作者感谢洛桑大学医院的M.菲切提供的隆乳照片，M.维尔特和瑞士研究所实验癌症研究能力的研究分子肿瘤学国家中心，生命科学学院，巴黎高等联邦理工学院的A. Ayyanan英国曼彻斯特大学的洛桑技术援助和R·克拉克的批评。这项研究导致这些结果根据赠款协议已获得支持SNF3100A0-112090，Oncosuisse 531817，以及创新药物计划联合执行体没有。 115188，资源，都是由欧盟第七框架计划（FP7 / 2007-2013）和制药工业协会的实物出资公司的欧洲联盟财政贡献。创新药物行动的网址是<a href="http://www.imi.europa.eu/" target="_blank">http://www.imi.europa.eu/</a> 。&lt;/ P&gt;

一般考虑:提前，成立了一个道德的协议，准备调查问卷的患者，并请培训临床人员。使用的材料从乳房缩小成形术手术前，采购授权，并确保病人的同意。组织可以是感染性和需要进行相应的处理。该协议被批准ISREC的伦理委员会 - 瑞士研究所的实验癌症研究。 1.组织回收，加工和生物银行以确保用于生物银行最佳样品的质量，一些步骤进行，在运送到实验室现有的医院。 医院: <ol><li>获得人乳房组织从乳房缩小成形术的手术和血液样本在无菌条件下，如果需要的话。组织需要由病理学家谁将会品尝以排除恶性病变的存在进行审查。 </li><li>将乳房吨发出引擎盖下的无菌板，并准备剪刀，手术刀，哪些是已经无菌蒸汽灭菌器钳。使在不同地点的乳腺组织深的伤口用手术刀将其打开。在人类的乳腺组织外观为白色股由导管和小叶，这是嵌在黄色的脂肪组织。 注:白色实质到黄色脂肪组织的比例不同的妇女;一般年轻的女性有更多的上皮组织。 </li><li>挑选白料件并把它们用钳子，然后用剪刀脂肪组织轻轻的将它们分开。避免服用血管。 </li><li>在磷酸盐缓冲盐水（PBS）pH 7.2的漂洗件的组织去除血液。转移组织到250毫升塑料瓶含有F12培养基（Dulbecco氏改良的Eagle氏培养基F12）无酚红，用2％青霉素/链霉素（包括5000单位/ ml青霉素和5000克/ ml链霉素）和1％的tibiotic /两性霉素B（包括10,000单位/ ml青霉素，万微克/毫升链霉素和两性霉素B的25微克/毫升）。商店组织中运输到实验室的媒介。 </li><li>准备3件 - 直径5mm用于RNA和蛋白质提取和闪光灯用冷（干冰）的异戊烷冻结它们在冷冻管。将它们转移到干冰盒运输到实验室。 </li><li>放的3个 - 5毫米入模具中并用一层最佳切割温度（OCT）覆盖它，然后将模具上的盒子充满冷的异戊烷的平坦底部。完成冷冻后的模具转移到干冰框。 </li><li>固定件5 3 - 直径5mm的多聚甲醛（PFA）的4％和另5个中的甲醛（FA）的在PBS中的4％在室温下的组织学分析以后。两种不同的固定剂被推荐为一些抗体与工作的一个或另一个的2的更好。 </li></ol>在实验室中: <ol start="8"><li>办cument关于乳房缩小成形术的所有信息，手术是否样品日期来源于左与右乳房。 </li><li>将冷冻的样品在-80℃和2小时内固定后转移固定组织成组织学盒中。然后把它们无论是在PBS或70％的乙醇，对O / N储存在4℃。第二天，转移组织学盒到组织学设施对石蜡包埋。 </li></ol> 2.组织消化和激素刺激<ol><li>传送含有组织样品的瓶层流柜中的P2的细胞培养室。将组织块在无菌砧板（29×19厘米）。握住钳材料刮去剩余的脂肪和船只远离乳腺实质（白色部分）与手术刀。将白色材料PBS在10cm的培养皿中。 </li><li>根据安全规则的所有作品去除脂肪物质和处置后，他们实验室S，把含有上皮组织放回案板的白色部分。使用相对的手术刀切割组织并将其砍至高达2毫米直径的碎片。 </li><li>将切碎的材料进足够的规模管:最多1个，2.5和10毫升到5，15或50ml试管，分别。通常有5毫升管产量足够的材料为石蜡包埋和激素刺激，和一个15毫升管产量足够的材料用于荧光激活细胞分选（FACS）。较大的管用于冷冻等分试样。 </li><li>准备消化主结构由介质（DMEM / F12无酚红）与1％青霉素/链霉素/两性霉素B和胶原酶A（梭状芽胞杆菌A以上的溶组织梭菌 ）。根据消化倍剂量胶原酶。 <ol><li>为12，24和36小时培养中，使用1.5毫克/毫升，1.0毫克/毫升和0.5毫克胶原酶的终浓度/毫升分别。加入消解预混高达4倍将消化的材料的体积，以确保有足够的搅拌和通气。 注意:根据实验设计和激素刺激的持续时间，期间或酶消化步骤和显微恢复后添加激素。 </li></ol></li><li>添加激素（s）的期望的浓度，以测试样品和所述车辆到控制。为刺激17-β雌二醇，和promegestone（R5020）的使用为20nM的最终浓度。 注:R5020是合成孕酮受体激动剂，这比在介质天然孕酮更稳定。 <ol><li>地方管上的培养箱内的滚筒混合器，在37℃和40rpm下O / N。在放置样品的调音台，其中重悬很好地确保所有材料沿离心管分配。 </li></ol></li><li>作为消化至少需要12小时（O / N），对于小于12小时激素刺激倍，微结构的恢复后添加激素（德下步3尾）。添加激素，你悬浮组织和放置在超低附着板暂停。组织的微观结构，可以保持在超低附着板长达7天的生长因子的培养基中保持约70％的存活率。 </li><li>用于RNA和蛋白质分析，收集组织显微离心在400×g离心5分钟，并快速冷冻在离心管中。 </li></ol> 3，恢复组织微结构<ol><li>离心反应管（多个），在400×g离心5分钟。从离心管的顶部吸出脂肪和传输剩余的含水上清液，其富含成纤维细胞的上清液，向一个新的离心管中并离心，在1250×g离心5分钟。 注意:作为乳房组织的微结构是非常粘性的，对于所有后续步骤通过轻轻搅拌该管重悬沉淀。移液不因丢失的材料的危险，建议。 </l我&gt; <li>悬浮富集的微观结构和在在PBS，2％胎牛血清（FCS）的衍生自水性上清液的成纤维细胞的小球。接着旋两个管在1250×g离心5分钟，然后弃去上清液。 </li><li>悬浮颗粒在3 - 5毫升红血细胞裂解液和他们转移到清洗管。孵育他们5分钟，在室温。 </li><li>添加PBS，2％FCS中的2倍量和离心它们在1250×g离心5分钟。在1250×g离心洗用PBS，2％FCS和离心机的粒料5分钟。重复此步骤两次。 </li></ol> 4.样品的流式细胞仪<ol><li>加入1 - 2 ml胰蛋白酶0.25％至沉淀并通过吹吸用1000微升的Pipetman以解离细胞混合它和向下轻轻2分钟。 </li><li>通过加入9毫升的PBS，2％FCS中抑制胰蛋白酶活性。离心机在450×g离心5分钟，重悬沉淀在10ml的PBS，2％FCS中。 </li><li>细胞悬液转移到40微米的细胞滤网置于吨一50ml离心管中的运算以制备单细胞。如果细胞悬液几微保留在过滤器的顶部上，帮助他们通过过滤器从过滤器的另一侧吸移它们与一个吸管，并将它们添加到经过滤的微结构。 </li><li>分离免疫细胞，从微结构与抗CD45的鸡尾酒成纤维细胞和内皮细胞，抗FAP和抗CD31抗体，然后对其进行排序用FACS基于泛上皮标记的EpCAM和CD10的分离腔和基底细胞分别为10 。 </li></ol> 5.样品的组织学<ol><li>洗涤上述的机械和酶促消化材料之后，从固定1.5毫升初始材料粒料在1ml的4％PFA的PBS中在RT下30分钟。 </li><li>离心机在1250×g离心5分钟，并用1洗净 - 2毫升的PBS两次。 </li><li>离心在16000×g离心10分钟。在此期间，制备1％的多用途琼脂糖在Tris-醋酸盐，乙二胺四乙酸（TAE）缓冲液中并加热，在微波炉的溶液，直到该溶液来煮沸。 <ol><li>除去从微波炉的溶液，摇动烧瓶轻轻以混合溶液重悬任何剩余的琼脂糖颗粒。凉爽琼脂糖溶液至50℃，并倒在石蜡基模1毫升尺寸31×23×13.5毫米。 </li></ol></li><li>弃去上清液，然后将粒料在固化层的顶部 - 琼脂糖使用刮刀具有平面端部（1 3毫米）。覆盖所有琼脂糖的附加层（40°C）。 </li><li>琼脂糖凝固后，通常在5分钟内，将琼脂糖块放入纸盒组织学石蜡包埋。随着琼脂糖样品放入一个白色的办公用纸与有关乳房缩小成形术数，实验条件和日期写在铅笔小写字母的所有信息的磁带;写这篇文章承受所有后续处理步骤。 </li><li>把磁带中PBS O / N，或70％的乙醇储存在上周末，在4℃。我们有没有注意到这两个存储解决方案之间的任何差别。然后，组织学磁带转移到组织学设施石蜡包埋。 </li></ol> 6.冷冻微观结构（未来培养） <ol><li>重悬组织微结构在冷冻介质组成的FCS，用10％二甲基亚砜（DMSO），并冻结1ml等分在冷冻管。所得冷冻管的数量取决于从乳房缩小成形术获得的主材料的量。 </li><li>冻结含使用一个受控速率冷冻器与以下程序组织微结构的冷冻管:5分钟栖居在4℃时，温度为6℃/分速率降低到-7℃，降低温度至-12℃，用率的6℃/分钟，10分钟的住宅中，温度为6℃/分速率降低到-40℃，在温度下降到-80℃下用率的6℃/分钟。然后将离心管转移到-80℃的冰箱中。 </li><li>第二天，从-80℃转移至冷冻管液氮罐长期贮存。 </li></ol>

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C., Su, Y., Kuhn, I., Polyak, K., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+out+the+FACS:+a+devil+in+the+details.">Sorting out the FACS: a devil in the details.</a> Cell Rep. 6 (5), 779-781 (2014).</li></ol>

作者什么都没有透露。

此处所描述的体外培养提供了含有完整导管和小叶，连同在人类女性乳房通常存在的其它细胞类型的乳房组织的微观结构。在通常从降低mammoplasties获得的人类乳房组织的处理中，去除脂肪组织，基质基质的机械和酶消化，和血红细胞裂解的丰富的牛奶导管片段和终端导管小叶单位。轻柔酶促消化在合适的浓度，为右持续时间是至关重要，以确保组织的微结构保持不变，保留多种细胞类型，在很大程度上保持其细胞外基质。还应注意支付处理迅速，一旦从患者移除的组织。患者样品变化基本上与机械和酶消化可能需要被延长，和/或额外酶需要加入时的组织是特别高的胶原含量。 虽然微结构保持90％存活长达6天，该模型具有用于长期激素刺激的局限性。由于不同类型的细胞有不同的半衰期，蜂窝组合物很可能会随着时间而改变。而组织的微结构保持浸润免疫细胞，这些都不是补充作为所述组织是从身体的循环除去。此外，固体管线与临床伙伴谁提供的手术标本，需要为需要大量的样品，因为除病人的变异进行分析。组织微结构可以冷冻供以后使用。然而，到什么程度冻融影响细胞生存力，这可能差异为不同类型的细胞，以及这可能会改变激素反应，尚未表征。实验规划可以是困难的，因为对于任何隆乳组织的微结构将要获得的量是难以预料。 体外模型，提出了研究生理激素作用于人体乳腺癌的第一款车型。虽然已经开发了先进的三维培养系统的主要乳腺上皮细胞，这种体外系统保留了组织架构及其在数天的细胞的复杂性。其结果是，不仅在激素受体阳性靶细胞的反应可以被分析，但在其它类型的细胞旁分泌信号下游的事件成为适合进行学习。微观结构被保持在生长因子的培养基在整个实验的持续时间。因此，存在信号级联的无混杂外在活化。因此，乳房组织的微观结构提供了可能性，以解决在更接近地反映了人类乳腺癌的生物复杂性的设定的问题。 体外系统可以用来评估BR的响应东天然和合成激素。这是在光乳腺癌的发病率日益增加的非常重要的。此外，药物，小分子，肽，生长因子，和细胞因子可以应用和它们对细胞增殖，细胞凋亡和信令影响进行研究。 本详细描述旨在促进由其他研究者使用这种方法，并以帮助减少实验室间的变化。作为这种方法依赖于患者的样品，它是最重要的是一个标准化的方法用于使得结果可以开始不同的实验室和15更好地了解患者间变异性成为可能之间进行比较。笔者欣赏的反馈，将竭诚为改进融入这个协议的修订版。与人类样本的工作是非常具有挑战性的，作者希望能够刺激更多的转化研究与有趣的生物样品。

A correction was made to <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>. There was an error with the author, Marie Shamsheddin&#39;s, name. The author&#39;s name has been corrected to:
Marie Shamseddin
instead of:
Marie Shamsheddin

A correction to the author list has been made for the article <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>.

Erratum: An Ex Vivo Model to Study Hormone Action in the Human Breast

关于乳腺癌的突变的景观信息正以迅猛的速度。更少的受到人们的重视，以影响乳腺癌的发展全身因素。暴露于生殖激素对疾病进展1-3产生重大影响。然而，通过该生殖激素撞击在人乳腺癌的机制知之甚少。遗传工程小鼠模型的工作表明，它们涉及到一些下游效应4细胞的内在和旁分泌信号。 有关激素作用于人体乳房的知识有限，主要是由于缺乏足够的模型。大多数工作对雌激素受体（ER）和孕酮受体（PR）信令的机制已被执行激素受体阳性的乳腺癌细胞系，如MCF-7和T47D。这些来源于胸腔积液患者晚期乳腺癌谁曾alreaDY接受多次治疗5。在人乳腺癌的这种简单的体外模型的发现的生物相关性是在这些体外模型确定可疑和靶基因是不同于那些在动物模型6鉴定靶基因。当主人乳腺上皮细胞在体外培养它们往往会失去激素受体 ​​表达，因此激素反应7,8。这个问题可以通过使用基质胶尖端的3D方法来circumventd。以这种方式，克拉克和同事成功地建立了该保持激素受体 ​​的表达，并显示出对孕酮刺激9增殖反应乳腺上皮细胞。然而，2 体内孕激素受体 ​​的靶基因重要的是， 的Wnt-4和RANKL，均未在孕酮刺激诱 ​​导在该系统9。这种方法是最近采取进一步在体外 </EM&gt;激素预处理和RANKL诱导达到10。一个警告仍然是基质胶具有是批次依赖性的活动，是昂贵的，并且要求的实验设计，易于仅用于小细胞数。 基于这一发现， 在体内 ER和PR信号在很大程度上是由旁分泌相互作用11介导的，我们认为，细胞间的相互作用需要维护。即失去作为组织的另一个重要因素是解离为单细胞的体外培养是上皮细胞与细胞外基质之间的相互作用;但这些都是上皮细胞分化的关键和他们的破坏是肿瘤发生的重要12。考虑到这一点，我们建立了一个方法，从新鲜手术丢弃材料13分离乳腺组织的微观结构。乳房实质，由两层上皮内管腔和外myoepitheli人的细胞中，从脂肪组织解剖距离，并进行机械和酶解。洗涤和离心后，乳管的片段保留与许多基质细胞密切相互作用获得的。这些组织的微观结构保持激素反应。该模型在临床标本13进行了验证。这样，本程序可以帮助研究激素作用在乳房中的生物和临床相关的上下文。

<table><tbody><tr><td>Microlitre Centrifuge</td><td>Heraeus Biofuge Fresco</td><td>75005521</td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td>5810 000.327</td><td></td></tr><tr><td>Cryobox</td><td>Nalgene&amp;nbsp;&amp;nbsp;</td><td>5100-0001</td><td></td></tr><tr><td>Controlled Rate Freezer EF600 Grant</td><td>Asymptote&amp;nbsp;</td><td>EF600</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Hera Cell Heraeus</td><td>51022391</td><td></td></tr><tr><td>Roller Mixer SRT9D</td><td>Sturt</td><td>SRT9D</td><td></td></tr><tr><td>Histology Cassettes</td><td>Medizintechnik</td><td>81-0021-00</td><td></td></tr><tr><td>Cell Strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Strile Surgical Blade&amp;nbsp;</td><td>Aesculap</td><td>BB536&amp;nbsp;</td><td></td></tr><tr><td>Scalpel</td><td>Aesculap</td><td>BB084R</td><td></td></tr><tr><td>Forceps</td><td>Aesculap</td><td>BD047R</td><td></td></tr><tr><td>Scissors</td><td>Aesculap</td><td>BC374R</td><td></td></tr><tr><td>Paraffin base mold</td><td>SAKURA</td><td>4166</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Cryomatrix</td><td>Thermoscientific</td><td>6769006</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>Antibiotic/Antimycotic</td><td>Life Technologies</td><td>15240-062</td><td></td></tr><tr><td>DMEM F/12</td><td>Life Technologies</td><td>11039-021</td><td>Prewarm (37 &amp;deg;C)</td></tr><tr><td>Collagenase</td><td>Roche</td><td>11 088 793 001</td><td>Prewarm (37 &amp;deg;C)</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10270</td><td>Can be replaced with Fetal Calf Serum</td></tr><tr><td>Cell Blood Lysis Buffer</td><td>Sigma</td><td>R7757</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F1635</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Roth</td><td>335</td><td></td></tr><tr><td>Isopentane</td><td>Sigma</td><td>M32631</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1009831000</td><td></td></tr><tr><td>Cryogenic vials (5.0 ml)</td><td>VWR, International</td><td>479-0820</td><td></td></tr><tr><td>Ultra low attachment culture dish</td><td>Corning, NY 14831</td><td>3471</td><td></td></tr></tbody></table>

an ex vivo model to study hormone action in the human breast

的激素作用在人乳房的研究已经阻碍了缺乏适当的模型系统。经体外培养，初级乳腺上皮细胞往往会失去激素受体 ​​的表达。广泛使用的激素受体 ​​阳性的乳腺癌细胞系的体内情况相关性有限。在这里，我们描述了一个体外模型来研究激素作用于人的乳房。新鲜的人乳腺组织标本从外科废弃材料如还原mammoplasties或mammectomies被机械和酶促消化以获得包含导管和小叶和多种基质细胞类型的组织碎片。保持在基础培养基不含生长因子，这些组织的微结构保持它们的细胞间接触的组织架构，并保持激素反应数天。它们可容易地处理用于RNA和蛋白质的提取，组织学分析或存储在冷冻介质。荧光激活细胞分选（FACS）可以用来富集特定细胞群。该协议提供了具有高度复杂，多样的人体标本翻译研究一个简单的，标准的方法。

研究雌激素和孕激素的作用，更好地了解人类乳腺癌的分子功能，我们收集获得他们的知情同意后进行还原mammoplasties（ 图1）患者的新鲜人类乳腺组织标本。我们还获得了患者的医疗和生育史，以及血液样品，以确定血清孕酮水平在手术的时间。从新鲜还原mammoplasties组织被机械地和酶促解离。所得的组织微结构具有导管和小叶的原点（ 图2A）的乳腺组织的特性。琼脂糖和染色的肌上皮标记ΔNp63（ 图2B）的石蜡包埋组织的微结构的免疫组织化学分析表明，微观结构保留了正常乳房组织不仅形态特征，而且该CEL的分子谱升的人群。 为了评估激素反应，组织的微观结构进行处理或者与合成孕酮受体激动剂promegestone（R5020）或乙醇的控制。以丰富的激素受体 ​​阳性管腔细胞流式细胞仪根据进行的上皮（EpCAM的）和肌上皮标记;枯竭的内皮细胞，成纤维细胞和抗CD31，抗FAP和鸡尾酒免疫细胞后（CALLA 图3A）的反CD45抗体。的EpCAM +的定量RT-PCR分析，CD10-细胞显示出孕酮靶基因的Wnt-4（ 图3B）的调控中的管腔细胞富集的群体从R5020暴露组织的微观结构。 <img alt="图1" src="/files/ftp_upload/52436/52436fig1.jpg" /> 图1.乳腺组织标本。照片新鲜切开乳房缩小成形术的样品。箭头指向白色组织股包含乳腺实质， 即，乳管及终端导管小叶单位。在丰富的黄色脂肪组织嵌入。而脂肪的颜色是否是患者之间是一致的，脂肪组织的比例，乳房组织以体积计算的大致最大的部分，各不相同。比例尺:5毫米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52436/52436fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52436/52436fig2.jpg" /> 图2.组织显微结构。 （A）的微观结构中的明场图像上培养的低安装板用于机械解离和酶消化后的48小时。比例尺:40μm的（B）中对组织的微结构的组织切片包埋在琼脂糖和石蜡后3天的显微照片。文化。的部分进行免疫组织化学染色的肌上皮标记ΔNp63和用苏木。箭头指向内部，管腔细胞，箭头指向ΔNp63阳性肌上皮细胞。比例尺:40微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52436/52436fig2large.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/52436/52436fig3.jpg" /> 图3.流式细胞术分选分离和组织微结构的RNA分析。 （A）分离从组织显微结构不同的细胞群与R5020或乙醇流式细胞仪处理的。组织的显微组织解离和免疫亲免疫细胞，成纤维细胞和内皮细胞用抗CD45，抗FAP，和抗CD31抗体的鸡尾酒。细胞用antibo死亡对上皮细胞粘附分子（EpCAM的）（克隆HEA-125）以丰富的管腔细胞群（绿色）和常见的急性淋巴细胞白血病抗原（CD10 / CALLA）的肌上皮细胞（克隆SS2 / 36）。示的代表性散射印迹。（B）的棒图表示从诱导与乙醇或R5050 24小时组织的微观结构（绿点云，A组）的腔亚群的相对的Wnt-4 mRNA的表达水平。孕酮靶基因的相对mRNA表达水平， 的Wnt-4标准化针对次黄嘌呤-鸟嘌呤磷酸酶（HPRT）的mRNA水平。所示数据代表平均值±三次重复的SD。 RNA分离和定量RT-PCR方法如前面描述的14进行。引物序列: 的Wnt-4:正向5&#39; - GTGGCCTTCTCACAGTCGTT-3&#39;和反向5&#39; - ACCTCACAGGAGCCTGACAC-3&#39;，HPRT:前进5 &lt;EM&gt;"-GACCAGTCAACAGGGGACAT-3&#39;和反向5&#39; - CCTGACCAAGGAAAGCAAAG-3&#39;。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52436/52436fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about An Ex vivo Model to Study Hormone Action in the Human Breast at JoVE.com

An Ex vivo Model to Study Hormone Action in the Human Breast

我们已经开发出一种新的体外模型来研究激素作用在人的乳房。它是基于组织的微结构从外科乳腺组织标本其中保存组织结构，细胞间的相互作用，以及旁分泌信号隔离。

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

an-ex-vivo-model-to-study-hormone-action-in-the-human-breast

Nanyang Technological University

Research

JoVE Journal

Medicine

An Ex vivo Model to Study Hormone Action in the Human Breast

11.4K 次观看.  Ecole polytechnique fédérale de Lausanne. 我们已经开发出一种新的体外模型来研究激素作用在人的乳房。它是基于组织的微结构从外科乳腺组织标本其中保存组织结构，细胞间的相互作用，以及旁分泌信号隔离。 

视频: An Ex vivo Model to Study Hormone Action in the Human Breast

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

科研

癌症研究

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

乳腺癌临床前模型乳腺肿瘤的矫形移植

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

使用微生理系统模拟人体乳房组织中的乳腺癌

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

人类乳腺上皮细胞的激素反应3D文化模式

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

生物工程

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

医学

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

原发性肿瘤和/或转移相关的临床前和治疗的临床开发的离体处理响应

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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乳腺癌细胞原位注射到小鼠乳房脂肪板

乳腺癌临床前模型乳腺肿瘤的矫形移植

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

使用微生理系统模拟人体乳房组织中的乳腺癌

一种研究并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

人类乳腺上皮细胞的激素反应3D文化模式

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

原发性肿瘤和/或转移相关的临床前和治疗的临床开发的离体处理响应

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

一个

摘要

探索更多视频

此视频中的章节

乳腺癌细胞原位注射到小鼠乳房脂肪板

乳腺癌临床前模型乳腺肿瘤的矫形移植

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

使用微生理系统模拟人体乳房组织中的乳腺癌

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

人类乳腺上皮细胞的激素反应3D文化模式

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

原发性肿瘤和/或转移相关的临床前和治疗的临床开发的离体处理响应

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

一种研究并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型