The authors would like to acknowledge Alexandra Quackenbush for assisting with surgical procedures during filming of the video protocol, Hadley Holden for histological support, Sonali Jindal for input on tumor and liver pathology during method development, and Breanna Caruso for critical review of the manuscript. The D2A1 and D2.OR mammary tumor cells were a gift from Dr. Ann Chambers, the D2A1-GFP tumor cells were a gift from Dr. Jeffrey Green, and the 4T1 tumor cells were a gift from Dr. Heide Ford. The OHSU Advanced Light Microscopy Core at the Jungers Center was utilized for imaging. The work included in this manuscript includes funding from NIH/NCI NRSA F31CA186524 (to ETG) and NIH/NCI 5R01CA169175 (to PS).

本文中的所有动物的程序进行了审查，并在俄勒冈卫生科学大学机构动物护理和使用委员会的批准。 1.手术区及仪器的研制<ol><li>通过高压灭菌器在124℃制备的剪刀，镊子，以及止血30分钟，1 - 前计划手术2天。保证进入蒸压或无菌床上用品，笼具和食品手术后的恢复。 </li><li>制备无菌手术区，优选在层流罩。 <ol><li>擦拭手术区的所有表面用10％的漂白剂，包括加热垫，光源，麻醉管和鼻锥，以及外科套件，就可以在接近手术过程中的任何其他部分，而正在执行它。 </li><li>在无菌手术区，放置清洗加热垫无菌的悬垂性，光源，麻醉管和鼻锥，胰岛素注射器1毫升注射器，布比卡因，人工泪液，无菌生理盐水，2×2"无菌纱布海绵，4×4"无菌纱布，止血纱布切成0.5 -为1cm 2片，剪刀，镊子，止血钳，4-0薇乔缝线锥针，并在高压灭菌容器50×2％洗必泰。 </li><li>确保有空间在该空间用于储存在冰上制备肿瘤细胞。 </li><li>在邻近手术区的替补，准备与第二加热垫和干净笼子用无菌床上用品的回收区。 注:此区域也可房子件如珠灭菌。 </li></ol></li></ol> 2.门静脉注射<ol><li>一小时前计划打针，治疗的Balb / c小鼠年龄8 - 14周的100微升0.015毫克/毫升丁丙诺啡，皮下，疼痛管理。 注意:此注射方案可在任何年龄使用适当应用到雌性或雄性小鼠的任何菌株细胞系更改到应变。 </li><li>制备基于所述细胞系或选择的肿瘤外植体协议的肿瘤细胞注射。测试的鼠病原体存在给药前所有的肿瘤细胞系，以减少引入这种病原体到动物菌落的危险性。 <ol><li>对于同基因的Balb / c肿瘤细胞系，包括D2A1，D2.OR，和4T1肿瘤细胞，解冻细胞变成在10cm组织培养板中在注射前3天使得第二天细胞是在约90 - 100％汇合。 </li><li> 1天以下肿瘤细胞解冻洗涤细胞一次用1×磷酸盐缓冲盐水（PBS）和t​​rypsinize使用2毫升0.05％胰蛋白酶的汇合的肿瘤细胞在37℃下5分钟。加入8 ml完全培养基（DMEM高葡萄糖，10％胎牛血清，2mM的L-谷氨酰胺和1x青霉素/链霉素）和通道1:10到一个新的10cm皿用10ml完全培养基中。 </li><li>在注射当天，洗细胞一次用1×PBS和叔如上所述rypsinize。 </li><li>胰酶消化重悬细胞的8ml完整的媒体，旋转5分钟，在1500 XG，删除媒体并悬浮在5毫升1X PBS。 </li><li>指望使用可行性分析台盼蓝排除血球细胞。悬浮用于注射在1×PBS中的细胞以一个预先确定的浓度和体积。 注:5 - 推荐10微升较小的注射量防止肝脏造成不必要的损害。 </li><li>保持细胞在冰上注射的持续时间。以下注射完成后，在完全培养基返回细胞到实验室的样品，并放置在培养1天，以确保可行性。 </li></ol></li><li>放置麻醉下鼠标2 - 中的氧输送2.5％的异氟醚（2-氯-2-（二氟甲氧基）-1,1,1-三氟乙烷）。使用加热垫保持体温。通过评估到脚趾捏的反应确保完全麻醉，然后保持anestheSIA在2 - 2.5％异氟醚。 注:监测呼吸率的动物，并在整个过程相应地调整异氟烷流速是很重要的。 </li><li>放置人工泪液或兽医软膏少量每只眼上，以避免在手术过程中对眼睛的过度干燥。 </li><li>鼠标放置在仰卧位置，其与腹部露出背部。 </li><li>擦拭用化学脱毛剂的区域上的腹侧左侧从第二肋空间啮齿动物的下降到第 4腹股沟乳腺的乳头移除毛发。让脱毛坐1 - 2分钟，然后用纱布和H 2 O.这一步可以做到1完全删除-提前2天，如果众多的手术计划，以节省时间。 </li><li>需要一个2×2"无菌纱布海绵（2％洗必泰浸泡），在脱毛的部位擦拭鼠标。消毒的整个周边地区，包括尾巴，尽量减少细菌续仪器胺化。 </li><li>擦拭脱毛的部位及周边地区下用酒精消毒片。 </li><li>重复2％洗必泰和酒精步骤一次和最后完成洗必泰擦拭下来一共有三个2％洗必泰和两个酒精片洗涤。做最后的洗必泰擦拭，这样的化学品绕不手术部位滴落，避免内脏越来越洗必泰。注意:可能导致体温显著下降的大量氯己定和醇对皮肤和周围毛皮中的应用。不要使用过程中的步骤2.7-2.9过量体积擦拭。保持体温用加热垫。 </li><li>使用无菌手套，并用无菌刀片无菌手术刀，使一个单一的1英寸切口进入鼠标的左侧的中位数和矢状平面之间的皮肤，开始只是肋下方和刚刚第四腹股沟的平面上方结束乳腺乳头。 </li><li>使用高压灭菌或消毒珠剪刀和镊子，做一个类似的1英寸的切口进入腹腔。不要切到乳腺脂肪垫，确保不切肠，肝，或隔膜。 </li><li>放置浸泡在无菌盐水的小鼠，其中该切口制成，例如内脏可以放在纱布和不与周围的皮肤或外科区域接触的左侧4×4"纱布垫。 </li><li>通过上下吹打数次肿瘤细胞将制备鼠标期间沉降制备肿瘤细胞。制备25μl的可移动针注射器和32号针头与肿瘤细胞。推注射器直至肿瘤细胞是在针的顶端与柱塞是在注射适当的体积;避免气泡的注入。 </li><li>擦拭针的外面用无菌酒精垫以除去任何外部的肿瘤细胞。请小心，避免扎伤。 </li><li>按住中间的sid切口，包括皮肤和腹膜衬里，一边用钳子e和使用无菌棉签小心地将大，小肠子出来，将它们放置于灭菌生理盐水浸泡消毒纱布。拉出大肠和小肠，直到门静脉可视化。 </li><li>盖在盐水浸湿的纱布内脏，保持内部的水分和不育。 </li><li>有一个助理，还戴无菌手套，轻轻地握裹在盐水浸湿的纱布闪开用消毒棉肠子放倒棉签充分显露门静脉。另外，可能有必要使用蒸压止血钳或钳子到一旁保持组织上的切口的中间侧。 </li><li>插入装载有肿瘤细胞〜3的针 - 5毫米深处门静脉〜肝脏10毫米以下一角度&lt;5°到静脉，以斜角朝上。缓缓注入含有肿瘤细胞的最大音量。允许血液流过针ħEAD几秒钟，以避免后面的肿瘤细胞的流出静脉。最大限度地减少在注射过程中移动在静脉针。再次，谨慎使用，以免扎伤。 注意:门静脉的可视化是不放大完成，但如果优选的，可以使用立体显微镜。 </li><li>卸下针，同时放置一个无菌棉签在静脉压力。与助手仍持有肠子一旁放置一块0.5 - 1 平方厘米的止血纱布上静脉注射部位。 注:止血粉也尝试了这一步的协议，但并没有有效阻止以下注射静脉失血。 </li><li>从无菌棉签5分钟保持在注射部位有压力的止血纱布。 </li><li>通过仔细地提起止血纱布，如果纱布粘附到周围的组织，与不孕少量评估静脉闭合Ë盐水可以用来浸泡并解除了纱布。 </li><li>如果此时发生失血，放置在与压力站点止血纱布的附加一块额外5分钟。当血流完全停止，取出鼠标纱布。 注:在手术过程中失血必须仔细评估，如果总让出血量达到或超过（基于研究者的机构审查委员会的监管标准操作程序）鼠标必须同时在心脏灌注麻醉下实施安乐死。 </li><li>一旦注射部位被视为完整的，没有血液离开注射部位，放置内脏轻轻放回腹腔。 </li><li>缝合腹膜衬里，然后用简单的连续或间断缝合图案用无菌4-0薇乔缝合和锥形针的皮肤。通常情况下，关闭切口需要缝合10-15。 </li><li>注入100微升补脾的vacaine（5毫克/毫升）沿使用胰岛素注射器切口部位为局部疼痛管理。用1毫升注射器26号针头水化注入0.5毫升无菌生理盐水皮下。手术需要15 - 25分钟才能完成。 </li><li>在整个手术保持无菌条件下，确保所有的工具和材料进入鼠标，包括戴手套的手接触，进行清洁以适当联系前。在可能的情况使用的无菌材料和手套，或最低限度利用70％乙醇溶液或10％漂白溶液清洗。 </li><li>如果多次手术计划为单个会话重拍新鲜无菌的悬垂性，胰岛素注射器1毫升注射器，无菌生理盐水初期手术区，2×2"无菌纱布海绵，4×4"无菌纱布，止血纱布切成0.5 - 为1cm 2片，用锥形针4-0薇乔缝合线，和2％氯己定。珠消毒手术的剪刀，镊子，止血和在并允许到充分冷却前再次使用。 </li></ol> 3.恢复，监控鼠类健康和终点分析<ol><li>外科手术完成后，维持在无寝具-，洁净笼回收了至少20分钟的加热垫的小鼠。小鼠通常需要2 - 4分钟从麻醉中醒来。 </li><li>不要返回已进行手术，同居与其他动物，直到它已完全麻醉中恢复的动物。而它的苏醒不要让动物无人值守和监控，直到动物已经恢复，以保持自己在胸骨斜卧的能力。 </li><li>给小鼠0.05 - 0.1毫克/千克丁丙诺啡疼痛管理每6-12小时手术后，时间长达72小时。 </li><li>检查缝合线每天以确保它们愈合过程中保持不变。在这种缝合来撤消的情况下，将鼠标麻醉下，除去剩余的缝线，并更换。在感染或inflamm的情况下通货膨胀请教兽医人员。 注:感染或炎症尚未遇到此协议。 </li><li>对于转移研究，进行日常健康检查，直至观察到，包括转移性疾病的外在迹象，但不限于:&gt; 10％的重量损失/收益，scruffiness，注意输给周围，腹部水肿/腹水，苍白的眼睛和耳朵，或驼背的位置。 注:开发使用新的细胞系或细胞浓度使用模型时，直到转移的时间轴是很好理解的每日健康检查是特别重要的。 </li><li>在研究终点，用CO安乐死小鼠吸入2颈椎脱位。 注:由调查"监督委员会的批准安乐死的替代方法可以采用，包括用1×PBS灌注，而麻醉下。肝脏的灌注是通过cannulating门静脉，剪断下腔静脉，并推压1×PBS中第执行粗糙以4毫升/分钟为1的速率的肝脏血管 - 2分钟以除去循环血液和白细胞。 注意:根据端点的研究中，细胞系，和所使用的浓度，明显的肝转移可以或不可以在尸检是显而易见的。微转移病变可能与肝的组织学分析显现出来。端点会根据研究设计。 </li><li>通过首先去除胆囊提取用剪刀和镊子肝脏，然后通过下腔静脉优于肝脏切断。通过腔静脉，门静脉和肝动脉劣于肝脏切。 </li><li>轻轻取下整个肝脏和在1X PBS洗5倍。 </li><li>分离肝分为左，右，中位数，和尾状叶。 </li><li>同时在室温下振摇，在10％中性缓冲的福尔马林福尔马林修复肝48小时。流程组织中，通过增加浓度和二甲苯一系列醇;石蜡嵌入固定的组织71 </s了&gt;。 注意:可替换地，肝脏可以通过将组织与最佳切割温度（OCT）式的cryomold和冻结在95％乙醇中浸没干冰颗粒单元冷冻。 <ol><li>使用切片机，切成石蜡包埋的组织块，使得组织的匹配头大小显露。 </li><li>切五个4微米连续切片，这构成用于分析的第一级。 </li><li>通过组织250微米切，在垃圾扔这些部分。 </li><li>在250微米切割五4微米的连续切片第二级。 </li><li>在整个肝脏必要时重复节。 HE染色每一级的第一部分以评估转移72。 注:对于快速冷冻的组织使用低温恒温器切段。 注:微转移病灶的检测将需要肿瘤细胞的特异性染色。 </li></ol></li><li>从研究中移除老鼠如果在切口有肿瘤生长坐即，因为这表明肿瘤细胞渗漏到下面的注入腹腔。 注意:此问题还没有被观察到。 </li></ol>

The authors have nothing to disclose and declare no competing financial interests.

在Balb / c小鼠门静脉注射模型允许乳腺癌病灶的研究中在没有混杂多器官转移的肝脏，并在充分的免疫感受态宿主。我们的协议是允许进入门静脉对肿瘤细胞的直接注射到肝16,55,56先前公布的外科手术过程的进步。我们已经取得一个进步是显著减少注射肿瘤细胞的数量从≥1×10 5细胞/注射16,55,56降至≤万肿瘤细胞/注射。我们还扩展为乳腺癌转移到肝脏的研究模型。使用该协议，与已知的转移两个可能乳癌细胞系开发具有更短的延迟肝转移比更静态的乳腺肿瘤细胞系。此外，在早期时间点，肿瘤细胞在整个肝实质作为单个CE的单个或一小群分布肿瘤细胞注射后LLS。该模型有望解决转移效率问题，包括肿瘤细胞外渗，细胞存活，休眠，和增殖 - 有助于微转移和公开转移性疾病的肝脏中发展的所有表型。 考虑之前发起的研究门静脉注射方案的许多方面是很重要的。强烈建议小心决定细胞系，细胞浓度，总细胞数，和基于较小探索性研究兴趣端点。另外，使用的免疫能干主机和同基因细胞系是对于理解宿主 - 肿瘤细胞相互作用至关重要。新开发的"发光头"小鼠表达EGFP和荧光素酶从垂体前叶是用于消除宿主反应外感EGFP和荧光素酶的重要工具，蛋白质经常用来标记乳腺肿瘤系75 </sup&gt;。在"发光的头"鼠标和同系的使用标记的肿瘤细胞会方便识别单个细胞播散和微转移灶免疫组化，而不对绿色荧光蛋白或荧光素酶炎症反应的关注。同样，强烈建议仔细选择疼痛管理策略，以确保最小抗或药物治疗方案有利于肿瘤的影响。在这个协议中的关键步骤包括:整个手术无菌维护条件，确保感染不会发生，因为这会混淆任何结果。同样重要的是，该针被适当地放置在门静脉，以确保肿瘤细胞被递送到肝脏。践行着染料如墨汁的协议将有助于这个问题。在皮肤切口部位肿瘤的生长发生不当针的位置的最佳指标。最后，这是至关重要的来自门静脉血液损失充分控制，并且停止向suturi完全事先纳克动物。使用止血纱布的大大削弱来自门静脉注射后不受控制失血的风险。在我们手中，由于从门静脉注射后失血程序相关的死亡率从30％降低到小鼠2％与使用止血纱布。 值得注意的是，门静脉注射模型不复制的充分转移级联是很重要的，但不限于肿瘤细胞外渗，肿瘤细胞小生境相互作用以下外渗和肿瘤生长研究。模型准确复制的充分转移级联到肝脏，如发生在患者中，迫切需要。门静脉注射模型的一个额外的限制是，它是由手术的主机上的冲击混淆，与已知影响疾病的进展76,77伤口愈合。 门静脉注入模型代表了其他注射的改进模型来研究肝转移，包括心内和脾内的机型。具体地，门静脉注射模型允许更大范围的疾病进展比心内模型，这通常是由在其他组织中伴随转移限制的研究。此外，门静脉模型不被取出脾脏复杂的，因为在脾内模型完成。 门静脉注射模型可以证明对于肝转移研究一般的有用工具。肝转移是腺癌转移的最频繁的部位整体，在胰腺癌特别高率（转移85％的肝），结肠和直肠的腺癌（&gt; 70％），以及胃和食管（&gt; 30 ％）1。虽然胰腺癌和结肠癌腺癌的自发和原位原发肿瘤模型更容易转移到肝脏78,79，门静脉注入模型可以提供图片E要了解这些癌症的转移过程中肿瘤细胞的控制下交付有用允许生物化学，分子和组织学评估，在肿瘤细胞后到达指定的时间。总之，门静脉注射模型表示对乳腺癌的可用肝转移模型的一个重要改进，并且通常还可以是适用于肝转移字段。

乳腺癌转移至肝 肝脏是乳腺癌转移的一个共同的部位，骨和肺1-3沿。乳腺癌患者肝转移是非常差的结果4,5独立的预后因素，如乳腺癌患者肝转移范围中位生存期从4.8至15个月6-9。与此相反，乳腺癌患者的肺或骨转移有9至27.4个月8,9和16.3〜56个月8,10-12，分别平均存活率。转移是一个多步骤的过程，称为转移级联，这与在原发肿瘤的肿瘤细胞的传播的开始与患者的死亡率因远处器官13-15中循环的肿瘤细胞的播种和生长结束。转移的啮齿动物模型已经表明，转移级联是非常低效的，只有0.02 - 10％的循环肿瘤细胞建立显性转移16,17。转移性低效的一个主要瓶颈是由唯一的组织微环境在辅助站点决定的，称为转移性龛18，突出理解位点特异性转移的重要性。转移性利基是唯一的复发的部位，并且，在部分，其特征在于由不同的细胞外基质蛋白19,20的沉积，各种免疫细胞群21-23的渗透，和改变的组织稳态包括失调生产大量细胞因子，趋化因子和生长因子15,18,24,25。因此，组织特异性转移利基的理解之前的如何定位转移性疾病的认识。然而，肝转移的可靠的模型所缺乏的。此外，改进的肝转移模型将是识别用于乳腺癌患者的Wi新颖目标和有效的治疗方法必需日肝转移。 建立的模型，以乳腺癌转移研究肝 目前可用的模型来研究乳腺癌转移到肝脏包括在免疫缺陷小鼠的人肿瘤细胞异种移植物。这些模型通常使用充分研究的人乳腺癌细胞系如MCF-7和MDA-MB-231和裸体，RAG1 - / -或SCID免疫缺陷鼠主机26-29。异种移植模型提供涉及人来源癌细胞系的优点，但是，在转移30-32和在治疗性33-35给出了免疫细胞的最近升值，转移在一个完全的免疫感受态宿主的研究是至关重要的。模型来研究乳腺癌转移到在免疫活性宿主的肝脏包括原位注射同基因的肿瘤细胞（ 例如 ，4T1和D2A1细胞系）的到乳腺脂肪垫，有或没有外科切除原发肿瘤和转移36-38的后续评估。值得注意的是，肝转移从原位移植模型的速率是非常低的或相对于其他转移部位，如肺39,40不存在的，或者是建立肺转移后发生，肝特异性转移37,39的研究并发。 从自发遗传工程乳腺癌模型肿瘤外植体可以被重新注入到幼稚主机作为同基因肿瘤细胞的乳腺脂肪垫。例如，最近有报道，从K14 的Cre ECAD F / F P53 F / F小鼠自发性肿瘤哪个型号浸润性小叶乳腺癌，肿瘤发展原位时注入野生型主机。下列手术切除这些肿瘤一旦达到15 mm 2时，小鼠的18％发展为肝脏转移40,41。第三种方法来模拟肝转移开发前景自发转移在遗传工程小鼠。迄今为止，这很容易扩散到肝脏乳腺癌转移的自发小鼠模型的报告是少见。例外情况包括H19-IGF2，p53的FP / FP MMTV-Cre重组酶的Wap-Cre重组酶和K14 的Cre ECAD F / F P53 F / F遗传工程小鼠模型，其中肝转移小鼠的比例很低开发38,41- 43。因此，虽然基因工程小鼠模型有助于转移级联，提供强大的临床相关模型的各个阶段的研究中，他们是有限的，由于肝转移38的低利率。 几个转移模型绕过转移级联的初始步骤包括从原发肿瘤和血管内的肿瘤细胞的传播。这些模型允许调查的后续步骤转移级联，从外渗到二级站点建立肿瘤。的intracardiac喷射模型提供肿瘤细胞进入左心室，它通过主动脉瘤细胞分配到循环系统。心内注射需要注射部位或其他成像方式，如荧光素酶的生物发光的超声引导下的成像标记为细胞，以确认成功的注入。通过左心室肿瘤细胞注射可能导致骨，脑，肺，和/或肝脏转移，除其他器官44-48。因为多器官转移，这些小鼠经常需要公开肝转移显影之前进行安乐死，否定充分调查肝脏内的转移性生长的能力。该多部位转移的发展显著最小化的另一种方法是在脾内注射模型。脾内注射经由与肠系膜上静脉联接成为门静脉49,50脾静脉提供肿瘤细胞。动物可以outgr被监控owth在肝脏转移性病灶因为其他部位形成转移的是罕见的，并且作为结果，动物的总体健康状况被保持49,50。然而，要注意的是，脾内模型要求脾切除，以避免脾肿瘤49,50是很重要的，一个过程，影响免疫功能。例如，心肌缺血再灌注损伤的特征在于的Ly6C +单核细胞亚群，从脾脏起源和是缺血51,52伤口愈合过程中负责吞噬细胞和蛋白水解活性的浸润。与脾切除，有在单核细胞群体所观察到的减少了有助于伤口愈合52。另外，脾切除已显示减少原发肿瘤的生长和肺转移的非小细胞肺癌模型中，特别是通过在循环内和肿瘤CCR2 +细胞CD11b +的Ly6C +单核髓的数量减少ID细胞53。另外，脾切除以下结肠癌细胞的脾内注射导致肠系膜淋巴结和肝转移升高54抗肿瘤自然杀伤细胞的水平降低。总之，这些发现表明，脾切除与转移性细胞命运随后后果损害免疫系统的作用。 肝转移的门静脉注入模型 调查乳腺癌转移到肝脏在完全的免疫感受态宿主，其中小鼠不损害由于多器官转移的条件下，门静脉注射模型。静脉内注射模型此前已用于研究的大肠癌55,56和黑素瘤16细胞系肝转移;在这里，我们描述了门静脉注射的应用同基因乳腺肿瘤细胞转移模型。该模型可用于研究转移级联的后期阶段，包括乳腺癌细胞外渗和播种，关于死亡/增殖/休眠肿瘤细胞命运的决定，并生长成显性病变。在这种模式下，同基因乳腺肿瘤细胞系通过免疫活性的Balb / c雌性小鼠的门静脉，即首先和直接将肿瘤细胞到肝脏不除去脾脏的方法注入。为了开发这种模式，利用被雇用的范围内的转移能力，从低到高四乳腺肿瘤细胞系:D2.OR，D2A1和4T1，并采用D2A1标记的绿色荧光蛋白（D2A1-GFP），以探讨肿瘤细胞注射后早期的时间点。 4T1是从410.4肿瘤，在一个MMTV +的Balb / c雌性小鼠36,37自发产生和转移至肺，肝，脑，和骨从乳房脂肪垫的原发性肿瘤39,57,58衍生的高转移性细胞系。 D2A1肿瘤细胞WERE也最初从D2的增生的肺泡结节细胞移植之后在一个只Balb / c主机引起的自发乳腺肿瘤衍生的，并证实为从原发肿瘤转移至肺59,60。 D2.OR肿瘤细胞是一种非转移性姐姐线到D2A1线，虽然他们逃脱原发肿瘤及辅助站点到达时，他们很少建立远处转移60,61。 另外，为了避免使用通常用于疼痛管理的药物包括非甾体类抗炎药（NSAIDs）期间或之后的手术过程是重要的。的NSAIDs有在某些乳腺癌62-65的抗肿瘤活性，和NSAID的一些类增加肝66,67的风险，潜在地损害肝脏转移的研究和肝转移利基。此外，研究表明，NSAIDs的直接影响组织的微环境，降低亲转移性分机racellular基质蛋白肌腱蛋白-C 68和纤维状胶原62,65。可替代地，使用阿片样物质衍生物，丁丙诺啡，被使用，因为其在啮齿类动物疼痛管理69功效由于缺乏证据和阿片类具有抗肿瘤活性的70。这个门静脉注射模型为5小注射体积优化 - 10微升，以避免对肝造成不必要的损害。该模型也被优化，以包括具有较小直径（≥32表压），利用止血纱布的立即注射的过程中，以减少失血以下针。相对于这些优化注射参数，细胞数目应的个体基础上确定，基于所述细胞系的致瘤潜力。但是，建议开始≤10,000个细胞/注射长期研究。对于更短的端点（ 例如，24小时后注射）相当多的肿瘤细胞（ 例如 ，1×10 5 -如有必要1×10 6个 ），可以使用。总之，这里详述的门静脉注射模型表示为乳腺癌转移到肝脏研究的有用的工具和规避其他一些肝转移模型的局限性。这种模式有利于肿瘤细胞外渗，播种，免疫小鼠主管主机存活，增殖和休眠和转移性生长早期的命运决定的研究。

<table border="0" cellpadding="0" cellspacing="0" width="1001">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">1 ml Syringe w/ 26-gauge Needle</td>
			<td style="width:159px;">BD Syringe</td>
			<td style="width:152px;">309597</td>
			<td style="width:435px;">For subcutaneous buprenorphine injection; use caution, sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Alcohol Prep Pads</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:152px;">06-669-62</td>
			<td style="width:435px;">For cleaning of abdomen prior to surgical incision</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">All Purpose Sponges, Sterile</td>
			<td style="width:159px;">Kendall</td>
			<td style="width:152px;">8044</td>
			<td style="width:435px;">4&#34; x 4&#34;, use dipped in sterile saline to keep large and small intestines protected and hydrated during surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Artificial Tears</td>
			<td style="width:159px;">Rugby</td>
			<td style="width:152px;">370114</td>
			<td style="width:435px;">Mineral oil 15%, white petrolatum 83%; use to protect eyes during surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Buprenorphine HCl, 0.3 mg/ml</td>
			<td style="width:159px;">Mfg. by Reckitt Benckiser</td>
			<td style="width:152px;">NDC-12496-0757-1</td>
			<td style="width:435px;">Use at 0.05 - 0.1 mg/kg body weight, 1-2x daily for 72 hours, injected subcutaneously</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Bupivacaine HCl, 0.5% (5 mg/ml)</td>
			<td style="width:159px;">Mfg. by Humira Inc</td>
			<td style="width:152px;">NDC-04091163-01</td>
			<td style="width:435px;">Use at 0.5%, 1x immediately after surgery, 10 ul injected subcutaneously at incision site&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">anti-CD45 antibody</td>
			<td style="width:159px;">BD Pharmingen</td>
			<td style="width:152px;">550539</td>
			<td style="width:435px;">Use in multiplex immunofluorescence to exclude leukocytes in identification of metastatic foci</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Celox&#8482; Rapid Hemostatic Gauze</td>
			<td style="width:159px;">Medtrade Products Ltd.</td>
			<td style="width:152px;">FG08839011</td>
			<td style="width:435px;">Cut into 5mm&#178; pieces, use to stop blood flow out of the portal vein with pressure following injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Chemical Depilatory</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Use to remove hair from surgical area; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Chlorhexidine, 2% Solution</td>
			<td style="width:159px;">Vet One</td>
			<td style="width:152px;">1CHL008</td>
			<td style="width:435px;">Use caution, do not get chlorhexidine in mucous membranes or ears of the mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">anti-CK18 antibody</td>
			<td style="width:159px;">Abcam</td>
			<td style="width:152px;">ab53118</td>
			<td style="width:435px;">Use in multiplex immunofluorescence to identify metastatic foci</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Cotton Tipped Applicators, Sterile</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:152px;">23-400-114</td>
			<td style="width:435px;">6&#34; Wooden Shaft 2 pc/envelope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">DMEM, High-Glucose</td>
			<td style="width:159px;">HyClone</td>
			<td style="width:152px;">SH30243.01</td>
			<td style="width:435px;">Cell culture media base for use with D2A1, D2.OR, and 4T1 mammary tumor cell lines</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Dry Glass Bead Sterilizer</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Use between surgeries to sterilize stainless steel tools, use caution, extremely hot; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Ethanol, 70% solution</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Use caution flammable; use to clean surgical area as needed; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fetal Bovine Serum</td>
			<td style="width:159px;">HyClone</td>
			<td style="width:152px;">SH30071.03</td>
			<td style="width:435px;">Cell culture media additive for use with D2A1, D2.OR, and 4T1 mammary tumor cell lines, use at 10% in DMEM high glucose</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Gauze, Sterile</td>
			<td style="width:159px;">Kendall</td>
			<td style="width:152px;">2146</td>
			<td style="width:435px;">2&#34; x 2&#34;, use dipped in chlorhexidine 2% solution for cleaning of abdomen prior to surgical incision</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">anti-Green Fluorescent Protein antibody</td>
			<td style="width:159px;">Vector Laboratories</td>
			<td style="width:152px;">BA0702</td>
			<td style="width:435px;">Use to stain for GFP tagged D2A1 or other mammary tumor cell lines</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">L-Glutamine 200 mM (100X)</td>
			<td style="width:159px;">Gibco</td>
			<td style="width:152px;">25030-081</td>
			<td style="width:435px;">Cell culture media additive for use with D2A1, D2.OR, and 4T1 mammary tumor cell lines, use at 2 mM (1x) in DMEM high glucose</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Heating Pad, x2-3</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Use to maintain body heat during surgery and recovery; multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Hemocytometer</td>
			<td style="width:159px;">Hausser Scientific</td>
			<td style="width:152px;">1483</td>
			<td style="width:435px;">For use in cell culture to count cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Hemostatic Forceps</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Stainless steel; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">anti-Heppar-1 antibody</td>
			<td style="width:159px;">Dako</td>
			<td style="width:152px;">M7158</td>
			<td style="width:435px;">Use in multiplex immunofluorescence to exclude hepatocytes in identification of metastatic foci</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Insulin Syringe, 0.3 ml, 29-gauge</td>
			<td style="width:159px;">BD&#160;&#160;</td>
			<td style="width:152px;">324702</td>
			<td style="width:435px;">For bupivacaine injection at suture site; use caution, sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Isoflurane</td>
			<td style="width:159px;">Piramal</td>
			<td style="width:152px;">NDC-66794-017-25</td>
			<td style="width:435px;">Administered at 2.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Isoflurane Vaporizer</td>
			<td style="width:159px;">VetEquip</td>
			<td>911103</td>
			<td style="width:435px;">Use caution, vaporizes anesthetic gases</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Light Source</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Use for visualizing the surgical field; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Neutral buffered formalin, 10%</td>
			<td style="width:159px;">Anatech Ltd.</td>
			<td style="width:152px;">135</td>
			<td style="width:435px;">Use caution, toxic; use as a tissue fixative for metastasis endpoints and assesment of metastatic burden by histology</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Opal&#8482; 4-color fIHC kit</td>
			<td style="width:159px;">PerkinElmer, Inc.</td>
			<td style="width:152px;">NEL794001KT</td>
			<td style="width:435px;">Use for multiplex immunofluorescence of Heppar-1/CD45/CK18 to detect metastases in murine liver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Operating Scissors</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Stainless steel; use caution, sharp; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Penicillin/Streptomycin, 100x</td>
			<td style="width:159px;">Corning</td>
			<td style="width:152px;">30-002-Cl</td>
			<td style="width:435px;">Cell culture media additive for use with D2A1, D2.OR, and 4T1 mammary tumor cell lines, use at 1x in DMEM high glucose</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Phosphate-buffered Saline</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Use at 1x for resuspending tumor cells prior to injection, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Removable Needle Syringe, 25 ul, Model 1702</td>
			<td style="width:159px;">Hamilton</td>
			<td style="width:152px;">7654-01</td>
			<td style="width:435px;">For portal vein injection; use caution, paricularly while working with tumor cell-loaded needles, sharp when needle is attached</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Scalpel handle</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Stainless steel; multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Scalpel blade, #15</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Carbon steel, sterile, size 15; multiple suppliers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Small Hub Removable Needles, 32-gauge</td>
			<td style="width:159px;">Hamilton</td>
			<td style="width:152px;">7803-04</td>
			<td style="width:435px;">For portal vein injection, 1&#34; length, point style 4, 12&#176;angle, 33- to 34-gauge reusable needles can also be used; use caution, paricularly while working with tumor cell loaded needles, sharp</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Sodium Hypochlorite</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Use caution, corrosive; use at 10% to disinfect workspace and surfaces, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Sterile Saline</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:152px;">BP358-212</td>
			<td style="width:435px;">0.9% NaCl solution; alternatively, can be homemade and sterile filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Surgical Gloves, Sterile</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Sutures, Sterile&#160;</td>
			<td style="width:159px;">Ethicon</td>
			<td style="width:152px;">J310H</td>
			<td style="width:435px;">4-0 27&#34; coated vicryl w/ 22 mm 1/2c taper ethalloy needle; use caution, sharp</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Table Top Portable Anesthesia Machine</td>
			<td style="width:159px;">VetEquip</td>
			<td>901801</td>
			<td style="width:435px;">Use with isoflurane vaporizer for mouse anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Thumb Dressing Forceps</td>
			<td style="width:159px;"></td>
			<td style="width:152px;"></td>
			<td style="width:435px;">Stainless steel, serrated, blunted; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Towel Drapes, Sterile&#160;</td>
			<td style="width:159px;">Dynarex</td>
			<td style="width:152px;">4410</td>
			<td style="width:435px;">18&#34; x 26&#34;, to cover heating pad and provide a sterile workspace during surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Trypan Blue</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:152px;">T10282</td>
			<td style="width:435px;">For use in cell culture to assess viability, use 1:1 with cells in 1x PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Trypsin/EDTA, 0.05% (1x)</td>
			<td style="width:159px;">Gibco</td>
			<td style="width:152px;">25300-054</td>
			<td style="width:435px;">Use in cell culture to detach tumor cells from tissue culture plates</td>
		</tr>
	</tbody>
</table>

a portal vein injection model to study liver metastasis of breast cancer

乳腺癌是全世界妇女癌症相关死亡的主要原因。肝转移是参与乳腺癌转移的病例的30％以上，并导致仅为4.8中位生存率不良后果 - 15个月。乳腺癌转移的电流啮齿动物模型，包括原发性肿瘤细胞异种移植物和自发肿瘤模型，很少转移到肝脏。心内和脾内注射模型并导致肝脏转移，但这些模型可以通过二级伴随现场转移混淆，或免疫力受损，由于切除脾脏，以避免在注射部位肿瘤的生长。以解决需要改进的肝脏转移模型，鼠门静脉注射方法，该方法首先和直接将肿瘤细胞到肝脏的开发。该模型提供了肿瘤细胞向肝脏而不在其他器官或取出脾脏并发转移并发症。该选择在注射部位以控制失血10微升，≥32号针头，和止血纱布 - imized门静脉协议使用5的小注射体积。在Balb / c小鼠通过关于变转移潜能测试了三款同基因乳腺肿瘤行门静脉注射的方法;高转移性4T1细胞，温和转移性D2A1细胞和低转移性D2.OR细胞。在〜20中的等待时间的≤10,000个细胞/注射的结果的浓度 - 40天的肝转移具有较高转移性4T1和D2A1线的发展，而对于不太积极D2.OR线&gt;55天。这个模型是一个重要的工具，以乳腺癌转移研究到肝脏，并且可以适用于频繁转移至肝脏，包括结肠直肠癌和胰腺癌等癌症。

门静脉注射模型，其中肿瘤细胞是通过在外科手术过程直接递送至肝，允许肿瘤细胞注射进入门静脉。下防腐剂的条件下，在麻醉小鼠，〜1英寸手术切口是在中位数和矢状平面之间的鼠标的左侧制成，开始就在第四腹股沟乳腺奶头的平面上方和刚肋骨下方结束。大，小肠子通过切口轻轻拉到提供门静脉（ 图1A）的可视化。门静脉和成功帧内门户注射的准确解剖识别可以通过实施与印度油墨或类似染料注射方案来确认。通过门静脉正确注入将导致被立即和特异性递送到肝脏的墨水，并在印油墨扩散到肺不会导致（Figu重新1B）。此外，使用具有标记的GFP D2A1小鼠乳房肿瘤细胞，肿瘤细胞的扩散在整个肝脏以90分钟表观注射后，确认门静脉注射递送到肝脏（ 图1C）。在更高的放大倍率变得明显的是，在90分钟注射后，肿瘤细胞被发现正弦内，以及内靠近门户三和弦，其中，门静脉血液进入肝脏（ 图1C）肝实质。这些数据表明，活性肿瘤细胞外渗是在90分钟肿瘤后细胞注射的发生。总之，这些数据证实，门静脉注射模型直接将注射体积至肝，与分散在整个肝和注射量没有明显的运输至肺墨水或肿瘤细胞。 为了评估门静脉注入模型的鲁棒性，三组合通道吃同基因小鼠乳腺肿瘤细胞系在成年雌性Balb / c小鼠进行测试。被选择的基础上在乳房脂肪垫型号其特征行为这些乳腺肿瘤细胞系，并且包括高度侵袭性和转移性4T1细胞系，所述不太积极转移性D2A1线和低/非转移性D2.OR线37,61,73 ，74。每次注射2000和10,000个细胞与在时间明显转移的发展与这些低细胞浓度没有显着的差异进行了测试。这些研究转移的替代标记物，使用如缺乏修饰的，苍白，和体重减轻来证明剖检，在其肝脏转移瘤的存在或不存在是由肝脏的视觉评估和其他器官证实，以确认帧内门户输送。这些数据证实了先前的报告，相对于注射的小鼠的4T1和D2A1细胞系代表更积极的乳腺肿瘤线，短转移无瘤生存率观察与较少侵略性D2.OR线（ 图2A）。小鼠〜30 4T1或D2A1肿瘤细胞开发公开肝转移注射- 40日龄注射后，以及一些发达转移早在18天后注射（ 图2A），而只有一个鼠标D2.OR细胞注射过65天肿瘤后的细胞注射 - 通过研究结束时，这是60发达明显的肝转移。转移随后通过在250微米水平的肝脏切片和分析伊红（H＆E）染色切片（ 图2B-D）确认。 除了检测在小鼠肝脏明显的转移病灶后，门静脉模型也可用于研究早期事件在转移级联包括检测单个细胞/细胞簇以下外渗，并形成微转移灶。采用多重免疫荧光染色检测4T1，D2A1，和D2.OR乳腺肿瘤细胞在肝脏的时候都存在作为单细胞或微转移灶，以H＆E是不足以证实小病灶的存在。 图3A示出与阳性的上皮角质CK18，负为泛免疫标记物CD45，和负的肝细胞标记Heppar -1- D2A1肿瘤细胞的推定的微转移灶的代表性的Balb / c小鼠的肝脏。肝细胞也染色阳性，CK18，在这种染色面板迫使使用Heppar-1。一个重要的说明是，胆管上皮细胞和肝祖细胞染色阳性CK18，要求肿瘤细胞和胆管之间仔细辨析，特别是评估汇管区（ 图3A）时。另一种复用免疫荧光识别单一传播的细胞和微小转移灶是利用标有绿色荧光的同源乳腺肿瘤行蛋白（EGFP）和/或萤光素酶和执行IHC为标记（ 图1C）。由于eGFP的，萤光素酶，和其它蛋白质的免疫原性，它是必须使用被耐受这些蛋白质小鼠模型，如新颖的免疫能干"发光头"表达eGFP的鼠标和萤光素酶在垂体前叶75。对于未标记的同基因系macrometastatic病变如D2A1肿瘤细胞系的鉴定，CK18 / Heppar-1 / CD45的多重免疫是理想的（ 图3B）。 <img alt="图1" src="/files/ftp_upload/54903/54903fig1.jpg" /> 图1: 门静脉注射直接将肿瘤细胞对肝脏。 A）门静脉注射;的切口中位数和左矢状平面之间做出，从第四腹股沟乳腺奶头的平面上方和刚里下方结束b笼子。 B）的Balb / c肝脏用PBS注射（左）。通过门静脉以下墨汁喷射，肝脏（中），但不是肺（右）占用墨水。 C）通过门静脉以1×10 6个肿瘤细胞90分钟注射后具有标记在Balb / c小鼠肝GFP D2A1小鼠乳房肿瘤细胞的代表性薄片图像。肝脏进行福尔马林固定，石蜡包埋，切片，并用抗GFP抗体对肿瘤细胞的检测染色。顶部面板显示分散于整个肝脏，星号表示中央静脉周围的非特异性肝细胞染色暗褐色染色的肿瘤细胞（箭头）;比例尺= 300微米。下图显示，在90分钟注射后仍与脉管密切相关的肿瘤细胞的代表性图像;比例尺= 50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54903/54903fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p类="jove_content"FO: - together.within页保留="1"&gt; <img alt="图2" src="/files/ftp_upload/54903/54903fig2.jpg" /> 图2: 在肝脏经过门静脉注射小鼠乳腺肿瘤细胞系的生长。 A）显示小鼠无转移生存率的Kaplan-Meier曲线注射2000-10000 4T1，D2A1，或D2.OR小鼠乳腺肿瘤细胞。小鼠用肿瘤细胞注射并监测转移的迹象包括缺乏修饰的，苍白的眼睛，和重量的变化。在转移的尸检时间和H＆E对肝脏组织学切片证实。 N = 2 4T1 2000细胞; 2 4T1 10,000个细胞。 N = 2的D2A1 2,000个细胞; 3 D2A1 10,000个细胞。 N = 3 D2.OR 2000细胞; 3 D2.OR 10,000个细胞。 的B代表H＆E图像）4T1病变以10,000细胞，C），D2A1病变的21天内注射后以10,000肿瘤细胞26天注射后，和D）D2.OR皮损处10,000个细胞59天注射后;比例尺= 75微米。当2000 4T1或D2A1肿瘤细胞注射观察到类似的病变。 2,000 D2.OR细胞中没有检测到病变。在其他器官或手术切口部位转移的证据在这些研究中任何鼠标是明显的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54903/54903fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54903/54903fig3.jpg" /> 图3:使用多路免疫单个细胞和转移灶在小鼠肝脏的检测。 A）中使用门静脉注射模型，在90分钟后注入D2A1肿瘤细胞在Balb / c小鼠肝脏的代表性多路复用免疫荧光。染色使用多重工具包完成的。从左至右显示DAPI; CD45标记白细胞; Heppar-1玷污ķ肝细胞;和CK18标记肿瘤细胞，肝细胞和胆管上皮细胞。合并的图像显示CK18 + Heppar-1推定的簇- CD45 -用入肝密切相关D2A1肿瘤细胞。箭头= D2A1肿瘤细胞，星号=胆管上皮细胞;比例尺= 25微米。 B）代表公开D2A1转移灶使用相同的染色板作为A.肿瘤细胞CK18 +，而相邻的肝细胞CK18 + Heppar-1 +;比例尺= 25微米。图像被抓获的20×0.8显微镜，40×1.3，和60×1.4的目标和CCD摄像头，采用显微镜软件。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54903/54903fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer at JoVE.com

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

a-portal-vein-injection-model-to-study-liver-metastasis-breast

Nanyang Technological University

Research

JoVE Journal

Cancer Research

40.3K 次观看.  Oregon Health and Science University. A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver. 

视频: A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

内髂动脉注射显微骨转移的效率和选择性建模

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

科研

癌症研究

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

肝大肠癌转移的高级动物模型:成像技术和转移性克隆性质

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

颈动脉肿瘤细胞注射，产生脑转移瘤小鼠模型

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

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 ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

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 ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

利用计算机图像分析提高4T1乳腺癌模型中肺转移的定量

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

研究利普托梅宁格疾病直接靶向治疗的穆林·奥马亚异种格拉夫特模型

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

通过炎症性乳腺癌细胞的尾静脉注射模拟脑转移

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...