进行这项研究的凯克医学院，美国加州大学和伦敦大学学院，英国。 KL是由国家卫生研究所获得5 U19 CA148112-02。 BG是由前夕上诉妇科肿瘤慈善机构（英国）项目的资助。 / UCL在UCLH开展这项工作的一些部分部的健康的的NIHR生物医学研究中心资助计划的资助。

图1示出了概述下面描述的工作流程。 1。正常卵巢成纤维细胞在体外寿命的延长，过表达的端粒酶全酶的催化亚基分离<ol><li>卵巢组织可以收集患者有知情同意和批准的机构审查委员会（美国机构）。腹式全子宫切除术或腹腔镜子宫切除术，双侧输卵管卵巢切除术的正常卵巢组织可以收集。在这项研究中，组织由病理学家和卵巢基质活检的细胞培养部的检查。约2-5毫米2的组织样本，从中央区域的卵巢收获试图减少也采样卵巢上皮细胞的机会。 </li><li>新鲜卵巢组织运输的正常卵巢成纤维细胞永生细胞培养实验室（INOF），辅以额外的抗生素和抗真菌的生长培养基。 INOF增长的介质（INOF-GM）包括：中以1:1的比例混合，补充有15％牛胎儿血清，2mM的L-谷氨酰胺，50微克/毫升庆大霉素和250毫微克/毫升两性霉素B 199和MCDB105 </li><li>生物安全柜内部工作：删除任何保持松散连接的上皮细胞，组织，用5毫升的PBS洗涤样品。吸出，重复两次以上。 </li><li>样品加PBS转移到一个干净的直径10厘米的培养皿（P100）。使用无菌镊子处理的组织，并放入第二，干燥的P100干净的培养皿中。使用无菌手术刀切割组织0.5厘米2件。将组织每一块成一个单独的P100的菜和进一步削减成碎片&lt;1毫米2的大小。 GM（20毫升INOF加用抗生素和抗真菌药），并置于37°C，5％CO 2。使用相显微镜监测细胞的生长。可以看出细胞附着的菜机智<html>欣24小时。 </li><li>再进给在一个星期后，通过抽吸除去任何非粘附组织。此后，再供给培养，每周两次。细胞会形成菌落，通常在1-3周。一旦这些菌落是足够大，以继代培养（一旦达到大约500微米），隔离使用胰蛋白酶-涂层克隆磁盘的克隆。 确认细胞是100％的成纤维细胞和其他细胞污染物通过电镀到盖玻片上的细胞系的样品，和一个成纤维细胞的标记（FSP），上皮细胞的标记（泛细胞角蛋白）和血管内皮细胞的标志进行免疫荧光染色（冯Willenbrand因子VIII）。 </li><li>到3 ​​P100菜前一天的端粒酶逆转录病毒转导通道主要的正常卵巢成纤维细胞（NOF）细胞株从一个单一的P100菜。前病毒上清可以购买，或自行生产，使用标准协议，共转染HEK293T细胞（目标=“_blank”&gt; www.stanford.edu/group/nolan/）。 逆转录病毒上清可以使用的pBabe的HYGRO-hTERT载体（Addgene）。从培养箱中移除NOFS，然后吸出细胞培养基中。加入3毫升的病毒上清，每道菜：一盘接收端粒酶逆转录酶病毒， 第二届菜接收GFP-反转录病毒和第三道菜没有接收到病毒。叠加与含INOF通用的聚凝胺（polybrene），使最终浓度为8微克/毫升的病毒上清液。翌日再供给细胞用新鲜培养基。感染两天后，开始选择用低剂量（10 U / ml）的潮霉素B七天后，此剂量可提高到20 U / ml的选择应该是在10-14天完成。 </li><li>表达hTERT或GFP的克隆可以通过环克隆分离一旦达到大约500微米。细胞系需要额外的特性，以确认成功地表达hTERT和保留的主要细胞功能。这包括：（a）采用PCR-ELISA和端粒长度确认端粒酶活性，通过Southern印迹或PCR 6,7;（b）确认的关键标志的表达（ 如抗泛识别上皮细胞的细胞角蛋白抗体的免疫染色，用抗成纤维细胞成纤维细胞表面蛋白抗体）是小学和端粒酶永生化的细胞之间的相似;（c）确认的单元格不显示的肿瘤转化的属性（例如，使用锚地独立生长实验），（d）确认在体外寿命的延长观察细胞表达hTERT基因但不GFP的。 的 hTERT-表达永生化的正常卵巢成纤维细胞（INOFs）应该绕过复制衰老，但不被瘤形成转化;另外INOF细胞应维持成纤维细胞表面蛋白的表达，但没有上皮标记如细胞角蛋白（ 图2） 。 </li></ol> 2。涂层的组织文化视角Ë菜聚HEMA非贴壁细胞培养<ol><li>要准备聚HEMA的解决方案，权衡了1.5克聚HEMA，并放置到一个无菌瓶。加入分子生物学95毫升级无水乙醇和5毫升细胞培养级的蒸馏水。聚HEMA水凝胶会溶解在该溶液中的水含量，和乙醇将消毒制剂。 </li><li>将聚HEMA的溶液在65℃水浴中，直到完全溶解。这可能需要长达8小时。请注意，聚HEMA溶液不能被保存很长一段时间，所以应始终新鲜配制。 故障排除：解聚HEMA需要很长的潜伏期在65°C。定期反转聚HEMA混合的解决方案。的玻璃瓶的底部观察到的粘度表示聚HEMA没有完全溶解，并进一步培养在65℃下需要。 </li><li>层流罩，外套组织培养皿内聚HEMA的解决方案。任何SIZe的组织培养皿中，烧瓶或多孔板可以涂覆有聚HEMA。 表2中给出的推荐量的聚HEMA的解决方案需要为每个容器。 </li><li>允许干细胞培养皿内的层流罩。这可能需要2-3天。可以使用温和的摇摆的摆动平台上，以确保均匀的涂层的更大尺寸的菜肴或烧瓶。如果聚HEMA的解决方案干燥太快了表面上可能不会一帆风顺。为了避免这种情况，确保餐具/烧瓶的盖子保持在整个干燥过程。 </li><li>聚HEMA涂上第二层，晾干上述。 故障排除：在聚HEMA涂层的孔可能发生，特别是当板干燥得太快。双涂层板聚HEMA有助于弥补缺口或裂纹在涂层覆盖后方可使用。板通常需要2-3天，为每个聚HEMA涂层干燥，所以应该准备至少一个星期前，他们将需要细胞培养。 </li><li>聚HEMA涂层钢板可存储在室温下直至使用。 </li></ol>注：1％琼脂糖的解决方案，，溶解在1X PBS中并高压灭菌来消毒，也可用于涂层的细胞培养皿中，以创建一个非粘附表面为短期的三维培养（小于1周）。琼脂糖多井板涂层创建的凹形表面，促进形成一个单一的旋转椭球体的许多细胞系每孔。然而，更长的3D培养聚HEMA涂层被推荐为琼脂糖涂层往往崩解后较长的培养期间。 3。生成三维（3D）异型细胞球体和再喂养的三维培养<ol><li>恢复和扩大INOF和上皮细胞体外培养，准备足够的细胞建立三维异型球体。对于“大众文化”，我们通常种子2 - 4×P100菜和6 INOFs每0.5〜1×10 6上皮细胞一间质上皮岭比4:1。 INOF细胞生长在无庆大霉素或两性霉素B INOF生长培养基（INOF-GM）如果需要的话，成纤维细胞可以进行预处理之前的三维培养（ 如用于诱导衰老，细胞可以用亚致死剂量的过氧化氢）。 </li><li>干燥的聚HEMA板洗净，用5ml磷酸盐缓冲盐水（PBS）中5分钟。吸取PBS和15毫升INOF的-GM更换。 </li><li>同时，正常卵巢trypsinize永生化的成纤维细胞中分离它们从培养皿3-5分钟后，在450×g离心5分钟，用等体积培养基和沉淀细胞通过离心分离中的胰蛋白酶。 </li><li>弃上清。创建一个单细胞悬液重悬细胞沉淀在5毫升INOF的通用，，上下吹打或轻轻涡旋式拌匀。混合10微升细胞悬浮液和10μl台盼蓝（trypan blue）和枚举使用血细胞计数器测定细胞浓度。台盼蓝拒可使用d，来确定活细胞。 </li><li>加入4×10 6 INOF细胞的介质已经分配到聚HEMA涂板。文化传媒量在37〜20毫升，用适当体积的新鲜INOF-GM，并培育°C，5％CO 2。细胞在24小时内开始聚集成多细胞球体。旋转椭圆体的形成可以通过相显微镜监测。 大众文化条件下是难以计数准确的球体数字，但我们估计，我们获得50-100接种4×10 6时，间质细胞球体。球体的尺寸范围一般为80-250微米。 </li><li>重新放入球体培养，每2-3天休息的角度上移液管放置在层流罩板。允许解决的球体。这可能需要长达20分钟。 5毫升的移液管小心地卸下疲惫的媒体，直到留在盘子里只有约4毫升。以关心不吸的球体。饲料用16毫升新鲜INOF-GM和返回的孵化器。 </li><li>在第7天，添加上皮细胞，成纤维细胞球体创建异型如下文化。卵巢上皮细胞（表型正常或转换）的胰蛋白酶消化的准备悬浮液中描述的部分3.4-3.5。使用血球计数仪测定细胞浓度。洗一次在INOF-GM，以防止任何生长因子的上皮生长介质进行。 在我们的实验室中，正常卵巢表面上皮细胞（OSEC）线的收集与知情同意从卵巢切除腹式全子宫切除术或腹腔镜子宫切除术，双侧输卵管卵巢切除术的病人。卵巢一个的无菌cytobrush收集上皮细胞刷的。原代细胞系的建立和转导与 hTERT使用上述方法。详细信息的OSEC文化在2D，3D和OSEC的永生可以发现在参考文献10和11。 </li><li>要形成异型SPHeroids，首重饲料文化单独成纤维细胞球体。的比例为4:1的成纤维细胞，上皮细胞，然后接种成纤维细胞与1×10 6个上皮细胞的旋转椭球体的文化。异型球体在INOF-GM培养。 </li><li>再喂异型文化额外的14天，每2-3天。 </li></ol> 4。处理成像和分子分析<ol><li>可以使用许多不同的技术来分析异型球体。用于成像，它是推荐的，球体与25毫升移液管从培养皿中收获。移液慢慢将扰乱旋转椭圆体的体系结构的剪切力的机会降到最低。在PBS中，沉淀细胞球体然后可以被洗涤由轻轻在100×g离心10分钟离心;更高的离心力可能会损坏球体。另外，文化可以被转移到50ml falcon试管，静置，并在生长培养基中，然后除去。 <ol><li>对于免疫组化化学，固定球体与中性缓冲福尔马林在室温下30分钟。颗粒的球状体和取代的福尔马林，用70％的乙醇。石蜡使用标准协议，用于人体组织的过程。石蜡包埋的球状体，可以是切片，并用苏木精和曙红或特定的分子标记物，使用标准的免疫组化（ 图3）染色。 </li><li>另外，在PBS中洗涤的球状体可以固定在4％多聚甲醛（其制备在PBS中），嵌入在最佳切削温度OCT介质和快速冷冻的冰冻切片和免疫荧光染色。 </li></ol></li><li>分子分析，可以收获的球体，颗粒和之前在PBS洗涤两次，裂解细胞，DNA，RNA或蛋白质的提取。 </li><li>用流式细胞仪分析，球体可以在PBS和分离的由胰蛋白酶或accutase的消化洗涤，在37℃要做到这一点，淹没Falcon管中含有球状我NA水浴，以促进胰蛋白酶消化。到单细胞悬浮液中的球状体的完全分离，可以促进该溶液通过移液向上和向下与1毫升移液管尖端部。小心不超过trypsinize的细胞。 </li></ol>用等体积的培养基中，并中和反应，通过通过40-70微米的细胞过滤网的悬浮液中除去细胞团块。沉淀细胞，用PBS洗涤，枚举和悬浮所需的FACS缓冲液中的细胞数。根据制造商的说明染色细胞。 5。代表性的成果这种方法可以被用于广泛的应用，并且为正常卵巢生物学研究，以及卵巢癌发起的研究。在创建的三维培养，使用市售的细胞外基质凝胶这种方法的一个主要优点是，正常的卵巢成纤维细胞产生的细胞外基质，使üp为核心的球粒。根据我们的经验，正常卵巢成纤维细胞所产生的基质材料是异质的，更接近于比任何市售基质凝胶卵巢的细胞外基质。在我们的实验室中，我们已利用已部分转化使用定义的遗传元件（ 例如CMYC过度）和共培养这些细胞在体外模仿早期卵巢癌卵巢正常和衰老成纤维细胞，在绝经后卵巢的上皮细胞。我们的数据表明，老化的微环境促进肿瘤的功能（ 例如增生）的部分转化的上皮细胞（ 图3）中，而正常的微环境抑制肿瘤进展8。我们的实验室和其他人也用类似的方法来模拟正常卵巢上皮细胞9，输卵管上皮细胞，逐步模型的肿瘤进展<sup&gt; 10，卵巢癌和子宫内膜癌的细胞株11-13同质球体文化。我们已经观察到，都不能检测时，将细胞作为单层培养的正常和恶性细胞的组织学特征的被恢复时，细胞被转换为旋转椭球体的文化。例如，透明细胞性卵巢癌的组织学特征的可检测到的透明细胞性卵巢癌的细胞系（ 图4）在三维培养的，但不是在相同的细胞的单层培养物。此外，三维培养的细胞更密切地反映组织的主要组织中的特定蛋白的表达相比，在相同的细胞在传统的二维单层培养9-11。这些方法的建模正常的和恶性的卵巢组织，也代表了新的候选卵巢癌的肿瘤抑制基因和癌基因和分子生物标志物的发现和功能分析的有价值的工具，可能与二sease发展10,14。 <img alt="图1" src="/files/ftp_upload/4206/4206fig1.jpg" /> 图1。协议的示意性概览三维异型培养。组织培养烧瓶两次用1％的聚HEMA溶液涂布，并使其干燥。聚HEMA涂覆的塑料用1×PBS在使用前立即洗涤。永生化的正常卵巢成纤维细胞（4×10 6个细胞）接种到烧瓶中，用20毫升生长培养基中。球体的形成和生长监测超过7天。此时，接种1×10 6个上皮细胞，成纤维细胞的旋转椭球体的文化，和两种类型的细胞共培养14天前的多细胞球体收获和加工用于下游应用程序（分子分析，成像，等等）。 <img alt="图2" src="/files/ftp_upload/4206/4206fig2.jpg" /> 图2。 端粒酶逆转录酶免疫荧光染色&lt;/ em&gt;的永生化正常卵巢成纤维细胞，原发性卵巢正常成纤维细胞（箴NOFS）和永生化的正常卵巢成纤维细胞（INOFs）快递成纤维细胞特异性蛋白（FSP），不表达细胞角蛋白7（CK-7）和快速波形蛋白（VIM） 。利益的标记显示为绿色，DNA用DAPI染色显示为蓝色，细胞质复染与埃文的蓝色，并以红色显示。 <img alt="图3" src="/files/ftp_upload/4206/4206fig3.jpg" /> 图3。共培养的间质细胞和上皮细胞的免疫组化。左侧面板中，正常卵巢成纤维细胞球体单养，由苏木精和曙红染色。正常卵巢单独培养成纤维细胞在3D形式球体组成一个梭形的形态和丰富的细胞外基质的细胞。右侧面板：正常卵巢成纤维细胞暴露于过氧化氢诱导衰老。部分转化的卵巢上皮细胞（IOSE <su对&gt; cmyc）加入到7天的衰老成纤维细胞的球体。额外的14天共培养后，球被固定，石蜡包埋，切片，双染的增殖标记（MIB1，粉红色），来区分两种类型的细胞，上皮标记（泛角蛋白，棕色） 。曙红用作探讨2，未染色的成纤维细胞在旋转椭圆体的核心是清晰可见（星号）。下面板：上皮细胞增殖在双染色的共培养物中的量化。永生卵巢表面上皮（IOSE）细胞系从三个不同的患者，在这项研究中使用（IOSE4 IOSE11和IOSE19）。衰老成纤维细胞（SEN）促进增殖的合作培养的IOSE cmyc细胞的显着超过在同一细胞的衰老的感应（预衰老，PSN）。 CMYC高表达的的家长IOSE细胞没有PSN SEN成纤维细胞共培养时的增殖没有什么区别。这些数据表明我们在体外共培养模型，可促进老化过程中的卵巢间质的发展，业主立案法团11。 * P≤0.05，双尾配对t检验。 <img alt="图4" src="/files/ftp_upload/4206/4206fig4.jpg" /> 图4。三维培养的细胞组织学特征反映的主要组织。（A）卵巢透明细胞癌透明细胞结构显示特性，这些都是不存在当透明细胞EOC线是生长在塑料，但类似的结构时，同样的细胞生长时检测到的3D球体（的B）。 （C）的正常卵巢上皮细胞生长14天作为球体的扫描电子显微镜照片。表面的微绒毛都可见（箭头）。 （D）浆液性卵巢癌的细胞球体，在文化。 （A，B）苏木精和曙红染色切片石蜡包埋的肿瘤组织或三维球体（C）扫描电子显微镜（D）相差显微镜。的细胞球体可以是球形的（虚线圆圈）或不规则的形状，如在左侧的图像中看到的结构的情况下。 <table border="1"><tbody><tr><td> 碟式/板尺寸 </td><td> 体积的聚HEMA（要施加两次） </td><td> 文化传媒的最终含量 </td></tr><tr><td> 6 - 孔板</td><td> 3毫升</td><td> 5-7毫升</td></tr><tr><td> 12孔板</td><td> 1.5毫升</td><td> 2毫升</td></tr><tr><td> 24 - 孔板</td><td> 500μL </td><td> 1.5毫升</td></tr><tr><td> 48孔板</td><td> 250μL </td><td> 500μL </td></tr><tr><td> 96 - 孔板</td><td> 50微升</td><td> 200微升</td></tr><tr><td> P100培养皿</td><td> 4-5毫升</td><td> 20毫升</td></tr><tr><td> P60培养皿</td><td> 2-3毫升</td><td> 5-7毫升</td></tr><tr><td> F25烧瓶</td><td> 2-3毫升</td><td> 7毫升</td></tr><tr><td> F75烧瓶</td><td> 4-5毫升</td><td> 20-30毫升</td></tr><tr><td> F175烧瓶</td><td> 15毫升</td><td> 30-50毫升</td></tr></tbody></table> 表2。聚HEMA涂料的体积和三维培养。

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没有利益冲突的声明。

早期的上皮性卵巢癌（EOC）的生物学知之甚少。也许多年在这一领域的主要障碍之一是缺乏组织特异性疾病的起源，EOC发展的微环境中的作用的重要性的认识。在过去的几年中，已经很清楚，EOC是一种异质性疾病与多个的不同histophathological亚型，可能有不同的细胞起源不同亚型。例如，当前的数据表明，高品位的浆液EOCS可能源于双侧输卵管分泌性上皮细胞和卵巢表面上皮细胞，至少一定比例被认...

<table border="1"><tbody><tr><td> 试剂 </td><td> 提供者 </td><td> 目录编号 </td></tr><tr><td>聚HEMA，合适的细胞培养</td><td> Sigma Aldrich公司</td><td> P3932 </td></tr><tr><td>分子生物学级乙醇</td><td> Sigma Aldrich公司</td><td> E7023 </td></tr><tr><td>用于细胞培养的无菌水</td><td> VWR </td><td> 12001-356 </td></tr><tr><td> MCDB105 </td><td> Sigma Aldrich公司</td><td> M6395 </td></tr><tr><td>中199 </td><td> Sigma Aldrich公司</td><td> M2154 </td></tr><tr><td> Hyclone公司胎牛血清</td><td> Thermo Scientific的</td><td> SH30088.03 </td></tr><tr> &lt;TD&gt;庆大霉素</td><td> Sigma Aldrich公司</td><td> G1397 </td></tr><tr><td>两性霉素B </td><td> Sigma Aldrich公司</td><td> A2942 </td></tr><tr><td> pBabe的温湿度计端粒酶逆转录酶</td><td> Addgene </td><td> 1773 </td></tr><tr><td> PBS </td><td> VWR </td><td> 12001-766 </td></tr><tr><td> 0.25％胰蛋白酶-EDTA </td><td> Invitrogen公司</td><td> 25200-072 </td></tr><tr><td>细胞过滤器（40或70微米） </td><td> VWR </td><td> 21008-949 21008-952 </td></tr><tr><td>的反成纤维细胞表面蛋白的抗体，克隆1B10 </td><td>西格玛</td><td> F4771 </td></tr><tr><td>反泛角蛋白抗体（C11） </td><td>圣克鲁斯</td><td> SC-8018 </td></tr><tr><td>聚凝胺（溴化己二甲铵溴） </td><td>西格玛</td><td> 107689 </td></tr><tr><td> TeloTAGGG端粒酶PCR ELISA PLUS </td><td>罗氏公司</td><td> 12013789001 </td></tr><tr><td> TeloTAGGG端粒长度的测定</td><td>罗氏公司</td><td> 12209136001 </td></tr></tbody></table> 表1。试剂和仪器在这项研究中。

heterotypic three-dimensional in vitro modeling of stromal-epithelial interactions during ovarian cancer initiation and progression

卵巢上皮细胞癌（EOCS）是在西方社会的妇科恶性肿瘤死亡的首要原因。尽管手术治疗和改善铂类为基础的化疗方案，很少有改善在EOC存活率，超过四十年的1,2。虽然第一期肿瘤5年生存率&gt; 85％，Ⅲ/Ⅳ期疾病的存活率为40％。因此，平机会的高发生率，死亡率可显着减少，如果检测到肿瘤在早期阶段，更可治疗，3-5。目前，疾病早期发展的分子遗传学和生物学基础，更是知之甚少。更具体地说，很少有人了解的微环境在肿瘤发生中的作用，但已知的危险因素的EOCS（ 如年龄和奇偶校验）表明，微环境中起着关键的作用的早期起源EOCS的。因此，我们开发了三维异型模型既正常卵巢和早期卵巢癌。而在正常卵巢中，我们共培养正常卵巢表面上皮细胞（IOSE）和正常间质成纤维细胞（INOF）细胞，永生化的retrovrial转导的催化亚基端粒酶全酶（hTERT）的延长寿命这些细胞在培养。为了模拟卵巢上皮细胞转化的CMYC IOSE细胞癌基因的过度表达，再次合作，培养与INOF细胞的最早阶段。这些异型上皮细胞的转化和侵袭的模型被用来研究老化和衰老的影响。在这里，我们描述了在发展的这些三维模型的方法步骤，这些方法是不正常卵巢和卵巢癌组织的发展，可用于研究其他类型的组织基质和上皮细胞间的相互作用是一个根本方面的组织维护和二sea​​se发展。

Watch this Scientific Journal Video about Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression at JoVE.com

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

我们描述的方法建立<em在体外</em&gt;异型三维模型，包括卵巢成纤维细胞和正常卵巢表面或卵巢癌上皮细胞。我们讨论了使用这些模型来研究间质 - 上皮细胞相互作用过程中发生卵巢癌的发展。

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

heterotypic-three-dimensional-vitro-modeling-stromal-epithelial

Research

JoVE Journal

Medicine

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

15.0K 次观看.  University of Southern California. 我们描述的方法建立

视频: Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

科研

医学

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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 ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三维文化：一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.