Name: creating anatomically accurate and reproducible intracranial xenografts of human brain tumors
Uploaded: 2014-09-24T15:00:00.000+00:00
Duration: 11 min 14 s
Description: Watch this Scientific Journal Video about Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors at JoVE.com

基廷博士是由美国国防部资助CA100335资助，是圣Baldrick基金会学者。

所有上述程序进行审查和批准我们的机构动物照顾及使用委员会。 1，规划实验<ol><li>选择的细胞被注入。从各种来源的细胞是候选注射:贴壁细胞培养系，遗传修饰的克隆，神经干细胞，原代培养物，或分类肿瘤。模型所需的类型将确定注射的最适当的站点。 <ol><li>确定细胞数的注射。形成肿瘤所需的细胞数量与细胞系而不同，必须根据经验确定;肿瘤生长速率很大程度上依赖于细胞类型，细胞数目和组织培养传代次数。细胞数为1×10 4〜2×10 5以上每次注射都产生了不同的生长率的肿瘤。 </li><li>限制用于注射的体积。细胞应在无血清培养基或磷酸盐缓冲S的体积最小暂停艾琳（PBS），允许通过＆G针光滑，不易通过。较小的体积实际上输送到大脑，更精确的和所限定的所得肿瘤;最佳的结果是通过注入体积为3至6微升获得。打算制备至少50微升细胞悬浮液的最终的总体积，而不论计划注射的次数的，允许精确测量，混合和采样。 </li></ol></li><li>选择合适的老鼠模型。人类肿瘤的小鼠模型中通常使用年轻免疫妥协小鼠以避免免疫介导的移植物排斥。这是没有必要的工作，以发生在生物安全柜中，如果​​适当的预防措施，包括去污，消毒，以及对材料和设备灭菌。这里示出的工作已在无胸腺裸鼠，雄性和雌性已经完成，6周和12周的年龄之间。该称重小于20g可以是困难的小鼠到位置上进行tereotaxic帧，并且可以更容易受到低温。 </li></ol> 2，装配设备<ol><li>获取并净化鼠标立体框架，调整控制台，鼠标气体麻醉头部固定器，微量注射泵，热板机，麻醉机，光纤工作灯，和变速回转钻机。清洁和消毒所有设备。 </li><li>编程的注射参数（包括注射体积，流速，速度的单位，注射器型）和其他变量所需的注射器泵的操作。优化设置，为每个特定的模式。 注意:在这些注射的设置分别为3000 NL样品在400升/分钟的速度（速度单位"M"）用25微升注射器（设备类型"E"），在非分组（"N"）模式。 </li><li>清洁精密微量与＆G针头无菌去离子水冲洗几次（DIH 2 O）和70％的乙醇（乙醇），做最后的RINSE与DIH 2 O。一些但不是所有的模型被设计成可高压灭菌。确保柱塞运动是平滑和自由。 </li><li>获得麻醉交运设备。异氟醚是优选的麻醉剂，因为它很容易调节麻醉深度和持续时间。确保该立体定位单元配备一个鼠标麻醉气体附着和必要的管道和连接器的位置，从麻醉机提供的气体混合物的小鼠。 </li><li>高压釜中的外科手术工具，包括精尖的剪刀，二镊子/止血剂（有齿），用于缝合，中等大小的剪刀，中期和细尖镊子，以及至少两个1毫米牙科钻头。用70％乙醇，消毒液，或珠灭菌器在操作过程中保持仪器的清洁。 </li><li>从获得的异氟烷，镇痛药，眼用软膏/润滑剂，抗生素软膏，盐水，30％过氧化氢 ​​（H 2 O 2），防腐剂，和骨蜡（如果需要的话）兽医和医疗用品。准备无菌DIH 2 O和70％的乙醇。一次性用品如纱布，无菌敷料，缝合线，乙醇棉签，棉签拭子，无菌手术手套，手术刀，更是详细列出材料清单。的细尖永久性标记用于标记头骨应进行消毒，并专门用于外科手术中使用。 </li></ol> 3，准备细胞注射<ol><li>在这里所示的实验中，选择一个人永生化的GBM细胞系。确定细胞的数量（2×10 5）和悬浮液的体积被注入（3微升）经验;细节可以与细胞类型而有所不同。使用至少50微升的体积，方便精确的测量，混合和注射。 </li><li> Trypsinize细胞重悬在媒体或PBS。执行以下步骤之前，以细胞注射: <ol><li>通过抽吸去除细胞介质和冲洗用10毫升的PBS，每100毫米的钢板;除去PBS。 </li><li>广告d 1的ml胰蛋白酶以每次100毫米的钢板并在室温下孵育足够长的时间以得到单细胞悬浮液。 </li><li>停止胰蛋白酶的活性用5毫升贝科的改良Eagle培养基（DMEM）+10％胎牛血清（FBS）每100毫米的钢板。 </li><li>离心沉淀细胞在450 XG重悬在每100毫米的钢板〜5ml无血清的DMEM（SF-DMEM）。 </li><li>计数使用血球计数器或自动细胞计数活细胞。 </li><li>调节活细胞密度为2×10 5个细胞/ 3微升（或通过实验测定）通过制粒的细胞和再悬浮在SF-DMEM。 </li></ol></li><li>保持细胞冷冻在冰或冷包（不要让细胞冻结）。通过手指轻弹，轻轻混匀。 </li></ol> 4，麻醉并准备鼠标外科手术<ol><li>诱导麻醉用〜5％的异氟醚（氧流量〜2升/分钟）或按照兽医。电磁炉应取2〜3分钟。鼠标保持在适当的深度的麻醉〜3％的异氟烷​​。 <ol><li>检查麻醉深度脚趾捏，如果显示调整异氟烷，并持续监控整个手术过程中的呼吸和脚趾捏响应。覆盖鼠标，用纱布垫保暖并在整个过程中，这可能需要45分钟到1小时，这取决于注入的速率定期监测小鼠。 </li></ol></li><li>通过仔细地滑口感棒在舌头和放入口中放置鼠标在立体框架。挂钩的前门牙到牙齿洞。使用耳棒以稳定头部，与耳朵到眼睛表面尽量接近水平越好。不要强行进入酒吧耳道。关键是要牢固地定位头:轻轻检查一侧到另一侧与向上和向下运动，用指尖。利用立体定位装置的调节控制，优化配合。 </li><li>润滑眼睛眼药膏。清洁耳朵和眼睛之间的皮肤用聚维酮碘比交替应用的两倍Ë防腐剂和70％的乙醇。 </li><li>选择注射止痛皮下（SQ）为沿（或遵医嘱）。 </li></ol> 5，注射执行<ol><li>确定注射部位。注射部位可在大小和小鼠品系与细胞的类型而变化。细胞注射太靠近心室可能导致疾病的颅脊椎蔓延。细胞注射太浅，可能会生长出通过针轨道。 请注意:在这里所示的实验中，目标被定义为大脑皮质的额叶区域的站点2.5毫米横向（右），1.5毫米前壁，和3.5毫米腹侧相对于前囟点被选择（ 图1），产生中脑肿瘤的小鼠，从20至30g。 </li><li>使用无菌技术通过头部的皮肤，以暴露前囟和注射部位的一小切口（〜长8毫米）。的斜切口，从中线轴线并朝向右耳右眼（之间的点<strong&gt;图2）允许将前囟门和注射部位。 </li><li>牵开皮肤，用无菌棉签擦干头骨表面。吸干骨与另一个棉签蘸有H 2 O 2形象化前囟门。的H 2 O 2将产生氧气气泡，而使细线的白色泡沫状物沿相交于前囟的冠状面和矢状缝。 </li><li>锁针空的注射器上的微型泵和机动针直接在囟门的顶端;对准控制台上设置的坐标为0.0毫米横向，0.0毫米前/后，以及0.0毫米腹/背。 </li><li>移动针至2.5mm的横向（右）至1.5mm前壁相对于前囟（或所希望的位置）用的控制旋钮的立体定位装置上。提高注射器略有并标记上配有专用毡尖笔头骨的确切位置。如果头骨的外表面不与前囟门（I A级平面。即，在背/腹坐标不再读数为零），复位腹侧/背侧读取到0.0，以确保注射的深度不偏移。 </li><li>钻一小孔与手持式回转钻机配备在标有步骤5.5钢笔相应部位的无菌牙尖的头骨。保持钻头以一个角度向颅骨和非常轻微接触尖端到骨。根据需要重复。钻几乎但不完全穿过骨，留针以实际穿透该层的头骨，结果在最干净的注射。 </li><li>去除骨扬灰，用棉签蘸在PBS或生理盐水。回到注射器泵和降低针直线下降到钻孔中，以确认位置。如果钻孔不居中的针，然后用电钻扩大开放。 </li><li>轻轻混合细胞悬液和绘制的细胞分化成使用泵控制器的注射器。避免气泡和团块，然后擦拭针头机智ħ酒精棉签去除的外表面污染的细胞。如果注射器/针头堵塞或冷冻，取出，并迅速干净，用无菌水和70％乙醇。 </li><li>降低针的颅骨表面的水平（0.0毫米腹/背部）。然后慢慢地（经过大约1分钟）降低针头刺穿颅骨的整个厚度和穿透脑向4毫米ventrall的深度。慢慢地拔出针头至3.5mm腹侧。如果鼠标头在立体部牢固地保持，应该有颅骨几乎没有移动。 </li><li>确认正确的参数输入到泵控制器（2.2节），然后按"运行/停止"（或按照产品说明书），以autoinject细胞。监视鼠标和指示，如果调整​​的异氟醚。观看的注射器，并确保该柱塞被朝着桶​​。冷冻或堵塞移动可导致弯曲/断柱塞。 </li><li>等待1到2分钟，在B的针雨，然后非常缓慢地（超过3至4分钟）从组织收回针头。总时间以除去针注射后是完整的是5分钟。用棉签吸干周围钻孔的面积;离开皮肤开的边缘，以允许所述骨干。 </li><li>从泵中取出注射器和快速冲洗3次，用无菌H 2 O，3次用70％乙醇和3倍，用无菌水（或按照制造商）。擦拭针头乙醇，备用。 </li><li>应用无菌骨蜡（相当于1或2微升）到所述钻孔并使用无菌棉签的木端夯实蜡上并进入骨骼。如果表面被充分地干燥，则应该坚持。 </li><li>散布无菌覆盖在鼠标周围的工作区和缝合切口;三针应该是足够的。或者，可使用外科手术粘合剂。涂抹外用抗生素。 </li><li>异氟醚降低至0％，并从立体定位装置移开鼠标，是Careful保护牙齿。检查耳朵，嘴巴和舌头。 </li><li>监控鼠标密切关注注射。在5〜10分钟的热垫鼠标的地方;然后转移到笼子里（上隔热垫），并观察到鼠标的唤醒，是门诊。不要让鼠标无人值守，直到它重新获得足够的意识来维持胸骨recumbence。不要返回鼠标其他动物的公司，直到完全康复。考虑使用额外的镇痛如果疼痛的迹象注意到。 </li></ol> 6，完成并监控恢复与肿瘤发展<ol><li>清洁所有采用70％乙醇，老鼠之间的珠灭菌或消毒仪器和设备。 </li><li>对于疼痛，感染或其他并发症的征兆的步骤后监测小鼠注射两天。注入镇痛在24和48小时（或指示）过程之后。如果有必要在7〜10天取出缝线。 </li><li>评估老鼠的疾病，paralysi临床症状秒，活性降低，体重减轻，癫痫发作，或一般疾病。 </li><li>监测肿瘤发展通过适当的方法[磁共振成像（MRI）， 体内成像系统（IVIS）等]。 </li></ol>

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作者什么都没有透露。

人脑肿瘤的原位小鼠模型可以是一个很好的工具，用于评估临床治疗的效果，但必须小心，以优化布局细胞的脑组织中。研究表明，过量的等分试样体积，次优的喷射技术和匆忙的注射速率可导致泄漏和肿瘤细胞中不希望的位置（脑室，脊髓硬膜外区域等 ）的外观和高变异在肿瘤大小为2（个人观察）。非细胞样品的微处理器驱动输送的分析发现，使用的微型泵的制造更focalized交货...

在脑肿瘤细胞的体外研究是非常宝贵的解剖驾驶生长，存活，迁移和侵袭癌细胞的分子机制;培养细胞实验中可以定义信号传导通路，提出潜在的治疗靶点，并描述对药物治疗的细胞反应。但在体外系统过于简单化预测到药品有机体反应;他们缺乏的生理反应，免疫反应，细胞微环境和活生生的动物系统的整体异质性。基因工程模型可以是无价的，当可用的，但不同的分子种类和鼠细胞之间存在着可能无法概括人类进程的事件，造成显著差异比较动物模型的临床观察1时。鼠标涉及皮下（SQ）注射人脑肿瘤细胞系的侧翼的皮肤下异种移植模型中很容易就可以执行和衡量;它们可以被用来处理基因修饰和药物施用/输送，代谢和毒性的影响。显著缺点，但是，限制SQ模型的实用性。微环境并没有概括了一个自然发生​​的脑肿瘤:不同类型的细胞和组织的相互作用;局部血管，以及无数的其他因素所特有的大脑是无法复制的。为了更准确地再现了自然发生脑肿瘤的独特氛围和测试药物干预的效果，鼠标原位模型应该加以利用。此外，原位技术可以被用来作为一种基因工程的方法，其中人类初级的非癌性细胞（分化的细胞或祖）的一部分被遗传修饰，并注射到小鼠的相关部位，具有或不具有人的基质细胞，导致肿瘤发生在人中1所见相似的。 本文介绍一种方法来精确和可重复产生脑肿瘤的小鼠。使用该技术，用户可以准确地注入少量等份悬浮细胞到小鼠大脑皮质的额叶后部顶颞区的指定位置。鼠标死亡率极低;在我们的手中，没有小鼠的后185程序从手术并发症死亡。将所得的肿瘤的特征可以与典型的人类临床肿瘤进行比较;例如:生长，坏死的程度，浸润程度的细胞类型，有丝分裂细胞的存在下，细胞增殖和细胞凋亡等的标志物的异质性快速性的细胞系或分类的人体组织或肿瘤样品随后可以根据自己的能力进行评价以模拟实际的临床表现。医药品，选定基于它们在细胞培养物的性能，可以在一个正常运作代谢，循环系统，和血 - 脑屏障的情况下进行测试，因为它们在动物背负机智存在医管局肿瘤，在所有相关的建筑环境。此外，选择用于注射的细胞可以被遗传修饰以探讨特定击倒，缺失的影响，敲插件，突变等，对肿瘤生长和存活。 许多出版物文档中使用的各种颅内肿瘤的技术研究。 Yamada 等人做了注射U87细胞的染料和进行详细研究，结果发现最小体积和喷射率产生最佳的肿瘤2。 布鲁克斯等人发现使用微处理器控制的喷油器，而不是人工的方法来实现病毒载体卓越的重现性和效率;他们对最佳注射参数的结论也适用于小区交付3。 Shankavaram 等人发现，胶质母细胞瘤（GBM）细胞系注射原位（使用手动方法）进入大脑概括了CL的基因表达谱inical肿瘤更加紧密地比在体外或倚移植，配套使用颅内模型的临床前研究4。尼尼等注入从已持续在裸鼠 ​​的侧腹通过连续传代至另外的小鼠的脑中人类手术标本的细胞，并表明，这种方法保藏患者肿瘤基因改变模型中的5。类似的结果，报告毅等[6]。采用立体设置，仔细界定注射部位，并缓慢而稳定的注入量，他们获得可重复的脑瘤一致的增长率和高（100％）植入率。此技术的有效性也因此被确立;文献检索表明，这一技术的应用非常广泛。卡蒂等人使用颅内注射成功提供病毒载体表达治疗基因进入transgeni的额叶皮质阿尔茨海默氏病7的C型号。特哈契等人描述了使用颅内注射递送治疗的溶瘤腺病毒在神经干细胞的基础载体为已携带原位注射的GBM肿瘤8裸鼠中。显然，颅内注射是一种多用途的和有效的工具，临床前研究。在可视化实验杂志此前出版物描述的基本方法9-11，但我们采用易于掌握技术把颅内肿瘤注射原位模型的概念，精度更高的水平。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Small Animal Stereotaxic Instrument with Digital Display Console</td><td>Kopf</td><td>Model 940</td><td></td></tr><tr><td>Mouse Gas Anesthesia Head Holder</td><td>Kopf</td><td>Model 923-B</td><td></td></tr><tr><td>Mouse Ear Bars</td><td>Kopf</td><td>Medel 922</td><td></td></tr><tr><td>Fiber Optic Illuminator</td><td>Fisher</td><td>12-562-36</td><td></td></tr><tr><td>UltraMicroPump III</td><td>WPI</td><td>UMP3</td><td></td></tr><tr><td>Micro4 microprocessor</td><td>WPI</td><td>UMC4</td><td></td></tr><tr><td>Variable speed hand-held rotary drill</td><td>Dremel</td><td>Model 300</td><td></td></tr><tr><td>Dental drill bit, 1.0 mm</td><td>Spoelting</td><td>514554</td><td></td></tr><tr><td>Adaptor for dental drill bit: 3/32 inch collet</td><td>Dremel</td><td>481</td><td></td></tr><tr><td>Heating pad</td><td></td><td></td><td>for mice</td></tr><tr><td>Isoflurane vaporizer system</td><td></td><td></td><td>for mice</td></tr><tr><td>Medical tubing and connectors</td><td></td><td></td><td>to connect isoflurane vaporizer with stereotaxic frame</td></tr><tr><td colspan="4">&lt;strong&gt;Instruments&lt;/strong&gt;</td></tr><tr><td>Precision 25 &amp;mu;l microsyringe</td><td>Hamilton&amp;nbsp;</td><td>7636-01</td><td>Model 702, without needle</td></tr><tr><td>Microsyringe needles, 26s G</td><td>Hamilton&amp;nbsp;</td><td>7804-04</td><td>RN, 25 mm point style 2</td></tr><tr><td>Fine-tipped scissors (straight, sharp/sharp)</td><td></td><td></td><td></td></tr><tr><td>Medium-sized standard scissors</td><td></td><td></td><td></td></tr><tr><td>Standard serrated forceps</td><td></td><td></td><td></td></tr><tr><td>Serrated hemostats (2)</td><td></td><td></td><td></td></tr><tr><td>Fine-tipped forceps</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Supplies&lt;/strong&gt;</td></tr><tr><td>Sutures 5-0 vicryl P-3 13 mm (Ethicon)</td><td>MWI</td><td>J463G</td><td></td></tr><tr><td>Surgical blades #10, stainless (Feather)</td><td>Fisher</td><td>296#10</td><td></td></tr><tr><td>Isoflurane (Fluriso)&amp;nbsp;</td><td>VetOne&amp;nbsp;</td><td>NDC 13985-528-60</td><td>Item #502017. Liquid inhalation anesthetic. federal law restricts this drug to use by or on the order of a licensed veterinarian.</td></tr><tr><td>Carprofen (Rimadyl Injectable 50 mg/ml)</td><td>Pfizer</td><td>NDC 61106-8507-01</td><td>dilute in saline</td></tr><tr><td>Ophthalmic ointment (artificial tears)</td><td>Rugby</td><td>NDC 0536-6550-91</td><td></td></tr><tr><td>Topical antibiotic (AK-Poly-Bac )</td><td>Akorn</td><td>NDC 17478-238-35</td><td></td></tr><tr><td>Povidone-iodine topical antiseptic, 10% (Betadine)</td><td>Betadine</td><td>NDC 67618-150-04</td><td></td></tr><tr><td>Hydrogen peroxide, 30%</td><td>Fisher&amp;nbsp;</td><td>H325-100</td><td>for visualizing skull landmarks</td></tr><tr><td>Sterile saline</td><td>VetOne&amp;nbsp;&amp;nbsp;</td><td>NDC 13985-807-25</td><td>for diluting solutions, cleaning tissue</td></tr><tr><td>Bone wax</td><td>WPI</td><td>Item #501771</td><td></td></tr><tr><td>Sterile drapes</td><td>McKesson</td><td>25-517</td><td></td></tr><tr><td>Sterile surgical gloves</td><td>McKesson</td><td>(to fit)</td><td></td></tr><tr><td>Sterile gauze pads, 2 x 2</td><td>Fisherbrand&amp;nbsp;</td><td>22028556</td><td></td></tr><tr><td>Sterile gauze pads, 4 x 4</td><td>Fisherbrand&amp;nbsp;</td><td>22-415-469</td><td></td></tr><tr><td>Alcohol prep pads (medium)</td><td>PDI</td><td>B603</td><td></td></tr><tr><td>Sterile cotton-tipped applicators</td><td>Fisherbrand&amp;nbsp;</td><td>23-400-114</td><td></td></tr><tr><td>Sterile 0.5 ml screw cap tube with caps for cells</td><td>USA Scientific</td><td>1405-4700</td><td>for cells</td></tr><tr><td>Individually wrapped sterile dispo pipettes</td><td>Fisher</td><td>BD 357575</td><td>for needle cleaning solutions</td></tr><tr><td>BD insulin syringes with needles&amp;nbsp;</td><td>Fisher</td><td>329461</td><td>for analgesic</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td>for cleaning</td></tr><tr><td>Sterile diH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>for cleaning</td></tr><tr><td>Microfuge tubes for cleaning solutions</td><td></td><td></td><td>for needle cleaning solutions</td></tr><tr><td>Felt tip pen (dedicated)</td><td></td><td></td><td>for marking skull</td></tr></tbody></table>

creating anatomically accurate and reproducible intracranial xenografts of human brain tumors

原位肿瘤模型目前正在研究一种肿瘤类型的特点，使用和不干预的最佳方式，在一个活的动物的情况下-尤其是在遗址具有独特的生理和建筑品质，如大脑在体外和异位模型不能。考虑功能，如血管，血脑屏障，代谢，药物输送和毒性，以及一系列其他相关因素。原位模型有其局限性太多，但与感兴趣适当技术的肿瘤细胞能够正确地植入到组织，在人类大脑最密切模仿的条件。通过采用能够提供精确测量的量在一个一致的速率和压力下，人脑肿瘤具有可预测的增长率的小鼠模型可再现地创建准确的限定位置，并适合各种干预的可靠的分析方法。这里描述的方案着重于德设计和准备的颅内注射，进行了手术，并确保肿瘤的生长成功的和可重复的技术注释细节并提供出发点的各种情况，可定制各种不同的脑肿瘤模型。

可靠的颅内异种移植物可在此描述的技术来创建。识别小鼠头盖骨的关键结构（ 图1）将允许对识别的前囟和引导研究者以一个精确的和可重复的注射位置。在这些研究中的U251亲本系，转染的荧光素酶（U251-吕克）或U87 U251细胞永生化人GBM组织培养细胞在4至6微升SF-DMEM中悬浮，并注射2.5毫米横向（右），1.5毫米前，和3.5毫米腹侧相对于前囟点（ 图2）。 <p class="jove_content...

Watch this Scientific Journal Video about Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors at JoVE.com

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

大脑与那些没有得到很好的利用体外或异位的分析代表素质唯一的站点。原位小鼠模型重现的位置及生长特点可以用立体定位仪固定和低压注射泵颅内注射可靠地创建。

创建精确的解剖学和人脑肿瘤的重现性颅内移植瘤

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

creating-anatomically-accurate-reproducible-intracranial-xenografts

Research

JoVE Journal

Medicine

15.5K 次观看.  University of Colorado School of Medicine. 大脑与那些没有得到很好的利用体外或异位的分析代表素质唯一的站点。原位小鼠模型重现的位置及生长特点可以用立体定位仪固定和低压注射泵颅内注射可靠地创建。 

视频: Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

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

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

一个简单的指南螺杆颅内移植瘤小鼠的研究方法

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

医学

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 ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

癌症研究

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

原正畸胶质血异草回顾渗透生长和同源脱氢酶 I 突变

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

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 ...

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

多形性胶质母细胞瘤的转化原位模型

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of neuro-oncology. One of the most widely used techniques is the orthotopic intracranial injection, a method that allows investigating of tumor growth, invasion, and dissemination within a controlled setting. This technique involves transplanting tumor cells from a specific patient region into the corresponding anatomical site in an animal. By doing so, these orthotopic brain tumor models offer a unique advantage, as they more accurately replicate cancer's biological behavior and its interactions with the brain environment seen in human patients. This makes them especially valuable for preclinical therapeutic testing, where a close resemblance to the clinical scenario is essential for evaluating potential treatments. This protocol shares experiences in developing patient-derived xenograft (PDX) models for pediatric brain tumors, including diffuse midline glioma (DMG), glioblastoma (GBM), medulloblastoma, and ependymoma. This method delineates the procedure for conducting intracranial stereotaxic injections in mice, ensuring the correct targeting of the injection site within the brain. Additionally, we describe the post-procedural monitoring system employed to detect signs of successful tumor engraftment. Following tumor injection, a rigorous monitoring system is implemented to observe the animals for any signs of neurological impairment, behavioral changes, and/or weight loss, which are common indicators of tumor progression. This system allows for timely intervention and provides critical data regarding the tumor's growth dynamics. By refining these models and protocols, we aim to enhance the reliability and translational potential of preclinical studies, contributing to the development of more effective treatments for pediatric CNS tumors.

The development of orthotopic pediatric brain tumor models requires meticulous precision, using a stereotaxic device to implant cancer cells precisely. The methodology presented here outlines the steps involved in preparing brain tumor cells, performing intracranial injections, and implementing a post-operative monitoring system to assess brain tumor engraftment.

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

免疫与感染

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...