这项工作是由补助卫生研究院（NIH）全国学院为RCB，CA122985和前列腺孢子CA90386，并JMP，美国国立卫生研究院的T32 AG000260"药物发现培训中年龄相关性疾病"，以及由沃尔特·S.和LUCIENNE支持德里斯基研究生课程在生命科学在西北大学。我们还要感谢鼠标分型和组织学实验室及病理核心设施在西北大学。

对于涉及动物的所有程序，协议获得批准的机构动物护理和使用委员会（IACUC）在西北大学。手术技术和动物保健条件由兽医人员进行了观察和修改，以尽量减少动物的紧张和死亡率。个别机构可能有不同的要求，并与IACUC和动物的工作人员制定和执行这种手术方法时，工作是很重要的。 1。细胞注射液的制备<ol><li>这一步应该进行尽可能接近到开始手术。胰蛋白酶消化细胞被用于该实验中，从板取下，并用中和介质。的PC3-M人类前列腺癌细胞株稳定转染GFP已经被成功地用于这些实验中，由于PC3-M细胞的快速生长的条件。 GFP加入额外的易用性检测肺组织样本，与快速测定肺转移的癌细胞与从血液和骨髓，以确定循环肿瘤细胞中获得的免疫细胞在培养物。如果修改的感兴趣的特定基因，然后建立稳定的细胞系与该基因改造的首先执行，然后转染GFP。如果GFP将改变感兴趣的该基因的功能，GFP它可以省略。这将使检测肺转移比较困难，但还是可以实现的。此外，如果使用特定的成像技术使用荧光标记物，如荧光素酶为基础的IVIS，可以使用其它荧光蛋白代替。 </li><li>离心5分钟，在225×g的。 </li><li>分离2.5×10 5个细胞，并离心5分钟，在225×g下。 </li><li>在20微升无菌盐水除去上清，重悬细胞。 </li><li>吸出细胞悬液加入0.5 ml注射器具有永久28½号针头（推荐:肯德尔MONOJET，其他品牌造成的问题），确保没有空气进入针人翁的悬浮液。 </li><li>注射器包装在无菌铝箔和冰上储存直至使用。 </li></ol> 2。人类前列腺癌细胞原位移植<ol><li>雄性6-8周龄的BALB / c无胸腺小鼠的使用。理想的小鼠体重的手术是19-21克，和鼠标应该被允许在手术前将增长到至少有18克小动物在技术上比较难操作的。较大的动物往往会遇到较慢的肿瘤生长和转移的动力学。动物应该被安置在至少一个星期在手术前，尽量减少动物应激的动物设施。根据实验设计，药物治疗可能1周开始在手术前。 </li><li>根据动物设施的指示，注射手术前的止痛药。使用连接到1毫升注射器30½号针头0.1毫克/千克体重皮下注射BUPRENEX建议。 </li><li>将动物进入异氟醚室，等到动物是完全一nesthetized。肌张力无趾反射应该出席了这一点。建议使用此方法，但如果无法使用，麻醉等方法可以根据动物设施的使用说明书操作。 </li><li>移动动物出来的异氟烷室的进入无菌程序罩。将动物入鼻锥设备，并重新确保动物是在全麻醉，然后再继续。 </li><li>用优碘擦洗消毒棉球消毒下腹部区域，用酒精擦拭擦拭，并用聚维酮碘溶液最终喷雾。晾干。 </li><li>无论是使用消毒的手术刀或削尖的无菌手术剪，约3-4毫米低中线腹部切口而成。使用镊子轻轻提起膀胱和识别前列腺腹叶。这两个波瓣位于膀胱正下方。有些鼠标将有一个小的脂肪层覆盖，可轻轻移动一边用消毒棉签前列腺。 Minimiz机关电子所有的运动和肌肉组织，如果可能的。 </li><li>注入20微升体积的细胞溶液进入前列腺，最大限度地减少泄漏，并确保小泡被观察到。 </li><li>更换膀胱和使用4.0薇乔可吸收缝合线的单丝在一个简单的中断模式关闭肌肉层。 </li><li>使用无菌9毫米钉合皮肤层。 </li><li>从异氟醚麻醉取出动物和监控，直到清醒和正常转动。在恢复期放置在加热垫。 </li><li>如果多次手术正在执行，手术，清洁所有工具之间用70％的乙醇，并用消毒的玻璃珠灭菌。让工具下的动物在充分冷却。请勿重复使用缝线，棉球或无菌拭子。 </li></ol> 3。监测动物<ol><li>基于动物设施的指示管理止痛药。皮下美洛昔康的一剂4小时手术后施用1毫克/千克体重ü唱连接到1毫升注射器30½号针头，建议，由一个附加的剂量为每12-24小时在未来48小时。 </li><li>史泰博可以从动物7-10天手术后取出，一旦伤口愈合。 </li><li>监测动物体重，摄食量，和触诊老鼠的肿瘤，每周两次，直至终止实验。增加频率可达每一天，当肿瘤变成可见的检查下显著肿瘤负荷或胁迫的动物。早期死亡从这个模型是由于大的原发肿瘤的负担，因为原发肿瘤可以阻止尿流，因此具有大于15％的体重，或原发肿瘤达到直径为1.5厘米的损耗的任何动物，应立即进行尸体剖检，以防止尿路梗阻和动物死亡。 </li><li>监测需要对动物环境的进一步浓缩。手术的压力增加动物的明争暗斗。而不是一个和其他行为增加了两个塑料小屋每笼人的富集可以减少这些事件。讨论与可在动物被安置在该设施的兽医工作人员的选择。由于缺乏对这个品系小鼠的睫毛，小屋和富集纸做的，不推荐，因为它们会刺激眼睛。 </li></ol> 4。剖检程序<ol><li> 4-6周后，肿瘤完全明显，动物开始减肥，由于增加肿瘤负荷。肿瘤通常可触诊和/或在视觉上是明显的。尸体剖检应在此时进行。在使用连接到1毫升注射器30½针头260毫克/公斤体重进行腹腔注射戊巴比妥，并让动物成为完全无意识，无趾反射或肌张力存在。 </li><li>一旦动物被麻醉，用无​​菌手术剪刀或手术刀，剪水平穿过直接在左肋动物的躯体，然后垂直地沿着动物的侧腋下，暴露心脏。执行使用连接到1毫升注射器用4％枸橼酸钠的DPBS冲洗，防止凝血，获得尽可能多的血液尽可能从动物30½号针头终端心脏穿刺。 </li><li>通过切割气管手术剪刀或手术刀从动物取出肺，并立即放置到组织培养盒和在10％福尔马林中。 </li><li>通过单独地切断附连到任何肿瘤的血管并确保没有额外的器官如输精管或精囊从动物取出原发性前列腺肿瘤相连。 </li><li>记录肿瘤的重量和体积。测量肿瘤的重量在以实验室规模克。用卡尺测量肿瘤的以厘米为单位的最长径的长度，以及相应的垂直轴。乘这些值来获得肿瘤的大小。根据不同的使用目的，马上急速冷冻在液氮中，和/或地方到组织盒放入10％福尔马林。 </li><li>暴露髋关节和膝关节手术剪刀或手术刀，并断开腿关节，注意要保持股骨完好。从动物中取出股骨并放入无菌盐水。 </li><li>取得任何其他器官或感兴趣的材料，并根据您的机构的指示处置动物。特别地，区域淋巴结往往有转移，往往如果窝藏它们被放大，并且可以收获。然而，应该注意，因为这可能会受到所引起的外科手术过程中的变化的静水压力。 </li></ol> 5。加工和肺组织免疫染色样品<ol><li>在24-48小时将组织成PBS中10％的甲醛，嵌入肺石蜡。 </li><li>第肺部在45微米步路段，采取2-3个相邻的4微米的肺组织切片。 </li><li>如果使用GFP阳性细胞（推荐），进行免疫染色绿色荧光蛋白。如果没有，执行标准苏木精和曙红（H＆E）染色。 </li></ol>注:Dako公司设想+套件结合来自Invitrogen的GFP抗体已成功地用于根据制造商的说明，但是这可以被修改为任何免疫程序。 <ol start="4"><li>以盲分数转移。如果GFP阳性，肿瘤细胞会弄脏褐色除了拥有庞大而独特的细胞核。当第一次得分转移，咨询病理学家，以确保正确的得分，并用H＆E染色的肺组织，以确认肿瘤细胞的存在，并没有浸润的免疫细胞。 </li></ol> 6。从血液和骨髓循环肿瘤细胞的识别<ol><li>添加从尸检收集到1.5 ml离心管中所有的血。离心5分钟，在800×g的。 </li><li>去除血浆和加入1 ml的ACK裂解缓冲液（154.95毫米氯化铵，9.99毫米钾Bicarbo内特，0.0995毫米EDTA）。让血液在室温下放置3-5分钟。 </li><li>离心5分钟，在800×g的。 </li><li>去除上清，加入1 ml含抗生素的细胞培养基。加细胞含有9毫升细胞培养基的T75烧瓶中。 </li><li>培养细胞在37℃，在含5％CO 2中培养10天，检查的CTC的存在下，每2-3天的潮湿气氛中。 </li><li>对骨髓，从使用剪刀，刀片或手术刀股骨移除所有的肌肉组织。除去骨的端部，尽可能靠近端部尽可能。 </li><li>使用23¾号针头，轻轻弹出骨髓从股骨到1.5ml的离心管中。 </li><li>添加到1ml细胞培养基，并轻轻吸取良好混合。 </li><li>加细胞含有9毫升细胞培养基的T75烧瓶中。 </li></ol> 7。肿瘤的分子特征<ol><li>用于聚合酶链式反应（PCR）测定秒，肿瘤样品是快速冷冻在液氮中首先被粉碎的干冰上，然后使用组织匀浆3-5分钟，用1ml TRIzol试剂均化。的TRIzol提取是根据制造商的说明书进行。根据制造商的说明使用RNeasy RNA提取试剂盒纯化的RNA。进行cDNA的合成和定量实时根据制造商的说明书，使用TaqMan试剂PCR（定量RT / PCR）的建议，但是可以修改为任何目前使用的PCR方法。 </li><li>对于Western印迹分析，使用组织匀浆3-5分钟用1ml裂解缓冲液中匀化组织:PBS（137 mM氯化钠，10mM的磷酸钠，2.7毫米氯化钾），0.5％的Triton X-100，1mM EDTA中，的2.5mM焦磷酸钠，1mM的β-磷酸甘油，并加入蛋白酶抑制剂混合物，磷酸酶抑制剂鸡尾酒2和3，10mM的氟化钠，和1mM原钒酸钠。细胞裂解物混合物置于1.5毫升离心管中。 </li><li>一分钱rifuge细胞裂解液混合物在13,000×g的10分钟。 </li><li>除去上清液并置于一个新的1.5毫升离心管中，以13,000×g下再离心10分钟。如果细胞碎片仍然存在，另外的离心步骤可以被执行。 </li><li>除去上清液，进行蛋白质印迹按正常的实验室条件。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

在本文中，我们提供了人类前列腺癌转移的一种新的小鼠模型。在这个模型中，人前列腺癌细胞系PC3-M被原位直接植入的BALB / c无胸腺小鼠的肿瘤和允许发展4-6周的前列腺。在代表性的结果部分中，我们表明，可以被收集的数据，包括小鼠体重和食物消耗，肿瘤大小和体重，肿瘤的分子特征，并形成明显的转移到肺的例子。此外，各种各样的额外输出可以根据具体的研究领域进行研究。一个例子是CTCs的存在下，在血液和骨髓中。由于车辆检验中心是一个比较罕见的事件（我们观察到的CTC控制动物的约5-20％），我们并不感到惊讶，没有五鼠在这些实验中开发的CTC。我们实验室也采用这种模式通过测量核细胞形态的判断改变细胞黏附前列腺组织10。我们还利用该模型使用Ki67和前列腺肿瘤14 TUNEL染色以评估原发肿瘤的增殖和凋亡状态。 除了各种各样的能够得到的数据输出，该模型可用于确定两个转变蛋白表达以及小分子治疗的效果。相比于人前列腺癌转移的许多自发的和诱发的模型中，存在着只4-6周更高的周转时间。其他型号的高周转时间，如尾静脉或intercardiac注射模型，有没有原发肿瘤的限制，从而不能充分扼要诊所转移级联。在我们的模型中，肿瘤细胞必须逃离原籍主站点，输入和循环系统存活，并植入到辅助站点。这提供了额外的步骤，在该蛋白表达的治疗干预或改变可能带来的影响。 ADDI倚重，原发肿瘤的存在应该可以很容易地应用该模型对新的成像技术，如荧光素酶为基础的IVIS 16-17。 尽管有各种​​各样的这种模式的好处，也有一些限制来考虑。越来越多的研究表明免疫系统在肿瘤微环境和转移18的发展中的重要性。在这个模型中，由于无胸腺啮齿动物的使用方法，以评估免疫系统的效应的能力是不可能的。第二个局限是缺乏在PC3-M细胞雄激素反应性。当前列腺癌的初步诊断，患者经常会接受雄激素治疗作为第一线治疗。然而，患者最终将成为雄激素耐药和肿瘤将开始恢复增长。由于PC3-M细胞缺乏雄激素受体，这种模式只能衡量药物治疗或蛋白调制对后期抗雄激素c中的作用ancer。虽然这是一种限制，雄激素响应性前列腺癌是目前很好管理的，并具有多种有效的治疗方案，并因此雄激素耐药性癌症已变得更加突出了研究。这种模式还特意使用了小鼠的近交系，其中以鼠变异最小化鼠标。但是，该菌株可以特别响应于特定的蛋白质或小分子，从而照顾外推这些数据来诊时，应采取。 虽然这个模型提供了4-6周的快速周转时间药物疗效的有效测量，这可能没有考虑到长期的药物给药的效果。长时间暴露在许多目前可用的治疗后，患者可能会返回与抗药性的癌症多年后处理。周转快的这种技术的不允许的肿瘤成为抵抗治疗的能力有效建模。然而，这位前调制实验中，治疗性人前列腺癌细胞可以被植入，并且在预防前列腺癌的肿瘤生长和转移的第二代治疗的有效性可以被建模。此外，如果一个小组试图研究一个原发肿瘤随着时间的分子变化，一个较长远的模式，如流浪汉模式将可能是更有效的为这些研究。 该模型的另一个限制是不同的转移只到淋巴结和动物的肺中的传播。这两个网站都是转移频繁和临床相关的网站，就证明了人类温暖的尸检研究19。然而，临床上骨转移构成了人类前列腺癌的一个突出特点，因而模型扼要重述这是利益。不幸的是，这些都是很难在一个小鼠模型来概括，除了极少数的模型显示无尾静脉注射intercardial，或者直接植入骨转移到骨20。因此，如果定位到骨是关键实验重要性，另一种模式可能更有效。然而，这种模式确实提供了交通的一些措施，对骨的骨髓循环肿瘤细胞的形式。 尽管有这些限制，这种技术是人类前列腺癌的一个强大的模型。以测量在​​两个原发肿瘤以及在很短的周转时间转移形成的影响的能力提供了广泛的应用。在这个模型中，细胞必须躲避的主要机构，输入并在血液中存活，并在植入物的辅助站点，扼要过程在人类。的原发肿瘤的分子特征的附加测量，细胞形态的变化，和循环肿瘤细胞的存在提供的信息，广泛地从一个模型。此过程既可以在药物发现中的上下文以及学习改变在肿瘤生物学中使用。

前列腺癌（PCa）是最常见的癌症诊断的男人，和癌症死亡的美国1的第二大原因。从前列腺癌的死亡不是由于形成的原发肿瘤，而是转移的形成。因此，防止患者转移是非常重要。前列腺癌的小鼠模型提供了选项来揭开这种疾病的关键生物信息的多样性。 多种前列腺癌的小鼠模型存在，每个固有的优势和局限性。而前列腺癌在人体中的频率高，自然发生前列腺癌的小鼠2极为少见的，尽管小鼠等于整体的易感性癌症3。一种啮齿动物异常是前列腺癌的Lobund Wistar大鼠的发展，它可以通过感应methylnitrosourea和睾酮4达到90％前列腺癌的比率由12月龄。出于这个原因，诱发模型系统中，如在TRAMP模型（小鼠前列腺的腺癌的转基因）是常用的。流浪汉模型可以特异性地诱导转基因表达在前列腺癌和前列腺癌发生的正常发展，从增生前列腺上皮内瘤（PIN），淋巴和肺转移5-6。这些模型提供了能够测量的全方位肿瘤进展，以及包含一个完整的免疫系统的益处。然而，相关前列腺癌发展的分子事件可以小鼠和人类之间的差异，以及小鼠和人体临床研究之间的相关性表明变性。此外，这两种模式都是费时，作为一个例子的TRAMP模型需要约28周，以发展转移。 在研究转移，经常一尾静脉或左心室注入模型被使用。这个模型受益于快速周转时间，并且还可以测量骨头metastas的存在是使用特定的细胞系和条件。 Yang 等人报告说，皮下注射GFP阳性PC3细胞可引起广泛传播骨转移7，和尾静脉注射intercardiac也产生骨转移发展8-9。这些模型的主要限制与缺乏居住前列腺本身内的原发肿瘤。此外，对于模型依赖于注射癌细胞进入血液循环，这也避开了转移级联的整个上半年。因此，它排除了考试的最初步骤，包括通过入侵的主要器官，它是生物转移性转型的关键措施。转移性转型的许多监管机构直接影响早期细胞的侵袭。在转移级联早期步骤构成的高优先地点的靶向治疗，因为一旦癌细胞传播，克隆变异极大地扩展，从而增加生物人的多样性和减少有效的靶向治疗。 在试图对许多这些模型的局限性回应，我们的实验室已经开发人类前列腺癌的原位模型​​中的人类前列腺癌PC3-M细胞系被直接植入的BALB / c无胸腺小鼠的前列腺。 4-6周后，肿瘤大小，循环肿瘤细胞（CTCs的）的存在下，与对肺和淋巴结转移都可以量化。我们已经有效地使用这个模型来评估4的功效&#39;，5,7 -三羟基异黄酮（染料木素）能抑制人类前列腺癌转移10。染料木素的饮食消费一直挂在前列腺癌转移和死亡11-12的减少，但以前没有研究已确定染料木素的施用是否会改变前列腺癌转移在动物或人。在这个研究中，我们证明了用染料木黄酮处理大大降低肺转移的数目。此外，我们确定染料木素人tered几个重要的促转移蛋白在原发肿瘤，包括粘着斑激酶（FAK），p38促分裂原活化蛋白激酶（p38蛋白），和热休克蛋白27（HSP27）的活化和表达。 这些结果与对应在临床观察。使用从小鼠获得的血液，我们能够精确地测量染料木素的血药浓度，并观察这些是类似于人类与染料木黄酮经常饮食消费水平。此外，本集团所进行的II期研究确定，在处理与染料木素，经验丰富的人在减少与细胞的侵袭和转移相关基因的前列腺组织中的表达，特别是基质金属蛋白酶型蛋白酶-2（MMP-2）13。 我们也使用这个模型来评估对人前列腺癌转移14原发肿瘤的改变的基因产物的表达的效果。肿瘤supprESSOR内皮糖蛋白是TGF-β超家族的一员，并通过改建Smad信号15抑制人前列腺细胞体外侵袭。我们扩展了这些研究，以确定内皮糖蛋白对人前列腺癌转移的效果。稳定内皮糖蛋白敲除，矢量控制或内皮糖蛋白过表达细胞株植入小鼠。内皮糖蛋白敲低的细胞显示在小鼠的38％的最大数目肺转移，以及的CTC。小鼠植入控制向量呈中等响应，每只小鼠少肺转移，并在车辆检验中心仅18％的小鼠。高内皮糖蛋白植入小鼠表现出肺转移几乎完全抑制，车辆检验中心的完全抑制。 这些是在各种各样的应用中，这种技术具有的两个例子。从药物发现，在分子生物学模型的变化，这种模式提供评估对肿瘤生长和摩尔各种功能的效果的高通量方法丘拉尔变化的CTC的存在，且在肺和淋巴结形成鲜明的转移。

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>1 ml Syringe, Tuberculin, Slip-Tip</td>
 <td>Becton Dickinson</td>
 <td>309659</td>
 <td></td>
 </tr>
 <tr>
 <td>23 G 3/4 Precison Glide Needle</td>
 <td>Becton Dickinson</td>
 <td>305143</td>
 <td></td>
 </tr>
 <tr>
 <td>30 G 1/2 Precision Glide Needle</td>
 <td>Becton Dickinson</td>
 <td>305106</td>
 <td></td>
 </tr>
 <tr>
 <td>Alcohol Wipes</td>
 <td>Triad</td>
 <td>10-3001</td>
 <td></td>
 </tr>
 <tr>
 <td>Autoclip Applier, 9mm, Stainless Steel</td>
 <td>Becton Dickinson</td>
 <td>427630</td>
 <td></td>
 </tr>
 <tr>
 <td>Clips, 9mm</td>
 <td>VWR</td>
 <td>15431-673</td>
 <td></td>
 </tr>
 <tr>
 <td>Convertors Polyline Towel (Sterile)</td>
 <td>Cardinal Health</td>
 <td>3520</td>
 <td></td>
 </tr>
 <tr>
 <td>Cotton-Tipped Applicators, 6 inch, Sterile, Wooden Shaft</td>
 <td>Fisher</td>
 <td>23-400-125</td>
 <td></td>
 </tr>
 <tr>
 <td>Curad Sterile Cotton Balls</td>
 <td>VWR</td>
 <td>500043-544</td>
 <td></td>
 </tr>
 <tr>
 <td>Derf Needle Holder, Integra Miltex, 121 mm (4 3/4")</td>
 <td>VWR</td>
 <td>95039-192</td>
 <td></td>
 </tr>
 <tr>
 <td>Duraprene SMT Sterile Neoprene Powder-Free Surgical Gloves</td>
 <td>Cardinal Health</td>
 <td>2D72PN70</td>
 <td></td>
 </tr>
 <tr>
 <td>Germinator 500</td>
 <td>Cell Point Scientific</td>
 <td>SN 7030</td>
 <td></td>
 </tr>
 <tr>
 <td>Kendall Monojet Needles, 1/2 cc syringe with permanent 28 G 1/2 needle</td>
 <td>Tyco</td>
 <td>1180528012</td>
 <td></td>
 </tr>
 <tr>
 <td>Medium Heating Pad </td>
 <td>VWR</td>
 <td>100229-094</td>
 <td></td>
 </tr>
 <tr>
 <td>Merit Iris Scissors, Sklar, 11.4 cm (4 1/2")</td>
 <td>VWR</td>
 <td>94000-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Metric/English Vernier Caliper</td>
 <td>VWR</td>
 <td>19155-057</td>
 <td></td>
 </tr>
 <tr>
 <td>PDS*11 Violet Monofilament Sutcher 4-0, 27"</td>
 <td>Ethicon</td>
 <td>Z304H</td>
 <td></td>
 </tr>
 <tr>
 <td>VetEquip Inhalation Anesthesia System</td>
 <td></td>
 <td></td>
 <td>Contact Your Animal Facility for Availability</td>
 </tr>
 <tr>
 <td>VWR Premium Tissue Cassettes</td>
 <td>VWR</td>
 <td>18000-010</td>
 <td></td>
 </tr>
 <tr>
 <td>VWR Specimen Forceps, Serrated, Straight, 114 mm (4 1/2")</td>
 <td>VWR</td>
 <td>82027-440</td>
 <td></td>
 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Betadine Surgical Scrub</td>
			<td>Fisher Healthcare</td>
			<td>19-027132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Buprenex</td>
			<td></td>
			<td></td>
			<td>Controlled Substance - Obtain from Animal Facility</td>
		 </tr>
		 <tr>
			<td>Dako DAB + Chromagen</td>
			<td>Dako</td>
			<td>K3468</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dako Envision + System HRP-Labeled Polymer, Anti-Rabbit</td>
			<td>Dako</td>
			<td>K4003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dako Envision Kit Protein Block</td>
			<td>Dako</td>
			<td>X0909</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Formalin</td>
			<td>VWR</td>
			<td>95042-908</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GFP Antibody</td>
			<td>Invitrogen</td>
			<td>A11122</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hematoxylin</td>
			<td>Mayer</td>
			<td>MH580-2.5L</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Meloxican</td>
			<td></td>
			<td></td>
			<td>Obtain from Animal Facility</td>
		 </tr>
		 <tr>
			<td>Nembutal</td>
			<td></td>
			<td></td>
			<td>Controlled Substance - Obtain from Animal Facility</td>
		 </tr>
		 <tr>
			<td>Rneasy RNA Isolation Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sterile Saline</td>
			<td></td>
			<td></td>
			<td>Obtain from Animal Facility</td>
		 </tr>
		 <tr>
			<td>Taqman Reverse Transcription Reagents</td>
			<td>Life Technologies</td>
			<td>N8080234</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Life Technologies</td>
			<td>4304437</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>10296</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

an orthotopic murine model of human prostate cancer metastasis

我们实验室已经开发人类前列腺癌（PCa）的一种新的原位移植模型。作为前列腺癌的死亡不是由于原发性肿瘤，而是不同的转移的形成，以有效地预临床模型这个级数的能力是很高的价值。在这个模型中，将细胞直接注入到在BALB / c无胸腺小鼠的前列腺腹叶，并允许4​​-6周的进展。在实验终止时，多个不同的端点可以被测量，如大小和分子特征的原发肿瘤，血液和骨髓中的循环肿瘤细胞中的存在和定量，并形成转移至肺。除了各种端点，这种模式提供了入侵和逃避的主要器官，进入并在循环系统存活，植入和生长在辅助站点一个细胞的能力的图片。这种模式已经被有效地用来测量转移性响应这两个变化的蛋白表达以及对响应的小分子治疗剂，在很短的周转时间。

在这个实验中，我们将展示一组有代表性在这些手术过程中获得的小鼠。五个小鼠植入GFP阳性含有对照载体的PC3-M细胞。肿瘤被允许生长了六个星期，然后多个参数进行了评价。在图1A和1B中，我们显示体重和小鼠的食物消耗的改变，分别。有因麻醉一个小坑体重和周围手术日期的食品消费。在实验的过程中，体重缓慢增加手术后，然后开始下降朝向实验结束时作为肿瘤负荷达到临界水平。这将与在这些小鼠的食物消费。 在图2A和2B，示出了获得的代表性肿瘤的大小。个别肿瘤大小各不相同，但平均我们达到约1克肿瘤，具有之间0.5-1.5克，以及1厘米2的肿瘤大小标准方差，以0.5-1.5厘米2的正常变异。虽然肿瘤的大小而变化，这些不相关与所得的转移的数量，无论在本文中与我们先前发表的作品10所示。然而，从这个模型，可以确定药物治疗或肿瘤的重量和体积的分子变化的影响。在该模型中的一个重要考虑是，当用于实验的相应端点。在图2C和2D中，我们显示在一个特定的PC3-M细胞系在第4周稳定转染GFP和对照载体和6周时的改变在肿瘤重量和肿瘤的大小。在过去的两个星期，平均瘤重增加2.7倍，肿瘤大小1.9倍。这说明在实验中加入额外的1-2周可以显着地影响结果。由于多种因素可以改变肿瘤的生长，包括年龄和小鼠的大小，该细胞的传代次数等建议不要终止实验，直到可见的肿瘤中大多数小鼠均观察到。 在图3A-3C，转移灶的数量进行定量三种不同的方式。在图3A中 ，GFP阳性的人类前列腺癌细胞的总数来表示。在图3B中 ，细胞基因座，或转移性沉积物存在的位置的数目，被示出。最后，在图3C中，不同的转移的数量，由一个清晰的约束组的细胞示出5个或更多的GFP阳性人前列腺癌细胞的定义，被显示出来。这些不同的条件代表性图片示于图4A-4D。在图4A中 ，在40倍的数量级的单个单元格被突出显示带有箭头。注棕色染色和独特的大核。使用H＆E染色染色相邻肺段如图4B所示 ，证实了没有GFP，检测癌细胞仍是容易实现的。此照片是在40倍的数量级和该信元被用箭头突出显示。在图4C中 ，将显示不同的细胞数在10倍大小的几个位点，每个位点突出显示一个箭头。 。最后，在图4D，示出了10个细胞的转移性沉积物。这些方法如何影响数据所表现出的鼠标1和鼠标3的差异。鼠标1具有总细胞的数量较少的肺，平均每肺段21.5的细胞相比，小鼠3，其具有平均每肺部700细胞。然而，鼠标具有3个位点少，或细胞存在比鼠标1的位置。鼠标3具有转移的相对较少的位点，但每单位面积细胞的数量，是由于一些非常大的细胞数目的转移非常高，每个位点82的细胞。相比之下，鼠标1具有更加独特的基因位点，但显著f每个地点壶细胞只是一个平均每个位置两个单元格的。这些不同的参数，可以对细胞贩运到肺和他们开始生长和增殖能力的动力学揭示。 另外，在图3D中，我们显示在尸体剖检在4与6周小鼠变化每肺总转移性细胞。如在图2C和2D描述的，显著变化观察到的肿瘤重量和大小中的最后两周的实验。这被概括在图3D中。剖检小鼠4周均未见转移的发展，而小鼠6周呈转移性细胞在评估所有小鼠。这进一步表明了确保小鼠尸检是在后期阶段端点执行，以确保发生形成转移的重要性。 在这个模型中添加的测量是在里面发生的分子变化<html>原发肿瘤。在图5A-5C我们展示三个示例定量RT / PCR实验测量的转移进程的兴趣三个基因，基质金属蛋白酶型蛋白酶-2（MMP-2），基质金属蛋白酶类蛋白酶9（MMP-9）和热休克蛋白27（HSP27）分别。可以使用这种技术来检测利益通过定量RT / PCR检测的任何基因。此外，蛋白质水平可使用免疫印迹方法进行量化。 <img alt="图1" src="/files/ftp_upload/50873/50873fig1.jpg" /> 图1。观察到体重和在动物中的食物消耗。AB）的体重（克），或在每克小鼠每天平均摄食量，被记录在整个实验和在A中所示）和B）分别。 fig2highres.jpg"SRC ="/ files/ftp_upload/50873/50873fig2.jpg"/&gt; 图2。肿瘤大小和肿瘤重量在个人和小鼠组。 AB）肿瘤重量（g）和肿瘤大小平方厘米的五个有代表性的对照组小鼠和5只小鼠在六个星期结束时，平均都显示在A）和B）分别光盘）的肿瘤重量以克为单位的比较和以厘米为单位的小鼠组间平方肿瘤大小在尸体剖检四到六个星期。 <a href="https://www.jove.com/files/ftp_upload/50873/50873fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50873/50873fig3highres.jpg" src="/files/ftp_upload/50873/50873fig3.jpg" /> 图3。转移扩散的个人和组小鼠。 AC）的平均转移性sprea每五个人的小鼠和5只小鼠在5 +细胞中的定义表示为细胞的任一数目（A），转移细胞的位置（B）中 ，或不同的转移六周端的平均肺D节明确定义群集（C）D）每鼠肺段小鼠之间转移细胞的平均数比较剖检在四个和六个星期。 <a href="https://www.jove.com/files/ftp_upload/50873/50873fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50873/50873fig4highres.jpg" src="/files/ftp_upload/50873/50873fig4.jpg" /> 图4。肺转移的代表性图像。 A）在40倍物镜的个人GFP阳性肺癌细胞与箭头:B高亮显示）的邻近肺从A）部分，显示在H＆E染色，C型相同的细胞）在10倍的目标的一个肺段与含有不同数量的细胞。 的D）一个明显的转移在10倍物镜含10癌细胞明确定义为一组七个人的基因位点。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/50873/50873fig5highres.jpg" src="/files/ftp_upload/50873/50873fig5.jpg" /> 图5。的三个需要关注的转移基因的PCR分析。定量RT / PCR技术从小鼠收集在6周个别肿瘤样本进行的。 MMP-2（A）的相对mRNA转录水平，MMP-9（B）和热休克蛋白27（C）计量，标准化为GAPDH。 <a href="https://www.jove.com/files/ftp_upload/50873/50873fig5large.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about An Orthotopic Murine Model of Human Prostate Cancer Metastasis at JoVE.com

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

人前列腺癌原位此模型允许对肿瘤大小进行量化，循环肿瘤细胞，并形成明显的转移到肺。由于细胞必须逃脱的主要器官，进入血流，并植入到辅助站点，这种模式有效地概括了情景在人。

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

an-orthotopic-murine-model-of-human-prostate-cancer-metastasis

Research

JoVE Journal

Medicine

35.0K 次观看.  Northwestern University. 人前列腺癌原位此模型允许对肿瘤大小进行量化，循环肿瘤细胞，并形成明显的转移到肺。由于细胞必须逃脱的主要器官，进入血流，并植入到辅助站点，这种模式有效地概括了情景在人。 

视频: An Orthotopic Murine Model of Human Prostate Cancer Metastasis

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

科研

医学

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

癌症转移，使用禽流感胚胎模型的定量分析

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

在原位小鼠脑肿瘤模型结合放疗

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

甲状腺未分化癌的原位小鼠模型

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

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

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

生物荧光原位模型胰腺癌进展

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

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.