Name: bioluminescence imaging of an immunocompetent animal model for glioblastoma
Uploaded: 2016-01-15T14:00:00.000+00:00
Duration: 9 min 17 s
Description: Watch this Scientific Journal Video about Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma at JoVE.com

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.

下面描述的所有程序已经审查和批准的机构动物使用及护理委员会在西北大学，并已在无菌条件下进行。 1.修改GL261细胞与萤火虫荧光素酶记者表达的注:HIV-1的基于慢病毒载体表达的脾脏病灶形成病毒（SFFV）启动子的控制下，萤火虫荧光素酶（Fluc）。光学报道基因被克隆到载体质粒pHRSIN-CSGW-dlNotI。 <ol><li>在含有95的潮湿气氛维持293-T细胞在补充有10％胎牛血清（FBS）和GL261细胞在补充有10％FBS和1％青霉素 - 链霉素的RPMI培养基中的Dulbecco改进的Eagle培养基（DMEM）中于37℃下％空气和5％二氧化碳（CO 2）。 </li><li>当293-T细胞培养物达到80％汇合在T-75烧瓶中，与pH值一次洗细胞osphate缓冲盐水（PBS）无Ca2 +和Mg2 +，通过保持该烧瓶稍微倾斜孵育细胞用3ml胰蛋白酶（0.05％）的在37℃下5分钟，磨碎用5毫升吸管和收集所有的细胞从烧瓶底部。传输3毫升胰蛋白酶细胞悬浮液入15ml锥形管中，并加入7 ml的肉RPMI培养基（共10毫升细胞悬浮液）的。 </li><li>用5毫升吸管混合细胞悬浮液很好。取100微升从管中的细胞悬浮液和计数用血球细胞的数目。为此，混合100微升的细胞悬浮液用300μl的台盼蓝溶液和插入2微升混合物成血球。计数细胞的数目，而不脱离在显微镜下四个独立的观点蓝染色。平均细胞数目从每个视图，根据该数代表10 4细胞/ ml。 </li><li>同时，离心含有细胞悬浮液30中的管0×g离心7分钟。吸媒体和重悬细胞沉淀用新鲜的RPMI培养基使一个GL261细胞悬浮液以1.0×10 6 / ml的密度。种子2.5×10 6个细胞GL261用2.5ml RPMI培养基中到带有T-175烧瓶中27.5毫升DMEM（1天）。 </li><li> 24小时后，更换用20ml新鲜的DMEM（2天）的介质。 </li><li>以产生慢病毒载体，混合54微升转染试剂用2ml无血清DMEM 5分钟。加入3微克的gag-POL的，3微克水泡性口炎病毒G包膜（VSV-G），以及4.5微克Fluc（PSIN-Fluc）的与转染试剂的培养基，孵育20分钟，在室温。删除整个DNA混合物到293-T的烧瓶（T-175），并在含95％空气和5％CO 2的48小时（2天）的潮湿室中在37℃。 </li><li>加入10ml新鲜的DMEM在24小时转染后（293-T细胞培养基总体积应为大约30毫升）（第3天）。 </li><li>要拆分GL261细胞成24孔板，一次用PBS通过保持烧瓶稍微倾斜洗细胞无Ca2 +和Mg2 +，孵育细胞用5ml胰蛋白酶（0.05％）的在37℃下5分钟，磨碎用5毫升吸管，收集所有的细胞从烧瓶底部，并且计数通过血球细胞如在步骤1.2。同时，离心管中，在300×g离心7分钟。吸媒体和重悬细胞沉淀用新鲜的RPMI培养基使一个GL261细胞悬浮液以2.5×10 4个/ ml的密度。种子2.5×10 4个细胞GL261用1ml RPMI培养基中在24孔板（第3天）。 </li><li>收集30 ml的慢病毒上清液，用10ml移液管从293-T的烧瓶（T-175）在48小时转染后，将上清液转移到50ml锥形管中。抽吸通过在50ml的注射器30 ml的慢病毒上清液，通过0.22μm注射器式滤器，并分装病毒上清进1ml等份（4天）。 </li><li>更换1ml的的R在GL261单元板（24孔）用1ml的慢病毒上清液和PMI介质，在37℃孵育，在湿润室（4天）。 </li><li> 48小时后的慢病毒感染的，用1毫升新鲜的RPMI（6天）更换培养基。 </li><li> 24小时后，重复GL261细胞（7天）的慢病毒感染。 </li><li>后第 2次48小时的慢病毒感染的，用1毫升新鲜的RPMI（9天）更换培养基。 </li><li>持续增长的萤光素酶改性GL261细胞（被称为GL261.luc细胞）和扩大细胞培养到T-75烧瓶中。 </li><li>当GL261.luc细胞培养物达到70％汇合在T-75烧瓶中，通过胰蛋白酶消化收获细胞并通过血细胞计数如在步骤1.2计数。板GL261.luc细胞到24孔板中于毕业密度（1.0×10 6，5.0×10 5，2.5×10 5，1.0×10 5，5.0×10 4，2.5×10 4个细胞/孔）和在一个加湿器孵育在37℃下田间室含95％空气和5％CO 2的3小时。 </li><li>测量荧光素酶活性相对蜂窝到荧光素酶变 ​​形的U-87 MG（U87.luc），使用生物发光成像（图1）的细胞。 <ol><li>启动图像处理软件（点击桌面上的图标），并初始化成像系统（点击"初始化"按钮）。初始化会自动开始并可能需要几分钟的系统检查和相机冷却至-90℃。 </li><li>添加25微升荧光素（D-萤光素钾盐，150毫克/千克）到每个孔中。 </li><li>孵育细胞荧光素为1分钟，在RT和图像的板为10秒。要设置成像系统，选择"发光"，"10秒"的曝光时间，而"中"分档。将24孔板在成像站上，并点击"获取"按钮，开始图像。 </li><li>成功后获得的图像，你会看到一个图像窗口和工具PA莱特在屏幕上。点击"ROI（感兴趣区域）的工具"，并从该狭缝图标选择6×4。创建6×4的狭缝，以覆盖24孔板的图像上的信号的区域，并点击测量标签（铅笔图标）。观察用ROI的测量结果的表中的一个窗口单元每每平方厘米（光子/秒/ SR / cm 2）的球面度第二光子。 </li><li>使用U87.luc细胞预先用这里用于GL261 变形例11相同的慢病毒改性，作为评估在转导的细胞GL261.luc萤光素酶活性的控制。 </li></ol></li></ol> 2.在体外增殖试验<ol><li>当GL261和GL261.luc细胞培养物达到70％汇合在T-75烧瓶中，通过胰蛋白酶消化收获两种细胞系和计数作为在步骤1.2，和板的细胞在96孔板中于1500个细胞的密度80微升每使用以及多道移液器，以减少时间和移液错误RPMI培养基。硒编每个细胞系成20个井总。为了限制在填充的细胞的孔的蒸发，填充空孔用100μl的新鲜RPMI培养基。 </li><li>评估用比色细胞增殖测定的细胞增殖。在这里，我们使用MTS细胞增殖试验。使用多通道移液管，添加20微升的MTS试剂入96孔板，包含细胞的每个孔中。 </li><li>孵育板在37℃下在含95％空气和5％CO 2的3小时的潮湿室中。用酶标仪测量490nm处的吸光度。这被重复，在天1，2，4，和7，电镀后。为了标准化吸光度值，每一天的值由在第1天（图2）获得的相应读数除以。 </li></ol> 3.肿瘤细胞移植注意:所有高压灭菌设备。通过喷洒消毒剂（2％洗必泰溶液）准备手术区，盖上absorben吨窗帘。为了保持无菌条件下，在手术过程中穿无菌手术手套，并划分手术区到由彩色带的两个领域。区域1标有"清洁"，并包含消毒用品;区域2被标记为"脏"，其中包含用于材料。 <ol><li>前动物手术，制备细胞悬浮液为颅内注射。从70％汇合的T-75烧瓶中通过胰蛋白酶消化并计数通过血球细胞如在步骤1.2，使细胞悬浮液以1.0×10 5个细胞/微升在Hanks&#39;平衡盐溶液的密度不含Ca 2+和收获细胞镁离子（HBSS）。 </li><li>麻醉小鼠腹膜内注射含0.9％盐氯胺酮甲苯噻嗪的100 mg / kg和10mg / kg的200微升混合物。要确认正确的麻醉，捏老鼠的脚趾。如果鼠标完全麻醉下，鼠标不应响应的刺激。 </li><li>辖0.1米通过皮下注射克/公斤丁丙诺啡，以减轻术后疼痛。 </li><li>剃使用推子和清洁用棉尖涂药蘸2％氯己定溶液的动物的头顶部。应用眼膏手术过程中保持足够的润滑。 </li><li>做一个切口矢状约为1厘米长比使用无菌手术刀顶枕骨。为了显现前囟，擦拭头骨表面用棉尖涂药浸泡在3％的过氧化氢溶液。 </li><li>钻颅骨，用无菌25-G针，使一个小孔3毫米至前囟门的右侧，仅落后于冠状缝。 </li><li>以确保颅内注射部位是3毫米从颅骨的深度，切割3毫米断用剪刀20微升枪头的尖端，并插入一个注射器插入枪头。 </li><li>垂直插入注射器之前创建的孔，慢慢注入肿瘤细胞SUS养老金含有3.0×10 5细胞在3微升HBSS，在1分钟内。留在原地针一分钟，然后慢慢拔出针。 </li><li>涂上棉尖涂药蘸3％双氧水和2％洗必泰溶液的头骨。干燥头骨表面与棉尖涂药。切出的无菌骨蜡约3毫米3大小，附加骨蜡上的棉尖涂药的端侧。应用骨蜡以支付与棉尖涂药的孔中。 </li><li>头皮的每一侧拉在一起过使用镊子颅骨，和订书钉到闭合伤口。清洁用棉尖蘸2％洗必泰溶液的伤口。 </li><li>监视所有小鼠手术后，直到他们成为走动，并保留正常活动。通常情况下，恢复时间约为30分钟。不要将小鼠与其他动物的笼子里，直到它们完全从麻醉中恢复。 </li></ol><ol><li>喷雾消毒剂对干净的纸巾大方量（0.05％二甲基氯化铵溶液）。使用消毒液浸泡过的纸巾彻底抹了黑片，鼻锥和分频器的成像室，在这里的动物将与该装置接触内部。彻底擦拭所有的表面用清洁，干燥的纸巾。重复此一次，并等待5分钟。 </li><li>初始化成像站如步骤1.14.1。 </li><li>麻醉小鼠用含有200微升氯胺酮/甲苯​​噻嗪和100μl萤光素（D-萤光素钾盐，150毫克/千克）通过腹膜内注射300微升混合物。 </li><li>等待10分钟，注射后确认，如果老鼠是完全在麻醉状态下按捏尾巴。要设置成像系统，选择"发光"，"自动"的曝光时间，而"中"分档。将小鼠在成像为static并点击"获取"按钮，开始图像。确保注射后的时间是与每个成像会议一致。 </li><li>成功后获得的图像，图像窗口和工具选项板应该会出现。点击"投资回报工具"，并从圆圈图标中选择"自动"。要定义投资回报，包围图像信号的区域，然后取投资回报率的测量单位为光子/秒/ SR /厘米2。 </li><li>就拿老鼠出来的成像室和监测小鼠，直到他们成为走动，并保留正常活动。通常的恢复时间是约15分钟。不要将小鼠与其他动物的笼子里，直到它们完全从麻醉中恢复。 </li><li>监测肿瘤生长通过生物发光成像每周两次直至动物呈现下列症状:重量损失（相对于20％以上的损失在植入前的重量），食欲不振（完整厌食24小时），和缺乏持续purposefu的●响应，以温和的刺激（两掌起床），此时他们应该被实施安乐死。安乐死的动物与这些症状使用CO 2室颈椎脱位。 <ol><li>将小鼠放入安乐死室（不要人满为患室），并流入压缩二氧化碳为5-6分钟。注意，所有的动物都昏迷，呼吸停止后大约5分钟。 </li><li>就拿老鼠出室。确保心脏没有跳动摸着你的拇指和食指之间的胸部和检查，没有眨眼反射。 </li><li>抑制鼠标在上平坦表面的俯卧位和把握颈部的尾部的后面，在颅底用一只手和碱用拇指和另一方面的第一手指。 </li><li>抑制头部，迅速拉尾巴落后。确保头骨是使用你的拇指和食指的第一颈椎分离。 </li></ol></li><li>规范阿利兹发光值的每一天对在生物发光成像的第一天获得如在步骤2.3（图3A）相应的读数。 </li><li>进行统计分析，生成用Kaplan-Meier估算12生存曲线，并比较使用对数秩检验13存活曲线之间的差异。 </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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+intracranial+brain+tumor+xenografts+with+subsequent+analysis+of+tumor+growth+and+response+to+therapy+using+bioluminescence+imaging.">Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging.</a> J Vis Exp. (41), (2010).</li><li>Baumann, B. C., Dorsey, J. F., Benci, J. L., Joh, D. Y., Kao, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotactic+intracranial+implantation+and+in+vivo+bioluminescent+imaging+of+tumor+xenografts+in+a+mouse+model+system+of+glioblastoma+multiforme.">Stereotactic intracranial implantation and in vivo bioluminescent imaging of tumor xenografts in a mouse model system of glioblastoma multiforme.</a> J Vis Exp. (67), (2012).</li></ol>

There are no conflicts of interest to disclose.

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。

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细胞用如步骤如上所述1.在体外生物发光成像演示类似于阳性对照U87.luc人恶性胶质瘤细胞系的水平健壮萤光素酶表达（图1）含有萤光素酶的慢病毒。正如预期的那样，未感染GL261细胞表现出没有背景荧光素酶表达。这一步很简单，但关键的执行之前，用被感染的细胞系，以确认稳定的荧光素酶表达进一步的研究。 颅内植入后荧光素酶的表达。 颅内植入前，我们展示了GL261.luc 体外生长速度没有差异相比，GL261 细胞 （图2）。颅内生长和存活分析，将细胞注射到BR对于生物发光成像使用协议步骤4 C57BL / 6小鼠的荷瘤小鼠的肿瘤GL261.luc AINS按照协议步骤3.肿瘤生长串联分析并证实检测的肿瘤生长（图3A）。荷GL261.luc肿瘤快速和持续成为奄奄一息，被实施安乐死。重要的是，在GL261.luc和GL261荷瘤动物（图3B）之间的总生存没有差别。 体内生物发光成像，因此可以用于监测响应于实验胶质母细胞瘤的治疗方法。快速可靠的肿瘤生长和短总生存所用的时间相对短的量，得到大量的数据。 <img alt="图1" src="/files/ftp_upload/53287/53287fig1.jpg" /> 在GL261.luc细胞图1萤光素酶活性。U87.luc，GL261.luc和GL261（阴性对照）的细胞铺板在密度1.0×10 6，5.0×10 5，2.5×10 5，1.0×10 5，5.0×10 4，2.5×10 4个细胞/孔的由左到右。发光（光子/秒/ SR /厘米2）由管腔成像站测得。 <a href="https://www.jove.com/files/ftp_upload/53287/53287fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53287/53287fig2.jpg" /> 图2.萤光素酶表达不影响GL261细胞增殖。GL261.luc细胞不引起 在增殖差就证明了MTS细胞增殖测定。该图显示增加倍数，相对于1天，如通过比较平均吸光度值来确定（平均值±SEM）在thespecified时间点到在第1天的平均值（比较非配对t检验值每个细胞系之间:P = 0.7796）14。误差棒代表平均值从每天四份样品（平均值±SEM）。 <a href="https://www.jove.com/files/ftp_upload/53287/53287fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53287/53287fig3.jpg" /> 图3.萤光素酶表达剂量不会对体内 肿瘤生长和动物存活的 影响。 （一）生物发光监测表明颅内GL261.luc瘤C57BL / 6小鼠进行性生长。（B）Kaplan-Meier生存分析表明总体生存与任何GL261（实线）或GL261.luc细胞植入颅内的小鼠没有区别（虚线）。请点击此处查看该图的放大版本。

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.

免疫活性动物模型胶质母细胞瘤的生物发光成像

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

bioluminescence-imaging-an-immunocompetent-animal-model-for

Research

JoVE Journal

Medicine

14.8K 次观看.  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.

视频: 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.

建立颅内脑肿瘤异种移植肿瘤的生长和对治疗的反应与后续利用生物发光成像分析

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

科研

神经科学

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.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

医学

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.

胶质瘤移植中体内成像的荧光分子层析成像研究

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 ...

癌症研究

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.

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

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.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

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.

生物发光细菌成像<em在体内</em

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 ...

免疫与感染

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

<meta charset="utf8"></head><body>氧化铁纳米颗粒用于小鼠胶质母细胞瘤的图像引导治疗递送

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.

<meta charset="utf8"></head><body>作者聚焦:使用氧化铁纳米颗粒治疗胶质母细胞瘤的创新癌症疗法

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.

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

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 ...

免疫活性动物模型胶质母细胞瘤的生物发光成像

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此视频中的章节

建立颅内脑肿瘤异种移植肿瘤的生长和对治疗的反应与后续利用生物发光成像分析

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

胶质瘤移植中体内成像的荧光分子层析成像研究

随后的三维颅内植入在体内

立体定向颅内植入和

生物发光细菌成像

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送