这项工作得到了国家神经疾病和中风研究所（R01 NS048353到SLC），美国国家癌症研究所（R01 CA122804到SLC，R01 CA134773答：凯文罗斯和SLC和CA13148 - 35到KRZ）和部国防部（X81XWH - 09 - 1 - 0086到SLC）。基金支持UAB的综合癌症中心的小动物成像共用设施的运作提供了一个国家癌症研究所的核心支援津贴（P30 CA13148 - 35; E.鹧鸪，有价证券）。我们感谢阿拉巴马州神经科学蓝图的核心中心（P30 NS57098）和UAB的神经核中心（P30 NS47466）提供的援助。内容完全是作者的责任，并不一定代表的美国国立卫生署或国防部研究院的官方意见。

1。非裸体的免疫缺陷小鼠（裸体株不必）的前期准备工作： <ol><li>所有的程序事先审查和批准由UAB的机构动物护理和使用委员会和适当训练的人员进行。我们已经成功地使用了这些实验的免疫缺陷小鼠株：A）Foxchase远交SCID小鼠（CB17/lcr- Prkdc SCID / lcrCrl小鼠）; B）美国国立卫生研究院第三小鼠（NIHS催化剂BG Foxn1 怒江 BRK XID小鼠）和C ）的NOD -SCIDγ小鼠（NOD.Cg Prkdc SCID IL2RG tm1Wjl / SZJ小鼠）。在我们的手中，原位移植瘤的生长，在美国国立卫生研究院第三和NODSCIDγ小鼠，进行这两种基因突变影响T细胞，B细胞和NK细胞的功能几乎是相同的，此协议，移植所需的天数增长，在治疗开始和时代嫁接后进行成像时的时间是指那些有经验，我们用的NODSCIDγ小鼠。 Foxchase接枝增长远交SCID小鼠，在T -和B -细胞功能缺陷，通常需要较长的时间。 </li><li>为了便于嫁接大量的非裸体的免疫缺陷小鼠，我们对下午的头发嫁接前从手术部位。小鼠麻醉用异氟醚诱导室，清除废气喷雾器。为了保持体温，动物在整个过程中的加热垫处理。快船队是用来去除头发从中期回落到膝盖就在身边，这是嫁接的后腿。任何剩余的头发，小心取出〜5分钟使用化学脱毛剂（如奈尔，尤其是敏感肌肤的芦荟产品）的网站。建议去除脱毛产品，因为它可以导致刺激皮肤，眼睛，以及它可能蔓延到其他身体表面。我们不移植坐骨神经在这两个实验，因为这可能会双边hindleg功能损害，造成损失的研究组在小鼠。 </li><li>小鼠可以完全恢复在没有被褥的隔离笼中的加热垫;，这既确保小鼠等，更充分地提醒动物受伤，并防止意外吸入床上用品。当老鼠是完全移动和显示没有证据证明的醉，他们可以重新进入一个与其他老鼠的笼子。 </li></ol> 2。嫁接日MPNST细胞注射液的制备<ol><li>在这个协议中，具体的数字细胞移植和移植后肿瘤生长所需的时代是我们有经验ST88 - 14细胞，是从5 NF1的相关肿瘤的一种常用的MPNST线确定。我们的嫁接使用的细胞已经包含一个巨细胞病毒荧光素酶- IRES -嘌呤磁带和克隆稳定表达萤火虫荧光素酶生物发光成像6所确定的慢病毒载体转导。我们还进行MPNST稳定与巨细胞病毒立即早期启动子的控制下表达萤火虫荧光素酶质粒转染细胞移植的实验。我们不建议这样做，因为我们发现，这些载体很容易经历表达以下嫁接的灭绝。 </li><li> ST88 - 14细胞是生长在贝科的最小必要辅以10％胎小牛血清，10μg/ mL的链霉素和10国际单位/毫升青霉素（DMEM10）介质; 5微克/毫升嘌呤是还包括​​以保持慢病毒载体的选择。我们一直保持着这些细胞在我们的实验室为50通道，并没有观察到任何变化，在细胞生长在这些通道中的任何。 9 × 10 5 ST88 - 14细胞镀在T175烧瓶，增长了3天，在这一点烧瓶约80％融合。移植35小鼠，需要一个单一的80％汇合T175烧瓶，在这个密度，ST88 - 14细胞的平均收益率是7.3 × 10 6细胞每T175烧瓶。不得细胞增长到合流之前嫁接的收获，这是极为重要的。我们发现，嫁接细胞融合烧瓶程度较高的移植失败的结果和收获往往延长体内移植增长当然。 </li><li> ST88 - 14细胞与室温汉克斯“平衡盐溶液冲洗一次。细胞从细胞剥线细胞分解解决方案为30秒至1分钟，在室温下使用的烧瓶。五毫升DMEM10（注： 不包括在这个媒介嘌呤）每人每毫升细胞用于剥线，然后加入到细胞。 </li><li>使用血球细胞计数。 5 × 10 3 14 ST88 - 3μL悬浮细胞，将每鼠注射。一个移液与津贴细胞移植整个队列足够的数量，错误（例如，35只小鼠，我们通常移植40只小鼠细胞足够的数量转移），转移到无菌的1.5 mL试管。这些细胞是在5分钟3000转离心。小心取出上清液，细胞沉淀悬浮在DMEM10（注：嘌呤是不包括在这个媒介）到终浓度为5 × 10 3每3μL细胞。细胞置于冰上。 </li></ol> 3。嫁接协议<ol><li>老鼠在异氟醚蒸发器的感应腔麻醉，直到他们不再撤回其后肢脚趾捏刺激。小鼠被放置在自己身边，和0.05毫克/公斤丁丙诺啡hydrocholoride皮下注入。麻醉，然后通过连续的异氟醚通过鼻锥交付的管理维护。请注意，安乐死的小鼠被用来证明这一点的视频程序，因此，油管，用于提供异氟醚异氟醚是在视频中不可见。 </li><li>为了保持体温，动物在整个嫁接过程中的加热垫处理。为预防角膜干燥，眼药膏的小水滴（Puralube兽医药膏）的管理，以每只眼睛。由于这将是整个审判过程中被唯一确定每个鼠标所必需的，裁剪了一个独特的识别号码的耳标是每鼠右耳。 </li><li>将动物胃，蔓延的后腿。鼠标盖，用无菌的悬垂性，揭露手术的窗口（侧面）移植会发生。无菌的悬垂性，应该允许可视化的头部，既保证了鼻子内锥，并允许监测呼吸和动物的颜色。优碘应用到手术部位用棉放倒撒施，开始在侧翼的中心，并逐步向外螺旋式手术窗口的边缘。 70％的乙醇，然后以同样的方式适用于手术部位。连续的优碘乙醇的应用程序重复两次以上。 </li><li>取出细胞​​从冰，要么轻轻涡旋或轻弹管重悬落户细胞。使用吸管，去除3.2μL的细胞悬液，并退出到一块的封口膜。删除比需要的更多，放在封口膜暂停，我们可以确保有暂停无气泡，我们有一个完整的3μL注入。的细胞悬液多少可能拉入10μL汉密尔顿配备了33号针头的注射器。泡沫轻弹注射器和驱逐多余的细胞悬液，整整3μL。保持在冰上的细胞和细胞含注射器，直到准备使用;确保保持无菌针，不容许用冰或桶的接触。可以放在一块保鲜膜直接接触冰块冰注射器的顶端。 </li><li>使用4号手术刀处理装备与第22号手术刀刀片，通过侧面的皮肤切口仅低于平行股骨;确保切口不超出手术擦洗领域的限制。打开皮肤切口，用手术钳或手动揭露底层肌肉。将会看到一个筋膜平面沿腿的长度;坐骨神经在于下面这两个相连的肌肉筋膜之间。用一双锐利的尖的剪刀，生硬的剖析，通过此筋膜暴露坐骨神经，这应该是一个粗线厚度为白色的线性结构明显。 </li><li>用一双锐利的尖弯钳，仔细剖析下神经，从底层肌肉松动，这既提升和隔离神经。保留下的神经镊子保持这一地位。小心地插入神经汉密尔顿注射器的针头，试图保持尽可能的神经的同时注射的角度，因此，针不通过神经底部的穿刺。应插入针对远端的神经年底脊柱我们发现注射到肿瘤细胞对脊髓神经，建立了接近脊髓移植的肿瘤细胞。当发生这种情况时，肿瘤细胞往往侵入脊髓，导致早产的动物研究组因脊髓功能妥协的损失。 </li><li>一旦针正确定位的神经，放松紧张的神经产生镊子，缓缓注入神经细胞从注射器超过45-60秒。它是必不可少的，这样做缓慢迅速驱逐注射器内容将导致在“反洗”周围注射部位的肿瘤细胞和他们的损失。所有的细胞已被成功地注入后，慢慢退出针注射材料，以尽量减少损失。 </li><li>删除了牛肝菌和仔细回到原来的位置放置到其下的肌肉神经。 Vetbond手术胶关闭手术切口。保持约20秒钳切口，让胶水干。 </li><li>鼠标是从异氟醚的鼻锥，并单独放置在一个床上用品，免费笼收回。这笼应保持在一个加热垫的顶部，直到动物已经完全康复。部分的回收笼，不应该在加热垫，这使得动物带走热量，如果他们太热情。以下嫁接，应定期评估动物嚼在现场，跛行或厌食;这些迹象可能表明，动物仍然在痛苦中，应给予适当的止痛药。在此过程中获得经验，我们发现30-40小鼠，可以很容易地在一个单一的一天嫁接。 </li></ol> 4。嫁接成功，并随机进入研究组的评估<ol><li>生物发光成像是用来评估的嫁接过程中的成功。我们使用一个IVIS - 100系统（原精诺真，这是目前已知卡尺公司）检测肿瘤表达萤火虫荧光素酶作为其底物，D -荧光素的荧光素酶的化学反应产生的光发射。对这些图像进行分析，确定具体嫁接小鼠是否适合进入治疗试验同伙。在初步实验中，我们有量化的生物荧光信号探测，牺牲嫁接小鼠，收获与地区的利益，使用制造商的生活图像软件分析，生物发光信号的肿瘤和相关肿瘤的重量。这些研究表明，有一个肿瘤移植物重量和生物发光的光发射之间的线性关系，表明生物发光成像是一种评估整个临床前试验过程中移植瘤生长的合适的替代手段。通常，我们的形象，在同一时间5只小鼠。生物发光成像的更多细节， 察看了7。 </li><li>为了评估移植成功，生物发光成像进行1天，3天后嫁接。图片收集小鼠在相同的位置，10分钟后腹腔注射2.5毫克的D -荧光素为导向。在成像过程中，老鼠都保持在异氟醚麻醉和自己的体温保持在37 ° C加热垫在精诺真成像。图像采集时间是在2秒至10分钟的范围内，确保没有像素图像采集过程中饱和的数据采集软件。肿瘤区域（相对光子计数/秒）的发光是利用精诺真专有软件。发光强度为代表的生物发光是重叠的黑白照片，是在同一时间收集的小鼠上的伪缩放。 </li><li>要列入在临床前试验队列的资格，老鼠必须有一个检测的生物发光信号1和3天，嫁接后的生物荧光信号的强度必须增加1和第3天之间。有一个信号减少或静态的动物，被排除在外。此外，嫁接在同一天的小鼠中检测到的信号应该是为了彼此的幅度内1;与生物发光信号，是一个幅度低于或高于嫁接在同一天的动物检测的小鼠也被排除在外。 </li><li>一旦小鼠已确定，符合这些标准，他们必须随机分为治疗组。要做到这一点，嫁接小鼠中检测到的荧光素酶信号嫁接后3天首次下令从最高到最低强度。小鼠，然后随机分配的力度，治疗组的顺序，扭转这个顺序，然后重复这个过程，直到所有的老鼠被分配到组，成组。例如，如果有4个治疗组小鼠（例如，安慰剂组和3个试验药物的浓度）被分配到各组的顺序1，2，3，4，4，3，2，1，1，2，3 4，4，3，2，1 ..等。在开始治疗之前，平均分配组的每个成员的生物发光检测信号，以确保所有组内的平均起点信号，是尽可能接近。候选治疗药物管理是在第3天开始。 </li><li>为了评估治疗剂已移植在治疗过程中的增长上的效果，生物发光成像是一次开始治疗后的一周（10天，17，24，30后的嫁接）。对于非裸体的免疫缺陷小鼠，这是极为重要的动物再有新的毛皮增长与奈尔删除之前执行每个成像会议。这是因为头发块发光信号，与外套颜色决定多少的信号丢失。例如，苔白如发现瑞士Webster小鼠产生发光信号强度8的减少18％，而黑色的皮毛可以减少信号高达10倍9。我们还要注意，不能假设的，统一的毛皮再生会发生在所有治疗组和治疗组之间的“正常化”信号减少。这是因为治疗药物本身可能会影响头发生长。例如，我们已经注意到，他莫昔芬治疗抑制毛发再生，从而最大限度地减少安慰剂治疗的小鼠相比，认为本剂对肿瘤生长的效果。 </li><li> ST88 - 14 MPNST细胞，小鼠就可以进行移植后30天。此时，肿瘤通常有成长为机构动物护理和使用委员会所允许的最大尺寸，必须牺牲。最终成像会议之后，小鼠死亡，治疗结果的进一步分析（例如，血液循环的治疗剂，重量测定肿瘤组织，组织学检查，Ki67的决心和TUNEL标记指数水平的决心和必要的标本收集生化指标的评估）。正是标本的收集将取决于实验设计的需要。 </li><li>应每天监测与肿瘤的小鼠和终止，如果他们成为病（缺乏正常的疏导和避税行为），不能吃或喝，或若肿瘤过大，妨碍正常的身体运动。肿瘤的负担，不应该被允许超过10％的动物的正常体重。肿瘤重量为体重的百分比是比较荷瘤动物的年龄/性别匹配的对照组动物的体重的重量计算。恶病质不应该被允许成为临床意义。在荷瘤动物的重量损失不应超过正常体重的20％。 </li></ol> 5。代表性的成果： 图1显示了典型的生物发光逐步增加观察1至18天，嫁接后在正确建立原位移植瘤裸第三研究院（NIH）鼠标（AC）。生物发光信号的量化观察嫁接后1-24天，虽然这些信号的逐步增加，肿瘤生长明显加速在研究期间的后期阶段（图1D）。要严格表明，肿瘤的生长发生在移植小鼠，我们定期收集坐骨神经，如果它出现，肿瘤已经破裂的外膜，并侵入邻近组织，周围的软组织和骨骼肌肉。鉴于MPNSTs积极的增长，这并不奇怪，我们常常发现，嫁接肿瘤细胞有违反正常的神经障碍，并侵入邻近组织（图1E）。我们也常常发现，嫁接MPNST细胞移植到神经嫁接部位的近端和远端积极。 <img src="/files/ftp_upload/2558/2558fig1.jpg" alt="图1" /> 第三鼠标美国国立卫生研究院的生物发光成像原位MPNST荧光素酶标记的细胞嫁接，移植后1天 （图1）。请注意，为了彼此的幅度内，所有的感兴趣区域（ROI）内检测到的信号在不同小鼠。重新制作映像10天（B）和18天（三）嫁接后的发光信号的显示，在个人小鼠移植网站逐步增加。 （四）图表说明在相对光子计数/ 1-24天嫁接后在嫁接部位发现的第二个逐步增加。 （五）从这个鼠标的嫁接部位的显微照片显示出肿瘤的生长，到邻近的骨骼肌和联络入侵。

<ol>
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详细原位xenografting这里介绍的方法是我们开发使用ST88 - 14 MPNST细胞，这些NF1的相关的神经鞘瘤的研究中被广泛使​​用的行。然而，这种方法很容易与其他MPNST细胞株的临床前研究的适应性。举例来说，我们也进行原位STS - 26T 10个细胞，从一个偶尔发生的MPNST和T265 - 2C 11细胞，这是从NF1的相关MPNST派生派生行xenografting，类似的成功，我们与ST88观察-14细胞。 尽管如此，我们注意到，我们这里描述为ST88 - 14细胞原位xenografting的确切条件，不能直接通过嫁接其他MPNST线。相反，必须凭经验确定关键参数的方法为每个细胞株。这些参数包括初步嫁接，移植的发展，并在较小程度上允许的时间MPNST细胞的数量，体积，其中的肿瘤细胞被注入。要建立一个新行的这些参数，我们10 3到5 × 10 6个肿瘤细胞的小鼠移植组（5％组小鼠），检查每个量级（即10 3细胞，5 ×两种细胞浓度10 3细胞，10 4个细胞，5 × 10 4细胞等）。必须注入肿瘤细胞（&gt; 10 6）的数目越多，量也较大;至5毫升，可以不注射移植神经细胞的损失，我们已经找到。我们还发现，不超过5 × 10 6每5 mL体积的肿瘤细胞可以被注入密集悬浮变得越来越容易杀死肿瘤细胞的剪切。我们遵循这些老鼠，直到至少有三个内各组动物明显的肿瘤，在这一点，我们终止该组和神经病理检查，以确认移植增长。很明显，所需的时间，达到这一点会有所不同，这取决于注射细胞数​​量;在一般情况下，我们正在寻求将达到30-60天之内最大允许肿瘤生长的细胞浓度。可以更快速的增长难以达到有效治疗浓度前终止试验和/或防止在实验过程中收集足够的生物发光成像数据点。一个时间过程超过60天的调整实验参数，笨重和不必要地延长了工期计划的实验。 最后，我们也注意到，我们已经使用了嫁接到美国国立卫生研究院第三小鼠证明疗效的他莫昔芬6 ST88 - 14细胞原位xenografting方法。一个我们经常使用的方法，以评估候选人原位移植瘤的治疗药物的影响的详细描述，可以发现在该手稿。它也已经证明，神经纤维瘤细胞可以成功地嫁接到周围神经12，这表明，在本议定书中所述的程序进行了修改，可用于执行对神经纤维瘤的候选治疗药物临床前试验。

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<td>Foxchase outbred SCID mice</td>
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<td>Charles River </td>
<td>#236</td>
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<td>NIH III mice</td>
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<td>Taconic </td>
<td>#NIHBNX</td>
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<td>NOD-SCID&gamma; mice</td>
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<td>Jackson Laboratories </td>
<td>#005557</td>
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<td>Cell Stripper</td>
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<td>Fisher Scientific</td>
<td>25-056-cl</td>
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<td>Vetbond surgical glue</td>
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<td>Hamilton syringe</td>
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<td>Hamilton Company</td>
<td>#80030</td>
<td>10 &mu;L volume</td>
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<td>Hamilton syringe needles</td>
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<td>Hamilton Company</td>
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<td>33 Ga., 0.5 inch, PT4 (custom made)</td>
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<td>Ophthalmic ointment</td>
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orthotopic xenografting of human luciferase-tagged malignant peripheral nerve sheath tumor cells for in vivo testing of candidate therapeutic agents

虽然在体外屏幕必不可少的候选治疗药物的初步鉴定，药物的能力，抑制肿瘤的生长，必须在一个合适的动物模型进行严格的评估。动物模型的类型是最适合这个目的的激烈讨论的话题。一些证据表明，在转基因小鼠产生的肿瘤药物效果研究的临床前试验，临床结果1预测，所以往往是在这些模型中测试候选治疗药物。不幸的是，转基因模型并不适用于许多类型的肿瘤。此外，转基因模型往往有其他限制，如小鼠肿瘤模型以及人力，不完全外显的肿瘤表型和无法预测时，肿瘤会发展的关注。 因此，许多研究者使用对临床前试验（人类肿瘤细胞移植到免疫缺陷小鼠）移植瘤模型，如果没有提供适当的转基因肿瘤模型。即使转基因模型是可用的，他们往往是合作与异种移植模型，因为后者方便的治疗范围的快速测定。此外，这种伙伴关系允许的代理人在转基因肿瘤和真正的人类肿瘤细胞的有效性进行比较。从历史上看，xenografting经常被执行注射肿瘤细胞皮下异位移植瘤。这种技术是快速，重现性好，价格相对低廉，并允许连续定量肿瘤的生长，在治疗期间2。然而，皮下空间是不正常的微环境对大多数肿瘤和异位xenografting取得如此结果可能会产生误导，由于因素，如缺乏器官特异性表达宿主组织和肿瘤基因。因此强烈建议，异位移植术的研究，更换或嫁接到他们的组织的原产地（原位xenografting）2在人类肿瘤细胞的研究补充。不幸的是，这项建议的执行往往是挫败原位xenografting方法尚未发展为许多类型的肿瘤。 恶性外周神经鞘瘤（MPNSTs）高度侵略性的肉瘤，在协会或偶尔发生型神经纤维瘤病1 3，最常出现在坐骨神经 4 。在这里，我们描述了一个技术上的简单方法orthopically到坐骨神经免疫缺陷小鼠移植瘤中，萤火虫荧光素酶标记的人类MPNST细胞。还讨论了我们的方法来评估在动物个体和不带偏见的后续随机进入研究组成功的嫁接过程。

Watch this Scientific Journal Video about Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents at JoVE.c

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

一种可靠荧光素酶标记的人类恶性外周神经鞘肿瘤细胞移植到免疫缺陷小鼠坐骨神经的方法是描述。利用生物发光成像显示肿瘤移植和动物为研究组随机分离的标准进行了适当的建立进行了讨论。

原位Xenografting人类荧光素酶标记的恶性外周神经鞘瘤细胞在体内测试候选治疗药物

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

orthotopic-xenografting-human-luciferase-tagged-malignant-peripheral

Research

JoVE Journal

Medicine

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

18.5K 次观看.  University of Alabama at Birmingham - UAB. 一种可靠荧光素酶标记的人类恶性外周神经鞘肿瘤细胞移植到免疫缺陷小鼠坐骨神经的方法是描述。利用生物发光成像显示肿瘤移植和动物为研究组随机分离的标准进行了适当的建立进行了讨论。

视频: Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

一次性充填机对治疗药物的肺吹入小动物

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...