We would like to thank Dr. Konstantin Dobrenis for providing murine microglia for these studies.

1.细胞荧光标记注:标签胶质母细胞瘤细胞系和小胶质细胞用荧光染料9。可替换地，产生如在14中所述组成型表达的荧光蛋白如GFP / RFP的细胞系。 <ol><li>在6孔板板细胞，他们将70 - 对染色天80％融合。对于鼠恶性胶质瘤细胞系GL261和人成胶质细胞瘤细胞系U87，板1×10 6和1.5×10 6个细胞，分别在染色前一个6cm皿24小时。 </li><li>在DMSO准备荧光细胞染色染料溶液，加入5μM染料媒体，无论是罗斯威尔公园纪念研究所培养基（RPMI）或巨噬细胞无血清培养基（MSFM），涡好。 </li><li>孵育细胞用染料在37℃，30分钟和5％ 的 CO 2。 </li><li>除去含染料的介质和新鲜培养基（RPMI或MSFM）添加到细胞中。 </li><li>孵育细胞30分钟。注:细胞现在染色并准备在实验中使用。 </li></ol>预涂2矩阵商会共同培养侵袭试验<ol><li>区分使用佛波醇肉豆蔻乙酸酯（PMA）15 THP-1细胞。 <ol><li>板2 - 3×10 5个 THP-1细胞在1.5ml的RPMI / 10％FBS中的在6孔培养板上。电镀后立即添加100nM的PMA和在37℃，5％CO 2孵育48小时。 注:48小时后，已成功地进行分化的THP-1细胞将粘附并扩散到井服用上的圆形形态的底部。 </li><li>通过用1×PBS洗涤一次除去PMA和用新鲜的RPMI / 10％FBS更换。再等48小时，直到细胞准备在实验中使用。 </li></ol></li><li>通过将它们放置在一个井仅含有500微升的培养基（无血清）和添加的媒体500微升单独顶端平衡矩阵预涂腔室。孵育室在37℃，2小时和5％ 的 CO 2。 <br/&gt;注:预涂室是从制造商那里购买;见材料/设备的表 。 </li><li>轻轻取下细胞（荧光标记的胶质母细胞瘤细胞系和小胶质细胞/巨噬细胞），通过抽吸从细胞中取出介质。洗与2mM EDTA的/ PBS培养皿的细胞。加入500μl2mM EDTA的/ PBS中并孵育5 -在37℃下在细胞培养箱10分钟，5％的CO 2。 </li><li>悬浮细胞在5ml的培养基含有0.3％的牛血清白蛋白（BSA）。 </li><li>离心机在120×g下5分钟。 </li><li>吸上清，重悬沉淀在MSFM / 0.3％BSA的介质（GL261细胞和小胶质细胞）或RPMI / 0.3％BSA中（为U87和THP-1细胞），使得细胞浓度为1×10 6个细胞/ ml 。使用血细胞计数器计数细胞。 </li><li>添加每24孔板的孔500微升媒体/ 0.3％BSA中。 </li><li>从已平衡至少2小时（步骤2.2）腔室移除介质。地方chambeRS以及中含有500微升媒体/ 0.3％BSA（步骤2.7）。 </li><li>如果正在使用，研究其对巨噬细胞作用的抑制剂/小胶质细胞刺激神经胶质瘤侵袭，适当的浓度添加到该底部和腔室的顶部部分两者。 注:CSF-1R的浓度的抑制剂来抑制充分刺激小胶质细胞胶质瘤GL261入侵可以参考9中找到。 </li><li>根据所使用的细胞系，增加细胞在接下来的两个步骤中描述的室顶:鼠标胶质母细胞瘤（2.10.1）和人脑胶质瘤（2.10.2）。 <ol><li>混合1.5×10 5 GL261（成胶质细胞瘤）细胞（150微升）和5×10 4小鼠小神经胶质细胞（50微升）（参见步骤2.6媒体详细信息）。加入300微升MSFM / 0.3％BSA带来体积为500微升。细胞混合物添加到顶部室中，并在37℃，5％的CO 2孵育48小时。 注:如我所描述的小胶质细胞的小鼠从新生小鼠中分离N + 1 6。 </li></ol></li></ol>混合7.5×10 4 U87（胶质瘤）细胞（75微升）和2.5×10 4 THP1（巨噬细胞）细胞（25微升）。加入400微升RPMI / 0.3％BSA带来500微升的体积。细胞混合物添加到顶部室中。孵育细胞在37℃，5％CO 2的24小时。 <ol><li>孵化后，从室顶部细胞轻轻吸并在PBS在3.7％的甲醛将修复室。允许腔室保持在固定剂在室温下（或过夜，在4℃）15分钟。轻轻吸删除固定液，用500微升1×PBS代替。继续进行图像分析第4节。 注:实验可以在这一刻被暂停，并保存在1X PBS成像。荧光染料信号可以为固定后长达2周进行检测。 </li></ol> 3.侵袭试验"3D"-embedding胶质瘤和巨噬细胞/小胶质细胞在矩阵<ol><li>在4℃下过夜解冻矩阵组合。对执行中的冰罩采用矩阵所有进一步的操作。 </li><li>制备10毫克/毫升基质在介质上冰的0.3％的BSA浓度（MSFM或RPMI）。 注:基质的库存浓度通常为大约15毫克/毫升。 </li><li>以制备用于悬浮细胞（标记为在步骤1）中的矩阵，通过抽吸从细胞中除去介质。洗与2mM EDTA的/ PBS培养皿的细胞。加入500μl2mM EDTA的/ PBS中至细胞并孵育5 -在37℃在培养箱中10分钟，5％的CO 2。 </li><li>悬浮细胞在5ml含0.3％BSA的介质。 </li><li>离心机在120×g下5分钟。 </li><li>从颗粒吸出上清液。在媒体/ 0.3％BSA中重悬沉淀，使得细胞浓度为1×10 6个细胞/ ml。使用血细胞计数器计数细胞。 </li><li>添加1.5×10 5 GL261细胞（150微升）+ 5×10 4小胶质细胞（在50μl）至1.5 ml微量离心管中。加入2×10 5个 GL261细胞（200微升）到一个单独的1.5毫升离心管。 注:单独的没有小胶质细胞GL261作为对照。 </li><li>自旋细胞离心，在120×g下5分钟。 </li><li>悬浮细胞在200冰微升冷的矩阵。 </li><li>板50微升（50,000个细胞）到8微米孔径室刀片的顶室的中心。 </li><li>孵育在37℃下30分钟为发生聚合反应。 </li><li>加入200μl无血清培养基的上部腔室和700微升含有血清的细胞生长培养基到下孔。 </li><li>孵育在37℃，5％CO 2的48小时。 </li><li>孵化后，从室顶部细胞轻轻吸并在PBS在3.7％的甲醛将修复室。允许腔室保持在固定剂在室温下（或过夜，在4℃）15分钟。轻轻吸删除固定液，用500微升1×PBS代替。请进入第4 FOR图像分析。 </li></ol> 4.成像测定<ol><li>卸下3.7％的甲醛商会和洗井含有新鲜500μl的1X PBS。 注意:不要让室内干燥。 </li><li>使用棉签轻轻刮过滤表面清洗从过滤器（侧朝向腔室的内部）的顶部部分的非侵入性的细胞。轻轻地启动和逐步增加的压力。做每室这至少3次。 </li><li>放置室中任一玻璃底培养皿中或在24孔板离开。 </li><li>将样品在配备有摄像头和图像采集软件9,10啶或激光共聚焦荧光显微镜的阶段。 </li><li>就拿几个有代表性的领域10X 20X或图像。 </li><li>使用图像分析软件，如ImageJ的，计数已越过过滤器的底面9,10胶质母细胞瘤细胞的数量。 </li><li>如果成像"3D上述4.6 - 4.2"侵袭测定（在第3节中描述），使用荧光激光共聚焦显微镜5-6产生为Z堆叠一系列要图像上的过滤器的下侧的侵入性细胞群，遵循协议的步骤。 </li></ol>

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The authors have nothing to disclose.

高级别星形细胞瘤和胶质母细胞瘤的高度创伤性使这些脑癌非常致命的。因此至关重要它是理解胶质母细胞瘤侵袭的分子和细胞机制。很多人都了解胶质母细胞瘤入侵已经17的过程。使用本文详述的测定形式，我们的实验室已表明在小鼠和人模型肿瘤相关巨噬细胞可以通过5刺激神经胶质瘤细胞侵袭 - 10倍。这个共培养模型忠实复制成胶质细胞瘤细胞的物理上与巨噬细胞/小胶质细胞相互作用的能力将允许对旁分泌和近分泌相互作用（通过配体 - 受体系统，例如Notch的三角洲和肝配蛋白-EPHS），它们可能参与之间的双向通信肿瘤细胞和巨噬细胞。其他分析形式而寻求发现巨噬细胞对神经胶质瘤细胞侵袭的影响通常有细胞作为巨噬细胞接种在孔的底部和胶质母细胞瘤细胞是在腔室3,4物理分离。因此，这些研究表明巨噬细胞刺激神经胶质瘤的入侵（2倍）较为温和上升。此共培养试验改进了这种效果（5 - 10倍）可能表明近分泌相互作用的巨噬细胞中的重要性/小胶质细胞刺激胶质母细胞瘤入侵。 在这个文件中提出的协议包括，必须小心进行，以达到满意的效果几个步骤。如果实验进行如这里描述的和无侵入细胞被看见，所述荧光染料可能已经过期，这将导致非常弱或根本不存在染色。对于使用预涂覆室侵袭测定法，它是必须使用新鲜的侵入腔室不属于过去的到期日期和一直保持冻结的全部时间，直到进行实验。这留给钱伯斯出在室温下超过12小时被认为是在该基质屏障令人满意不再完整并于比正常高得多的频率侵入细胞。对于3D分析，这是绝对至关重要的，以保持所有的移液管和管冰。如果在室温下的时间显著长度，基质将聚合并不能用来悬浮细胞中它。建议在小托盘装满冰在引擎盖用于容纳其中将与基质接触的所有材料。该协议可以被修改成包括一个更高的细胞密度，然而，这可能会影响时间看到胶质瘤侵袭的最佳效果所需的长度。这将是感兴趣的，看看其他细胞类型可以在共培养测定法来加入，它们可能对入侵什么潜在影响。最后，我们承认，大脑微环境是唯一的，难以在体外复制。此协议的一个限制是该入侵CH这里使用的琥珀缺乏正常的脑胶质母细胞瘤而将细胞入侵期间遇到（ 即 ，密密麻麻的神经细胞和星形胶质细胞）的典型组件。虽然与该警告，巨噬细胞的刺激作用/小胶质细胞在该测定是用什么是在体内观察到的，并且可以用于预测哪些抑制剂和/或蛋白质可以与此过程干扰一致。 模仿在培养的肿瘤微环境是给定类型的细胞和基质蛋白多少不同的是存在于恶性肿瘤的不同阶段肿瘤的艰巨任务。然而， 在与微环境的组成部分肿瘤相互作用的体外模型合理准确将极大地促进新的治疗途径的发现。它现在认识到，细胞如巨噬细胞中与进展为恶性，包括血管生成，侵入，免疫逃逸和耐药性相关的多个方面发挥重要的作用。中国奥委会这里描述ulture侵袭测定使用的肿瘤细胞的比例是在患者中观察到的高级别胶质瘤的巨噬细胞（约3:1; 5）。它已经表明，巨噬细胞/小胶质细胞的刺激胶质母细胞瘤侵袭的能力依赖于CSF-1R和EGFR信号作为使用这些途径的药理学抑制JNJ-28312141，PLX3397（CSF-1R）和吉非替尼（EGFR）的强烈衰减的增加在入侵9。此外，使用嵌入式矩阵分析，证实该塞马莫德，已知靶向巨噬细胞和小胶质细胞，也可以阻止小胶质细胞的小分子刺激成胶质细胞瘤侵袭10。从这些共培养实验的结果提供了动力，以验证使用体内模型这些化合物。与来自本文中所描述的体外测定数据一致，CSF-1R与抑制剂PLX3397（其穿过血脑屏障）的封锁很大程度上阻止了GL261胶质瘤细胞的能力，在C57BL / 6小鼠9的大脑侵入。同样，塞马莫德递送到脑显示抑制入侵和大大延长小鼠窝藏GL261肿瘤10的存活与标准的放射治疗协同。这两种方法都可以证明是有效的诊所。 它现在是公理解，巨噬细胞系统是用于各种癌症的进展为恶性3-6，11-13相当重要。已经清楚地确定在乳腺癌和脑癌模型肿瘤相关的巨噬细胞是肿瘤细胞侵袭和转移是至关重要的。此外，巨噬细胞很可能是介导免疫逃逸如巨噬细胞（和如树突细胞相关谱系的细胞）功能作为协调适应性免疫响应于感染18个专业的抗原呈递细胞非常重要。它现在是正常的PH明确一个适应性免疫系统的ysiological功能是防止不受控制的增殖通过破坏异常细胞。向肿瘤细胞免疫可能是由于该癌细胞的高度诱变性质生成由适应性免疫系统识别为外来新抗原的事实。确实是"免疫逃避"的过程中被假定为恶性肿瘤的重要组成部分癌细胞需要防止免疫系统（特别是细胞毒性淋巴细胞，自然杀伤细胞和炎性巨噬细胞）从破坏肿瘤。希望本文中所描述将有利于胶质母细胞瘤的相互作用与巨噬细胞的研究，并且可能允许的类型的问题，我们可以解决有关使用体外培养测定恶性癌症的膨胀测定。

胶质母细胞瘤是一种积极的人脑肿瘤与来自诊断1,2的时间的大约12个月的中位生存期。胶质母细胞瘤是最致命和临床具有挑战性的癌症之一，因为它是难治标准化疗和手术切除。胶质母细胞瘤的扩散性质，使肿瘤细胞在整个大脑的正常传播使晚期肿瘤几乎不可能完全手术切除。这种高度侵入性的方面是胶质母细胞瘤等先进星形细胞瘤的标志特征。因此，许多研究的重点是胶质母细胞瘤细胞侵袭的分子机制。胶质母细胞瘤肿瘤微环境在建立恶性肿瘤3-6至关重要的作用。肿瘤相关巨噬细胞/被证明小胶质细胞负责促进胶质母细胞瘤侵袭7,8。大部分这些研究然而用测量的巨噬细胞/小胶质细胞的作用测定其物理上胶质瘤细胞将它们分开。我们的实验室已着手产生提高检测这让我们能够研究胶质母细胞瘤入侵是如何依赖于共同培养巨噬细胞/小胶质细胞和入侵期间使我们能够像它们之间的物理相互作用。 经典实验测定细胞侵袭包括"标准"Boyden小室趋化和化学侵袭格式。这里待研究的细胞铺在含有对具有指定尺寸（一般为0.4和8微米的直径）的孔的底部的聚碳酸酯过滤器的塑料室中。细胞侵袭的方法，包括一个物理屏障，通常由细胞外基质蛋白质。在化学侵袭测定中，所使用的优选的基质是Matrigel中（以下简称为"矩阵"），通过Engelbreth-Holm的-群（EHS）分泌的胞外基质蛋白混合物小鼠肉瘤细胞和大部分由山口的拉根Ⅳ型和层粘连蛋白。然后将腔室放置在包含有或没有被怀疑刺激侵袭生长因子的细胞培养基的组织培养孔。具有较高侵袭能力通过细胞外基质涂覆的过滤器在较高的频率会侵入并附着到过滤器的下侧的细胞。我们已在以评估小胶质细胞和肿瘤相关巨噬细胞对成胶质细胞瘤细胞侵袭的作用改性该测定。 我们已经能够确定使用该纸张胶质可由5刺激两成胶质细胞瘤细胞系的侵袭中描述的共培养测定法- 10倍9,10。这反映了在胶质母细胞瘤的动物模型中观察到。此外，我们开发了一种三维侵袭测定，其中成胶质细胞瘤细胞和巨噬细胞之间的相互作用/小胶质细胞，可以更直接地检测。胶质母细胞瘤细胞浸润由macropha刺激的程度在3D试验GES /小胶质细胞可媲美什么是使用矩阵涂层的腔体的方式看到。类似的分析是以前开发的入侵在11-13研究与巨噬细胞乳腺癌的相互作用。本文中所描述这两种方法应在解剖胶质母细胞瘤细胞的巨噬细胞/小胶质细胞刺激的入侵的分子机制（S）的能力帮助。

<table><tbody><tr><td>Corning BioCoat Matrigel Invasion Chamber: With BD Matrigel Matrix</td><td>Corning/Fisher Scientific</td><td>Cat: 354481</td><td></td></tr><tr><td>Macrophage Serum Free Media (MSFM) (500 ml)</td><td>Life Technologies</td><td>12065-074</td><td></td></tr><tr><td>CellTracker Red CMTPX Dye</td><td>Life Technologies/Molecular Probes</td><td>C34552</td><td></td></tr><tr><td>CellTracker Green CMFDA Dye</td><td>Life Technologies/Molecular Probes</td><td>C2925</td><td></td></tr><tr><td>GL261 cell line</td><td>National Cancer Institute (NCI)</td><td></td><td></td></tr><tr><td>U87 cell line</td><td>American Tissue Type Culture Collection</td><td>HTB-14</td><td></td></tr><tr><td>THP-1 cell line</td><td>American Tissue Type Culture Collection</td><td>ATCC TIB-202</td><td></td></tr><tr><td>RPMI 1640 Medium (500 ml)</td><td>Life Technologies/Gibco</td><td>11875-093</td><td></td></tr><tr><td>Formaldehyde solution</td><td>Sigma Aldrich</td><td>F1635</td><td></td></tr><tr><td>Corning Transwell polycarbonate membrane cell culture inserts (8 &amp;micro;M pore) 48 per pack.</td><td>Corning</td><td>CLS3422</td><td></td></tr><tr><td>Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, PathClear</td><td>Trevigen</td><td>3445-005-01</td><td></td></tr><tr><td>Fetal Calf Serum (FBS)</td><td>Life Technologies</td><td>Cat: 10500064</td><td></td></tr><tr><td>Bovine Serum Albumin, Fraction V, Heat Shock Treated</td><td>Fisherscientific</td><td>BP1600-100</td><td></td></tr><tr><td>0.5 M EDTA</td><td>ThermoFisher Scientific</td><td>15575-020</td><td></td></tr><tr><td>phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma Aldrich</td><td>P8139-1MG</td><td></td></tr></tbody></table>

coculture assays to study macrophage and microglia stimulation of glioblastoma invasion

胶质母细胞瘤（IV级胶质瘤）是一个非常积极的人类癌症1年后诊断中位生存期。尽管引起胶质母细胞瘤的分子事件的了解增多，这种癌症仍然是非常难治常规治疗。高档脑肿瘤的手术切除是很少完整由于胶质瘤细胞的浸润性高的性质。因此，这削弱胶质母细胞瘤细胞侵袭的治疗方法是一个有吸引力的选择。我们的实验室和其他人已经表明，肿瘤相关巨噬细胞和小胶质细胞（脑居民巨噬细胞）强烈刺激胶质母细胞瘤入侵。本文中所描述的协议被用来模拟使用体外培养测定胶质母细胞瘤，巨噬细胞/小胶质细胞的相互作用。这种方法可以极大地方便，使这个恶性behav的扰乱与巨噬细胞的沟通药物的开发和/或发现IOR。我们已经建立了两个强大的共培养入侵检测，其中的小胶质细胞/巨噬细胞刺激5胶质瘤细胞侵袭 - 10倍。不具有和具有巨噬细胞/小胶质细胞接种于涂覆基质 - 聚碳酸酯室插入标记有荧光标记物或组成型表达的荧光蛋白胶质母细胞瘤细胞或嵌入在三维基质。细胞的侵袭是通过使用荧光显微镜图像和计数仅侵袭细胞在过滤器的下侧进行评估。使用这些测定，若干药理学抑制剂（JNJ-28312141，PLX3397，吉非替尼，和塞马莫德），已经鉴定阻断巨噬细胞/小胶质细胞刺激胶质母细胞瘤侵袭。

使用此处所述的方法中，我们已经表明，小胶质细胞和巨噬细胞可基本上刺激成胶质细胞瘤细胞的侵袭。两种不同的侵袭测定法采用与图1中描绘在图2中，组成型表达的荧光蛋白mCherry GL261细胞涂布在有和没有小胶质细胞48小时预涂布腔室。然而，当培养与小胶质细胞的侵袭能力增加了大约10倍，GL261细胞在自己的微创。 我们使用人细胞系观察到类似的效果。在图3中 ，U87细胞（与5-氯甲基二乙酸酯（CMFDA）绿染色）示出5-6倍的侵入时分化的THP-1细胞共培养。 THP-1是一个人单核细胞系（从急性单核细胞白血病患者来源），可以是二fferentiated到使用佛波脂（PMA）15巨噬细胞样状态。分化的THP-1细胞显示许多的人巨噬细胞的特性，如细胞因子的分泌，并进行吞噬作用的能力。 可替代地，细胞侵袭可以通过在矩阵它们嵌入并直接电镀这到在腔室中插入的滤光器来测量。激光共聚焦显微镜可以用于产生一系列图像产生三维表示（Z堆叠）。相似的是，在涂覆基质室法观察（示于图2 - 3），小胶质细胞（染色CMPTX红色）的共培养刺激GL261细胞（CMFDA绿色）侵入由已越过细胞的数目测得的到过滤器的底侧（ 图4）。这种格式具有能够形象化glio之间的潜在细胞 - 细胞相互作用的额外优点入侵期间母细胞和小胶质细胞。事实上，小胶质细胞似乎紧密与悬浮于3D矩阵（ 图5）的神经胶质瘤细胞相关联。如果只需要端点分析和激光共聚焦是不可用，电池腔室可以作为基质涂覆腔室中所述进行清洗。然后将剩余的（侵入型）的细胞的数目可以通过从标准啶显微镜获得使用图像计数来确定。 电影1示出了这样的测定的三维组件。 <img alt="图1" src="/files/ftp_upload/53990/53990fig1.jpg" /> 图 1: 示意的两种不同的协议用于测定小胶质细胞/巨噬细胞刺激的成胶质细胞瘤细胞侵袭顶板示预涂矩阵室侵袭测定法（第2部分）。底部面板显示3D矩阵侵袭实验（第3节）。图改编自9， <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53990/53990fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53990/53990fig2.jpg" /> 图2.小胶质细胞（MG）加强GL261胶质母细胞瘤细胞侵袭。 （A）在不存在入侵GL261细胞（表达mCherry）的代表性图像（左面板;单独GL261）或小胶质细胞的存在（右面板; GL261 + MG）合并，并接种于包被基质室中，然后孵育48小时，然后该已越过过滤器单元的数量的评估。使用10X物镜拍摄的图像。比例尺= 400微米。（B）的试验只计算侵入GL261细胞（红色）定量。所示数据来自三次独立实验的平均值±SEM表示。（℃）在presenc小胶质细胞刺激GL261入侵的定量药理学抑制剂吉非替尼（5微米），JNJ-28312141（10μM），PLX3397（1μM），PLX5562（1μM）电子。显示的数据是平均从六个独立实验±SEM。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53990/53990fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53990/53990fig3.jpg" /> 图3. THP1巨噬细胞增强U87胶质母细胞瘤细胞侵袭。 （A）的侵入与CMFDA绿色染色的U87细胞的代表性图像镀上涂覆基质室单独（左面板; U87）或分化THP1巨噬细胞（右面板; U87 + THP1）中，随后培养24小时，然后评价已越过过滤器单元的数量。图片使用10X物镜拍摄。比例尺= 400微米。（B）的试验只计算定量微创U87细胞（绿色）。显示的数据是平均从十独立实验±SEM。 <img alt="图4" src="/files/ftp_upload/53990/53990fig4.jpg" /> 。所示图4. 3D侵袭试验GL261和小胶质细胞共培养 （A）图像从Z系列嵌入式独自矩阵GL261细胞的图像（与CMFDA绿染色）的预测（左图; GL261）或鼠小胶质细胞（标有CMPTX红色，右侧面板; GL261 + MG）。的Z预测是在底面4片其包括腔室过滤器的底部的图像栈的并因此表示侵袭细胞。比例尺=200μM的（B）的 GL261细胞（绿色）的定量测定为在过滤器的下侧，如上所述。测定并归一化到在单一GL261细胞入侵GL261细胞的总数。显示的数据代表平均±S表示3个独立实验，一式两份（图改编自10）进行EM。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53990/53990fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53990/53990fig5.jpg" /> 图5. 图像从GL261（绿色）和小胶质细胞（红色）在共培养基质，GL261和小胶质细胞相互作用嵌入式的3D堆栈中的一个切片突出显示与白色箭头。比例尺=100μM的。 <img alt="图1" src="/files/ftp_upload/53990/53990movie1.jpg" /> 电影1: 成胶质细胞瘤和小胶质细胞在3D旋转相互作用围绕整个Z系列从GL261（绿色）和小神经胶质细胞（红色）的共培养所拍摄的图像的3D投影的X轴嵌入在马TRIX。切片是在5微米的增量。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53990/Movie1.avi" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion at JoVE.com

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

coculture-assays-to-study-macrophage-microglia-stimulation

Nanyang Technological University

Research

JoVE Journal

Cancer Research

13.9K 次观看.  Kean University. Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

视频: Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

科研

癌症研究

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

与器官型脑片和癌细胞的3D球体共培养系统

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

医学

<meta charset="utf8"></head><body>检查 3D 胶原基质设置中的巨噬细胞-肿瘤球状体相互作用

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

<meta charset="utf8"></head><body>巨噬细胞-肿瘤球状体共侵袭测定

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

巨噬细胞-肿瘤球状体共侵袭测定

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

Direct-Contact Co-culture of Astrocytes and Glioblastoma Cells Patterned using Polyelectrolyte Multilayer Templates

Glioblastoma Multiforme (GBM) is the most abundant and fatal malignant brain cancer. There are more than 13,000 cases projected in the United States in 2020 and 2021. GBM tumors most often arise from astrocytes and are characterized by their invasive nature, often recruiting healthy tissues into tumor tissue. Understanding communication between astrocytes and glioblastoma cells is vital for the molecular understanding of tumor progression. This protocol demonstrates a novel patterned co-culture method to investigate contact-mediated effects of astrocytes on GBM employing layer-by-layer assembly and micro-capillary-force driven patterning. Advantages include a protein-free cell culture environment and precise control of cellular interaction dictated by the pattern dimensions. This technique provides a versatile, economical, reproducible protocol for mimicking cellular interaction between glioma and astrocytes in glioma tumors. This model can further be used to tease apart changes in GBM molecular biology due to physical contact with astrocytes or with non-contact mediated soluble cofactor communication.

This protocol describes a patterned direct contact glioma-astrocyte co-culture utilizing micro-contact printing on polyelectrolyte multilayers (PEMs) to pattern U87 or A172 GBM cells and primary astrocytes.

Glioblastoma Multiforme (GBM) is the most abundant and fatal malignant brain cancer. There are more than 13,000 cases projected in the United States in ...

生物工程

Matrix-Based Tumor Invasion Model Using Microglia Glioblastoma Co-Culture

Source: Coniglio, S., et al.&#160;<a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53990">Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion.</a> J. Vis. Exp. (2016)
In this video, glioblastoma and microglia cells are mixed with a polymer matrix solution and transferred to a porous chamber insert where a gel forms, embedding the cells. Microglia stimulate the migration of glioblastoma cells through the porous insert, mimicking tumor cell invasion into surrounding tissue, Cells are then fixed and prepared for further analysis.

Source: Coniglio, S., et al.&#160;Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion. J. Vis. Exp. (2016)
In this ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Tumors

Co-Culturing Glioblastoma Cells on Patterned Neurons: A Technique to Monitor the Migration of Glioblastoma Cells on Rat Hippocampal Neurons In Vitro

Source: Guyon, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/62213" target="_blank">Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions.</a> J. Vis. Exp. (2021)
This video describes a co-culture assay of glioblastoma cells on micropatterned neurons, which mimic the myelinated neuronal tracts invasion by the glioblastoma stem-like cells as they migrate on neurons in the peritumoral spaces. This assay can help analyze the effects of pharmacological drugs that inhibit glioblastoma cells&#39; migration on neurons in a glioma.

在图案化神经元上共培养胶质母细胞瘤细胞:一种在体外监测胶质母细胞瘤细胞在大鼠海马神经元上迁移的技术

Source: Guyon, J. et al. Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions. J. Vis. Exp. 
...

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

摘要

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此视频中的章节

体外测定研究肿瘤-巨噬菌体相互作用

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

与器官型脑片和癌细胞的3D球体共培养系统

巨噬细胞-肿瘤球状体共侵袭测定

巨噬细胞-肿瘤球状体共侵袭测定

巨噬细胞-肿瘤球状体共侵袭测定

Direct-Contact Co-culture of Astrocytes and Glioblastoma Cells Patterned using Polyelectrolyte Multilayer Templates

Matrix-Based Tumor Invasion Model Using Microglia Glioblastoma Co-Culture

在图案化神经元上共培养胶质母细胞瘤细胞:一种在体外监测胶质母细胞瘤细胞在大鼠海马神经元上迁移的技术