This research was supported by grants from the New Jersey Commission on Cancer Research (Pre-Doctoral Fellowship DFHS13PPCO17), the National Institutes of Health (CA049062), and the National Aeronautics and Space Administration (NNX15AD62G).

1.文化传媒和细胞的制备<ol><li>制成500毫升之Eagle氏最低必需补充有12.5％（体积/体积）热灭活的胎牛血清（FBS），100单位青霉素培养基，2mM的L-丙氨酰-L-谷氨酰胺和的和每毫升100微克的链霉素。 注:生长培养基和补充物（多个）可以为其它的细胞株或细胞系的生长要求很容易地交换。 </li><li>制备70微升细胞培养基的每个插入物（6孔格式插入）:Eagle氏最低必需培养基补充有50％（体积/体积）的热灭活FBS，2mM的L-丙氨酰-L-谷氨酰胺，和100单位的青霉素和100μg每ml的链霉素。 注:该培养基补充有50％FBS的，以促进细胞附着于刀片的底侧（即下侧）。生长培养基和补充物（多个）可以为其它的细胞株或细胞系的生长要求很容易地交换。 </li><li>制备往在刀片的底侧被镀该细胞的细胞悬浮液。 注意:在这里，用AG1522在75cm 2的细胞培养瓶中生长的正常人成纤维细胞所获得的结果，因此，这些细胞将细胞悬浮液的制备的描述的重点。 </li><li>以收集细胞，去掉生长培养基并用5毫升1×磷酸盐缓冲盐水（PBS）洗涤细胞单层2倍。 </li><li>移除PBS并传播将1ml的0.25％（体积/体积）用2.21毫乙二胺四乙酸（EDTA），预升温至室温（RT）下在细胞上2分钟在RT（室温）胰蛋白酶。 </li><li>猝灭胰蛋白酶活性用9ml完全生长培养基（在步骤1.1中制备），并通过将烧瓶10倍的表面上轻轻吹打细胞悬浮液收集细胞。 </li><li>通过使用血球或电子计数器测定细胞浓度和吸取细胞悬液体积将providÈ250,000细胞每插入到无菌15ml离心管中。 </li><li>沉淀通过离心细胞悬浮液（800 XG，2分钟）。暂停与生长培养基（在步骤1.2中制备）将细胞沉淀以创建每70微升250,000个细胞的浓度。 注:由于在可渗透微孔膜插入细胞培养物是基于二维基材，同样地进行细胞接种，除了细胞最初应悬浮于含有50％（体积/体积）胎牛血清，以方便附着介质。 </li></ol> 2.插入的制备注:为了确保无菌条件下，工作专注于细胞培养层流生物安全柜。 <ol><li>为0.4微米的膜孔径，1微米，或3微米选择插入物（由实验利息（多个）确定，如孔径大小可以用来探索间通讯的不同模式的作用）。 注:0.4微米孔径是SMaL公司升，足以限制形成的小区之间的功能性间隙连接在插入膜的任一侧，并且可以用于研究由分泌的因子间通讯。然而，在1微米和3微米的孔大到足以允许形成功能性间隙连接和可用于研究由两个间隙连接和分泌的因子间通讯。 </li><li>从包装中取出单独插入并放入一个同样大小的多井菜。 注意:插入可与各种膜的表面区域，以满足特定的应用需求（ 如 ，6孔，12孔，24孔插入）。这里，使用6孔插入物所获得的结果，并因此将是模型描述的重点。 </li><li>盖菜，含刀片的菜盖子。持有多井的菜用双手轻轻颠倒的菜，这样插入底部（ 即底部）现在朝上。 </li><li>删除次多井培养皿电子底部，露出刀片的底侧。有一次一个插入工作，用无菌钳子，轻轻举行的地方插入。随着放开手脚，用吸管用宽口尖划出70微升细胞悬液（步骤1.3-1.8编写的），含有250,000个细胞。 </li><li>到板上的细胞，轻轻放在插入件的膜的中央枪头，并慢慢地吸取70微升细胞悬浮液，同时缓慢移动所述膜的表面附近的枪头。小心不要枪头和细胞悬液延伸到刀片的边缘。 注:触摸刀片的边缘，可能会导致该细胞悬浮液，导致附着在干燥细胞的毛细管张力的损失。 </li><li>重复剩余的插入，小心被行使不可直接在裸露的插入工作，以避免潜在的微生物污染。 </li><li>轻轻返回多井盘底部，以覆盖插入。保持与插入的盘反转，孵育在5％（体积/体积）CO 2加湿的空气气氛中，在37℃下30-45分钟。 注:如果在插入接触孔底部的细胞悬浮液，小心地将培养皿底部和安放在盘顶的边缘，以便它形成一个很小的角度的盘的表面之间产生更大的分离和刀片的底侧（其中细胞接种）。这将防止细胞附着到孔的，而不是插入的表面上。 </li><li>继30-45分钟的培养时间，轻轻地取下菜含刀片，并将其放置在层流生物安全柜。 </li><li>用两只手和保持盘盖严密，轻轻重新颠倒菜使得含有贴壁细胞插入件的底侧现在朝下。 </li><li>慢慢地，小心地加2ml（1毫升3微米孔径插入避免migrati通过预热完全培养基的插入孔）的细胞（见步骤1.1），以每孔，从而使插入件的底部被浸入。 </li><li>在包含插入的井加入生长培养基后，将盘子放回培养箱。 </li><li>允许插入到在孵化器保持不受干扰48小时。 48小时后，通过从井的底部抽吸2毫升培养基并加入2ml新鲜生长培养基的进料的细胞。 </li><li>加入新鲜培养基到井，种子将1ml第二细胞群悬浮液（〜250,000个细胞/毫升）上的插入件，其已经具有第一细胞群上的底侧生长的顶侧的底部（为这些后实验中，AG1522（对照）细胞或癌（实验）细胞接种在插入，即已经有AG1522细胞，注定要成为的CAF，上插入件的底侧生长）的顶部。放置培养皿放回培养箱。 注意:如果工作与不很好地粘附于刀片的底部细胞ING，这些细胞可被替换地生长在刀片的顶侧。 </li><li>通过吸从井的插入件和底部的顶部介质播种上刀片的顶侧的细胞的第二群后改变为24，72的介质，和96小时。轻轻1毫升新鲜培养基到插入件的顶侧和2ml添加到井的底部。允许两个细胞群体保持在共培养上可渗透微孔插入物膜的任一侧为120小时。 </li></ol> 3.从胰蛋白酶消化收集插入细胞注意:除了易于获得富集的细胞群，该插入TME模型还允许类似实验处理（ 例如 ，孵育化学品，暴露于氧的条件是高于或低于环境气氛，电离辐射处理等 ）作为其他体外 </em&gt; TME共培养模式。此外，可渗透插入共培养基底可以通过已经为标准的2D组织培养模型开发程序进行分析。例如，细胞可以从插入膜，以获得高度富集的人群，然后可以用于端点分析的任一侧被收获（ 例如，原位免疫检测，免疫印迹）或繁殖用于随后的实验36。 <ol><li>为了收集细胞，去除生长培养基中插入，并将其放入含1毫升的PBS 35毫米的细胞培养皿。用1ml PBS洗刀片1倍的底侧和顶侧。 </li><li>移除PBS并加入200微升的0.25％（体积/体积）与2.21毫摩尔EDTA胰蛋白酶，预温热至室温。 <ol><li>如果收集生长在插入件的底侧的细胞，将胰蛋白酶溶液放入35mm培养皿和所述插入件的底部轻轻放入在室温2分钟的胰蛋白酶溶液。 </li><li>如果收集细胞S于刀片的顶侧生长，将插入到35mm培养皿和胰蛋白酶溶液加入到刀片的顶和在室温下孵育2分钟。 </li></ol></li><li>通过加入800微升完全生长培养基中的猝灭胰蛋白酶活性。 <ol><li>如果收集生长在插入件的底部的细胞，生长培养基添加到35mm培养皿并轻轻吹打1ml的细胞悬浮液的刀片10倍的表面上，与插入被以较小的保持收集细胞角，使得该细胞悬浮液收集到盘中。 </li><li>如果从刀片的顶侧收集的细胞，生长培养基添加到顶部侧和插入件10倍的表面上轻轻地吸取1ml的细胞悬浮液。 </li></ol></li></ol> 4.流式细胞仪鉴定细胞纯度的注意:为了表征可渗透微孔膜插入物保持的t纯度的能力他两个细胞群体（ 即，顶部和底部）长达120小时，节1-3进行，用绿色荧光蛋白（GFP）-tagged被电镀的MDA-MB-231细胞上的刀片的顶侧，并且被镀上0.4μm-底侧非GFP标记AG1522细胞，1μm-，或3微米孔径的插入。 <ol><li>一旦从所述插入件的底侧收集细胞（过程在第3节中描述），沉淀通过离心细胞悬浮液（500克，30秒）。 </li><li>除去上清液，并暂停在补充有1％（体积/体积）FBS的400微升的Hanks&#39;平衡盐溶液（HBSS）中的细胞沉淀。 </li><li>关于流式细胞仪配备有488nm的激光的流程进行流式细胞仪分析，以激发GFP和530/30的带通滤波器以检测GFP的光子发射37。 </li><li>对于细胞识别的目的，使用AG1522控制的细胞群，调整前向和侧向散射电压的流式细胞仪。调整将GFP通道电压来设置蜂窝自发荧光值作为GFP信号较低检测阈值。 </li><li>使用确定对照细胞门控阈值。评估基于GFP阳性细胞相对于对照的存在下各细胞群的纯度。 注:纯粹的人口将是反射没有检测到GPF阳性细胞。 </li></ol> 5.通过原位表征免疫细胞<ol><li>从插入下列细胞的胰蛋白酶处理（见第3），收集细胞，板5×10 4个细胞在250μl的生长培养基（参见步骤1.1），进入无菌玻璃盖玻片，并允许进行1小时中的潮湿空气的气氛附加5％（体积/体积） 的 CO 2在37℃培养箱中。 </li><li>在温育结束时，轻轻取出含有盖玻片培养皿并将其放置在层流生物安全柜。 </li><li>小心加2ml预热的完全培养基（见步骤1.1）向每个皿，使盖玻片浸渍。 </li><li>加入生长培养基含有盖玻片的所有的菜后，在恒温箱在37℃下返回菜的5％（体积/体积） 的 CO 2的潮湿空气的气氛48小时。 </li><li>以下的48小时温育后，用PBS洗样品3次，用4％（重量/体积）甲醛的PBS固定在室温10分钟。 </li><li>冲洗5次用Tris缓冲盐水（TBS:50毫摩尔Tris-氯，pH值7.5，150mM的NaCl）中，然后用0.25％（体积/体积）的Triton X-100，0.1％（重量/体积）皂苷透化在5分钟RT。 </li><li>阻断非特异性结合位点用2％（体积/体积）正常山羊血清（NGS），1％（重量/体积）牛血清白蛋白（BSA），0.1％（体积/体积）的Triton X-100的TBS（阻塞溶液）在室温1小时。 </li><li>隔夜阻挡在4℃解决方案:一抗（1000抗小窝蛋白1（CAV1），1）孵育。 </li><li>用0.2％NGS（体积/体积），1％（重量/体积）牛血清白蛋白，0.1％洗掉未结合的初级抗体（3×，5分钟/洗涤）（沃升/体积）的Triton X-100的TBS（洗涤溶液）。 </li><li>在室温下伴随在封闭液1小时的二级抗体孵育（见第5.4节）。 </li><li>洗掉未结合的二级抗体（3倍，5分钟/洗）用洗涤液（见步骤5.6）。 </li><li>安装盖玻片上用含有4&#39;，6-二脒基-2-苯基吲哚（DAPI）抗褪色封固剂的滑动，并用指甲油密封。 </li><li>观察上配备有外部光源荧光激发倒置显微镜使用63X油倍率物镜的细胞，并使用连接的数码相机进行后续分析拍摄图像。 </li></ol> 6.表征Western印迹细胞注:Western blot检测过程在别处描述38。一个简短的概要说明如下: <ol><li>通过胰蛋白酶消化从插入以下的细胞脱离（见第3部分），通过离心（500克，30秒）沉淀细胞悬浮液。 </li><li>除去上清液，并中止与100微升PBS细胞沉淀2倍。 </li><li>在50μl改性放射免疫测定法除去上清液，裂解细胞（RIPA）缓冲液（的Tris pH为7.5的50mM，NaCl的150mM的，NP40 1％（体积/体积），脱氧胆酸钠一水合物（DOC）的0.5％（体积/体积），十二烷基硫酸钠（SDS），0.1％（体积/体积）），含有蛋白酶和磷酸酶抑制剂混合物。 </li><li>孵育在冰上并离心20分钟以19,000rpm克于4℃15分钟。 </li><li>确定使用蛋白的光密度测定法中，与在RIPA缓冲液的洗涤剂兼容上清液中的蛋白质浓度。 </li><li>分离蛋白通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和electroblot到0.2μm的硝化纤维素膜。 </li><li>孵育膜在含有0.1％（体积/体积）吐温20（TBST）和4％（重量/体积）的TBS脱脂60分钟乳（封闭溶液），并用一级抗体在4℃反应过夜（抗CAV1，1:1000）。 </li><li>在室温（5分钟/洗）用TBST洗膜3倍。 </li><li>孵育膜30分钟在含有与辣根过氧化物酶（HRP）（1:5000）缀合的抗小鼠第二抗体阻断溶液。 </li><li>在室温（5分钟/洗）用TBST洗膜3倍。 </li><li>使用免疫印迹检测系统，化学发光可视化反应产物。 </li></ol> 7.表征间通讯的流式细胞仪注意:为了表征可渗透微孔膜的适应性插入以检查细胞间通讯（ 例如，分泌的因子，间隙连接）的不同模式。切片1-2.12用于0.4μm-底侧，1μm-，或3微米的微孔插入物被电镀非荧光AG1522细胞进行。 <ol><li>文化非标记AG1522细胞35毫米的菜使用第1.1节中描述的90％汇合。 </li><li> - [R樱雪细胞1倍用1毫升的HBSS。 </li><li>称重传感器与钙黄绿素AM在HBSS含有钙黄绿素AM30μm的培养细胞。 </li><li>孵育在37℃，5％（体积/体积） 的 CO 2的潮湿空气的气氛15分钟。 </li><li>洗涤细胞2倍用1毫升的HBSS。 </li><li> Trypsinize细胞通过添加2.21毫摩尔EDTA胰蛋白酶在RT 2分钟的0.25％200微升溶液（体积/体积）。 </li><li>通过加入800微升含12.5％FBS的完全培养基（体积/体积）的猝灭胰蛋白酶活性。 </li><li>把细胞注入悬挂反复吹打。 </li><li>通过使用血球或电子计数器测定细胞浓度和板装载钙黄绿素AM到已经AG1522细胞在底侧生长刀片的顶侧250,000个细胞。 </li><li>孵育在5％CO 2的加湿空气气氛中的插入，在37℃ 6小时。 </li><li> 6小时后，收集细胞，从插入件的底侧，如上所述第3节。 </li><li>沉淀通过离心将细胞悬浮液（500克，30秒）。 </li><li>除去上清液，并暂停于400μl的HBSS补充有1％（体积/体积）FBS的细胞沉淀。 </li><li>分析细胞上配有激光一仪和筛选能够激励和检测钙黄绿素（分别为496纳米和516纳米，）的排放，根据制造商的协议。 </li><li>对于细胞识别目的，使用未染色AG1522控制细胞群进行调整正向和侧向散射电压的流式细胞仪。调整钙黄绿素通道电压来设置蜂窝自发荧光值作为钙黄绿素信号较低检测阈值。 </li><li>确定使用钙黄绿素加载的对照细胞的上阈值限制。评估基于钙黄绿素阳性细胞相对于对照的存在下各细胞刀片通信。 </li></ol>

作者宣称，他们没有竞争或利益冲突。

此处所描述的协议是简单的， 在体外过程适应（ 图1），其利用一个可渗透微孔膜插入物，以产生从异型细胞的共培养高度富集个体的细胞群。显著，该模型适用于研究细胞间通讯的各种模式。的关键步骤包括选择适当的孔径插入特定实验利息（多个），在补充有50％FBS的培养基中插入件的底侧播种所述第一细胞群上的顶侧接种第二细胞群插入，并共培养两种不同的细胞群的时...

肿瘤微环境（TME）是由该共存和进化旁边宿主基质癌细胞的高度复杂的系统。此基质组分通常由成纤维细胞，肌成纤维细胞，内皮细胞，各种免疫组件，以及细胞外基质1。一个显著组分，往往大部分这种基质，被激活的成纤维细胞，经常被称为癌症相关的成纤维细胞或癌相关成纤维细胞（CAF）2,3-。不同于正常的，未活化的成纤维细胞，的CAF有助于肿瘤发生，发展，血管生成，侵入，转移和复发4-11在多种癌，包括乳腺癌，前列腺癌，肺癌，胰腺癌，皮肤癌，结肠癌，食道癌，和卵巢5,6,12-17。然而，CAF的整个发病机制的贡献的确切性质仍然不好界定。此外，临床证据已证明的CAF的预后价值，他们的存在以高品位的恶性肿瘤，治疗失败，和总体预后较差10,18,19相关。 显然，增强我们在CAF发展的始发事件，以及细胞间通讯介导的TME内的作用的理解，可以提供能改善患者的治疗效果令人兴奋的新治疗靶点和增强策略。为实现这一目标，一些在体内和体外模型已经制定出来。而在体内的方法是多个反射病人TME的，它们具有局限性，包括内和肿瘤之间的巨大复杂性和非均质性。此外，从人类受试者的肿瘤样本通常代表着高度发达的TME和不允许TME始发事件的理解。实验动物研究提供了一定的优势，但动物数据对人类的概括应该谨慎进行，由于在physi差异人类和动物如啮齿动物（ 例如，硫醇化学20，代谢速率21，胁迫耐受性22 等 ）之间易学。此外，与人类群体，它在本质上是遗传异质性，实验室动物通常饲养至均一。另外，通常难以检查瞬时生理变化和细胞表型的变化，以及以控制如啮齿类使用动物特定的实验参数。因此， 在体外 2-和3-维（2D和3D）的组织培养模型经常用于推进TME发展的基本理解。尽管他们缺乏体内系统的复杂性的准确写照，这些模型提供了极大的方便机理研究的优势， 在体外模型允许TME更简化，重点突出，具有成本效益的分析，即统计显著数据可以被生成在细胞免费的动物出现的系统性的变化。 有几个品种在体外系统。两种最常用的TME 体外模型由混合单层或球体的细胞培养物。两种培养方法对于细胞间的相互作用（ 例如，正常细胞与肿瘤细胞）和用于各种TME特定细胞表型的变化进行分析的基础研究有利（ 例如 ，从正常成纤维细胞癌相关成纤维细胞的出现）。此外，该球状体是能够创建TME的多个反射组织样结构，并且可以是代表性的肿瘤的异质性23。然而球体往往会产生跨层广泛变化的氧张力梯度，这可能复杂化实验的结论24。不幸的是，这两款车型在他们的隔离作进一步鉴定和研究以下共同的纯细胞群的能力是极其有限文化。这样做将需要至少一种细胞类型是荧光标记的或具有一个识别制造者标记，然后使该混合共培养来大量处理和细胞分选来分离细胞群。而细胞分选仪能够分离出，而纯的细胞群，其中一个必须认识到细胞应激和可能的微生物污染的风险25。 为了促进细胞间通讯的理解，伟大的努力，一直致力于对发展和体外系统紧密模拟体内环境，同时允许一个简化的方法进行优化。一个这样的工具是渗透微孔插入，这是第一次在1953年26研制并随后适用于不同的应用和研究（ 如细胞极性27，内吞28，运输毒品29，组织建模30，叔膜基材ilization 31，旁观者效应32,33 等 ）。这个系统允许在体内样解剖和功能分化的细胞在体内的生长，以及许多的表达标记物未观察到当在不可渗透的塑料制品培养34,35。此外，极薄的多孔膜（10微米厚）允许分子和平衡时间的快速扩散，它模拟体内环境，并允许在两个顶端和基底外侧细胞结构域独立蜂窝运作。插入件的效用作为TME系统的一个附加的优点是上生长在相同的环境条件下在膜的两侧的两个异型细胞群的其物理分离，同时保持通过膜孔间通讯的各种模式。虽然物理上分开，这两个细胞群体的代谢经由分泌的元件联接，正如日这里刻划，也可以通过间隙交界的通道。此外，通过保持在体内局部氧气张力（PO 2）的插入，该模型可以减少在其他系统中观察到的氧气和化学梯度的并发症。相反，它增加了自然机制控制TME的理解。值得注意的是，这两个细胞群体可容易地以高纯度分离，没有荧光标记和/或细胞分选共培养后的长时间。 在这里，我们通过膜孔描述由人乳腺癌细胞和生长，分别在一个可渗透微孔膜插入件的任一侧人成纤维细胞的体外 TME协议，但还没有在连续的双向通信。我们表明，通过使用膜不同孔径，特定类型的细胞间通讯的贡献（ 例如 ，分泌的因子与间隙连接）到显影在TME可以进行调查。

<table><tbody><tr><td>&lt;strong&gt;For Cell Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AG01522 (&lt;em&gt;i.e.&lt;/em&gt;, AG1522) human diploid fibroblast</td><td>Coriell</td><td>107661</td><td>Passage 8-13</td></tr><tr><td>MDA-MB-231-luc-D3H1 breast adenocarcinoma cell line</td><td>PerkinElmer</td><td>119261</td><td>Parental line: ATCC (#HTB-26)</td></tr><tr><td>MDA-MB-231/GFP breast adenocarcinoma cell line</td><td>Cell Biolabs</td><td>AKR-201</td><td></td></tr><tr><td>Eagle&#039;s minimal essential medium (MEM)</td><td>Corning Cellgro</td><td>15-010-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), Qualified</td><td>Sigma</td><td>F6178-500mL</td><td></td></tr><tr><td>Corning Glutagro Supplement (200 mM L-alanyl-L-glutamine)</td><td>Corning Cellgro</td><td>25-015-Cl</td><td></td></tr><tr><td>Penicillin Streptomycin Solution, 100x</td><td>Corning Cellgro</td><td>30-002-Cl</td><td></td></tr><tr><td>Transwell Insert (&lt;em&gt;i.e.&lt;/em&gt;, permeable microporous membrane insert) (0.4 &amp;mu;m pore)</td><td>Costar</td><td>3450</td><td></td></tr><tr><td>Transwell Insert (&lt;em&gt;i.e.&lt;/em&gt;, permeable microporous membrane insert) (1 &amp;mu;m pore)</td><td>Greiner bio-one</td><td>657610</td><td></td></tr><tr><td>Transwell Insert (&lt;em&gt;i.e.&lt;/em&gt;, permeable microporous membrane insert) (3 &amp;mu;m pore)</td><td>Costar</td><td>3452</td><td></td></tr><tr><td>6-well Culture Plate</td><td>Greiner Bio-One Cellstar</td><td>657160-01</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; cell culture flask</td><td>CellStar</td><td>658 170</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS), 1x</td><td>Corning Cellgro</td><td>21-040-CV</td><td>without calcium &amp;amp; magnesium</td></tr><tr><td>0.25% (vol/vol) Trypsin, 2.21 mM EDTA, 1x</td><td>Corning Cellgro</td><td>25-053-Cl</td><td></td></tr><tr><td>15 ml Centrifuge Tube</td><td>CellTreat</td><td>229411</td><td></td></tr><tr><td>35 mm x 10 mm Cell Culture Dish</td><td>Greiner bio-one</td><td>627 160</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Immunofluorescent Microscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse anti-Caveolin 1</td><td>BD Transduction Laboratories</td><td>610406</td><td>&lt;em&gt;In situ&lt;/em&gt; Immunofluorescence - 1:5,000</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>ThermoFisher Scientific</td><td>A-11029</td><td>&lt;em&gt;In situ&lt;/em&gt; Immunofluorescence - 1:2,000</td></tr><tr><td>Bovine Serum Albumin - Fraction V</td><td>Rockland</td><td>BSA-50</td><td>Immunoglobulin and protease free</td></tr><tr><td>16% (wt/vol) Formaldehyde Solution</td><td>ThermoFisher Scientific</td><td>28908</td><td>Dilute to 4% with 1x PBS</td></tr><tr><td>Premium Cover Glass (22 mm x 22 mm No.1)</td><td>Fisher</td><td>12548B</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-50ML</td><td></td></tr><tr><td>SlowFade Gold antifade reagent with DAPI</td><td>Invitrogen</td><td>S36938</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Flow Cytometric Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Calcein, AM</td><td>Molecular Probes</td><td>C3100MP</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14025-076</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Western Blot Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse anti-Caveolin 1</td><td>BD Transduction Laboratories</td><td>610406</td><td>Western Blot - 1:1,000</td></tr><tr><td>Tween-20</td><td>BioRad</td><td>170-6531</td><td></td></tr><tr><td>Nitrocellulose Membrane (0.2 &amp;mu;m)</td><td>BioRad</td><td>162-0112</td><td></td></tr><tr><td>Western Lightning Plus-ECL</td><td>PerkinElmer</td><td>NEL104001EA</td><td></td></tr><tr><td>BioRad DC Protein Assay</td><td>BioRad</td><td>500-0116</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>BioRad</td><td>161-0302</td><td></td></tr><tr><td>Sodium deoxycholate monohydrate (DOC)</td><td>Sigma</td><td>D5670</td><td></td></tr><tr><td>IGEPAL CA-630 (NP40)</td><td>Sigma</td><td>I8896</td><td></td></tr><tr><td>30% Acrylamide/Bis Solution, 37.5:1</td><td>BioRad</td><td>161-0158</td><td></td></tr></tbody></table>

a mimic of the tumor microenvironment: a simple method for generating enriched cell populations and investigating intercellular communication

了解肿瘤细胞和周围非癌性基质之间的早期异型相互作用是阐明导致基质激活和建立肿瘤微环境（TME）的事件非常重要的。几个在体外和在 TME的体内模型已经开发;然而，在一般情况这些模型不容易允许个别的细胞群的分离，非扰动的条件下，进行进一步的研究。为了避免这个困难，我们使用由可渗透微孔膜插入件，其允许简单代高度富集的细胞群的紧密生长，但单独的一个细胞生长衬底采用体外 TME模型，对用于扩展共插入的膜的任一侧 - 文化时代。通过使用这种模式，我们能够产生极大的丰富癌相关成纤维细胞（CAF）的正常二倍体人成纤维细胞的人口以下共培养（120小时）与高转移人乳腺癌细胞，而无需使用荧光标记和/或细胞分选的。此外，通过调节刀片的孔隙大小，就可以控制两个异型细胞群体之间细胞间的通信（ 例如 ，间隙连接通讯，分泌的因子）的模式，它允许的发展基础的机制的调查TME，包括间隙连接渗透性的作用。该模型可作为在增强我们最初的事件导致癌症基质开始时，TME的早期演化，和基质对肿瘤细胞对治疗剂的应答的调节作用的理解的宝贵工具。

在这里，我们适于可渗透微孔膜插入物来开发模仿体内肿瘤微环境（ 图1） 在体外异型细胞共培养体系。该系统允许在插入的多孔性膜的任一侧待生长的时间过长（高达120小时，在我们的使用）两个不同的细胞群体。重要的是，系统能够保持细胞群的纯度，如通过对0.4〜，1-或3间孔隙刀片的顶侧镀GFP标记的MDA-MB-231细胞，并允许它们生长中测定共?...

Watch this Scientific Journal Video about A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication at JoVE.com

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

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Cancer

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10.2K 次观看.  Rutgers University. We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

视频: A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

基于细胞外基质提取物 (ECME) 的三维系统中单核细胞与原代乳癌细胞的通讯分析

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

科研

癌症研究

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

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 ...

医学

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

利用微阵列研究微环境对癌症细胞表型的影响

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

基于流式细胞术的癌症免疫周期监测 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

监测癌症免疫周期并探索肿瘤微环境动力学

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

体外共培养系统以重现癌症免疫循环

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

<meta charset="utf8"></head><body>使用微生理系统进行肾癌和健康组织共培养

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating intricate morphological features in three-dimensional environments with dynamic interactions lacking in conventional models. We implemented a renal cell carcinoma (RCC) co-culture model to recreate the cross-talk between healthy and malignant renal tissue. 
This model is based on the referenced multi-organ platform and consists of co-culturing a reconstructed renal proximal tubule with RCC spheroids. Custom-designed 3D-printed chambers were used to culture human renal epithelial proximal tubule cells (RPTEC) and facilitate their self-assembly into a renal tubule contained in a collagen type I matrix. Caki-1 RCC cells were embedded in an agar collagen matrix, subsequently forming cancer spheroids. Both collagen and agar/collagen gels were optimized to maintain their integrity during cyclic perfusion and withstand shear stress during a minimum culture period of 7 days. 
The gels also enable adequate nutrient supply and cell secretions. Moreover, the agar/collagen gels limit the overproliferation of RCC cells, ensuring relatively homogeneous spheroid size. The MPS chip microfluidic circuits comprise two independent culture chambers with the size of a standard 96-microplate well. The renal tubule and RCC gels populate separate chambers and share the same culture media, which is recirculated approximately twice per minute. Under these conditions, we observed upregulation of immune factor expression and secretion in the renal tubules (interleukin-8 and tumor necrosis factor-alpha). The renal tubules also shift their metabolic activity towards glycolysis under the influence of RCC. This novel approach demonstrates that a co-culture-based MPS can amplify the complexity of RCC in vitro and be employed to study the impact of cancer on non-tumor cells.

<meta charset="utf8"></head><body>肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

Here, we present protocols that detail instructions for implementing 3D cell cultures using collagen and collagen-agarose matrixes in a microphysiological system. These protocols support renal proximal tubule and renal cell carcinoma spheroids co-culture, simulating in vivo conditions and enabling advanced investigation of kidney cancer cell interactions.

肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating ...

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

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基于细胞外基质提取物 (ECME) 的三维系统中单核细胞与原代乳癌细胞的通讯分析

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

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

利用微阵列研究微环境对癌症细胞表型的影响

监测三D培养中的癌细胞入侵和T细胞毒性

体外共培养系统以重现癌症免疫循环

体外共培养系统以重现癌症免疫循环

体外共培养系统以重现癌症免疫循环

肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展