Name: fluorescence molecular tomography for in vivo imaging of glioblastoma xenografts
Uploaded: 2018-04-26T14:00:00.000+00:00
Duration: 7 min 52 s
Description: Watch this Scientific Journal Video about Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts at JoVE.com

我们感谢弗雷德里克. 朗博士, PDX neurospheres 肿瘤中心。这项工作得到了失败的研究合作的支持, 美国脑肿瘤协会 (弗兰克 Furnari) 的子公司, R01-NS080939 (弗兰克 Furnari), 詹姆斯的麦基金会 (弗兰克 Furnari);贝尼特斯获得美国脑肿瘤协会 (ABTA) 奖的支持;Zanca 被美国-意大利癌症基金会博士后研究奖学金部分支持。弗兰克 Furnari 接受了来自路德维希癌症研究研究所的工资和额外支持。

使用实验研究动物和传染性药物, 如慢病毒北疆传感器癌细胞, 需要得到机构动物保育计划和机构生物安全委员会的事先批准。本议定书遵循加州大学圣地亚哥 (UCSD) 的动物保育指南。
1. FP635 或 FP720 结构对胶质瘤细胞的标记
<ol><li>根据 Tiscornia et描述的协议生成和纯化慢病毒北疆。12
</li>
	<li>培养大约 2.0 x 106细胞从人胶质瘤细胞线与 DMEM 加上10% 胎牛血清 (血清) 在 15 cm 盘;或文化 2.0 x 106人胶质瘤患者衍生 (肾小球-PDX) 球体与 DMEM/F12 1:1 培养基与 B27 补充和人类重组表皮生长因子 (EGF; 20 ng/毫升) 和在 T75 烧瓶 (10 ng/毫升)。</li>
	<li>保持37°c、5% CO2和100% 相对湿度的所有单元格。</li>
	<li>分离细胞。<ol>
<li>对于胶质瘤细胞: 从盘子中取出上清, 并在胶质瘤细胞中加入3-5 毫升的胰蛋白酶1X。</li>
		<li>对于肾小球球: 通过将细胞吹打成15毫升管来收集细胞。<ol>
<li>在室温下使用台式离心机, 在 200 x g 处旋转球膜球体, 5 分钟。</li>
			<li>取出上清液并添加3-5 毫升的细胞脱离溶液。</li>
		</ol>
</li>
		<li>孵化所有细胞在37°c 10-15 分钟。</li>
	</ol>
</li>
	<li>通过多次吹打, 仔细地分离细胞 (确保球体和胶质瘤细胞分离完成), 并在 200 x g 处旋转5分钟。</li>
	<li>取出上清液, 用5毫升培养基并用重悬细胞, 吹打数次。用台盼蓝排除法确定细胞的生存能力。</li>
	<li>板 1.0 x 106的目标细胞在 10 cm 盘与5毫升培养基由吹打。</li>
	<li>传感器细胞与慢病毒北疆表达荧光蛋白在多重感染 (语言) 5 如所述的Tiscornia et。12并孵育37摄氏度。</li>
	<li>24小时后, 将培养基从转基因细胞中取出, 加入5毫升新鲜培养基, 再孵育48小时37摄氏度。</li>
	<li>将受感染细胞与步骤 1.4-1.6 所述的单细胞悬浮液分离。在500µL 的分类溶液 (磷酸盐缓冲盐水 (PBS) 和1% 的血清) 下, 在 200 x g 的细胞上旋转5分钟和并用重悬。</li>
	<li>将细胞悬浮液注入到外地管里。用1µL/毫升 4 ', 6-diamidino 2-苯基吲哚 (DAPI) 二盐酸盐对细胞进行染色, 排除死细胞。需要负控制来设置流式细胞仪门 (非转基因和非转基因细胞/DAPI 染色)。</li>
	<li>将荧光阳性/DAPI 阴性细胞归类为排序解决方案, 如巴苏et所述。13, 并将已排序的单元格收集到15毫升圆锥管中。</li>
	<li>以 200 x g 为5分钟, 将已排列的细胞向下旋转, 移除上清, 并在5毫升培养基中并用重悬。用吹打将10厘米的盘子中排列的细胞播种。孵育37摄氏度至少48-72 小时。</li>
	<li>通过游离步骤 1.4-1.6 中的单元格并将其电镀到体内实验中的多个菜肴中, 展开已排序的单元格。 注: 有关细胞分类准备和纯化的详细信息, 请参阅巴苏et 。13
</li>
</ol>2. 颅内注射 iRFP 标记的胶质瘤细胞为 Immunodeficient 小鼠
注意: 在开始手术前, 要确保手术室和工具都是干净的。使用 immunodeficient 裸裸 (Foxn1女) 雄性或雌性小鼠, 介于4-5 周大和17-19 克颅内注射。动物应该被安置在至少3天在到达和在手术之前。
<ol><li>细胞制剂<ol>
<li>将荧光阳性胶质瘤细胞分为单细胞悬浮 (步骤 1.4-1.6) 和并用重悬0.5 或 1.0 x10 6 细胞, 每个动物每次注射µL 的 PBS。将细胞悬浮液注入1.5 毫升的离心管并放置在冰上。</li>
	</ol>
</li>
	<li>麻醉准备与管理<ol>
<li>准备200µL 含氯胺酮 (100 毫克/千克) 和甲苯噻嗪 (10 毫克/千克) 每20克小鼠体重的盐水溶液。权衡动物, 计算适当的麻醉剂量。</li>
		<li>提供腹腔注射麻醉, 并将眼膏应用于眼部, 以防止脱水。有关此步骤的详细信息, 请参阅凯瑟琳et 。14将动物放在预热的水热垫上37摄氏度。</li>
		<li>检查动物是否被麻醉通过监测脚趾捏反应 (踏板反射), 通常在5-10 分钟以内。</li>
	</ol>
</li>
	<li>颅内注射<ol>
<li>加载一个5µL 汉密尔顿注射器 (钝尖针) 与细胞悬浮和安装在探头持有人。</li>
		<li>将鼠标放在一个小动物立体定向的框架中, 并用耳棒和门牙适配器在描述的细节上塞金et . 来修复头部。15
</li>
		<li>用70% 乙醇和 betadine 溶液对头部进行消毒。确保手术部位完全不育。用小手术刀进行中间切口, 分离皮肤和结缔组织。</li>
		<li>使用机器人将哈密尔顿注射器放在 bregma 点上。</li>
		<li>移动探头支架1.0 毫米前后和2.0 毫米侧面从 bregma 点。用铅笔标记这个位置。</li>
		<li>在这个位置, 小心地用微电机手持钻头在头骨上打一个洞, 将轻微的压力向下, 直到血管变得可见。不要扰乱蛛网膜和脑实质。</li>
		<li>将针引入毛刺孔中, 并在脑膜表面下向前3.0 毫米。</li>
		<li>使用电动立体定向喷油器设置体积 (2-5 µL) 和流速 (1 µL/分钟), 或手动执行注射。</li>
		<li>注射后, 用1.0 毫米每1分钟提升针, 并用70% 乙醇清洗注射部位。</li>
		<li>用手术钉或做外科缝合 (通常称为针), 并看到凯瑟琳et 等, 关闭皮肤伤口。14了解更多详细信息。</li>
	</ol>
</li>
	<li>动物恢复<ol>
<li>将动物转移到水热垫 (37 °c), 并监测体温, 呼吸率和心率, 直到完全恢复。管理每标准协议的镇痛。 注: 有关立体定向过程、手术和坐标的详细信息, 请参阅其他报告5,14,15。</li>
	</ol>
</li>
</ol>3. 裂变材料成像
注: 根据实验目的, 在治疗之前、期间或之后, 可对 iRFP 标记的胶质瘤细胞进行体内监测。为了成像目的, 麻醉动物使用异氟醚感应腔, 在扫描过程中保持成像盒, 以及在裂变材料成像仪的坞站中的图像。
<ol><li>将动物从笼中移除并放置在异氟醚感应腔内。关闭会议厅, 并打开氧气流量和蒸发器到3-5%。</li>
	<li>监视动物, 直到它完全被麻醉, 通常在1-2 分钟以内. 无意识的动物不应该对外部刺激做出反应。</li>
	<li>把被麻醉的动物从房间里取出, 把动物放在成像盒里, 首先放置头部适配器, 然后将动物朝下。确保头部位于卡带中心。</li>
	<li>将卡带的顶部板放在顶部, 拧紧调整旋钮直到17毫米。</li>
	<li>一旦这种动物在成像盒中是安全的, 就进行成像, 将卡带插入到裂变材料成像仪的内部坞站, 在那里泵浦异氟醚, 以保持动物麻醉。</li>
	<li>通过双击软件图标来运行成像器和分析器软件。</li>
	<li>在 "实验选项卡" 窗口中, 选择 "数据库" 和 "学习组"。</li>
	<li>转到 "扫描选项卡" 窗口, 然后单击 "选择主题", 选择要查看的主题。另外, 在 "激光通道" 面板中选择激光通道。对于 FP635-labeled 细胞, 选择 "激光通道 635 nm", 并为 FP720-labeled 细胞, 选择 "激光通道 680 nm"。</li>
	<li>通过单击 "扫描选项卡" 窗口中的 "捕获" 选项获取图像。然后, 通过单击并将捕获图像中的扫描字段拖动到确定数量的源位置 (通常在同侧侧的20-25 个来源) 中, 调整扫描字段。</li>
	<li>检查 "添加到重建队列" 选项并在 "扫描选项卡" 窗口中命中 "扫描"。</li>
	<li>等待扫描完成, 然后从坞站中取出成像磁带。</li>
	<li>取下卡带的顶部板, 把动物放回笼子里。</li>
	<li>对其余的动物重复步骤 3.1-3.12。</li>
</ol>4. 图像分析
<ol><li>从图像和分析器软件中, 选择 "分析选项卡" 窗口。</li>
	<li>通过单击 "分析选项卡" 窗口中 "数据集选择" 面板中的 "+" 按钮, 加载数据集和要分析的主题。</li>
	<li>在 "分析选项卡" 窗口中加载图像后, 通过选择位于 "分析选项卡" 窗口左上角的椭圆形图标来执行感兴趣区域 (ROI) 分析。</li>
	<li>在 "统计数据" 面板中, 右键单击 "分析选项卡" 窗口中的 "阈值" 列, 将阈值调整为零。</li>
	<li>"统计数据" 面板中的总值代表了植入小鼠大脑的荧光标签胶质瘤细胞的信号。</li>
	<li>对其余的动物重复步骤 4.2-4.4。在 "分析选项卡" 窗口的 "数据集选择" 面板中, 分别单击 "+" 或 "−" 按钮, 加载或删除新主题。 注: 有关裂变材料成像和软件操作的详细信息, 请参阅20.
</li>
</ol>

<ol>
	<li>Ostrom, Q. T., 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=CBTRUS+Statistical+Report:+Primary+Brain+and+Other+Central+Nervous+System+Tumors+Diagnosed+in+the+United+States+in+2009-2013.">CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2009-2013.</a> Neuro-Oncology. 18 (suppl_5), v1-v75 (2016).</li><li>Pauli, C., 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=Personalized+In+Vitro+and+In+Vivo+Cancer+Models+to+Guide+Precision+Medicine.">Personalized In Vitro and In Vivo Cancer Models to Guide Precision Medicine.</a> Cancer Discovery. 7 (5), 462-477 (2017).</li><li>Stewart, E. L., 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=Clinical+Utility+of+Patient-Derived+Xenografts+to+Determine+Biomarkers+of+Prognosis+and+Map+Resistance+Pathways+in+EGFR-Mutant+Lung+Adenocarcinoma.">Clinical Utility of Patient-Derived Xenografts to Determine Biomarkers of Prognosis and Map Resistance Pathways in EGFR-Mutant Lung Adenocarcinoma.</a> Journal of Clinical Oncology. 33 (22), 2472-2480 (2015).</li><li>Gao, H., 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=High-throughput+screening+using+patient-derived+tumor+xenografts+to+predict+clinical+trial+drug+response.">High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response.</a> Nat Med. 21 (11), 1318-1325 (2015).</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), e1986 (2010).</li><li>Kondo, A., 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=An+experimental+brainstem+tumor+model+using+in+vivo+bioluminescence+imaging+in+rat.">An experimental brainstem tumor model using in vivo bioluminescence imaging in rat.</a> Child's Nervous System. 25 (5), 527-533 (2009).</li><li>Nyati, S., Young, G., Ross, B. D., Rehemtulla, A., Kozlov, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ATM Kinase: Methods and Protocols. , 97-111 (2017).</li><li>Kondo, A., 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=Longitudinal+assessment+of+regional+directed+delivery+in+a+rodent+malignant+glioma+model.">Longitudinal assessment of regional directed delivery in a rodent malignant glioma model.</a> J Neurosurg Pediatr. 4 (6), 592-598 (2009).</li><li>Badr, C. E., Badr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioluminescent Imaging: Methods and Protocols. , 1-18 (2014).</li><li>Shcherbakova, D. M., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescent+proteins+for+multicolor+in+vivo+imaging.">Near-infrared fluorescent proteins for multicolor in vivo imaging.</a> Nat Meth. 10 (8), 751-754 (2013).</li><li>Filonov, G. S., 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=Bright+and+stable+near-infrared+fluorescent+protein+for+in+vivo+imaging.">Bright and stable near-infrared fluorescent protein for in vivo imaging.</a> Nat Biotech. 29 (8), 757-761 (2011).</li><li>Tiscornia, G., Singer, O., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+lentiviral+vectors.">Production and purification of lentiviral vectors.</a> Nat. Protocols. 1 (1), 241-245 (2006).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Specific+Cell+Population+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS).</a> J. Vis. Exp. (41), e1546 (2010).</li><li>Pritchett-Corning, K. R., Luo, Y., Mulder, G. B., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+rodent+surgery+for+the+new+surgeon.">Principles of rodent surgery for the new surgeon.</a> J Vis Exp. (47), (2011).</li><li>Cetin, A., Komai, S., Eliava, M., Seeburg, P. H., Osten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nat. Protocols. 1 (6), 3166-3173 (2007).</li><li>Benitez, J. A., 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=PTEN+regulates+glioblastoma+oncogenesis+through+chromatin-associated+complexes+of+DAXX+and+histone+H3.3.">PTEN regulates glioblastoma oncogenesis through chromatin-associated complexes of DAXX and histone H3.3.</a> Nature Communications. 8, 15223 (2017).</li><li>Kirschner, S., 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=Imaging+of+Orthotopic+Glioblastoma+Xenografts+in+Mice+Using+a+Clinical+CT+Scanner:+Comparison+with+Micro-CT+and+Histology.">Imaging of Orthotopic Glioblastoma Xenografts in Mice Using a Clinical CT Scanner: Comparison with Micro-CT and Histology.</a> PLOS ONE. 11 (11), e0165994 (2016).</li><li>Mannheim, J. G., 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=Standardization+of+Small+Animal+Imaging-Current+Status+and+Future+Prospects.">Standardization of Small Animal Imaging-Current Status and Future Prospects.</a> Molecular Imaging and Biology. , (2017).</li><li>Engblom, C., 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=Osteoblasts+remotely+supply+lung+tumors+with+cancer-promoting+SiglecFhigh+neutrophils.">Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils.</a> Science. 358 (6367), (2017).</li><li>Lauber, D. T., 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=State+of+the+art+in+vivo+imaging+techniques+for+laboratory+animals.">State of the art in vivo imaging techniques for laboratory animals.</a> Laboratory Animals. 51 (5), 465-478 (2017).</li><li>Zanca, C., 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=Glioblastoma+cellular+cross-talk+converges+on+NF-&#954;B+to+attenuate+EGFR+inhibitor+sensitivity.">Glioblastoma cellular cross-talk converges on NF-&#954;B to attenuate EGFR inhibitor sensitivity.</a> Genes &amp; Development. 31 (12), 1212-1227 (2017).</li><li>Villa, G. R., 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=An+LXR-Cholesterol+Axis+Creates+a+Metabolic+Co-Dependency+for+Brain+Cancers.">An LXR-Cholesterol Axis Creates a Metabolic Co-Dependency for Brain Cancers.</a> Cancer Cell. 30 (5), 683-693 (2016).</li><li>Liu, F., 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=EGFR+Mutation+Promotes+Glioblastoma+through+Epigenome+and+Transcription+Factor+Network+Remodeling.">EGFR Mutation Promotes Glioblastoma through Epigenome and Transcription Factor Network Remodeling.</a> Molecular Cell. 60 (2), 307-318 (2015).</li></ol>

作者声明没有利益冲突。

肿瘤移植已被广泛应用于癌症研究中, 已经建立了一些成熟的成像技术: BLI;磁共振成像 (MRI);正电子发射层析成像 (PET), 计算机断层扫描 (CT);Fmt。每种方法都有利弊, 但最终会与提供的信息类型相辅相成。最常用的体内成像技术之一是 BLI5,6,7,8。BLI 和裂变材料条约都要求细胞工程 (与荧光素酶或红?...

癌症是工业化世界人类疾病相关死亡的主要原因之一。由于死亡人数极高, 迫切需要新的治疗方法。胶质瘤多形性 (肾小球) 是一种极其致命的脑癌类型, 由脑肿瘤、基质和免疫细胞的异质群组成。根据美国中央脑肿瘤登记处的数据, 原发性恶性和非恶性脑肿瘤的发病率约为 10万, 约为22例。大约1.1万新的案件预计将被诊断在美国在 2017年1。
前临床研究调查的可能性, 药物, 程序, 或治疗是有效的之前, 在人类的测试。临床前研究的最早的实验室步骤之一是通过植入宿主生物体中的癌细胞来确定药物治疗的潜在分子靶点, 定义为人移植模型。在此背景下, 利用患者源性异体移植 (PDXs) 的颅内脑肿瘤移植模型已被广泛应用于研究脑肿瘤生物学、进展、进化和治疗反应, 最近对生物标志物的开发、药物筛选和个性化医学2,3,4。
最负担得起和无侵入性的体内成像方法之一监测颅内移植是生物发光成像 (BLI)5,6,7,8。然而, 一些 BLI 限制包括基质的管理和可用性, 酶的稳定性, 和光淬火和散射在成像采集9。在这里, 我们报告红外裂变材料作为一种替代成像方法, 以监测前胶质瘤模型。在这种方法中, intracranially 植入 PDXs 的信号采集和量化, 表达近红外荧光蛋白 iRFP72010,11 (从今以后称为 FP720) 或 turboFP635 (从今以后称为 FP635),是用裂变材料成像系统执行的。使用裂变材料技术, 可以在治疗前、期间或之后以无创性、无基质和定量的方式监测原位肿瘤的体内。

<table><tbody><tr><td>DMEM/High Glucose&amp;nbsp;</td><td>HyClone/GE</td><td>SH30022.1</td><td></td></tr><tr><td>DMEM/F12 1:1&amp;nbsp;</td><td>Gibco</td><td>11320-082</td><td></td></tr><tr><td>FBS</td><td>HyClone/GE</td><td>SH30071.03</td><td></td></tr><tr><td>Accutase</td><td>Innovative cell technologies</td><td>AT-104</td><td></td></tr><tr><td>Trypsin</td><td>HyClone/GE</td><td>SH30236.01</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>human recombinant EGF&amp;nbsp;</td><td>Stemcell Technologies</td><td>2633</td><td></td></tr><tr><td>human recombinant FGF</td><td>Stemcell Technologies</td><td>2634</td><td></td></tr><tr><td>DPBS</td><td>Corning</td><td>21-031-00</td><td></td></tr><tr><td>FACS tubes</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>62248</td><td></td></tr><tr><td>Blasticidin</td><td>ThermoFisher Scientific</td><td>A1113903</td><td></td></tr><tr><td>p24 ELISA&amp;nbsp;</td><td>Clontech</td><td>632200</td><td></td></tr><tr><td>Xylazine</td><td>Akorn</td><td>NDC 59399-110-20</td><td></td></tr><tr><td>Ketamine</td><td>Zoetis</td><td>NADA 043-403</td><td>Controlled substance</td></tr><tr><td>Ointment</td><td>Dechron</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Absorbable suture</td><td>CpMedical</td><td>VQ392</td><td></td></tr><tr><td>5 ul syringe</td><td>Hamilton</td><td>26200-U</td><td>Catalog number as sold by Sigma-Aldrich</td></tr><tr><td>Cell Sorter</td><td>Sony</td><td>SH8007</td><td></td></tr><tr><td>Mouse stereotaxic frame&amp;nbsp;</td><td>Stoelting</td><td>51730</td><td></td></tr><tr><td>Motorized stereotaxic injector</td><td>Stoelting</td><td>53311</td><td></td></tr><tr><td>Micromotor hand-held drill</td><td>Foredom</td><td>K1070</td><td></td></tr><tr><td>Mouse warming pad&amp;nbsp;</td><td>Ken Scientific Corporation</td><td>TP-22G</td><td></td></tr><tr><td>Fluorescence Tomography System&amp;nbsp;</td><td>PerkinElmer</td><td>FMT 2500 XL</td><td></td></tr><tr><td>TrueQuant Imaging Software&amp;nbsp;</td><td>Perkin Elmer&amp;nbsp;</td><td>7005319</td><td></td></tr><tr><td>Ultra-centrifuge Optima L-80 XP</td><td>Beckman Coulter</td><td>392049</td><td></td></tr><tr><td>Tissue Culture 100mm Dishes</td><td>Olympus Plastics</td><td>25-202</td><td></td></tr><tr><td>Tissue Culture 150mm Dishes</td><td>Olympus Plastics</td><td>25-203</td><td></td></tr><tr><td>Tissue Culture Flasks T75</td><td>Corning</td><td>430720U</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>15 mL conical tubes</td><td>Olympus Plastics</td><td>28-101</td><td></td></tr><tr><td>Centrifuge Avanti J-20</td><td>Beckman Coulter</td><td>J320XP-IM-5</td><td></td></tr><tr><td>Tube, Polypropylene, Thinwall, 5.0 mL</td><td>Beckman Coulter</td><td>326819</td><td></td></tr><tr><td>Tube, Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>Athymic nude mice</td><td>Charles River Laboratories</td><td>Strain Code&amp;nbsp; 490 (Homozygous)</td><td>Prior approval by the Institutional Animal Care Program and by the Institutional Biosafety Committee required.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

fluorescence molecular tomography for in vivo imaging of glioblastoma xenografts

致瘤性是癌细胞形成肿瘤肿块的能力。一种广泛使用的方法来确定细胞是否是癌变是通过注射 immunodeficient 小鼠皮下肿瘤细胞和测量肿瘤肿块后, 它变得可见和明显。原位注射癌细胞的目的是在微环境中引入异种异体, 最接近于所研究的肿瘤起源组织。脑癌研究需要颅内注射癌细胞, 以使肿瘤形成和分析在独特的微环境中的大脑。颅内移植的体内成像能瞬间监测原位嫁接小鼠的肿瘤质量。在这里, 我们报告的使用荧光分子层析成像 (裂变材料) 的脑肿瘤移植。癌细胞首先转基因近红外荧光蛋白, 然后注入免疫缺陷小鼠的大脑。然后对这些动物进行扫描, 以便在较长的时间内获得肿瘤肿块的定量信息。细胞预标记允许对每只老鼠的肿瘤负担进行有效、可重复和可靠的定量。我们消除了注射成像基质的需要, 从而减少了对动物的压力。这种方法的局限性表现为无法检测到非常小的质量;但是, 它比其他技术更能解决较大的质量问题。可用于评价胶质瘤细胞系和患者源性样品的药物治疗或基因改变的疗效。

根据步骤 1.2, 对胶质瘤细胞 U87EGFRvIII (U87 细胞表达表皮生长因子受体变体 III) 进行了培养。慢病毒北疆是根据步骤1.1 生产和纯化的。p24 ELISA 法测定病毒浓度。细胞转基因与慢病毒北疆携带红外荧光蛋白根据步骤1.8。俄罗斯联邦普京 Verkhusha 博士亲切地提供了 FP720 10、11的质粒编码, FP635 向量是从商业供应商那里购买的.目标单元?...

Watch this Scientific Journal Video about Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts at JoVE.com

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

原位颅内注射肿瘤细胞已用于癌症研究, 研究脑肿瘤生物学, 进展, 进化和治疗反应。本文提出了肿瘤移植的荧光分子层析成像技术, 为临床前的胶质瘤模型提供了实时活体成像和肿瘤质量定量。

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 ...

fluorescence-molecular-tomography-for-vivo-imaging-glioblastoma

Research

JoVE Journal

Cancer Research

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

8.9K 次观看.  Ludwig Institute for Cancer Research. 原位颅内注射肿瘤细胞已用于癌症研究, 研究脑肿瘤生物学, 进展, 进化和治疗反应。本文提出了肿瘤移植的荧光分子层析成像技术, 为临床前的胶质瘤模型提供了实时活体成像和肿瘤质量定量。

视频: Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

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

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 ...

医学

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

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 ...

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 ...

在小鼠中使用 2P-IVM 实时跟踪脑肿瘤中的纳米颗粒

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

作者聚焦：优化胶质母细胞瘤治疗中药物递送和早期检测的多模式成像策略

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

小鼠模型中胶质母细胞瘤的双光子活体显微镜

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

癌症研究

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 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 ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

侵入斑马鱼大脑的胶质母细胞瘤细胞微管动力学的实时成像

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...

胶质瘤移植中体内成像的荧光分子层析成像研究

摘要

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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

随后的三维颅内植入在体内

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

立体定向颅内植入和

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

小鼠模型中胶质母细胞瘤的双光子活体显微镜

小鼠模型中胶质母细胞瘤的双光子活体显微镜

小鼠模型中胶质母细胞瘤的双光子活体显微镜

免疫活性动物模型胶质母细胞瘤的生物发光成像

侵入斑马鱼大脑的胶质母细胞瘤细胞微管动力学的实时成像