We thank Peter Johnson of the Georgetown University Microscopy Core for assistance with imaging the zebrafish embryos. The Microscopy &amp; Imaging Shared Resource and the Zebrafish Shared Resource are partially supported by NIH/NCI grant P30-CA051008. This work was also supported by NIH/NCI CA71508 (AW) and CA177466 (AW).

伦理学声明:根据经批准的协议IACUC产生了斑马鱼的胚胎。这些实验是在遵守进行与由乔治城大学动物护理和使用委员会的建议。 1.组织胚胎注射和创建库存解决方案<ol><li>生成必要的斑马鱼来评估癌细胞血管浸润。 <ol><li>设置成对或组交叉交配与TG（kdrl:grcfp）的Zn1; mitfa b692; ednrb1 B140鱼。 注:我们产生的Tg（kdrl:grcfp）的Zn1; mitfa b692; ednrb1 B140斑马鱼，通过杂交的Tg（kdrl:grcfp）的Zn1 25，表达绿色礁珊瑚荧光蛋白在血管内皮细胞，与缺乏色素细胞线，mitfa b692; ednrb1 B140，在斑马鱼国际资源中心开发的。 </li><li>有限公司llect鸡蛋，清洁，清除未受精或胚胎畸形22的第二天。 </li><li>孵育28.5℃，胚胎直到准备注射癌细胞，发生在斑马鱼的胚胎是2天受精后（2 DPF）。 </li></ol></li><li>使注射板。 <ol><li>熔体25毫升1.5％琼脂糖在卫生署2 O每个板。 </li><li>倾12毫升琼脂糖成100毫米×15毫米培养皿中，并让它变硬。 </li><li>再融化，然后倒入盘剩余的琼脂糖。 </li><li>所以，它是在一个30度角的琼脂糖和定位在板的中心立即放置一个切玻璃模具（3毫米×7.2厘米宽×7.5cm的长）。 注意:这将创建一个陡峭的60°墙和一个30°的倾斜坡道。 </li><li>胶带代替玻璃模具和让琼脂糖变硬。 </li><li>轻轻地取出玻璃模具，小心不要撕破琼脂糖。 注:模具可在4℃保存在卫生署2 O。 </li></ol></li><li>平衡注射板鱼水（0.4克/升海盐）。 <ol><li>漂洗板用蒸馏水两次。 </li><li>通过加入10ml鱼水到板并置于摇床进行10分钟平衡板。 </li><li>平衡板的第二时间与鱼的水。 </li></ol></li><li>他们转移到含有鱼水22菜肴拆分2天受精后（DPF）的胚胎成注射液组。 </li><li>准备恢复菜肴为每个组注射后利用。确保恢复培养皿含有10毫升鱼水，加青霉素（25微克/毫升）和链霉素（50微克/毫升）。 </li><li>通过加入4毫升缓冲三卡因库存（4毫克/毫升，10毫摩尔Tris，pH 7）中的50毫升鱼水加青霉素和链霉素的制备2倍三卡因溶液。 </li><li>溶解15mg的低熔点琼脂糖在10毫升的2×三卡因溶液以生成将固定为IMAGIN活胚胎的安装麻醉介质G。 注意:安装介质由1.5％琼脂糖。 </li><li>拉显微注射针。 <ol><li>将玻璃毛细管垂直吸管拉马。拉，用20毫安的电流和一个2线圈的加热元件长锥形移液器。 </li></ol></li></ol> 2.标签癌细胞与亲脂性荧光染料<ol><li>维持癌细胞推荐的培养条件。 注意:这些系在DMEM中维持+ 10％FBS:BT-474，MCF-7，MDA-MB-231，MDA-MB-468，和SK-BR-3。这些系维持在RPMI + 10％FBS:肝癌1806和T-47D。所有细胞系培养过程中维持在 37℃和5％ 的 CO 2。 </li><li>产生由解离的癌细胞的贴壁培养的单细胞悬浮液。 <ol><li>第一洗涤细胞用PBS，然后用0.05％胰蛋白酶-EDTA溶液处理。 注:胰蛋白酶的曝光时间将取决于细胞系。 </li><li>中和本身具有的胰蛋白酶溶液细胞培养基细胞分离后含朗姆酒。 </li></ol></li><li>离心胰蛋白酶中和的细胞悬浮液5分钟，在200 xg离心，然后重新悬浮于细胞计数在新鲜培养基中的细胞沉淀。 </li><li>使用自动计数器计数细胞悬液，并在200微升的细胞培养基的制备1百万个细胞。 <ol><li>验证与注入斑马鱼胚胎前，台盼蓝染色细胞活力。 注:只有活细胞群应注入斑马鱼胚胎，注射死细胞不会反映真实血管侵犯。 </li></ol></li><li>添加2微升的红色亲脂性染料的癌症细胞悬浮液以1:100稀释，拌匀，然后在37℃孵育该混合物20分钟。 注:的染料和标记时间浓度可能需要对每个细胞系进行优化。 </li><li>继温育后，加入1毫升的新鲜培养基的试管中，然后离心5分钟200×g的。 </li><li>从肿瘤细胞洗去残留荧光染料。 <ol><li>抽吸从细胞沉淀上清，重悬在1ml新鲜培养基的沉淀，并离心，在200×g下5分钟。 </li><li>再次吸出上清液，重悬细胞沉淀在1ml新鲜的培养基，然后离心机在200×g下5分钟:重复洗涤步骤的第二时间。 </li><li>再次吸出上清液，重悬细胞沉淀在1ml新鲜的培养基，然后离心机在200×g下5分钟:重复洗涤步骤三次。 </li></ol></li><li>吸出上清液，重悬含有百万标癌细胞于500μl新鲜培养基的细胞沉淀。 注:0.5mM EDTA的可以被添加到媒体，以防止细胞聚集。 </li></ol> 3.注射癌细胞进入斑马鱼胚胎的预心脏窦<ol><li>附上显微注射配制系统到加压空气源和打开第Ë显微注射配送系统电源。 <ol><li>通过压下脚踏板测试压力。空气的简单脉冲应持针器发射。 </li></ol></li><li>与2X三卡因液平衡注射板的两倍。 <ol><li>对于每一个平衡步骤，加入20毫升2×三卡因溶液的注入板和地点上摇动10分钟。 </li></ol></li><li>用塑料吸管一组胚胎转移到含有2个三卡因溶液一小碟。 </li><li>回填显微注射注射针与使用凝胶加载吸管尖癌细胞。 <ol><li>放置针与朝下使细胞定居尖端尖端的电极存储罐。 </li></ol></li><li>转移20 - 30麻醉胚胎到注入板通过收集用大量2X三卡因的胚在一个塑料吸管。 <ol style="margin-left: 40px;"><li>允许胚胎在吸管尖沉降。 </li><li>根特LY驱逐胚胎进入注入板的槽，沿所述槽的长度传播的胚胎。 </li><li>对准朝上对着槽的陡壁头和肚子的胚胎。 注:三卡因溶液应包括两个槽长度和平坦琼脂糖的表面上，胚胎仅居住在低谷。现在胚胎准备注射。 </li></ol></li><li>注入50 - 100癌细胞（2 - 5 NL）到使用显微注射配制系统的斑马鱼胚胎的precardiac窦。 <ol><li>针连接到一个显微的针保持器。 </li><li>立体镜下注射板60度墙的左侧位置，并专注于顶级胚胎在25倍的放大倍率。 </li><li>显微操作的位置，这样，当延长，中针会刺破胚胎。 </li><li>由眼延长针，直到它接近触摸胚胎。 </li><li>在显微镜下看，对准针这么牛逼帽子就会刺破后，进一步延伸胚胎。 </li><li>皮尔斯通过卵黄囊胚胎放置尖端刚好在，但不是在中，预心脏窦。 </li><li>通过压下脚踏注入细胞。喷射的力排出该细胞进入心脏窦。退针。 </li><li>使用右手，伸出和缩回的注射针。用左手，进行微调下胚胎定位。 </li><li>返回针电极瓶子储存在设置注入另一个板块。 </li></ol></li><li>胚胎转移到恢复菜一旦整个板被注入。 <ol><li>倾斜喷射板的底部汇集的胚胎，洗涤任何剩余的胚胎出槽，并与该塑料吸管收集它们。 </li><li>允许胚胎在移液管的底部沉降。 </li><li>胚胎转移到恢复盘中三卡因的最小体积。 </li></ol></li><li> Incuba在28℃下1小时TE恢复盘。 <ol><li>独立可行的斑马鱼胚胎的死胚等杂物。 </li></ol></li><li>孵育33°C盘，直到准备进行打分，通常为24 - 96小时。 注意:此温度被确定为37℃，对癌细胞的理想温度，和28.5℃，斑马鱼提供了理想的温度之间的折衷。 </li></ol> 4.外渗评分<ol><li>麻醉批次胚胎通过将它们放置在与三卡因溶液菜进行评分。 </li><li>广场上的凹陷显微镜载玻片的麻醉幼虫三卡因的下降。 <ol><li>东方幼虫为横向尾地区的最佳成像。 </li></ol></li><li>算已经成功通过聚焦上下通过尾区域清楚地辨别完整细胞侵入出脉管的癌细胞的数目。 注意:最好是有参与T至少有两个人他的过程，其中个人得分鱼是盲目的实验条件下被评估。 <ol><li>分数与10倍物镜的复合荧光显微镜的幼虫。使用20倍的目标任何困难的呼叫。 </li></ol></li></ol> 5.安装胚胎到幻灯片和随后的荧光成像<ol><li>熔融1.5％琼脂糖/三卡因溶液，并把到37℃。 </li><li>麻醉胚胎通过将其放置在三卡因溶液进行成像。 </li><li>在三卡因溶液到该成像表面一滴转移胚胎。 （可选）使用玻璃底盘或显微镜载玻片。 </li><li>使用玻璃吸管以除去过量的三卡因溶液，保持在成像表面上的胚胎。 </li><li>叠加在胚胎熔化琼脂糖溶液一滴。 </li><li>很快，琼脂糖聚合之前，用细腻的工具，比如睫毛刷，横向定位胚胎成像，给予额外的照顾以确保胚胎沿着成像表面平坦化。 </li><li>淹没在三卡因溶液聚合现在琼脂糖下降。 </li><li>若使活斑马鱼胚胎显微成像。 </li></ol> 6.修改:注射癌细胞进入斑马鱼胚胎卵黄囊<ol><li>如先前在本协议第（3）所述，准备显微注射配送系统和注射板。 </li><li>标签中的两个细胞群与先前在本协议第（2）所述的对比荧光染料。 </li><li>注入5 - 10 NL的2×10 ^ 7个细胞/ ml到卵黄囊。保持注射体积恒定注入相同的细胞数目 - 从每个细胞群体（100 200癌细胞） 注:我们可以用荧光缺乏血管这个实验透明斑马鱼的胚胎。颗粒的被动进入脉管可通过注入荧光珠（&lt;10微米），或者被控制为，改变本身，即不intravsate的细胞系。 </li><li>如前面部分所述（3）此协议中，然后筛选成功注射恢复注射的胚胎。 <ol><li>使用立体镜筛选并传送被成功注入了新的活力菜的胚胎。 注:所有的胚胎应该位于卵黄细胞的一贯大小的肿块。如果质量尺寸不同或胚胎被丢弃，如果任何细胞位于卵黄以外。 </li><li>转移存活的胚胎到新的培养皿如果癌细胞在卵黄囊清楚地看到。 </li></ol></li><li>孵育33°C盘，直到准备进行打分，通常为24 - 48小时。 注意:此温度被确定为37℃，对癌细胞的理想温度，和28.5℃，斑马鱼提供了理想的温度之间的折衷。 </li><li>得分血管内，按照一节所述的指导原则（4）本协议的，而是算数cancer细胞已经成功地侵入到尾区的脉管。 </li></ol>

<ol>
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T., Mumm, J. S., Cowell, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+human+cancer+cell+metastasis+in+zebrafish.">Evaluating human cancer cell metastasis in zebrafish.</a> BMC Cancer. 13 (453), (2013).</li><li>Cross, L. M., Cook, M. A., Lin, S., Chen, J. -. N., Rubinstein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+angiogenesis+drugs+in+a+live+fluorescent+zebrafish+assay.">Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay.</a> Arterioscl Throm Vas. 23 (5), 911-912 (2003).</li><li>Sharif, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+growth+density+modulates+cancer+cell+vascular+invasion+via+Hippo+pathway+activity+and+CXCR2+signaling.">Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling.</a> Oncogene. 34, 5879-5889 (2015).</li><li>Novoa, B., Figueras, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+model+for+the+study+of+inflammation+and+the+innate+immune+response+to+infectious+diseases.">Zebrafish: model for the study of inflammation and the innate immune response to infectious diseases.</a> Adv Exp Med Biol. 946, 253-275 (2012).</li><li>Kitamura, T., Qian, B. -. Z., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+promotion+of+metastasis.">Immune cell promotion of metastasis.</a> Nature Rev Immunol. 15 (2), 73-86 (2015).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-mediated+experimental+metastasis+is+enhanced+by+VEGFR+inhibition+in+a+zebrafish+xenograft+model.">Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model.</a> J Pathol. 227 (4), 431-445 (2012).</li><li>Tulotta, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+signaling+between+human+CXCR4+and+zebrafish+ligands+by+the+small+molecule+IT1t+impairs+the+formation+of+triple-negative+breast+cancer+early+metastases+in+a+zebrafish+xenograft+model.">Inhibition of signaling between human CXCR4 and zebrafish ligands by the small molecule IT1t impairs the formation of triple-negative breast cancer early metastases in a zebrafish xenograft model.</a> Dis Model Mech. 9 (2), 141-153 (2016).</li><li>Renshaw, S. A., Loynes, C. A., Trushell, D. M. I., Elworthy, S., Ingham, P. W., Whyte, M. K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108 (13), 3976-3978 (2006).</li><li>Berens, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratin-associated+protein+5-5+controls+cytoskeletal+function+and+cancer+cell+vascular+invasion.">Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.</a> Oncogene. , (2016).</li></ol>

The authors have no conflicts of interest to disclose.

这种技术利用了斑马鱼模型来有效地测试癌症细胞（参见图1）的血管侵袭能力。在这里，我们采用的技术的乳腺癌细胞系的组，以提供一个基线到其上其他研究者然后可以建立他们自己的研究（ 见表1; 图2 - 3）。该MDA-MB-231细胞很容易地侵入到斑马鱼胚胎的尾部区域观察会使该细胞系适用于测试可能抑制血管浸润剂。相对较少创伤的细胞系，像HCC1806或MDA-MB-468...

转移性疾病是癌症死亡率和许多机制，使癌细胞传播有待发现1的一个主要原因。为了使癌细胞成功转移，它首先必须通过围绕一个原发肿瘤中，输入（intravasate）进入循环系统基质侵入，生存在从循环中转，出口（渗出），最后建立一个可行的菌落在远处器官的网站2。血管内和外渗因此在转移级联关键步骤，但每一个癌细胞是不是破坏，并通过内皮连接3迁移本身娴熟。事实上，有一系列的周围癌细胞的血管侵袭和方法可以通过多种4内源性和外源性因素的进一步影响独特的选择压力。由于这些原因，该探头的晚期癌症的攻击行为技术通常专注上的vascular浸润能力作为一种手段来预测转移扩散。 各种模型系统中，以方便癌细胞血管浸润的体外研究。 在体外测定法中最常用的涉及任一的transwell系统通过内皮屏障5或电动细胞-基底阻抗传感（ECIS）技术评估癌症细胞迁移并监控由癌细胞6完整内皮单层的实时中断。这些测定通常缺乏流体动力学和基质因素，否则将影响癌细胞附着到内皮壁上。这个问题在某种程度上被从内皮细胞的三维培养与出现支持基质细胞perfusable血管网络规避，而这些3D微流体系统现在代表目前在体外选项7,8前列。尽管如此，这些方法省略官能循环系统的鲁棒微因此只在体内模型中部分替代品。 最广泛使用的血管浸润的体内模型是鼠标，其中因为它们发生在相对短的时间尺度，并且一般指示转移能力9实验性转移测定法，通常进行。这些分析包括直接注入肿瘤细胞进入流通，因此转移，器官即外渗和癌细胞定植结束阶段模型。实验转移测定不同根据肿瘤细胞注射的部位和器官，最终分析。在第一测定类型，癌症的细胞注射到小鼠和癌细胞播种在肺部尾静脉监视10,11。第二测定涉及执行腔内注射直接朝向骨微环境12-14转移性播种，而且脑15。在第三个实验中，癌症Ç厄尔注入脾脏以允许肝脏16的定植而第四传送路径进入颈动脉进行癌细胞到大脑17,18。无论癌细胞交货方式，器官殖民化是接受实验的端点，一般是通过发光，组织学，或基于PCR的技术来确定。尽管小鼠主机中进行实验测定转移的生理优势，这些实验仍然需要几个星期到几个月才能完成和分析。 斑马鱼（ 斑马鱼 ）模型最近出现的一个新的系统来研究癌症进展19,20，并允许癌细胞血管浸润过短得多的时间尺度的功能循环系统内的评估时，小鼠相比，21-24。该方法利用有其内皮标记有一个绿色的珊瑚暗礁FLUO透明斑马鱼品系rescent蛋白报告由kdrl启动子，血管内皮生长因子25的斑马鱼受体驱动。在测定中，癌细胞被标记有红色荧光标记物，并注入2日龄胚胎precardiac窦。间48到96小时的任何地方的注射后，即已经侵入了脉管的和成胚胎的尾部区域癌细胞可以有效地在荧光显微镜评分。在这里，我们采用的技术中常用的人乳腺癌细胞系组成的小组，证明他们的血管侵袭能力明显的差异。此外，我们证明，改变注射部位的胚胎卵黄囊允许异质细胞相互作用的研究，如癌症的细胞群可以用荧光染料进行差异标记，并注射入缺乏荧光脉管斑马鱼的胚胎。在后一种测定法中，已经侵入蛋黄和癌细胞intravasated入vasculaturE分别打进尾区域24注射后48小时。由于该模型的有效性和方便性，斑马鱼日益用于快速测试下一个生理设置癌细胞的血管侵袭能力。

<table><tbody><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>100 mm Dishes</td><td>Corning Incorporated</td><td>3160-100</td><td></td></tr><tr><td>5 3/4&quot; Disposable Pastur Pipets, borosilicate Glass</td><td>Fisher Brand</td><td>13-678-20B</td><td></td></tr><tr><td>60 mm Dish</td><td>Corning Incorporated</td><td>3160-60</td><td></td></tr><tr><td>Agarose, Low Melting</td><td>Fisher</td><td>BP165-25</td><td></td></tr><tr><td>Agarose, Molecular Grade</td><td>Bioline</td><td>BIO-41026</td><td></td></tr><tr><td>Capillary Glass, Standard, 1.2 mm x 0.68 mm, 4&quot;</td><td>A-M Systems, Inc</td><td>627000</td><td></td></tr><tr><td>David Kopf 700C Vertical Pipette Puller</td><td>Hofstra Group</td><td>3600</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>Electrode Storage Jar, 1.0 mm</td><td>World Precision Instruments, Inc</td><td>E210</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine, MS-222)</td><td>Fluka</td><td>A5040</td><td></td></tr><tr><td>Eyelash Brush</td><td>Ted Pella, Inc</td><td>113</td><td></td></tr><tr><td>Fetal Bovine Serum, Heat Inactivated</td><td>Omega Scientific</td><td>FB-12</td><td></td></tr><tr><td>Fisherbrand Transfer Pipettes</td><td>ThermoFisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>Gel Loading Pipet Tips</td><td>Fisher Brand</td><td>02-707-181</td><td></td></tr><tr><td>Glass Bottom Dishes (12.0 mm)</td><td>ThermoFisher Scientific</td><td>150680</td><td></td></tr><tr><td>Glass Depression Slide</td><td>VWR</td><td>470005-634</td><td></td></tr><tr><td>Instant Ocean Salt, Sea Salt</td><td>Pentair</td><td>IS50</td><td></td></tr><tr><td>Latex Rubber Bulbs, 2 ml, Pack of 72</td><td>Heathrow Scientific</td><td>HS20622B</td><td></td></tr><tr><td>SP8 Confocal Microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td></td><td></td></tr><tr><td>Eclipse E600</td><td>Nikon</td><td></td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Penicillin-G Potassium</td><td>Fisher Biotech</td><td>BP914-100</td><td></td></tr><tr><td>Petri Plates, 100 mm x 15 mm</td><td>Fisher Brand</td><td>&amp;nbsp;FB0875713</td><td></td></tr><tr><td>Picospritzer II</td><td>General Valve Corporation</td><td></td><td></td></tr><tr><td>RPMI 1640 Medium&amp;nbsp;</td><td>Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>Streptomycin Sulfate</td><td>Fisher Biotech</td><td>BP910-50</td><td></td></tr><tr><td>Vybrant DiI</td><td>ThermoFisher Scientific</td><td>V22885</td><td></td></tr><tr><td>Vybrant DiO</td><td>ThermoFisher Scientific</td><td>V22886</td><td></td></tr><tr><td>Zebrafish&amp;nbsp;</td><td>Georgetown Zebrafish Shared Resources</td><td></td><td></td></tr><tr><td>Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and HCC18-6 cells, which were mantained in RPMI + 10% FBS.</td><td></td><td></td><td></td></tr></tbody></table>

testing the vascular invasive ability of cancer cells in zebrafish (danio rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

在这里，我们测试了在斑马鱼胚胎模型常用乳腺癌细胞系（ 图1）的血管侵袭能力。严格的标准在得分外渗被用于这些不同的细胞系，其中，如果癌细胞已经明确外渗积极事件只算的，这之中主要是做以限制可从得分细胞碎片出现的任何误报。 我们的分析显示，该线之间明显的差异时侵入被注入precardiac窦（ ...

Watch this Scientific Journal Video about Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio at JoVE.com

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

这种方法利用斑马鱼的胚胎以有效地测试癌细胞的血管侵袭能力。荧光癌细胞注射到发育中的胚胎的precardiac窦或卵黄囊。癌细胞血管浸润和渗出通过尾部区域的荧光显微镜24至96小时后评估。

测试癌细胞的斑马鱼的血管侵袭能力（<em&gt;斑马鱼</em&gt;）

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

testing-vascular-invasive-ability-cancer-cells-zebrafish-danio

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

11.9K 次观看.  Georgetown University. 这种方法利用斑马鱼的胚胎以有效地测试癌细胞的血管侵袭能力。荧光癌细胞注射到发育中的胚胎的precardiac窦或卵黄囊。癌细胞血管浸润和渗出通过尾部区域的荧光显微镜24至96小时后评估。 

视频: Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

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

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

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

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 ...

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 ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

斑马鱼中具有功能化荧光硅纳米粒子的异种移植人类癌细胞的Vivo靶向

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

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 ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

建立胰腺癌斑马鱼患者来源的异种移植物进行化学敏感性测试

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

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 ...

测试癌细胞的斑马鱼的血管侵袭能力（

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在斑马鱼胚胎人乳腺癌细胞的侵袭行为

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

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

斑马鱼中具有功能化荧光硅纳米粒子的异种移植人类癌细胞的Vivo靶向

斑马鱼拉瓦尔异种草和肿瘤行为分析的一代

建立胰腺癌斑马鱼患者来源的异种移植物进行化学敏感性测试

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