This work was funded by The National Centre for the Replacement, Refinement and Reduction of Animals in Research (G1000121 ID no.94513) and the Oracle Cancer Trust. SE is supported by The Institute of Cancer Research and Cancer Research UK (grant number C309/A8274). We acknowledge NHS funding to the NIHR Biomedical Research Centre.

1.代可重复大中球体瘤<ol><li>洗肿瘤细胞单层用磷酸盐缓冲盐水（PBS 5毫升的25cm 2或8 - 10毫升为一个75cm 2的烧瓶）中，加入细胞解离酶（1毫升的25cm 2或2毫升一75厘米2烧瓶），并孵育细胞，在37℃进行2 - 5分钟。 </li><li>检查细胞脱离，在显微镜下，中和细胞解离酶与完全生长培养基中（5毫升的25cm 2或8毫升一75cm 2的烧瓶中）。 </li><li>离心机细胞悬浮液在500×g离心5分钟。 </li><li>去除上清液，挖掘管和1毫升完全培养基用吸管P1000重新悬浮细胞沉淀。这应该产生单细胞悬浮液没有细胞簇。 </li><li>计数使用血球细胞和稀释的细胞悬浮液，以获得0.5 - 2×10 4个细胞/ ml（最佳细胞密度需要为在邻每个细胞系确定经过细胞种植12,13500μm的直径为4天） -的rDer获得300肿瘤球体。 </li><li>细胞悬液转移到无菌的贮存器，并使用多通道移液管，取200μl/孔到超低附件（ULA）96孔圆底板12。 </li><li>传送板向恒温箱（37℃，5％CO 2，95％湿度）。四天后，目视确认肿瘤球体的形成，并继续与3D侵袭实验。 </li></ol> 2. 3D球体肿瘤侵袭试验<ol><li> BMM解冻冰。 </li><li>保持一组无菌过滤嘴为P10，P200和P1000移液器和无菌试管（1.5毫升体积，或者根据所需的总体积在较大的）在-20℃下。 </li><li>上放置装有冰4天龄的球状体的ULA 96孔板中。 </li><li>使用多道移液器，轻轻地从球体板除去生长培养基的100μl/孔。对于这一步的角度对我的提示nside壁在U形底的，避免了与井和球状体的位置的底部接触;尽量减少球体的干扰。 </li><li>用冰冷的技巧，转移到BMM冰冷的管子。细胞因子诱导的入侵或药物评价研究，添加试剂（以2x终浓度）用冰冷的提示BMM。例如，使用表皮生长因子（EGF）刺激侵袭的CAL S和CAL - [R鳞癌细胞。纷飞轻轻，避免泡沫的形成拌匀。 </li><li>轻轻免除100微升BMM进入U型底部的孔，瞄准尖端朝向井的内壁。这个步骤是最关键的，以获得最佳的图像分析，球状体必须保持在阱12的中心。 </li><li> 6重复/条件 - 为所有需要的井，让5重复步骤2.6。如果必要的话，切换到一个新鲜冷冻的提示。 注意:当更多的经验，DISPENSE BMM使用多道移液器。 </li><li>用消毒针头，除去气泡，如果存在的话。 <ol><li>使用显微镜，目视检查球状体是在中心位置。如果不是这样，离心板在300×g离心3分钟，在4℃。这将确保球体居中位于每个孔中。 </li></ol></li><li>所述板转移到孵化器在37℃，并允许BMM固化。 </li><li> 1小时后，使用多道移液器，轻轻地加入100微升/完全生长培养基的好。对细胞因子诱导的侵袭或药物评价研究，包括在培养基中的细胞因子或抑制剂（1倍最终浓度）。替代地，如果试剂没有添加到BMM在步骤2.5中，将它们添加到所述介质在该点（3倍的所需最终浓度）。 </li></ol> 3.自动图像采集<ol><li>扫描板的流式细胞仪（用于规格见表设备和材料 ）每隔STA从时间rting零（T0 =成立的侵袭测定当天，对步骤2.10到72小时，或96小时为较慢侵入肿瘤细胞系后立即使用。 </li><li>选择" 合流"的应用程序。 </li><li>检查球体是重点，如果需要，手动调节和固定的" 焦点抵销"。 </li><li>在" 采样设置 "，选择要扫描的视图（FOV）一个中心领域。以下BMM此外，肿瘤球状体应该保持在中心位置，因此，就没有必要扫描整个井。 </li><li>在软件中，限定孔以通过突出显示它们在板地图上进行扫描。点击" 开始扫描 "。 注:扫描自动保存，可以在以后离线审查。 </li></ol> 4.自动图像分析<ol><li>选择" 合流"的应用程序。 </li><li>在"分析 "选项卡，调整应用程通货膨胀设置（ 例如 ，"精确":低，正常，高;"强度阈值":1 - 15），以产生围绕球体精确分割，包括细胞过程和侵袭伪足伸入BMM（ 见图1）。调整每一个肿瘤细胞系和/或在不同时间点的设置。在" 合流"的应用措施的井区覆盖的侵入细胞，在选择FOV的％。 </li><li>检查分析设置适当（ 即分割是准确的），在其他板块球体通过点击几个不同的井。如果分割不能准确地勾勒出一个球体，进一步调整应用程序设置。 </li><li>点击" 开始分析 "。 </li><li>在"结果 "选项卡，选中多口井，以验证质量分析。导出"嗯级数据&#39;到电子表格程序。 </li><li> Repea吨分析的所有时间点。计算平均值％汇合的重复井和情节入侵（％汇合）与时间的柱状图，用科学的图形和选择的统计软件。 </li></ol> 5.手动图像采集<ol><li>记录每个肿瘤球体的图像的时间间隔开始从t = 0到72 - 96小时，这取决于侵袭的细胞系中的问题，使用倒置显微镜（对于96孔板这需要约20的速度 - 30分钟）。用10倍的目标。使用4X物镜时侵入面积过大，无法完全视10X字段内捕获。 </li><li>保存获得的每一个人形象。 </li><li>为校准，同时使用10X和4X目标采取的一个阶段刻度（1毫米）的图像（和保存图像）。 </li></ol> 6.手动图像分析<ol><li>打开阶段刻度图像转换成所选择的图像分析软件。 </li><li>进入GRATicule测量，单位（μm）时，目标使用（10X或4X）和执行校准。此步骤是必需的仅一次。对于后续的图像分析，只需重新加载所需的校准设置。 </li><li>打开侵袭测定图像和选择取决于显微镜物镜（10X或4X）校准设置用于获得图像。 <ol><li>可替代地，获得的进口上的图像的成像仪（步骤3）和手动分析，如对于使用倒置显微镜获得的图像（参见图3和图4中所示的结果）。 </li></ol></li><li>测量所覆盖球体的区域。 <ol><li>在这里使用的代表性结果（规格见表设备和材料）的软件，进入"测量"，然后选择"计数/大小"。选择"文件"，然后"加载设置"，然后选择预先确定的ASSAÿ设置。然后选择"计数"。球体然后应准确分割。 </li><li>或者去"编辑"，然后"绘制/合并对象"，打开"魔术棒/跟踪"窗口。使用棒光标点击球体图像上，并获得分割。选择"跟踪"（在" 魔棒"窗口）使用鼠标手工勾勒球体。 </li></ol></li><li>不同的参数（面积，直径，周长等 ）的电子表格导出测量。在电子表格记录的图像信息（ 例如 ，井数，相对时间点）。 </li><li>重复绘制球体（入侵）与时间用科学的图表和统计软件的平均面积。可替代地，计算在每个时间点在球体区域中的变化，相对于在t面积= 0。然后情节入侵（％T0）与时间的关系为线性硅胶制作pH值。 </li></ol>

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所有作者宣称没有利益冲突的竞争。

这里介绍两个肿瘤模型（胶质母细胞瘤和SCCHN）是专门选择体现我们的3D试验，因为他们是临床上尚未满足的需求进行有效的治疗方法12项相关局部侵袭性癌症。下列药物治疗， 体外药效学（PD）生物标记物的变化的评估可以通过以下具体的使用市售的试剂，以允许从BMM和/或通过整体侵入肿瘤球状体的免疫荧光分析细胞回收蛋白印迹分析或裂解物的免疫测定法容易地进行坐骑染色。 这入侵检测是一个有用的技术对肿瘤细胞侵袭快速，重现评估变成半固体介质，因此在体外药物筛选尤其适用于未来。癌细胞侵入基质在三维方式，因为它们散开从"微肿瘤"，由肿瘤球体表示，且延伸到一个extracellulAR矩阵式的环境。真正的立体感的测定是显而易见的，在细胞的形态，这是从平坦的，粘附形态细胞假定在固体基质上移动时不同。侵入的程度使用任一的成像仪，让一个自动读出容易量化，或通过使用标准显微镜结合图像处理软件。此外，这种方法适合于荧光成像13（例如用表达荧光蛋白或细胞系预先用荧光染料标记）。 该方法简单，通过添加BMM，所代表的只是关键步骤来执行时，有干扰，在球体的中心位置的危险U形好。这可能会导致不理想的图像分析，以在不同的焦平面的球体。因此，关键是要小心缓慢地加入BMM到每口井。 BMM加入后，建议查询的板在显微镜下，并且如果定位被认为是不能接受的，这可以通过温和离心补救。随着经验的积累，这是几乎没有必要。虽然这种方法是稳健和重复性非常，除实验性的变化可能会出现BMM的不同批次。为了避免这种情况，最好是购买足够的BMM从单一批，完成了一系列的研究。 该方法的限制（如任何这样的测定法）是在侵袭和扩散，其中的肿瘤细胞在测定时间帧期间可能经受区分的困难。虽然细胞的倍增时间可以考虑，或细胞周期抑制剂诸如松弛素D推出​​，这是不容易控制或明显肿瘤进展的这两个不同的方面区分，特别是对快速生长的肿瘤细胞，并为那些有更多的"膨胀"，而不是浸润，浸润的格局。为此原因有人建议，一个平行三维生长测定中进行评估的任何抑制或刺激剂的具体的效果。如果仔细的剂量应答研究中进行，有可能选择浓度最小化对细胞增殖的影响。例如，我们已经表明，HSP90抑制剂17-AAG抑制U-87 MG 3D肿瘤球体入侵已经在24小时，在以下的浓度的浓度由50％（GI 50）12抑制三维成长。 另一方面，该测定的意义相对于其他标准入侵检测（ 例如 ，基于过滤测定或侵入单个细胞到三维矩阵1），是肿瘤细胞侵入到从球体周围基质，类似于一个"微肿瘤"或"微转移"，并因此考虑到重要肿瘤块的病理生理学的各个方面。我们目前的方法提供的信息肿瘤细胞，最初的时候离开球体，并且还单独，当单个细胞中的BMM达到更遥远的地区的集体行为。此外，肿瘤球状体内的细胞可以经历缺氧和营养缺乏，通过基因表达的变化，能促进迁移和侵袭;这些特征不存在于二维检测。此外，所有这一切都是在通过使用特定的96孔板的高通量格式和最新成像技术可用，允许更复杂的三维检测，以在靶验证和药物发现很容易地使用。 我们提出的方法，可容纳更多的应用程序，以解决肿瘤细胞侵袭的其它方面。特别地，额外的复杂性可通过加入宿主细胞，如成纤维细胞和/或内皮细胞的设想，进球体本身或周围基质。这还可以被修改，不仅在刚度方面（通过使用不同的共ncentrations BMM的），而且在组成方面（通过加入取决于所采用的肿瘤模型的特定的组织/器官其它组分: 例如，生腱蛋白为脑癌16和胶原为乳腺癌17）。 类似的建立（产生球状体和成像的）也可适于评估在3D，其中肿瘤球状体是共培养胚状体类似的复合组织12或与其它小区特定组织体（ 例如 ，星形细胞用于组织入侵胶质瘤18或隐窝文化为胃肠道癌），但需要进一步努力，使自动图像分析。

经典抗癌药物发展主要是着重于使用体外基于细胞的测定，以屏幕为细胞毒性剂是抑制肿瘤细胞增殖和/或促进程序性细胞死亡（凋亡）。更近来，努力走向靶向治疗与恶性细胞增殖的潜在的分子病因抑制剂的开发。尽管一些壮观结果，这些试剂通常与药物抗性的快速发展相关联。因为它是肿瘤细胞的侵袭性和转移潜力，负责大多数癌症死亡的，而且复发疾病往往呈现一个更积极的表型，这是合乎逻辑也考虑在体外实验模型1,2-鉴定新型互补代理商将抑制癌症这些附加键&#39;标志&#39;。 在恶性进展，肿瘤细胞获得侵入周围组织和/或扩散到远处器官（转移）的能力。癌细胞侵袭伪足由3,4形成穿透基底膜。这些结构是富含肌动蛋白丝，特定粘附蛋白和蛋白酶和共同负责肿瘤细胞运动和细胞外基质（ECM）5的劣化。侵袭伪足伸入ECM和被认为是对肿瘤细胞侵袭重要并且还外渗到血管通道，促进血行（或淋巴）传播和转移。 当前的标准方法来评估肿瘤细胞体外侵袭包括以下内容。 Transwell小的或基于Boyden小室测定法2,6，其中单细胞悬浮液接种在涂有一层厚厚的ECM来源的蛋白质的一个过滤器的顶部。细胞再侵入并移动到下腔室响应于化学引诱。常用的ECM蛋白质是I型胶原蛋白，或基底膜样基质（BMM， 例如 ，基质胶，从EHS肿瘤7派生），模仿基底膜的天然成分。 作为替代的基于滤波器的Boyden小室型测定8，细胞可以接种在一个ECM凝胶（向其中成纤维细胞可添加）在那里形成一个单层，然后单独或集体侵入到凝胶的顶部。入侵可以在从凝胶表面9行进侵入细胞和/或距离的数量来衡量。肿瘤细胞还可以完全嵌入到矩阵或者作为单细胞悬浮液，或作为球状体-通常的ECM凝胶-两层使细胞侵入出肿瘤块到周围的基质2,6,10,11之间放置。 三维（3D）肿瘤球体侵入这里描述测定提供了一种使用highl一种快速，自动化的入侵系统Ý再现的，标准化的方法12，在96孔板形式中，每孔1球体。 BMM直接加入到每个孔中，以提供半固体基质在其中肿瘤细胞从球体体侵入。 96小时 - 肿瘤细胞侵袭的程度每隔一段72监测。此方法提供了以下优点上述当前体外侵袭测定法:细胞被组织成一个三维结构模仿肿瘤微区或微转移的肿瘤;肿瘤球状体在尺寸上高度重复性;侵袭测定在原位进行在同一板作为肿瘤球体发展，而不需要将它们移动到次级板;的方法中，结合自动图像分析的最新技术，使兼具高含量和肿瘤细胞侵袭的高通量分析。 使用成像仪，其中扫描96孔进行的图像分析板在10分钟内。通过使用合流应用的程度和侵袭率或者通过单细胞或由细胞团从肿瘤球状体扩散出来并侵入到基质可以以动态的方式来测量实现。对于较低的吞吐量，用于图像分析的另一种方法，基于使用倒置显微镜和标准成像软件的呈现。

<table><tbody><tr><td>Corning Matrigel basement membrane matrix</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png" /> Corning</a></td><td>354234</td><td>Keep at 4 &amp;deg;C to prevent solidification and dispense using cooled pipette tips</td></tr><tr><td>Cultrex basement membrane extract, PathClear</td><td><a href="http://www.trevigen.com" target="_blank"><img src="/img/new_window.png" /> Trevigen</a></td><td>3432-005-01</td><td>Keep at 4 &amp;deg;C to prevent solidification and dispense using cooled pipette tips</td></tr><tr><td>Ultra-low attachment 96-well plate</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png" /> Corning</a></td><td>7007</td><td></td></tr><tr><td>EGF</td><td><a href="http://www.miltenyibiotec.com" target="_blank"><img src="/img/new_window.png" /> Miltenyi Biotec</a></td><td>130-093-825</td><td>Add 100 &amp;mu;l 1% BSA (w/v) in water to 100 &amp;mu;g EGF. To 25 &amp;mu;l of this, add 4,975 &amp;mu;l RHB-A medium to give a 5 &amp;mu;g/ml working stock. Aliquot and store at -20 &amp;deg;C</td></tr><tr><td>RHB-A medium</td><td><a href="http://www.stemcellsinc.com" target="_blank"><img src="/img/new_window.png" /> Stem Cells Inc.</a></td><td>SCS-SF-NB-01</td><td>For diluting EGF</td></tr><tr><td>DMEM</td><td><a href="http://www.lifetechnologies.com/" target="_blank"><img src="/img/new_window.png" /> GIBCO</a></td><td>31966-021</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fetal bovine serum</td><td><a href="http://www.biosera.com" target="_blank"><img src="/img/new_window.png" /> Biosera</a></td><td>1001/500</td><td>Heat at 56 &amp;deg;C to inactivate complement before use</td></tr><tr><td>TrypLE</td><td><a href="http://www.lifetechnologies.com/" target="_blank"><img src="/img/new_window.png" /> GIBCO</a></td><td>12605</td><td>Cell dissociation solution</td></tr><tr><td>Celigo cytometer</td><td><a href="http://www.nexcelom.com" target="_blank"><img src="/img/new_window.png" /> Nexcelom Bioscience</a></td><td>n/a</td><td>Specialist equipment for image capture and analysis. Manual image capture by microscopy and independent image analysis software is an alternative</td></tr><tr><td>IX70 inverted microscope</td><td><a href="http://www.olympus.co.uk/" target="_blank"><img src="/img/new_window.png" /> Olympus</a></td><td>n/a</td><td>Used with camera for manual image capture</td></tr><tr><td>Retiga Exi Fast 1394 digital camera</td><td><a href="http://www.qimaging.com/" target="_blank"><img src="/img/new_window.png" /> Qimaging</a></td><td>n/a</td><td>Attached to microscope for manual image capture</td></tr><tr><td>Image-Pro Plus 7.0</td><td><a href="http://www.mediacy.com/" target="_blank"><img src="/img/new_window.png" /> Media Cybernetics</a></td><td>n/a</td><td>Software used to control camera and microscope for manual image capture</td></tr><tr><td>Image-Pro Analyzer 7.0</td><td><a href="http://www.mediacy.com/" target="_blank"><img src="/img/new_window.png" /> Media Cybernetics</a></td><td>n/a</td><td>Stand-alone image analysis software for manual measurment of spheroid size</td></tr><tr><td>Excel 2010</td><td><a href="http://www.microsoft.com/" target="_blank"><img src="/img/new_window.png" /> Microsoft</a></td><td>n/a</td><td>Scientific graphing and statistical software</td></tr><tr><td>Prism 6.0</td><td><a href="http://www.graphpad.com/" target="_blank"><img src="/img/new_window.png" /> GraphPad</a></td><td>n/a</td><td>Scientific graphing and statistical software</td></tr></tbody></table>

three-dimensional (3d) tumor spheroid invasion assay

周围正常组织的侵袭，一般被认为是恶性肿瘤的重要标志（相对于良性）肿瘤。特别是对于一些癌症（ 如脑肿瘤，如胶质母细胞瘤和头部鳞状细胞癌颈部- SCCHN）是严重发病的原因，可危及生命在无远处转移均匀。此外，随后有复发的癌症治疗往往不幸出现带有更积极的表型。因此，有机会对目标的入侵的方法，以提供新的治疗方法可能是互补的标准抗增殖剂。到现在为止，这一战略已经阻碍了缺乏适合入侵的详细分析和药物筛选稳定，可重复检测。在这里，我们提供了一个简单的微平板方法（基于统一的，自组装3D肿瘤球体），其中有这样的STU潜力巨大死亡。我们举例说明使用人成胶质细胞瘤细胞系和也在有针对靶向表皮生长因子受体（EGFR）抑制剂抗性的发展与增强基质侵入潜在相关联的SCCHN模型试验平台。我们还提供半自动化定量的两种可供选择的方法:一种使用成像仪和一个第二，它简单地需要标准显微镜和图像捕捉数字图像的分析。

三维肿瘤球体入侵是使用一种简单的，可重复的，自动化的方法示意性地示于图1评估:再现地大小肿瘤球状体是通过电镀细胞向ULA 96孔圆底板中获得。后4天开始球体被嵌入BMM。这提供了一种半固体结构成肿瘤细胞侵入和摊开球体的。使用成像仪96小时 - 球状体在每个的基部井和侵入的BMM位于中央是很容易的间隔从t = 0到72开始监测。这可以提供完全自动化图像分析。 不同类型的癌细胞侵袭的实例示于图2。 - 4的高度恶性人胶质母细胞瘤 （GBM）细胞系U-87 MG，形成紧密的球形三维结构和这里是用来举例说明脑肿瘤，其中本地侵袭是一个关键的危及生命功能。一旦嵌入BMM，从U-87 MG球体流传着一个典型的"星爆"入侵模式12细胞。肿瘤细胞放射状延伸到它们保持三维形状的矩阵。该过程接着在一段72小时的， 如图2的U-87 MG球状体中，当肿瘤细胞侵袭的程度，与细胞过程的细节和侵袭伪足伸入BMM是显而易见的。全自动使用图像仪可使肿瘤细胞侵袭随着时间的精确定量图像分析。获得图2中的量化如图1，其中所述自动分析产生围绕从U-87 MG球状体本体延伸进入BMM细胞突起分割。注意，对于该细胞系，肿瘤球体体被排除在侵入区域的测量。 invas的不同模式离子被显示人类鳞状头颈癌的细胞系。 CAL S和CAL R 2是等基因但CAL S是对敏感，和CAL - [R耐EGFR酪氨酸激酶抑制剂14。在没有EGF的，既不细胞系侵入到BMM（ 图3）然而，如表皮生长因子的浓度增加（2.5纳克/毫升），该球状体出现&#39;芽&#39;。在较高浓度的EGF（40和80毫微克/毫升）侵袭程度的降低。这是与传统的钟形剂量响应于EGF的侵袭先前已报告了15方面是一致的。在20纳克/毫升的EGF，从CAL - [R球体而CAL S细胞主体挤压指状突起分别为72小时（ 图3）后创伤小。这个差动入侵是明显48小时后的球状体放入BMM（含有20纳克/毫升的EGF为细胞系之间最大的区别）和72小时后的最大（ 图4）。使用成像仪，作为U-87 MG被收购这些图像迅速。然而，在这种情况下，举例说明另一种方法，侵袭程度手动定量使用单独的图像分析软件，以及图形的方式呈现（ 图3和4）。这些结果表明，可以利用筛选将特异性拮抗药抗性，侵袭性表型的化合物对药物敏感和耐药细胞之间的表型差异。 <img alt="图1" src="/files/ftp_upload/52686/52686fig1.jpg" /> 图三维球体肿瘤侵袭实验1.示意图 。工作流示出了只是嵌入BMM后该方法涉及包括U型87 MG成胶质细胞瘤的球状体的代表图像的步骤（顶面板; T = 0）和入侵后<html>成BMM（底部面板; T = 72小时）与无测量涵盖侵入细胞的区域分割。酒吧= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52686/52686fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52686/52686fig2.jpg" /> 图2.时间过程的U-87 MG成胶质细胞瘤球体入侵到BMM U-87 MG球状体3D侵入BMM监测和使用成像仪定量达72小时（A）的代表性图像和（B）的定量肿瘤细胞侵袭被示出。酒吧= 500微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52686/52686fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 再3"SRC ="/文件/ ftp_upload / 52686 / 52686fig3.jpg"/&gt; 图3. CAL R（抗性）和CAL -S（敏感）肿瘤球体的侵袭。（A）的代表性图像和（B）的表皮生长因子的剂量依赖性的CAL R和CAL 瘤球体侵袭手册量化被示出。酒吧= 500微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52686/52686fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52686/52686fig4.jpg" /> 当放置在B图4。从CAL R细胞（耐药）球体比CAL S（敏感）的肿瘤球状体更具侵入性的MM。一旦EGF刺激的CAL - [R肿瘤球状体显示出更明显的侵入比CAL 第 （A）的代表性图像和（B）的入侵手册量化给出的值是。酒吧= 500微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52686/52686fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Three-Dimensional 3D Tumor Spheroid Invasion Assay at JoVE.com

Three-Dimensional 3D Tumor Spheroid Invasion Assay

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

三维（3D）肿瘤球体侵袭试验

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

three-dimensional-3d-tumor-spheroid-invasion-assay

Nanyang Technological University

Research

JoVE Journal

Medicine

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

57.0K 次观看.  The Institute of Cancer Research. Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

视频: Three-Dimensional 3D Tumor Spheroid Invasion Assay

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

球体的检测，以测量TGF -β诱导的入侵

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

科研

医学

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

三维流状入侵检测中的全人肿瘤基基质

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

癌症研究

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

通过高分辨率共聚焦显微镜对3D肿瘤球体入侵的影响表征

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

胶质母细胞瘤的3D球形模型

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

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 ...

癌症生物学中的 3D 模型:异球体中的侵袭动力学

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

作者聚焦:破译头颈癌进展过程中癌症和免疫细胞之间的串扰

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

分析 3D 培养物体外细胞侵袭的替代策略

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

癌细胞球体检测，以评估入侵的3D设置

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

三维（3D）肿瘤球体侵袭试验

摘要

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此视频中的章节

球体的检测，以测量TGF -β诱导的入侵

三维流状入侵检测中的全人肿瘤基基质

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

通过高分辨率共聚焦显微镜对3D肿瘤球体入侵的影响表征

胶质母细胞瘤的3D球形模型

监测三D培养中的癌细胞入侵和T细胞毒性

分析 3D 培养物体外细胞侵袭的替代策略

分析 3D 培养物体外细胞侵袭的替代策略

分析 3D 培养物体外细胞侵袭的替代策略

癌细胞球体检测，以评估入侵的3D设置