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鼠神经胶质瘤细胞,颅内植入同基因免疫C57BL / 6小鼠时,相比于人神经胶质瘤的异种移植动物模型中提供了若干优点。许多异种移植的肿瘤生长为封装病变不准确概括的蔓延性人类疾病。相比之下,GL261肿瘤不仅展现侵入邻近脑,而且新生血管形成,核分裂象,而深刻坏死7。最重要的是研究肿瘤免疫学或免疫治疗策略15,16中,C57BL / 6小鼠保留完整的免疫系统8时。以往的研究表明免疫抑制细胞因子TGF-β的表达健壮和颅内肿瘤含有免疫抑制的T调节性细胞类似于人类成胶质细胞瘤9,10。
此处所描述的简单的技术允许荧光素酶由GL261细胞稳定表达。我们最近报道西米拉尔在体外和 GL261.luc细胞相比GL261细胞体内生长 ,除了类似的肿瘤组织学特征和免疫细胞渗透17。 GL261.luc细胞稳定表达的荧光素酶可催化底物萤光素化学能转化为光子,因此,可检测的光的氧化。萤光素可以安全地施用于动物和十字架腹膜内或静脉内注射后的血脑屏障。在小型研究动物如小鼠,生物发光可以从外部以非侵入性方式11检测 。因此,肿瘤生长可以被串行评估,而无需牺牲动物或昂贵的MRI或CT成像。
在协议中的关键步骤涉及,不仅慢病毒转导,而且还验证萤光素酶的稳定表达的体外开始任何体内实验之前。传导损失可能requi再重复慢病毒感染。抗生素抗性基因插入到表达载体的引入也可用于选择萤光素酶表达。细致的技术是必需的颅内植入可再现地放置细胞的一致数目以精确的解剖位置,以允许不同的治疗组进行比较。目前的研究和萤光素酶表达GL261细胞以前的研究的主要区别是使用徒手技术用于植入的细胞相比,使用立体定位框架18的。之前我们已经证实一致的植入结果与徒手技术19。自由手技术的优点是,以执行不同的处理剂的高通量分析的能力。在我们的手中,我们能在1小时内植入大约60只动物。
掌握该技术后,该技术的未来的应用是无限的。由于GL261 demonstr茨升高有丝分裂和类似人类成胶质细胞瘤肿瘤生长迅速,抗增殖治疗进行评估。同样,抗侵袭和抗血管生成疗法,可以用作GL261肿瘤侵入和血管生成。评估化疗药物针对人体目标的效果时,一定要小心。相比利用表达荧光素酶20胶质瘤异种移植物的以前的研究中,这将被视为我 们的模型的一个限制。另外,肿瘤生长的免疫治疗策略的效果可以研究在GL261异种移植模型。
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 |