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>
	<li>Rosenberg, M. P., Bortner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+transgenic+and+knockout+animal+models+should+be+used+(for+drug+efficacy+studies+in+cancer).">Why transgenic and knockout animal models should be used (for drug efficacy studies in cancer).</a> Cancer Met. Rev. 17, 295-299 (1999).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Met. Rev. 17, 279-284 (1999).</li><li>Carroll, S. L., Ratner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+the+Schwann+cell+lineage+form+tumors+in+NF1?.">How does the Schwann cell lineage form tumors in NF1?.</a> Glia. 56, 1590-1605 (2008).</li><li>Woodruff, J. M., Kourea, H. P., Louis, D. N., Scheithauer, B. W., Kleihues, P., Cavenee, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology and Genetics of Tumours of the Nervous System. , 172-174 (2000).</li><li>DeClue, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+expression+in+neurofibromatosis+type+1-related+tumors+and+NF1+animal+models.">Epidermal growth factor receptor expression in neurofibromatosis type 1-related tumors and NF1 animal models.</a> J. Clin. Invest. 105, 1233-1241 (2000).</li><li>Byer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tamoxifen+inhibits+malignant+peripheral+nerve+sheath+tumor+growth+via+an+estrogen+receptor-independent+mechanism.">Tamoxifen inhibits malignant peripheral nerve sheath tumor growth via an estrogen receptor-independent mechanism.</a> Neuro-Oncology. , (2010).</li><li>Zinn, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+bioluminescence+imaging+in+small+animals.">Noninvasive bioluminescence imaging in small animals.</a> ILAR J. 49, 103-115 (2008).</li><li>Wu, J. C., Sundaresan, G., Iyer, M., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+optical+imaging+of+firefly+luciferase+reporter+gene+expression+in+skeletal+muscles+of+living+mice.">Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice.</a> Mol. Therapy. 4, 297-306 (2001).</li><li>Luker, G. D., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luciferase+real-time+bioluminescence+imaging+for+the+study+of+viral+pathogenesis.">Luciferase real-time bioluminescence imaging for the study of viral pathogenesis.</a> Methods Mol. Biol. 292, 285-296 (2005).</li><li>Dahlberg, W. K., Little, J. B., Fletcher, J. A., Suit, H. D., Okunieff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitivity+in+vitro+of+human+soft+tissue+sarcoma+cell+lines+and+skin+fibroblasts+derived+from+the+same+patients.">Radiosensitivity in vitro of human soft tissue sarcoma cell lines and skin fibroblasts derived from the same patients.</a> Int. J. Radiat. Biol. 63, 191-198 (1993).</li><li>Badache, A., DeVries, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibrosarcoma-derived+Schwann+cells+overexpress+platelet-derived+growth+factor+(PDGF)+receptors+and+are+induced+to+proliferate+by+PDGF.">Neurofibrosarcoma-derived Schwann cells overexpress platelet-derived growth factor (PDGF) receptors and are induced to proliferate by PDGF.</a> BB. J. Cell. Physiol. 177, 334-342 (1998).</li><li>Muir, D., Neubauer, D., Lim, I. T., Yachnis, A. T., Wallace, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenic+properties+of+neurofibromin-deficient+neurofibroma+Schwann+cells.">Tumorigenic properties of neurofibromin-deficient neurofibroma Schwann cells.</a> Am. J. Pathol. 158, 501-513 (2001).</li></ol>

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。

<table><tbody><tr><td>Foxchase outbred SCID mice</td><td>Charles River Laboratories</td><td>#236</td><td></td></tr><tr><td>NIH III mice</td><td>Taconic</td><td>#NIHBNX</td><td></td></tr><tr><td>NOD-SCID&amp;#947; mice</td><td>Jackson Laboratory</td><td>#005557</td><td></td></tr><tr><td>Cell Stripper</td><td>Fisher Scientific</td><td>25-056-cl</td><td></td></tr><tr><td>Vetbond surgical glue</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamilton Co</td><td>#80030</td><td>10 &amp;#x03BC;L volume</td></tr><tr><td>Hamilton syringe needles</td><td>Hamilton Co</td><td>#7803-05</td><td>33 Ga., 0.5 inch, PT4 (custom made)</td></tr><tr><td>Ear tags</td><td>National Band and Ear Tag Co.</td><td>1005-1</td><td></td></tr><tr><td>Ophthalmic ointment</td><td>Dechra</td><td>NDC 17033-211-38</td><td></td></tr></tbody></table>

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

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

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

癌症研究

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

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 ...

医学

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

生物荧光原位模型胰腺癌进展

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的 离体 消融来定义肿瘤发生的基因功能

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

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

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

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

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

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 ...

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

摘要

探索更多视频

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

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

立体定向颅内植入和

生物荧光原位模型胰腺癌进展

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的离体消融来定义肿瘤发生的基因功能

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

免疫活性动物模型胶质母细胞瘤的生物发光成像

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

摘要

探索更多视频

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

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

立体定向颅内植入和

生物荧光原位模型胰腺癌进展

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的 离体 消融来定义肿瘤发生的基因功能

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

免疫活性动物模型胶质母细胞瘤的生物发光成像

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的离体消融来定义肿瘤发生的基因功能