivo vekst av GL261.luc celler sammenlignet med celler GL261, i tillegg til tilsvarende tumor histologiske karakteristika og immunceller infiltrere 17. GL261.luc celler som stabilt uttrykker luciferase som katalyserer oksidering av substratet luciferin omdanne kjemisk energi til fotoner og derfor påvisbar lys. Luciferin trygt kan administreres til dyr og krysser blod-hjernebarrieren etter intraperitoneal eller intravenøs injeksjon. I små forsøksdyr så som mus, kan Bioluminescens påvises utad i en ikke-invasiv måte 11. Derfor kan tumorvekst bli serielt vurderes uten behov for dyreoffer eller kostbare MR eller CT-avbildning.
De kritiske trinnene i protokollen omfatter ikke bare den lentiviral transduksjon, men også kontroll av stabil ekspresjon av luciferase in vitro før begynnelsen noen in vivo-forsøk. Tap av transduksjon kan Kravre gjenta lentivirusinfeksjon. Innføring av en antibiotisk resistens-gen i ekspresjonsvektoren kan også brukes til å selektere for luciferase ekspresjon. Grundig teknikk er nødvendig for intrakraniell implantasjon til reproduserbart å plassere et konsistent antall celler i en presis anatomiske stedet for å tillate sammenligning av forskjellige behandlingsgrupper. En viktig forskjell mellom denne studien og tidligere studier av luciferase celler som uttrykker GL261 er bruken av en fri hånd teknikk for implantering av celler sammenlignet med anvendelsen av en stereotaktisk ramme 18. Vi har tidligere vist konsistente implantasjon resultater med den frie hånden teknikk 19. Fordelen med den frie hånd teknikk er muligheten til å utføre høy gjennomstrømning analyser av forskjellige behandlingsmidler. I våre hender, kan vi implantere ca 60 dyr i en time.
Etter å mestre teknikken, de fremtidige anvendelser av teknikken er ubegrensede. Som GL261 demonstrates forhøyede mitoser og rask tumorvekst lik menneskets glioblastom, kan anti-proliferative behandlinger vurderes. På samme måte kan anti-invasive og antiangiogene terapi brukes som GL261 svulster er invasiv og angiogent. Forsiktighet bør da vurdere effekten av kjemoterapeutika rettet mot menneskelige mål brukes. Sammenlignet med tidligere studier benyttet humane glioblastom xenografter uttrykker luciferase 20, vil dette anses som en begrensning av vår modell. Dessuten kan virkningen av immunterapeutiske strategier for tumorvekst bli studert i GL261 xenografted modell.
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 |