这项工作是由来自教育，青年，体育和（LO1304）捷克外交部和捷克共和国的技术局（TE01020028）资助。笔者想感谢拉克什曼瓦拉纳西博士采取环保盖的静止图像。

1.琼脂糖包被的平板的制备<ol><li>权衡0.75克低熔点琼脂糖和它的McCoy的5A培养基中的溶液（有或没有酚红）无血清添加到100。加热在微波的溶液，摇动每1-2分钟以完全溶解琼脂糖。高压灭菌的溶液消毒它。 </li><li>冷却该高压灭菌琼脂糖至约70℃，并通过500毫升，0.22微米过滤器顶部通过真空过滤在层流箱过滤。如果不使用整个解决方案立刻分装0.75％的过滤琼脂糖溶液（FAS）为更小的体积。在寒冷的房间或4℃冰箱中长达4周存储这个准备使用的琼脂糖溶液无菌。 </li><li>附加一个小管与胶面纸或金属嘴分配盒到的Combi试剂分配器和素盒与一个流浆箱70％的乙醇（乙醇），然后用无菌磷酸盐缓冲盐水（PBS）。 </li><li>在使用前，加热FAS的i存储的等分试样<html> n个微波融化它。总理与琼脂糖溶液和外套384孔，组织培养（TC）分配盒 - 处理"特殊"与微孔板FAS的15微升。允许在板琼脂糖接种细胞之前冷却15-20分钟。存储琼脂糖涂层板在无菌条件下，包裹在聚乙烯袋中在寒冷的房间或在4℃，并远离直射光。 注:避免0.75％的FAS的反复加热，以防止在琼脂糖中的储备溶液浓度的变化。当在上述条件下保存的琼脂糖包被的板可以存储最多2周。上FAS板的涂覆过程中不需要加热。 </li><li>通过用70-80℃无菌水吸它以除去任何剩余的琼脂糖在盒提示和管清洁分配盒。 </li></ol> 2.细胞培养和形成MCTS <ol><li>培养人结肠直肠癌HCT116细胞，如先前所描述姑娘="外部参照"&gt; 8。 </li><li>除去所需数量的琼脂糖包被，384孔，从冷库TC-处理微孔板和它们平衡至室温（RT）搅拌15分钟。 </li><li>通过吸用70％乙醇和无菌PBS中的标准管分配盒制备用于细胞接种的组合式试剂分配器。调节播种卷到所需微升和使用上的分配器手动设定按钮分配速度到介质。 注:在无菌条件下，并在层流箱中进行整个细胞接种过程。 </li><li>分离使用重组细胞解离酶从组织培养瓶中的贴壁细胞。使在与种子细胞的无菌烧杯的细胞悬浮液的库存在完全生长培养基的50微升一个的每孔2.5×10 4个细胞/ mL的密度。当播种多个384可湿性粉剂，使用磁力搅拌器以防止它们沉降到烧杯的底部搅动细胞。 </li><li>允许板休息在RT 30分钟，然后离心以4×g下15分钟。 </li><li>同时，采取蒸发减少环境盖和用8毫升无菌H型2 O或5％二甲亚砜（DMSO）中的短边填充它使用5毫升吸管（ 图1A）（左和右）。首先，分配通过扫描枪头慢慢上下充填液体进入左侧槽的4毫升。重复此步骤以右侧槽。 注:确保液体加入到侧槽的盖子的中心不会合并，并留下气体交换（ 图1A）的差距。加入的H成过量2 O的结果在H 2 O的渗流到盖的外部，并随后到384-孔TC板的外孔。 </li><li>填用无菌H 2澳384孔TC板中的液体贮存器和替换用充满液体的环境盖子的常规板盖（ 图2A）。 </li><li>放置在用95％的湿度，5％CO 2和20％O 2 37℃的旋转恒温箱板，并且允许细胞聚集成MCTSes 4天。避免为了防止突然跌落湿度水平打开培养箱门太久在随后的日子里。 </li></ol> 3.中使用外汇机器人系统<ol><li>在下面的MCTS形成的第4天，每口井使用自动洗板机饮水机加预热中的30微升。允许MCTSes中生长额外3天。更换培养基每3天定期，直到它们达到所需的大小进行实验。 </li><li>为了吸定义从每个介质的体积以及凭经验调整垫圈歧管的Z -height。吸30％以及介质μL，并与新鲜，预热介质的30微升更换。 注意:在该垫圈歧管向下传播设置配送速度和速度成以最低速率孔，以尽量减少在孔中的湍流。 </li></ol> MCTS 4.高内涵成像和半自动图像分析<ol><li>图像的MCTS在使用4X空中目标的内容高度自动化成像系统（NA 16）。 </li><li>设置曝光时间11毫秒和分级，以4×4，调节的Z -stacks的数目和间隔并根据需要进行实验以每孔捕获整个MCTS的像素组合。 </li><li>处理图像逐步，如在所描述的"自述"，使用写在编程语言中的内部程序。 注:.M代码和.txt自述文件可作为补充代码文件（ 图1B）。常规措施MCTS面积，长轴和短轴，周长和坚固由2D图像。 </li></ol><img alt="图1" src="/files/ftp_upload/55403/55403fig1.jpg" /> 图1:环境罐盖的制备和半自动程序的流程图。 （A）中填充有正确的（左）和过量（右）填充液体的量（这里，5％DMSO）中的蒸发减少对环境的盖的图像。箭头指示用于将液体中的短边槽。星号示出了加入DMSO的正确体积的以下在盖中间的间隙。 （B）中示出所涉及的半自动化图像分析程序中的步骤的工作流程。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55403/55403fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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涂层384孔TC板用过滤琼脂糖在很多的标准做法是使用1-1.5％低熔点琼脂糖涂覆板，这需要琼脂糖和/或分配单元也可以保持加热，以防止琼脂糖6的胶凝。琼脂糖的凝胶化是可能引起关注的，而使用具有小管的孔的范围直径在0.2和0.4之间的液体分配盒制备多块板。为了克服堵塞分配盒的潜在问题，0.75％的FAS被使用，因为它在分配期间不需要额外的加热。顺便说一下，还观察到0.75％的FAS不尽可能迅速未过滤的1-1.5％，或甚至0.75％，琼脂糖溶液胶凝。用0.75％FAS，只需不到35秒大衣整个384孔TC板无堵塞点胶盒。燃气罐在最后用热水吸它来去除残留的一清洗garose，这可能阻塞提示当盒带不使用。 环境闸和旋转式孵化器比普通钢板盖和孵化器介质的蒸发引起的不均匀损耗显著影响均匀MCTS形成的再现性，因而损害了分析14的结果。令人惊奇的是，组1的环境的盖/旋转培养箱组合下的384孔TC板孔呈OG吸光度（ 图2C）在统计学上显著变化。然而，OG吸光度在第1组孔低的CV表示在环境盖子旋转培养箱384孔TC板的外井的蒸发是不那么过度以影响MCTS生长（ 图2D）。这是从形成在第1组孔在板与环境盖/旋转培养箱组合均匀尺寸MCTSes明显。此外，减少损失介质的防止从384孔符合环保盖子8旋转培养箱TC板药物检测以及对良好的可变性。 所提出的方法还导致一些其他癌症和非癌性细胞系8的均匀MCTSes的形成。然而，由于所有细胞不能固有聚合成在非粘附表面MCTSes，这种方法的许多不适合的未均匀MCTS形成的再现性测试的其他细胞系。此外，重点是在组合使用了先进的技术孵化用蒸发减少国会议员和板盖主要是增加的MCTS形成再现性。另一种廉价和简单的解决方案，以增加MCTS形成重复性可能是使用胚胎级矿物油，透气的密封胶带或膜，它防止水分蒸发，但允许正常的气体交换15。虽然琼脂糖增加了额外的厚度与板底部，这可能是一个障碍，以高含量成像，场由在透射光成像琼脂糖底部产生的深度不妨碍MCTS大小的认定和药物治疗后成形。因此，该方法将是潜在的利益在MCTS文化LOT面临重复性的问题研究员。 半自动图像分析例程虽然有许多半自动化/自动化可MCTS图像分析软件，药物治疗完成到部分崩解MCTSes后的大小的测量是容易出错16,17。在所提出的程序的第一步骤，所述聚焦最好的图像被自动地从集合Z -stack图像通过计算图像的梯度的模，并选择一个与最大0.999 QUA选NTILE。这是最大的一个坚固的噪声不敏感的测量。 接下来，较暗的边界被裁切，经过处理的图像存储在可移植网络图形格式的原始分辨率。这个过程从2.5 GB降低总规模，从成像整个384孔，TC处理板，大约100 MB。随后，MCTS的中心是通过与用户定义半径的圆形过滤器的卷积实测值（我们使用20个像素; 图4A）。过滤图像的清晰局部最小值提供的中心（ 图4A）的位置。从这个中心同心的环移动，中位灰度值，M在每个壳被计算为壳半径的函数，R。此后，第一（主要）最大的函数M的数值衍生物的（R）是实测值（ 图4B）。这个最大的点M OPT被作为最佳阈值，并且图像是S通过阈值在这个级别egmented。 该MCTS被确定为图像的最大的中央段。此过程通常工作比标准阈值技术更好，因为它是能够将MCTS芯从崩解部分往往从药物治疗（ 图4C和4D）导致分离。此外，这里所采用的方法使MCTS的球形隐式使用。简单的阈值例程或算法基于主动轮廓，不利用先验知识球形物体是被发现。当MCTS清楚地检测和划定的最大的m OPT是明确的，单一的。例程然后测量MCTS的特性，如面积，长轴和短轴，周长，并拟合椭圆的坚固性，并保存分割图像的预览。如果最大是不明确的，和/或如果多个局部最大值存在时，预观点建议的分割保存到一个文件夹中的校正。 在倒数第二个步骤，程序指导用户通过校正夹通过移动阈向上或向下和选择感兴趣切断掩模的可能的冗余部分的多边形区域手动调整所建议的分割。此手动校正后，程序的措施，并保存MCTS特征，如上所述。 <img alt="图4" src="/files/ftp_upload/55403/55403fig4.jpg" /> 图4:图像分割过程的示意图。 （ 一 ）MCTS的中心标有星号的绿色。环的一个例子绘制于红色。 （B）在每个壳的中位灰度值被绘制为壳半径（蓝线）的功能。该曲线的数值衍生物被计算（红线）。第一个实质性的马数值衍生物ximum被找到（黑色星号）。 （C）图像的所得正确分割成MCTS核心和与崩解MCTS零件的背景。 （ 四 ）由大津算法获得不正确的分割。 4X目标;比例尺= 500微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55403/55403fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

在肿瘤癌细胞生理学布置在一个复杂的，三维（3D）细胞外基质和相互作用的细胞所包围的结构。作为近组织中的所有细胞驻留在3D环境中，有必要在该模拟肿瘤性状体外肿瘤模型更生理学相关导致的几个三维培养技术1，2，3的发展。这些模型正在成为基本的研究工具用于研究在三维2转移和细胞响应于治疗肿瘤微环境的作用。此外，相对于2维（2D）细胞培养物4，3D模型允许更好地了解肿瘤-基质的相互作用，从而影响细胞信号通路的。 的肿瘤细胞株多细胞肿瘤球状体（MCTSes）在三维细胞c的常用由于其相对接近体内肿瘤ulture车型。出的使用中的多种技术，MCTS代的琼脂糖包被的平板的液体覆盖技术（LOT）已经获得了铅的优化和靶标确认5，6，7，8，9，10，11显著兴趣。这是从最近的研究已成功能在使用LOT 6,7 MCTS文化运行化合物文库的导频屏幕明显。然而，由于介质的蒸发引起的不均匀损耗油井到井变异MCTS形态学和生长是伴随使用微量滴定板（MPS）的LOT常见障碍。因此，非均匀MCTSes形成危及意义和相关从药理学试验8，12，13的数据。除了再现性的问题，使用自动液体分配单元时影响基于LOT-高通量测定另一实际问题是在MP的涂布用琼脂糖。虽然分配单元可以保持加热，以防止琼脂糖的凝胶化，分配盒和管道的堵塞是机器人系统6的电位的关注。 为了克服这些挑战，我们最近在很多关于MCTS文化8设计了一些修改。这些修改主要是根据可能的方法来防止用户使用该工具在高通量筛查实验室中常见的国会议员中损失不均匀。修改后的很多跨3大小均匀，重现性MCTSes生成的详细过程84孔板（WPS）这里提出。手稿还提出了MCTS大小的评价半自动程序，特别是在不具有横截面面积的测量一个明确定义的边界部分破碎，药物处理MCTSes。

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			<td>Small Tube Dispensing cassette&#160;</td>
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			<td>HighRes Biosolutions</td>
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			<td>TrypLE&#8482; Express recombinant cell dissociation reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604021</td>
			<td>Phenol red free</td>
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evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates

肿瘤模型， 在体内条件密切模仿正成为药物发现和开发的潜在的抗癌药物的筛选日益流行。多细胞肿瘤球状体（MCTSes）有效模仿实体瘤的生理条件下，使它们在体外模型优良铅优化和目标验证。从可供MCTS文化的各种技术，在琼脂糖液体覆盖法是对MCTS一代最便宜的方法之一。然而，使用液体的覆盖为高通量筛选MCTS培养物的可靠传输可能由许多限制，包括微量滴定板（MPS）琼脂糖的涂层并在井均匀MCTS地层的不可再现性受到损害。国会议员是显著容易发生边缘从培养基从板的外部过度蒸发造成的影响，防止药物使用整个板块的试验。这个手稿提供到液体覆盖技术来增加均匀MCTS形成的可扩展性和可重复性的详细技术的改进。此外，药物治疗提出后，对MCTS的评价简​​单，半自动，和普遍适用的软件工具的细节特征。

不均匀的损失中，尤其是来自周边井，是小培养体积议员们经常遇到的问题。大大改善培养条件，如与良好控制的温度/加湿系统和蒸发减少国会议员和板盖孵化器，降低介质的整个井8的显著损失。测量相对蒸发，橙G（OG）等体积加入到每孔中，并用3天OG吸光度变化被记录在与在标准和旋转孵化定期和环境盖板。板孔被分成基于从板（ 图2A）的边缘的距离6组。在基团1-4，有在OG的吸光度随时间的在标准培养箱中（ 图2B）定期盖板一个显著变化。虽然也有在OG吸光度的变化组1的孔中的环境盖/旋转培养箱组合（ 图2C）下板，变异（CVS）的系数相比，在标准培养箱中（ 图2D）定期盖板的要低得多。 介质的蒸发引起的不均匀损失是在MCTS规模和国会议员8法读数为小康以及变异的潜在原因之一。然而，随着对环境的盖/旋转培养箱组合384-孔TC板导致横跨6组孔（ 图2E），形成均匀的MCTSes的。与此相反，在与普通盖/标准培养箱组合板MCTSes在大小和坚固（ 图3F）显著变化。密实度是MCTS球形的量度，并给出了MCTS的崩解程度。二维区域的坚固是像素中的共同的比例感兴趣区域（ROI）的区域和的nvex船体被由ROI的凸包的区域分割的ROI的区域来确定。圆形和椭圆形区域具有1的坚固性，并且作为MCTS崩解，所述坚固减小。在区域中的差异已经被报道在Das 等。 （2016年）8。体积从MCTSes的长轴和短轴计算。 <img alt="图2" src="/files/ftp_upload/55403/55403fig2.jpg" /> 图2:以最小的蒸发结果增加MCTS重复性板。 （A），提出了一种板地图，以显示井划分为6组。 （B和C）中有OG的相对吸光度从孔中，在与常规盖（B）的一个标准培养箱中培养板组1-4个显著依赖时间的变化，并从第1组，我们LLS与在旋转恒温箱（C）的环境盖板。每天3-5 OG吸光度归一化到0天（D）的平均每组的OG吸光度的CV从第3-5天1井示为两个板盖/培养箱组合。的CV是3个独立实验的平均值。在形成与板块环境盖子/旋转孵化器组合（E）MCTSes没碰到过的6组井显著不同。 （B）但是，在标准培养箱定期盖板形成可变大小的MCTSes。数据显示，每组n&gt; 60 MCTSes。这些盒子并在箱线图的箱子内的横条分别代表第 25 次和第 75百分位数和中位数。晶须代表5 次和第 95 次百分，和异常值是由在箱图的排列的黑点表示。的秩和检验分析的p值现介绍如下EAC^ h箱线图。这些数据是从每盘盖/培养箱组合至少3个独立的实验。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55403/55403fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 为了确定例程的潜在适用性7日龄MCTSes用紫杉醇（PTX），长春新碱（VCR），奥沙利铂（OXA），阿霉素（DOX）和5-氟尿嘧啶（5-FU）在3个不同浓度处理4天。这些毒品是从大学医院奥洛穆茨，帕拉茨基大学获得的。然后MCTS图像进行分析，并且将面积，长轴和短轴，周长和处理MCTSes的坚固性进行了比较，对照（CTL的）。的长轴和短轴被用来计算的几何体积。有在MCTS面积浓度依赖性降低和体积的药物治疗（ 图3）之后。虽然0.001微克/毫升VCR和0.4微克/毫升阿霉素和5-FU导致MCTS面积的增加，将体积显著增加只有以下0.001微克/毫升录像机。 MCTS周边只在紫杉醇，阿霉素和5-FU，完全影响了MCTS大小的含量最高是显著不同。 PTX和0.25微克/毫升和0.06微克/毫升和DOX和5-FU，在100微克/毫升录像机导致的牢固一个显著下降，表明该MCTSes（ 图3）的一个完整的对局部的崩解。 <img alt="图3" src="/files/ftp_upload/55403/55403fig3.jpg" /> 图3:在MCTSes药物作用的半自动化程序测量。 （A）和CTL药物治疗MCTSes图像呈现。比例尺= 10微米。在微克/毫升的浓度给出了每个图像。 （B）的条形图显示PTX，录像机，OXA，DOX的剂量依赖性作用，和5-FU的区域和处理后4天的7日龄MCTSes体积。 MCTS周长以下0.25微克/毫升的PTX和100微克/毫升阿霉素和5-FU被显著降低。了导致MCTSes的完整到局部崩解的药物浓度相比的CTL显著减少MCTS牢固性。这些数据是由3个独立的板至少5 MCTSes的平均值±标准差。 * P &lt;0.001，＃P &lt;0.01，φP &lt;0.05药处理过的与CTL，单向ANOVA和Dunnett的多重比较检验。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55403/55403fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates at JoVE.com

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

培养基从微量滴定板的凹凸损失影响均匀的多细胞肿瘤球体形成的再现性。改善培养条件，以减少显著介质损耗将提高球体形成的再现和基于球状体的测定的使用液体覆盖技术的结果。

蒸发减少培养条件的提高多细胞球体形成的微孔板重现

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

evaporation-reducing-culture-condition-increases-reproducibility

Nanyang Technological University

Research

JoVE Journal

Cancer Research

6.9K 次观看.  Palacky University in Olomouc. 培养基从微量滴定板的凹凸损失影响均匀的多细胞肿瘤球体形成的再现性。改善培养条件，以减少显著介质损耗将提高球体形成的再现和基于球状体的测定的使用液体覆盖技术的结果。 

视频: Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

γH2AX 和53BP1 的免疫荧光显微镜分析 DNA 双链断裂的形成和修复

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

用球形发芽法评价 Hemangioblastoma 新生血管的体外性能的综合程序

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

3D 脊髓黑色素瘤球形皮肤模型

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

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 ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...