Name: transplantation of zebrafish pediatric brain tumors into immune-competent hosts for long-term study of tumor cell behavior and drug response
Uploaded: 2017-05-17T14:00:00.000+00:00
Duration: 9 min 43 s
Description: Watch this Scientific Journal Video about Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response at JoVE.com

我们感谢两位审稿人对手稿的出色建议和改进。我们还感谢亨斯迈癌症研究所/犹他大学的畜牧和养护。这项工作由美国癌症协会（＃124250-RSG-13-025-01-CSM），NIH补助金（P30 CA042014 CRR计划），犹他州大学种子基金会和亨斯曼癌症基金会资助。

所有动物手术均获得批准，并遵循犹他州大学体育动物护理和使用委员会（IACUC＃16-03019）的动物护理指南。 1.准备显微注射设备<ol><li> 准备注射板。 <ol><li>准备溶解在蛋水中的50mL溶液的1.2％琼脂糖（反渗透水中为0.6g / L水族箱盐+ 0.01mg / L亚甲基蓝）。将溶液煮沸直到琼脂糖溶解，然后加入0.05mg / L亚甲基蓝补充。将25 mL最终溶液倒入10 cm培养皿中，使其固化。将剩余的25 mL琼脂糖溶液置于42°C水浴中，直至步骤1.1.2。 </li><li>将2英寸直径的烧杯置于凝固表面的中心，倒入剩余的25 mL 1.2％琼脂糖。 注意:这在板的外围产生一个注射表面，在中心形成一个核心提供了一个测试针头尺寸和肿瘤悬浮液一致性的区域。 <ol><li>在注射前将注射板保持在28°C至少30分钟，或在4℃保持长期储存。 </li></ol></li></ol></li><li> 准备注射针。 <ol><li>通过将外部尺寸为1.2毫米，内径为0.9毫米（无丝）的10厘米毛细管拉到针拔出器上的两个针中来制作针头。 注意:每个针头应测量总长度为6厘米，其中约1.5厘米的端部是锥形的。 </li><li>小心地将针放在用塑料石蜡包裹的显微镜载玻片上。使用剃刀刀片以45°的角度切割针头，以产生在尖端处具有开口的针。 注意:通常，针头末端的0.1mm至0.3mm被去除。不需要斜面尖端进行注射。 </li></ol></li><li>准备显微注射设置。 <ol> &lt;li&gt;安排标准的显微注射设置，包括立体显微镜，操纵器和移植用微量注射器。 </li></ol></li></ol> 2.准备胚胎移植<ol><li>收集多达500个mitfa w2胚胎44 （或如用户定义的任何其他斑马鱼基因型的胚胎），并保存在28℃的培养箱中。 </li><li>通过加入100μL在蛋水中稀释的10毫克/毫升蛋白酶鸡尾酒溶液，轻轻摇动约20分钟，使24 hpf的胚胎脱水。用新鲜的蛋水冲洗胚胎3次以除去残留的蛋白酶。 </li><li>筛选胚胎在48 hpf适当的发育阶段，如标准分期系列标准45所定义。 注意:只有正确的分期和形态正常的胚胎才能用于移植。 </li><li>麻醉在培养皿中含有0.002％Tricaine-S的蛋水中约20个48-hpf胚胎。通过观察停止运动来确认适当的麻醉。 </li></ol> 3.制作肿瘤细胞悬浮液注意:斑马鱼脑肿瘤模型可以通过基因工程组合癌基因和肿瘤抑制基因17,46,47产生。这里，如最近描述的，使用了sox10启动子驱动的NRAS WT ， p53 zdf1 / zdf1 CNS-PNET斑马鱼模型。 <ol><li>使用0.4％的Tricaine-S过量，然后根据IACUC认可的方案将其浸泡在冰水中，使肿瘤负荷约占总体重的20％来安乐死。 </li><li>用剃刀刀片和镊子在荧光下解剖荧光标记的肿瘤使用无菌技术的显微镜，并将其置于5mL 1x磷酸盐缓冲盐水（PBS）中。 注意:精确的解剖方法将由用户定义并依赖于肿瘤类型和组织位置。参见列出的参考资料，了解不同斑马鱼器官和脑肿瘤的详细程序17,48。 </li><li>使用p1000移液器手动破坏肿瘤块，直至形成均匀，多云的溶液。用移液管尖端或使用40μm的细胞过滤器去除大颗粒物（这是肿瘤依赖性的）。将悬浮液在室温下以290×g离心5分钟并除去上清液。 注意:不进行荧光激活细胞分选（FACS）以分离肿瘤细胞。这允许移植基质，免疫和肿瘤细胞的异质群体。 </li><li>将肿瘤细胞沉淀重悬于100μL无菌PBS中，并将其转移至1.5 mL微量离心管取5μL细胞悬液，稀释至250μLPBS。使用血细胞计数器计数细胞数。以290×g离心悬浮液3分钟，除去上清液，并将沉淀重悬于无菌PBS中以获得所需的细胞浓度。 注意:通常，将细胞重新悬浮于30-40μL无菌PBS中，产生粘性和不透明（细胞浓度≥100细胞/ nL）的溶液，并且倾向于产生最成功的移植物。细胞活力尚未评估。 </li><li>在移植过程中将肿瘤悬浮液储存在28℃的热块上。 </li></ol> 4.将肿瘤悬液注入2-dpf胚胎的第四脑室<ol><li>使用转移移液管将10-20个麻醉的胚胎（从步骤2.4）转移到注射板的周边;胚胎应横向落在注射板上，使心室清晰可见。使用有角度的探针根据需要调整胚胎，并将其放置在离注射板外缘的位置。 </li><li>使用凝胶加样移液管头将1-2μL肿瘤细胞悬浮液装入注射针头，并将针头插入机械手。 </li><li> 手动降下机械手，将针头保持在45°角。在x，y和z方向调整显微操纵器上的旋钮，直到针头刚好在胚胎头右侧约5 mm处。观察立体显微镜的目镜，并在x方向缓慢调整显微操纵器，直到针刺穿胚胎的第四脑室。不要让针刺穿心脏或蛋黄。 <ol><li>推动连接到微量注射器的脚踏板注射肿瘤细胞悬浮液。 注意:注射压力和时间将根据针孔尺寸而变化，但良好的起始点为5 - 10 PSI和50 - 100 ms。如果需要，注射肿瘤细胞的体积可以通过注射到矿物油中的标准油滴技术来评估。滴注直径为0.1mm的500pL注射通常给出最好的结果。 </li></ol></li><li>使用新鲜的蛋水轻轻地将注射的胚胎从注射板上冲洗成培养皿。保持胚胎在蛋水中剩余的程序或直到它们在罐中生长（如步骤4.9.1所述）。 </li><li>在荧光立体显微镜下检查注射的胚胎。确认注射压力，角度，针头尺寸和细胞悬浮液粘度导致肿瘤细胞填充25-50％的心室空间。根据需要调整这些参数。 </li><li>对任何剩余的胚胎重复步骤4.1-4.5。根据需要麻醉额外的胚胎（在步骤2.4中描述）。 注意:有经验的用户可以在3-4小时内注射300个胚胎。 </li><li>把胚胎放回来28℃孵育器过夜。 </li><li> 第二天，通过检查形态学和生理学特征来评估胚胎存活，如正常的心脏和大脑发育，如45所述。 </li><li>筛选后，如步骤2.4所述，在移植后第1天（dpt）麻醉胚胎。 <ol><li>使用转移移液管将4％甲基纤维素添加到陪替氏培养皿的中间，并将胚胎添加到甲基纤维素滴下。使用成角度的探针和筛网定向胚胎（腹侧面向光源），使用荧光显微镜确定一致的移植尺寸。用不限于脑室的肿瘤细胞或包含小于心室空间的25％的肿瘤细胞的胚胎移除任何胚胎。 注意:一致的植入被定义为具有单个肿瘤块的胚胎，其包含25-50％的心室空间17 。为了筛选，使用荧光具有1X物镜的立体显微镜，0.15数值孔径，0.7-1.6X的放大倍数和50ms至1s的曝光时间。对于所有图像，除了GFP或RFP通道之外，还要利用明场通道。 </li><li>为了维持肿瘤，将移植的胚胎放置在坦克中，以8 dpf（6 dpt）生长45 。 </li></ol></li></ol>成像移植胚胎细胞行为<ol><li>麻醉在3-8 dpf（1-6 dpt）的胚胎，并在1.2％的低熔点琼脂糖中装载（面向盖玻片的背面）。使用激光扫描共焦显微镜对所安装的胚胎进行延时成像达所需时间长度，以根据需要38,43,53显示细胞行为，如迁移，细胞分裂或细胞 - 细胞相互作用。 注意:使用宽高比为1,024 x 1,024，扫描速度为8＆＃181; S /像素。采用扫描平均并定义z深度尺寸，以确保捕获整个肿瘤块（1,000-1,500μm）。此外，执行4小时延时实验，使用40x目标每10-15分钟获取图像，以监测侵入行为。检测器灵敏度将根据肿瘤质量的强度而变化，激光功率将依赖于显微镜，但通常推荐在15％至40％之间。 </li><li>一旦成像完成，使用角度探针将琼脂糖上的胚胎释放出来，并将其转移到带有新鲜蛋水的培养皿中。 </li></ol> 6.移植胚胎的药物管理和肿瘤负担的评估17 <ol><li>麻醉3 dpf的胚胎并将其定向在4％甲基纤维素中，腹侧面对光源。使用荧光显微镜图像预处理胚胎17 。 </li><li>解放军在12孔板中以3dpf移植移植的胚胎，每孔10个，在0.5mL蛋水中。加入适量的药物以获得所需的最终浓度（ 例如 50μMMEK抑制剂），并将胚胎返回到28℃的培养箱中。第二天，用细孔转移移液管取出药物处理的蛋水，加入新鲜处理。每天重复一次，直到胚胎达到8 dpf。 </li><li>在8 dpf时，将胚胎从药物处理中取出，并将其置于新鲜的蛋水中。在4％甲基纤维素中麻醉胚胎和定向（面向光源的腹侧）。图像胚胎后处理使用荧光显微镜。使用适当的软件量化肿瘤区域，如17所述。 </li><li>每次处理适当分开胚胎，并将其置于罐中生长。 4-9周后，使用标准荧光立体显微镜麻醉鱼并评估其整体肿瘤负荷，如d受理17 。 </li></ol>

作者没有什么可以披露的。

该协议详细介绍了一种简单有效的移植测定方法，其涉及将斑马鱼肿瘤细胞注射到2-dpf胚胎的心室中，该胚胎将形成完全有效的免疫系统。到目前为止，斑马鱼CNS-PNET 17和黑素瘤（数据未显示）已成功移植，用于长期研究肿瘤细胞行为和侵袭。该方案的关键步骤包括确保适当的针孔尺寸和适当的肿瘤细胞悬浮液和充分麻醉宿主胚胎。对于每个研究人员，这种技术的进一步优化可能包括调整肿瘤细胞浓度，注射压力和胚胎取向。此外，源自不同肿瘤的悬浮液的粘度可能存在异质性，因此不同的肿瘤类型可能或多或少难以重新悬浮和移植。然而，通过调节针孔尺寸和使用diffe稀释肿瘤悬浮液，可以克服与肿瘤粘度相关的困难。 根据我们的经验，脑室稀疏细胞/细胞质量不一致的最常见原因如下:1）肿瘤细胞悬浮液太稀; 2）针孔尺寸太小; 3）注射时间和压力太低;和/或4）剩余的组织没有从肿瘤中过滤并且被放在针中。当肿瘤细胞悬浮液注射后流出脑室时，可能是由于:1）将针放置在心室的地板上; 2）高注射压力和时间;和/或3）过大的针孔尺寸。移植胚胎的低存活可能是由以下原因引起的:1）长时间麻醉胚胎; 2）将胚胎留在注射板上干燥; 3）将气泡注入脑室;和/或4）刺穿重要的器官，如大脑和心脏，因为针穿过心室。 成人或胚胎斑马鱼脑中以前的肿瘤移植方法依赖于成年人（遗传，药理学或经辐射）的免疫抑制，或仅限于在胚胎38,43,52,53,54中的人或小鼠细胞的短期研究。例如，小鼠脑肿瘤可以鼻内注射到地塞米松免疫抑制的30-dpf青少年斑马鱼中，用于短期（〜2天）临床前药物测定54 。然而，这些实验中的注射部位被颅骨和脑组织遮蔽，并可能导致对正常脑组织的损伤，这降低了宿主活力和植入效率。另一个最近描述的方法涉及注射人类gliob最终瘤细胞系进入斑马鱼胚胎的中脑区域，这使得能够对生长，入侵和药物反应进行短期分析43 。再次，注射部位的精确位置是可变的并且可能损害正常组织。因此，以前的胚胎脑移植方法通常会损害宿主的生存能力，将这些研究限于短期分析（ 即 2至14天），导致由于注射部位模糊引起的单次注射之间的变异性增加，细胞行为和药物反应的长期分析或多代人的再移植。 这里描述的方法解决了斑马鱼胚胎和免疫受损的移植技术中的当前局限性，允许研究人员:1）可重复地注射到相同的位置，对周围组织的损伤最小; 2）直接可视化注射部位以最大化植入效率; 3）每天移植数百只胚胎; 4）允许肿瘤在免疫竞争动物中生长; 5）允许在斑马鱼的寿命期间监测肿瘤细胞行为和持久的药物反应;和6）允许肿瘤再次移植多代，用于肿瘤进展或药物复发机制的潜在研究。此外，该协议使研究人员能够利用任何斑马鱼基因型来评估宿主反应，包括不同的免疫群体。这些属性使得该方法可以容易地适应任何已经在斑马鱼中执行标准显微注射的实验室。最后，虽然这种方法对于斑马鱼脑肿瘤的原位注射是理想的，但当移植其他肿瘤类型（例如肝脏或胰腺）时，原位点可能比免疫能力更重要（ 例如，如果研究人员正在研究基质的影响微环境对肿瘤生长）。在这种情况下，新开发的免疫缺陷斑马鱼模型可能更适合于进行原位肿瘤移植11 。 该方案已被用于进行肿瘤细胞竞争测定并注射双重标记的肿瘤。还讨论了用于评估化合物对肿瘤发生的有效性的潜在治疗策略，其涉及通过将药物添加到水中来移植后的胚胎的治疗。之前报道了移植前肿瘤细胞离体治疗的方法17 。另外，移植肿瘤已经合并进行多轮重新移植，这将有利于肿瘤进展和化学耐药性的研究17 。目前，临床前研究依赖于小鼠异种移植物来评估潜在化合物的功效。然而，这些研究是耗时的并且成本高昂。考虑到斑马鱼与人类之间的致癌信号通路的高度保守性28 ，预计这种方法将补充常规的小鼠和人类细胞研究，以便更快速地鉴定进入临床前和临床试验的有效化合物。最终，这种方法可以证明对于初级患者肿瘤的快速化学筛选是有用的，这可以进一步推动个性化医学计划。然而，斑马鱼（成人或胚胎）中人类细胞长期生长的条件仍然需要确定。

肿瘤细胞移植到免疫受损的动物，特别是小鼠异种移植物中，是一种广泛使用的技术，用于研究控制癌细胞增殖1，2 ，存活，侵袭和转移的机制3,4以及提供平台用于筛选药物5,6,7 。最近，将原发肿瘤样本移植到免疫受损的小鼠中已经被用于产生患者来源的异种移植（PDX）模型用于诊断和临床前药物筛选目的，并且是个性化医学计划的主体8,9,10,11 。然而，重要的证据表明调节免疫系统茎可以对肿瘤行为和患者结局产生重大影响12,13 。这推动了基于异种移植物技术的重新设计，包括"人源化"小鼠，其中通过将肿瘤细胞共同移植人免疫细胞来重建小鼠的免疫系统。然而，这种方法在技术上仍然具有挑战性，具有与该技术相关的可变重现性和毒性，除了显着的成本14,15 之外 。因此，需要免疫竞争动物的新型移植技术来加速发现癌症进展和药物反应的免疫和肿瘤特异性机制。 斑马鱼是人类癌症研究的替代动物模型，目前已有超过20种癌症模型建立了16种 ，其中包括高度恶性的脑17 ，mela<html>神经18,19,20和胰腺癌21,22以及许多白血病23,24,25,26,27。斑马鱼系统的两个特征使其特别适合癌症研究:1）半透明动物的光学透明度允许使用简单的显微技术直接观察癌细胞行为（ 即增殖，存活，侵袭和传播），以及2）女性斑马鱼每天可以产生多达200个胚胎，从而以低成本快速缩放动物数量进行遗传或药物筛选。此外，斑马鱼和人类的癌症基因组是高度保守的（包括致癌基因和肿瘤抑制基因）28"，让机械和药物发现迅速转化为哺乳动物系统，这些属性也使得斑马鱼成为移植技术的理想动物模型，可以利用系统的成像，可扩展性和低成本。 免疫损害斑马鱼的以前的肿瘤移植研究有助于鉴定自我更新能力，肿瘤恶性肿瘤和入侵/传播11,29。肿瘤细胞行为的短期研究可以在移植到γ-照射的成年人之后进行，其免疫系统被有效地抑制约20天11,30。成人斑马鱼与地塞米松的治疗在排斥发生前抑制B细胞和T细胞多达30天31 。另一个较不普遍的策略是使用克隆斑马鱼菌株这使得能够在免疫能力较强的宿主中长期进行调查32 。然而，只有有限数量的克隆菌株已经产生并且由于低繁殖力而难以维持。此外，大多数已建立的斑马鱼肿瘤模型是在其他遗传背景下产生的，因此这些肿瘤不能移植到克隆菌株中而不会抑制免疫系统11,33,34。改进长期移植研究的最新方法包括开发rag2 E450fs突变体系，具有受损的B细胞和T细胞功能，已被用于成功移植多种癌症35,36 。为了规避克隆斑马鱼系或免疫受损宿主的要求，许多组使用早期胚胎（ 即 &gt; 72马力受精（hpf））用于人类肿瘤细胞移植，因为这些胚胎尚未完全开发适应性免疫系统37,38,39,40,41,42,43。然而，这些方法仅限于对肿瘤细胞行为或药物反应的短期分析（通常少于2周），因为人类癌细胞或移植技术本身会杀死宿主，防止长期研究和再移植。 该方案详细描述了一种修复的胚胎移植方法进入受精2 d后胚胎脑的第四脑室的腔。它可以最大程度降低对宿主的毒性，并可与斑马鱼脑肿瘤模型结合，以长期植入肿瘤细胞。因此，这种技术肿瘤细胞重新移植到新的宿主多代中的低水平，有助于今后研究肿瘤异质性，宿主/免疫应答，药物反应或转移潜能。这种方法也是简单，高效和可扩展的，每天可以由单个用户执行多达300次移植，最多可以进行90％的移植。这允许单个原发性肿瘤在2 dpf的基因或药物筛选项目中快速繁殖到数百个胚胎，或者直接在不同的宿主背景下直观显现出脑肿瘤细胞行为。

<table><tbody><tr><td>Egg water</td><td>in house</td><td></td><td>maintaining embryos, making injection plate&amp;nbsp;</td></tr><tr><td>Methylene Blue&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M9140</td><td>add to egg water to prevent fungal growth</td></tr><tr><td>Petri dish</td><td>Thermo Fisher</td><td>FB0875711Z&amp;nbsp;</td><td>housing embryos, making injection plate</td></tr><tr><td>50 mL&amp;nbsp; beaker (2 inch diameter)</td><td>Any commercial brand</td><td></td><td>making injection plate</td></tr><tr><td>Agarose</td><td>Denville</td><td>CA3510-8</td><td>making injection plate</td></tr><tr><td>Glass Container</td><td>Any commercial brand</td><td></td><td>making injection plate</td></tr><tr><td>Tweezers</td><td>Fine Science Tools</td><td>11295-10</td><td>tumor dissection</td></tr><tr><td>Razor Blade</td><td>Thermo Fisher</td><td>12640</td><td>needle preparation, tumor dissection</td></tr><tr><td>Glass slide wrapped in parafilm</td><td>Any commercial brand</td><td></td><td>needle preparation</td></tr><tr><td>Phosphate Buffered Saline 1x, pH 7.4</td><td>Life Technologies</td><td>10010023</td><td>tumor resuspension</td></tr><tr><td>Cell strainer, 40 &amp;micro;m</td><td>Corning Falcon</td><td>352340</td><td>tumor resuspension</td></tr><tr><td>1,000 &amp;micro;L filter tips</td><td>any commercial brand</td><td></td><td>tumor resuspension</td></tr><tr><td>100 &amp;micro;L filter tips</td><td>any commercial brand</td><td></td><td>tumor resuspension</td></tr><tr><td>50 mL conical tubes</td><td>Genesee Scientific&amp;nbsp;</td><td>21-108</td><td>tumor resuspension</td></tr><tr><td>15 mL conical tubes</td><td>Genesee Scientific&amp;nbsp;</td><td>21-103&amp;nbsp;</td><td>tumor resuspension</td></tr><tr><td>1.7 mL microtubes</td><td>Genesee Scientific&amp;nbsp;</td><td>24-281</td><td>tumor resuspension</td></tr><tr><td>Micropipettes</td><td>Any commercial brand</td><td></td><td>tumor resuspension and transplantation</td></tr><tr><td>Glass capillary (no filament)</td><td>World Precision Instruments</td><td>&amp;nbsp;TW120-4</td><td>tumor transplantation</td></tr><tr><td>Needle puller</td><td>Sutter Instruments</td><td>P-97</td><td>tumor transplantation</td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>930001007</td><td>tumor transplantation</td></tr><tr><td>Microinjector</td><td>Harvard Apparatus</td><td>PLI-90</td><td>tumor transplantation</td></tr><tr><td>Tricaine-S (MS-222)</td><td>Western Chemical</td><td>TRS1</td><td>tumor transplantation, anesthetic</td></tr><tr><td>Angled Probe</td><td>Fine Science Tools</td><td>10140-02</td><td>embryo manipulation</td></tr><tr><td>Transfer Pipette</td><td>Any commercial brand</td><td></td><td>embryo manipulation</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>required during tumor resuspension</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5424</td><td>required during tumor resuspension</td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZ61</td><td>tumor transplantation</td></tr><tr><td>Fluorescent Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td>imaging tumor transplants</td></tr><tr><td>Microscope Camera</td><td>Olympus</td><td>DP-72</td><td>imaging tumor transplants</td></tr><tr><td>Methylcellulose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M7140</td><td>imaging tumor transplants</td></tr><tr><td>Incubator</td><td>Any commercial brand</td><td></td><td>maintaining embryos, warming up injection plate</td></tr><tr><td>Microwave</td><td>Any commercial brand</td><td></td><td>making injection plate</td></tr><tr><td>Scale</td><td>Any commercial brand</td><td></td><td>making injection plate</td></tr><tr><td>Gloves</td><td>Any commercial brand</td><td></td><td>all aspects of the protocol</td></tr><tr><td>Low Melt Agarose</td><td>Any commercial brand</td><td></td><td>confocal imaging of embryos</td></tr><tr><td>Glass Bottom Dish</td><td>Mattek Corporation</td><td>P35G-1.0-20-C</td><td>confocal imaging of embryos</td></tr><tr><td>Laser-scanning confocal microscope</td><td>Olympus</td><td>FLUOVIEW FV1200</td><td>confocal imaging of embryos</td></tr><tr><td>Pronase</td><td>Roche Diagnostics</td><td>11459643001</td><td>dechorionate embryos</td></tr><tr><td>PBS</td><td>Any commercial brand</td><td></td><td>resuspend tumor/tumor cells</td></tr><tr><td>12-well plate</td><td>Any commercial brand</td><td></td><td>drug treatment of embryos</td></tr><tr><td>Thin-bore transfer pipette</td><td>Any commercial brand</td><td></td><td>drug treatment of embryos</td></tr><tr><td>Hemocytometer</td><td>Any commericial brand</td><td></td><td>For counting tumor cells in suspension</td></tr><tr><td>N2</td><td>Any commericial brand</td><td></td><td>For microinjector set up</td></tr></tbody></table>

transplantation of zebrafish pediatric brain tumors into immune-competent hosts for long-term study of tumor cell behavior and drug response

肿瘤细胞移植是确定控制癌细胞生长，迁移和宿主反应的机制的重要技术，并评估潜在的患者治疗反应。目前的方法很大程度上取决于使用同基因或免疫受损的动物，以避免肿瘤移植物的排斥。这样的方法需要使用通常阻止免疫 - 肿瘤细胞相互作用分析和/或限于特定遗传背景的特定遗传菌株。斑马鱼的替代方法是在3天之前利用胚胎脑中不完全发育的免疫系统，其中移植肿瘤细胞用于短期测定（ 即 3至10天）。然而，这些方法导致宿主致死性，这阻止了肿瘤细胞行为和药物反应的长期研究。该方案描述了长期原位移植斑马鱼脑肿瘤组织的简单有效的方法到2日龄免疫能力的斑马鱼的第四脑室。该方法允许:1）长期研究肿瘤细胞行为，如入侵和传播; 2）耐药性对药物的反应;和3）重新移植肿瘤用于研究肿瘤进展和/或不同宿主遗传背景的影响。总之，这项技术使癌症研究人员可以评估远端的移植，入侵和生长，以及在多个月内进行化学筛选和细胞竞争测定。该方案可扩展到其他肿瘤类型的研究，可用于阐明化疗耐药和转移的机制。

肿瘤移植和植入 图1中示出了该过程的示意图 。荧光标记的肿瘤细胞从主要器官部位提取，并且产生单细胞悬浮液用于移植到2-dpf mitfa w2斑马鱼胚胎44的第四脑室中。 图2显示了使用中枢神经系统原始神经外胚层肿瘤（CNS-PNET）的斑马鱼模型的这种方法的代表性结果，其中sox10启动子驱动p53缺失的寡核苷酸前体细胞中与mCherry融合的野生型NRAS或活化的NRAS的表达17 。使用缺乏黑素细胞的mitfa w2斑马鱼44突变体更容易可视化移植后和/或成年后的肿瘤细胞。其他颜料突变体也可用于提供额外的成像清晰度，例如Casper 50 。早在移植后24小时，可以看到肿瘤细胞侵入周围的脑组织（ 图2A ，3ddf，箭头）。在下周（ 图2A ，8dpf），肿瘤移植物在脑室和周围脑组织中继续生长。此时，也可以在肾脏区域观察到荧光（ 图2A ，星号）。这可能是由于肾从移植中过滤细胞碎片，这与使用荧光染料作为评估肾功能的测定的先前研究一致51 。肿瘤细胞继续增殖和侵袭，所以通过28 dpf，整个斑马鱼脑可以看到肿瘤块（ 图2A ，28dpf）。 图2B的顶部面板中。通常在80-90％的胚胎中实现肿瘤植入，肿瘤移植持续成年; 图2B的底部面板中显示了三个代表性的例子。这允许肿瘤移植物被收获并冷冻或重新移植数代。 肿瘤侵袭和药物反应 图3A显示了共移植方案，其中通过全部收获用GFP（肿瘤A）或mCherry（肿瘤B）标记的两个不同肿瘤或解离（注意，此处不使用FACS，但如果需要，可以使用FACS分选的肿瘤细胞）。肿瘤A和B可以来自不同的位置，诱导不同的癌基因，或诱导在不同的遗传背景。将每个肿瘤加工成单细胞悬液，然后将肿瘤细胞以1:1的比例混合在一起并移植到2-dpf胚胎中。然后可以在允许内部控制技术和遗传背景的同一宿主中观察肿瘤性质，如植入物，生长，侵袭和肿瘤B的扩散。 在图3B中 ，收获来自视神经的NRAS驱动，mCherry标记的斑马鱼CNS-PNET和来自小脑的GFP标记的CNS-PNET，并加工成单细胞悬液。对细胞进行计数，并将相同数目的来自每个肿瘤的细胞混合在一起并移植到2-dpf胚胎中。四d移植后的ays，用共聚焦显微镜成像胚胎。显示的是代表性的胚胎，证明Tectum肿瘤细胞（红色）比小脑肿瘤更广泛地迁移到宿主脑（箭头）中。 用荧光标记的肿瘤细胞移植的胚胎可用于药物治疗方案中以鉴定抑制肿瘤生长的化合物。 图3C是治疗和成像肿瘤移植物的典型时间表。移植后24小时，访问胚胎以保持一致的移植尺寸并分成治疗组。在提供的实施例中，用50mM二甲基亚砜（DMSO）或MEK抑制剂（AZD6244）处理用NRAS驱动的移植胚胎，mCherry标记的斑马鱼CNS-PNET。将胚胎处理5天，并以8dpf成像， 如图3C所示 。 图3D （左图）显示代表<html> e动物来自两个治疗组。 MEK抑制剂处理的胚胎具有比用DMSO处理的那些显着更低的荧光量。动物成像后，将其转移到没有药物的罐中，并监测肿瘤生长两个月。在提供的例子中，MEK抑制剂治疗在成年鱼中产生持久的反应（ 图3D ，右图）。有关药物治疗，成像和定量的更多细节，请参见参考文献17 。 <img alt="图1" src="/files/ftp_upload/55712/55712fig1.jpg" /> 图1:肿瘤细胞移植到2-dpf斑马鱼胚胎第四脑室的方案示意图。该方案的第3至第4节的一般原理图包括备选终点分析（第4.9-6节）。/55712/55712fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图2" src="/files/ftp_upload/55712/55712fig2.jpg" /> 图2:肿瘤移植和进展。 （A）将斑马鱼原代CNS-PNET 17肿瘤细胞用mCherry标记的细胞移植到2-dpf胚胎的第四脑室中。 3 dpf时，肿瘤细胞被侵入周围的脑组织（箭头）。在8 dpf，肿瘤细胞在心室内生长;红色肿瘤细胞也存在于周围脑组织和肾脏（星号）中。在28 dpf，肿瘤细胞已经侵袭并在整个宿主脑中生长。 （B）顶板。 7个3-dpf胚胎移植有mCherry标记的斑马鱼脑肿瘤细胞。肿瘤细胞可以一贯地移植到数百个胚胎中，具有高植入率（ 即通常为85/100胚胎）。中间和底板。三只代表性成年鱼与移植后4周施用受精（wpf）。肿瘤可通过荧光显微镜观察。 <a href="http://ecsource.jove.com/files/ftp_upload/55712/55712fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55712/55712fig3.jpg" /> 图3:比较肿瘤侵袭和药物反应。 （ A ）来自视神经或小脑的CNS-PNET 17分别用mCherry或GFP标记，加工成单细胞悬浮液，以1:1的比例混合在一起，并共同移植到2-dpf胚胎中。 （ B ）用GFP标记的小脑肿瘤细胞和来自视神经的mCherry标记的肿瘤细胞的混合悬浮液共同移植6-dpf胚胎。 t的每个肿瘤的侵入性质的差异他可以使用荧光显微镜观察相同的受体。在这个例子中，mCherry标记的肿瘤细胞比用GFP标记的那些（箭头）更广泛地迁移。 （ C ）移植肿瘤细胞的胚胎的药物治疗时间表。 （ D ）在治疗方案结束时（8dpf）和治疗后8周（8wpf）的代表动物的图像，用DMSO对照或50μMMEK抑制剂。可以通过定量荧光来测量肿瘤负担，如17所述。在该实验中，在MEK抑制剂处理的胚胎中，mCherry标记的肿瘤细胞的生长减少，并且在成人中转化为持久的反应。 <a href="http://ecsource.jove.com/files/ftp_upload/55712/55712fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response at JoVE.com

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

癌细胞的移植是识别癌症机制和治疗反应的重要工具。目前的技术取决于免疫力不足的动物。在这里，我们描述了一种将斑马鱼肿瘤细胞移植到免疫感受态胚胎中的方法，用于长期分析肿瘤细胞行为和体内药物反应。

将斑马鱼小儿脑肿瘤移植入免疫能力的宿主，用于长期研究肿瘤细胞行为和药物反应

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

transplantation-zebrafish-pediatric-brain-tumors-into-immune

Nanyang Technological University

Research

JoVE Journal

Cancer Research

11.4K 次观看.  University of Utah School of Medicine. 癌细胞的移植是识别癌症机制和治疗反应的重要工具。目前的技术取决于免疫力不足的动物。在这里，我们描述了一种将斑马鱼肿瘤细胞移植到免疫感受态胚胎中的方法，用于长期分析肿瘤细胞行为和体内药物反应。 

视频: Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

科研

癌症研究

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

在斑马鱼肿瘤异种移植物中宿主血管反应的Vivo成像和定量

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

斑马鱼拉瓦尔异种草和肿瘤行为分析的一代

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

斑马鱼神经母细胞瘤转移模型

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from &#945;-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

正常和恶性肌细胞移植到免疫受损成年斑马鱼

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

免疫与感染

利用斑马鱼幼虫进行高效的体内肿瘤研究

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

作者聚焦:利用斑马鱼肿瘤异种移植推进癌症治疗

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

成年斑马鱼的优化静脉注射

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

将斑马鱼小儿脑肿瘤移植入免疫能力的宿主，用于长期研究肿瘤细胞行为和药物反应

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此视频中的章节

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

在斑马鱼肿瘤异种移植物中宿主血管反应的Vivo成像和定量

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

斑马鱼拉瓦尔异种草和肿瘤行为分析的一代

斑马鱼神经母细胞瘤转移模型

正常和恶性肌细胞移植到免疫受损成年斑马鱼

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法

成年斑马鱼的优化静脉注射