Name: in vivo model for testing effect of hypoxia on tumor metastasis
Uploaded: 2016-12-09T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis at JoVE.com

This work was supported by National Institutes of Health (NIH) grants: UL1TR000101 (previously UL1RR031975) through the Clinical and Translational Science Awards Program, 1RO1CA123211, 1R03CA178809, R01CA197964 and 1R21CA198698 to JK. MRI was performed in the Georgetown-Lombardi Comprehensive Cancer Center's Preclinical Imaging Research Laboratory (PIRL) and tissue processing in the Georgetown-Lombardi Comprehensive Cancer Center's Histopathology &amp; Tissue Shared Resource, both supported by NIH/NCI grant P30-CA051008. The authors thank Dan Chalothorn and James E. Faber, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, for their assistance with postmortem x-ray angiography, and providing insight and expertise on collaterogenesis.

所有的程序是由乔治敦大学机构动物护理和使用委员会的批准。 1.细胞制备注射原位<ol><li>培养人类胚胎干细胞在标准条件下。使用大约15厘米细胞培养板不超过5只小鼠注射汇合的70％。 注意:对于该研究中，SK-ES1细胞的McCoy的5A培养基中培养，用15％胎牛血清（FBS）的胶原包被的板和TC71细胞培养在RPMI有10％FBS和1％的4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）。两种培养基均补充有抗生素 - 青霉素（100单位/ ml），链霉素（100微克/毫升）和两性霉素B（1微克/毫升）。 </li><li>用0.25％胰蛋白酶/乙二胺四乙酸（EDTA）洗涤ES细胞用磷酸盐缓冲盐水（PBS）和t​​rypsinize在70％汇合5分钟。 </li><li>从细胞培养中介板中取出胚胎干细胞一个，然后在室温下以200 xg离心离心5分钟。重悬的ES细胞在10ml冷PBS中，然后计数细胞数。 </li><li>离心机ES细胞在室温下200 xg离心5分钟，然后重新悬浮于2×10 7（SK-ES1）或每ml 10 7（TC71）细胞在冷PBS中。保持最终的细胞悬浮液在冰在执行注射。 </li></ol> 2.胚胎干细胞原位注入腓肠肌<ol><li>使用4-6周龄雌性SCID /米色小鼠。 </li><li>注入的ES细胞，轻轻握住鼠标和稳定在第四和第五手指之间的左腿，露出下部后肢的内侧。 </li><li>使用28 G½针，注入0.1包含任一2×10 6（SK-ES1）或10 6（TC71）ES细胞入腓肠肌（ 图2A）预先制备的细胞悬浮液中的溶液。 注意:虽然最大音量为肌内注射ections通常0.05毫升，对于此特定步骤的体积增加至0.1 m1为必要的，因为注入的高细胞数。此程序已获得乔治敦大学机构动物护理和使用委员会。 <ol><li>针插入腓肠肌前方约30 - 45度角在胫骨嵴/结节的方向。 </li><li>一旦倒是胫骨嵴/结节稍拔出针头。缓缓注入细胞悬液溶液，逐渐抽出针以释放压力。 </li></ol></li><li>在监控遇险的迹象，未来24小时的小鼠注射。 注:在动物的处理经验较少侦查人员应该考虑麻醉对肿瘤细胞注射小鼠。有些机构可能需要为安全起见麻醉。 </li></ol> 3.监测原发性肿瘤生长<ol><li>监测原发性肿瘤D的生长aily直到肿瘤大小达到所需的体积。 注意:在本研究中，为250mm 3小牛体积用作实验（ 图2B）的一个起点。通常情况下，需要约1 - 2周的肿瘤达到这个尺寸。 <ol><li>通过其内侧 - 外侧和前侧 - 后侧长度的数字卡尺每天测量小腿尺寸。 </li><li>确定由式（D XD 2/6）小牛体积×3.14，其中D是长直径，d是荷瘤低级后肢的短径。 注:在正常成年小鼠小牛的大小是约40 - 50 立方毫米。其体积会增加在稍后阶段小腿将主要由肿瘤组织由于肿瘤生长和。 </li></ol></li></ol> 4.股动脉进行结扎（FAL）在荷瘤后肢缺氧诱导<ol><li>准备所需此操作的手术工具:CURVED或尖细镊子，镊子尖，手术剪刀和一个持针​​器。之前使用高压釜或热珠灭菌手术消毒这些工具。此外，已准备好该手术精细棉签。 注:建议工具可以在手术期间根据需要的提示重新灭菌。 </li><li>注入镇痛剂（卡洛芬5毫克/千克）经皮下（SQ）。检测和确认缺氧，注入hypoxyprobe-1（pimonidazole，60毫克/千克）。 注意:此剂量相当于每只小鼠1.5毫克和通过注入0.1的PBS腹膜内（IP）的15毫克/毫升hypoxyprobe溶液毫升实现。该hypoxyprobe然后通过免疫动物组织中检测到死后。 </li><li>鼠标放置在含有3麻醉诱导腔 - 在100％的氧气5％异氟烷以1升/分钟的流速。 </li><li>留在感应腔室中的小鼠，直到它是响应于外部刺激。然后从我删除动物nduction室。放置动物在上放置的操作表面上的变暖垫顶上的无菌披盖仰卧位。使用鼻锥将其连接到1的连续流动 - 以0.8升/ min的流速3％异氟烷在100％的氧。 <ol><li>应用无菌非药物眼药膏每只眼睛预防角膜干燥。彻底脱毛手术区，应用脱毛膏，留在皮肤上的时间不超过10秒。然后擦去用乙醇消毒片脱毛膏。 </li></ol></li><li>延伸，并与来自小鼠的中线大约45度的一块胶带固定后肢。一旦后肢是安全的，擦拭用10％聚维酮/碘拭子/溶液，接着乙醇裸露的皮肤，重复各两次。对于手术过程的剩余部分，使用立体显微镜来获得后肢区域的放大图。 </li><li>用尖头镊子和手术剪，使SK的切口在大约长1厘米，从向腹股沟区，大腿中部。用盐水浸湿细棉签，轻轻刷掉围绕大腿肌肉皮下脂肪组织。 </li><li>小心露出通过皮下脂肪组织通过钝性分离底层的股动脉。稳住伤口，手术野暴露中上收肌的血管。 </li><li>用细镊子，轻轻捅破通过膜质鞘股暴露神经血管束。使用干净组细钳子，解剖并在腹股沟，远端的腹股沟韧带靠近近侧的位置分开从股静脉和神经股动脉。请小心，以免刺入股静脉壁。 </li><li>继夹层，通过6-0丝线缝合的股的股动脉和远端的旋股外侧动脉（LCFA）的分支下面。闭塞使用双结（ 图3）的股动脉。 </li&gt; <li>关闭使用6-0聚丙烯缝合切口。关闭切口后，注入流体平衡疗法温生理盐水SQ0.5毫升。广场上的恢复笼中披保暖垫的顶部动物和连续监测，直到清醒。 </li><li>监视第一6小时期间将动物手术后和注入的镇痛剂（卡洛芬5毫克/公斤，SQ）每天3天。删除缝线采用无菌剪刀10天手术后。 </li></ol>由截肢5.原发肿瘤切除术注:截肢荷瘤低级后肢时小腿尺寸达到250毫米3为对照组或FAL后3天的低氧组。 <ol><li>从荷瘤肢体从胫骨远端剃光头发骨盆区域的头发剪的同时轻轻地捧着动物。注入过程之前的镇痛剂（卡洛芬5毫克/公斤，SQ）。 </li><li>鼠标放置到麻醉诱导通道含有3琥珀色 - 在100％的氧气5％异氟烷以1升/分钟的流速。留在感应腔室中的小鼠，直到它是响应于外部刺激。然后取出从感应室的动物。 </li><li>放置在右侧卧位的动物上放置在操作表面上的暖垫的无菌披盖。使用鼻锥将其连接到异氟烷1的连续流 - 以0.8升/分钟的流速的3％在100％的氧。应用无菌非药物眼药膏每只眼睛预防角膜干燥</li><li>制备，用10％聚维酮/碘拭子/溶液中的手术部位，接着乙醇，重复3次。应用消毒纱布（ 如手术单）通过鼠标来获得无菌手术领域。 </li><li>制作股骨中段环形切开皮肤，其次是钝性分离和近端皮肤回缩。暴露在腿部的中间侧股骨内侧神经血管蒂，然后结扎使用附近的涂4-0（聚乳糖910）可吸收缝合线材料腹股沟韧带。 </li><li>执行肌肉群用剪刀，接着软组织的钝器解剖到coxofemoral接头的中间股骨横断。用骨刀，进行中期股骨截骨术。使用无菌细棉签或吸收性明胶海绵，轻轻按下截骨部位，以减少和防止出血。 </li><li>采用手术创口夹子关闭覆皮肤，注入0.5毫升温生理盐水SQ的流体平衡疗法。广场上的恢复笼中披保暖垫的顶部动物和连续监测，直到清醒。 </li><li>在手术，收集来自原发性肿瘤为RNA，DNA或蛋白分离组织样品，在-80℃管理单元在液氮中并储存冷冻。对原代细胞培养，收集组织样品在此步骤中，如在下面章节9所述。固定在10％中性缓冲的福尔马林剩余肢组织用于组织学和immunochemis尝试，包括hypoxyprobe-1检测。 </li><li>监测动物运动，疼痛和食品消费在首6个小时的手术后，每天都有3天，然后。注入每日镇痛剂（卡洛芬5毫克/公斤，SQ）3天。用夹子伤口卸妆截肢后取出伤口夹10天。 </li></ol> 6.监测小鼠转移的存在<ol><li>每日观察小鼠并且每周评估它们对转移的临床体征的至少两倍。 <ol><li>观察动物对macrometastases表现为，在各个位置，典型地肩，对侧腿和爪发展群众的存在。为此，仔细触诊头部，颈部和腋下部位，及对侧后肢。检查与MRI扫描一起通过腹胀内部器官转移。 </li><li>通过与印出按xyphoid处理（胸骨的下端）检查肺转移的存在点¯x手指。 24 注:这种压力减少膈肌呼吸的能力。晚期肺癌转移的小鼠显示了由呼吸费力表现呼吸困难的症状。 </li><li>观察动物对于神经症状，如腿瘫痪和共济失调提示转移到中枢神经系统。 </li><li>监测小鼠每周至少一次，体重减轻，作为潜在的疾病进展的指示。体重损失超过预程序重量的15％，被认为是一种人道终点。 </li></ol></li></ol> 7.磁共振成像（MRI），用于检测转移<ol><li>执行的MRI在所需的时间点来检测转移。 18 注:在目前的研究中，使用了7特斯拉水平分光计。 MRI是在15日进行，35后截肢的SK-ES1细胞，并在为TC71细胞15天。 </li><li>鼠标放置到麻醉诱导chambe在30％的氧气和70％一氧化二氮的气体混合物3％异氟烷 - 含1河</li><li>留在感应腔室中的小鼠，直到它是响应于外部刺激。然后取出从感应室的动物。 </li><li>与呼吸和温度monitorization用1.5％异氟烷的连续施用和30％的一氧化二氮传送麻醉鼠标到立体定位架。应用无菌非药物眼药膏每只眼睛防止角膜干燥。图像动物无论是在全身或脑成像，分别为40或23毫米布鲁克鼠标体积线圈。 </li><li>使用二维，T2加权RARE序列:TR = 3000毫秒，TE = 24毫秒，矩阵= 256，FOV = 4.35点¯x3.0厘米，层厚= 0.5毫米，稀有因子= 4和平均数= 4 18 </li><li>将动物在一个温暖的笼子里恢复并持续监测，直到清醒。 注:由于使用麻醉浅平面老鼠会迅速恢复。 </LI&gt; <li>监视第一6小时期间，动物成像后，以确保有所述麻醉无不良影响。 </li></ol> 8.安乐死和尸检<ol><li>安乐死一旦本与转移通过MRI和/或疾病进展的临床症状可检测动物的小鼠。 注:在目前的研究，小鼠在50天处死25后截肢动物轴承SK-ES1和TC71移植，分别。在某些情况下，较早的安乐死是必要的，因为高转移负担。 </li><li>通过暴露安乐死小鼠（在10 CO 2 -安乐死室体积/分钟的30％）1.5升/分钟到CO 2。为了确保动物死亡，CO 2的曝光之后进行颈椎脱位。 </li><li>喷用70％乙醇在整个鼠标，并将其放置到层流罩。用25 G½针用1ml注射器，并转移到一个收集血液涂通过心脏穿刺收集的血液是含有2mg EDTA中。 </li><li>收集以下组织:脾，肾上腺，卵巢，肾，肝，肺，脑，右腿，骨髓来自肱骨和脊椎，以及存在于其他地方的宏观转移。 25,26 </li><li>固定半在10％的中性缓冲的福尔马林每个组织用于组织学和免疫组织化学，包括hypoxyprobe-1检测。卡扣冷冻在液氮中的另一半，然后在-80℃下的RNA，DNA或蛋白质分离储存。 25,26对于原代细胞培养，采集组织样本，如下面第9节。 </li></ol> 9.原代细胞培养<ol><li>执行原代细胞培养，解剖在层流罩剖检（上述第8节）在无菌条件下在从截断肢（上述第5节）或组织。 </li><li>对于用于原位注射的细胞系，补充有青霉素（200单位/ ml）制备细胞培养基适当链霉素（200微克/毫升），两性霉素B（1微克/毫升），和0.2％支原体预防性抗生素。放置把2.5ml主培养培养基到6厘米细胞培养板。 </li><li>选择从原发肿瘤或转移瘤存活肿瘤组织的区域，然后在2-3毫米的每个使用无菌剪刀隔离两到三个段。 注:存活肿瘤组织通常是在肿瘤的边缘发现，可以从坏死由其带粉红色或红色色彩，显著光泽和整体湿外观来区分。相比之下，坏死组织通常出现在肿瘤中心，并提出与沉闷的，俗气的外观白色/奶油色质量。 27 </li><li>传送分离段包含在步骤9.2中描述的初级培养基6厘米细胞培养板。 </li><li>在标准条件下培养的细胞，如第1（NOTE）和9.2描述。 18,28检查从组织碎片，所产生的细胞生长的文化WHICH应在几天内进行观察。一旦细胞达到汇合时，trypsinize并根据标准细胞培养技术繁殖它们。 注:原代细胞培养可随后用于评估的生长和从对照和缺氧肿瘤，以及它们的分子特征衍生细胞的转移特性，如先前所述。 18 </li></ol>

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The authors have nothing to disclose.

我们的模型涉及转移的两个实验组的比较-对照组，其中，肿瘤被允许在后肢在达到250 立方毫米小牛体积随后截肢开发和缺氧暴露组，其中，肿瘤轴承后肢经受FAL在相同体积，随后截肢3天后。即使在这些实验中FAL治疗的肿瘤都具有稍微延迟的截肢，相比于对照肿瘤，其大小不期间结扎和截肢之间的3天期间增加。因此，这种方法能够相媲美的大小但显着不同程度的缺氧肿瘤的转移潜能的比?...

尤文肉瘤（ES）是影响儿童和青少年积极恶性肿瘤。 1肿瘤的软组织和骨骼发育，常见于四肢。虽然转移的存在是ES患者最强大的不良预后因素，其发展背后的机制仍不清楚。 2肿瘤缺氧是在ES进展牵连的几个因素之一。在ES患者，非灌注区域的肿瘤组织中存在与预后不良相关联。 3 在体外 ，缺氧增加ES细胞的侵袭，并触发亲转移性基因的表达。 4-6然而，尽管这些证据，对缺氧诱导的ES进展和蔓延没有直接的证据存在。此外，由缺氧产生如此效果，目前的机制尚不清楚。因此，我们已经创建了一个体内模型，以填补现有体外数据和临床安装前后之间的间隙vations。此模型系统使得在它们的天然环境中存在的肿瘤的缺氧的影响直接检测，利用磁共振成像（MRI），以按照与体外病理学和分子分析（ 图1）组合的体内肿瘤进展和转移。 自的ES没有建立转基因模型是目前可用的，这些肿瘤的转移特性的体内研究依赖于人的细胞注射到免疫缺陷的小鼠。而采用免疫受损的动物可能会低估对疾病进展的免疫系统的影响，用人类细胞的能力提高了这些研究的译。在不同的异种移植模型，全身注射到尾静脉是最容易执行，但他们忽略肿瘤细胞血管内的最初步骤，并从经济增长的主站点逃脱。 7-12另一方面，orthoto<html> PIC异种移植，其中涉及肿瘤细胞注射到骨头（股骨，肋骨）或肌肉，更多的是技术上具有挑战性，也更生物学相关的人类癌症。 13-16转移发展之前然而，在过去，与原发肿瘤的快速生长相关的高发病率常常必要动物安乐死。在这项研究中，我们采用的细胞注射的先前建立模型到腓肠肌接着将得到的原发肿瘤与通过MRI纵向监测转移进展的组合的切除。 17,18这种注射成靠近胫骨腓肠肌允许在两个天然ES环境肿瘤生长-肌肉和骨骼-并导致远处转移到通常受在人类中的位置。 18因此，这种模式概括准确疾病进展过程中ES患者发生转移的进程。 帐篷"&gt;原发性肿瘤中的下后肢的定位也有助于血液供应肿瘤组织的精确控制。股动脉结扎（FAL）是血管生成的研究用来阻止血液流向的远侧区域中的良好建立的技术腿和调查组织血管响应于缺血19,20重要的是，在血流量的初始下降之后是侧枝血管开口和组织灌注FAL后约3天观察到20因此，在荷瘤肢执行时，该模型再现自然出现缺氧/再灌注事件在迅速生长的肿瘤，使由于灌注到经由新打开的侧支血管的下后肢的恢复转移性肿瘤细胞的逃逸。21重要的是，当肿瘤大小是必须执行此程序足够小，以防止在对照肿瘤过度缺氧（通常在荷瘤小牛体积150 UME - 250 立方毫米），确保控制和FAL治疗组之间的肿瘤缺氧的水平显著的差异。 除了缺氧对ES延迟的影响和转移的频率纵向监测，这种模式也允许组织的收集和从原发肿瘤和转移两个新的细胞系的开发。重要的是，以前的工作确定，转移来源的细胞系后，再引入显示出增强转移潜力动物，这表明肿瘤传播与在肿瘤细胞中的表型永久改变，并由此确认使用这些细胞系的破译转移性进程相关联。 18总的来说，这些模型现在可以用于识别缺氧诱导转移性途径所需的遗传和分子分析。 由于缺氧是一个亲转移因子增强各种T的恶性肿瘤umors，我们的模型可以作为一个平台来调查缺氧在其他类型的肿瘤，在四肢自然发展，如骨肉瘤和横纹肌肉瘤的作用。 21-23此外，类似的方法可以适用于恶性肿瘤在其它解剖学位置具有定义良好的血液供应的路线发展。最终，模型可以被修改，并且它的效用进一步延长，这取决于个体的研究需要。

<table><tbody><tr><td>SK-ES1 Human Ewing sarcoma (ES) cells</td><td>ATCC</td><td></td><td></td></tr><tr><td>TC71 Human ES cells</td><td></td><td></td><td>Kindly provided from Dr. Toretsky</td></tr><tr><td>McCoy&#039;s 5A (modified) Medium</td><td>Gibco by Life Technologies</td><td>12330-031</td><td></td></tr><tr><td>RPMI-1640</td><td>ATCC</td><td>30-2001</td><td></td></tr><tr><td>PBS</td><td>Corning Cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F2442-500mL</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Gibco by Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco by Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Fungizone&amp;reg; Antimycotic</td><td>Gibco by Life Technologies</td><td>15290-018</td><td></td></tr><tr><td>MycoZap&amp;trade; Prophylactic</td><td>Lonza</td><td>VZA-2032</td><td></td></tr><tr><td>Collagen Type I Rat tail high concetration</td><td>BD Biosciences</td><td>354249</td><td></td></tr><tr><td>SCID/beige mice</td><td>Harlan or Charles River</td><td>250 (Charles River) or 18602F (Harlan)</td><td></td></tr><tr><td>1 ml Insulin syringes with permanently attached 28&amp;nbsp;G &amp;frac12; needle</td><td>BD</td><td>329424</td><td></td></tr><tr><td>Saline (0.9% Sodium Chloride Injection, USP)</td><td>Hospira, INC</td><td>NDC 0409-7984-37</td><td></td></tr><tr><td>Digital calipers</td><td>World Precision Instruments, Inc</td><td>501601</td><td></td></tr><tr><td>Surgical Tools</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Rimadyl (Carprofen) Injectable&amp;nbsp;</td><td>Zoetis</td><td></td><td></td></tr><tr><td>Hypoxyprobe-1 (Pimonidazole Hydrochloride solid)</td><td>HPI, Inc</td><td>HP-100mg</td><td></td></tr><tr><td>hypoxyprobe-2 (CCI-103F-250 mg)</td><td>HPI, Inc</td><td>CCI-103F-250mg</td><td></td></tr><tr><td>Povidone-iodine Swabstick</td><td>PDI</td><td>S41350</td><td></td></tr><tr><td>Sterile alcohol prep pad</td><td>Fisher HealthCare</td><td>22-363-750</td><td></td></tr><tr><td>LubriFresh P.M. (eye lubricant ointment)&amp;nbsp;</td><td>Major Pharaceuticals</td><td>NDC 0904-5168-38</td><td></td></tr><tr><td>VWR Absorbent Underpads with Waterproof Moisture Barrier</td><td>VWR</td><td>56617-014&amp;nbsp;</td><td></td></tr><tr><td>Oster Golden A5 Single Speed Vet Clipper with size 50 blade</td><td>Oster</td><td>078005-050-002 (clipper), 078919-006-005 (blade)</td><td></td></tr><tr><td>Nair Lotion with baby oil</td><td>Church &amp;amp; Dwight Co., Inc.</td><td></td><td></td></tr><tr><td>Silk 6-0</td><td>Surgical Specialties Corp</td><td>752B</td><td></td></tr><tr><td>Prolene (polypropylene) suture 6-0</td><td>Ethicon</td><td>8680G</td><td></td></tr><tr><td>Vicryl (Polyglactin 910) suture 4-0&amp;nbsp;</td><td>Ethicon</td><td>J386H</td><td></td></tr><tr><td>Fisherbrand Applicators (Purified cotton)</td><td>Fisher Scientific</td><td>23-400-115</td><td></td></tr><tr><td>GelFoam Absorbable Dental Sponges - Size 4</td><td>Pfizer Pharmaceutical</td><td>9039605</td><td></td></tr><tr><td>Autoclip Wound Clip Applier</td><td>BD</td><td>427630</td><td></td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Autoclip remover</td><td>BD</td><td>427637</td><td></td></tr><tr><td>Aound clip</td><td>BD</td><td>427631</td><td></td></tr><tr><td>MRI 7 Tesla</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>Paravision 5.0 software</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Euthanasia system</td><td>VetEquip</td><td></td><td></td></tr><tr><td>25 G 5/8 Needle (for heart-puncture)</td><td>BD</td><td>305122</td><td></td></tr><tr><td>0.1 ml syringe (for heart-puncture)</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>K&lt;sub&gt;3&lt;/sub&gt;-EDTA Micro tube 1.3 ml</td><td>Sarstedt</td><td>41.1395.105</td><td></td></tr><tr><td>10% Neutral Buttered Formalin</td><td>Fisher Scientific</td><td>SF100-4</td><td></td></tr></tbody></table>

in vivo model for testing effect of hypoxia on tumor metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

以下的ES细胞入腓肠肌注入，原发肿瘤被允许生长至250mm的3小腿尺寸（ 图1，2）。所需的肿瘤的时间来达到这个体积典型地从10范围 - 15天为TC71至20-25天SK-ES1异种移植物，分别。以250mm 3显示出内源性缺氧的相对低水平的小牛体积的肿瘤，根据hypoxybrobe-1（pimonidazole）（肿瘤组织的约3％）染色（ 图4A中的C）。重要的是，在这些对照肿...

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In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

体内模型对缺氧对肿瘤转移的影响测试

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

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Research

JoVE Journal

Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

12.4K 次观看.  Georgetown University Medical Center. This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

视频: In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

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.

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

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

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 ...

科研

癌症研究

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

医学

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

脱细胞和细胞肺模型研究肿瘤转移

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进<em&gt;在原位</em灌注肺组织和X-gal染色<em&gt; lacZ的</em&gt;标记的肿瘤细胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

缺血再灌注损伤中转移性肝肿瘤的模样

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

肝细胞癌原位小鼠模型中的无创PET/MR成像

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

体内模型对缺氧对肿瘤转移的影响测试

摘要

探索更多视频

此视频中的章节

测试癌细胞的斑马鱼的血管侵袭能力（

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

脱细胞和细胞肺模型研究肿瘤转移

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进

缺血再灌注损伤中转移性肝肿瘤的模样

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

肝细胞癌原位小鼠模型中的无创PET/MR成像

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内模型对缺氧对肿瘤转移的影响测试

摘要

探索更多视频

此视频中的章节

测试癌细胞的斑马鱼的血管侵袭能力（

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

脱细胞和细胞肺模型研究肿瘤转移

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进

缺血再灌注损伤中转移性肝肿瘤的模样

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

肝细胞癌原位小鼠模型中的无创PET/MR成像

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移