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 border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Cell Culture</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">AG01522 (i.e., AG1522) human diploid fibroblast</td>
			<td style="width:180px;">Coriell</td>
			<td style="width:139px;">107661</td>
			<td style="width:279px;">Passage 8-13</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MDA-MB-231-luc-D3H1 breast adenocarcinoma cell line</td>
			<td style="width:180px;">PerkinElmer</td>
			<td style="width:139px;">119261</td>
			<td style="width:279px;">Parental line: ATCC (#HTB-26)</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MDA-MB-231/GFP breast adenocarcinoma cell line</td>
			<td style="width:180px;">Cell Biolabs</td>
			<td style="width:139px;">AKR-201</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Eagle&#39;s minimal essential medium (MEM)</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">15-010-CV</td>
			<td style="width:279px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Fetal Bovine Serum (FBS), Qualified</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">F6178-500mL</td>
			<td style="width:279px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Corning Glutagro Supplement (200mM L-alanyl-L-glutamine)</td>
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			<td style="width:139px;">25-015-Cl</td>
			<td style="width:279px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Penicillin Streptomycin Solution, 100X</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">30-002-Cl</td>
			<td style="width:279px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (0.4 &#956;m pore)</td>
			<td style="width:180px;">Costar</td>
			<td style="width:139px;">3450</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (1 &#956;m pore)</td>
			<td style="width:180px;">Greiner bio-one</td>
			<td style="width:139px;">657610</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (3 &#956;m pore)</td>
			<td style="width:180px;">Costar</td>
			<td style="width:139px;">3452</td>
			<td style="width:279px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">6-well Culture Plate</td>
			<td style="width:180px;">Greiner Bio-One Cellstar</td>
			<td style="width:139px;">657160-01</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">75 cm2 cell culture flask</td>
			<td style="width:180px;">CellStar</td>
			<td style="width:139px;">658 170</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Phosphate-Buffered Saline (PBS), 1X</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">21-040-CV</td>
			<td style="width:279px;">without calcium &#38; magnesium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">0.25% (vol/vol) Trypsin, 2.21 mM EDTA, 1X</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">25-053-Cl</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">15 mL Centrifuge Tube</td>
			<td style="width:180px;">CellTreat</td>
			<td style="width:139px;">229411</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">35 x 10 mm Cell Culture Dish</td>
			<td style="width:180px;">Greiner bio-one</td>
			<td style="width:139px;">627 160</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Name</td>
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			<td style="width:139px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Mouse anti-Caveolin 1</td>
			<td style="width:180px;">BD Transduction Laboratories</td>
			<td style="width:139px;">610406</td>
			<td style="width:279px;">In situ Immunofluorescence - 1:5000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
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			<td style="width:139px;">A-11029</td>
			<td>In situ Immunofluorescence - 1:2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Bovine Serum Albumin - Fraction V</td>
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			<td>Immunoglobulin and protease free</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">16% (wt/vol) Formaldehyde Solution</td>
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			<td style="width:139px;">28908</td>
			<td>Dilute to 4% with 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Premium Cover Glass (22x22 mm No.1)</td>
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			<td style="width:139px;">12548B</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Triton X-100</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">T8787-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">SlowFade Gold antifade reagent with DAPI</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:139px;">S36938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Name</td>
			<td style="width:180px;">Company</td>
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			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Flow Cytometric Analysis</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Calcein, AM</td>
			<td style="width:180px;">Molecular Probes</td>
			<td style="width:139px;">C3100MP</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:139px;">14025-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Name</td>
			<td style="width:180px;">Company</td>
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			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Western Blot Analysis</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Mouse anti-Caveolin 1</td>
			<td style="width:180px;">BD Transduction Laboratories</td>
			<td style="width:139px;">610406</td>
			<td style="width:279px;">Western Blot - 1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Tween-20</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">170-6531</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Nitrocellulose Membrane (0.2 &#956;m)</td>
			<td style="width:180px;">BioRad</td>
			<td>162-0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Western Lightning Plus-ECL</td>
			<td style="width:180px;">PerkinElmer</td>
			<td style="width:139px;">NEL104001EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">BioRad DC Protein Assay</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">500-0116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">161-0302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Sodium deoxycholate monohydrate (DOC)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">D5670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">IGEPAL CA-630 (NP40)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">I8896</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">30% Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">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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10.1K 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

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

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In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

利用卡尔瓦里亚共同培养和教条法测量骨重塑和再造肿瘤-骨微环境

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

固体肿瘤细胞系侧面人口分析

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

分离近端液研究胰腺腺癌肿瘤微环境

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

工业化、人工智能引导的激光显微切割，用于肿瘤微环境的微尺度蛋白质组学分析

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...