作者希望在国家促进转化科学中心 (国家卫生研究院) 上承认他对这些化验的初步发展的支持和指导。此外, 我们要感谢艾莉弗里曼, Mariela Jaskelioff, 迈克尔 Acker, 雅各 Haling, Vesselina 库克作为项目团队成员的科学投入和讨论。

1. 1536年井3D 肿瘤球体的培养
<ol><li>将以下试剂加入500毫升的 Dulbecco 修饰鹰培养基 (DMEM/F12) 中, 制备新鲜的3D 肿瘤球形培养基茎 (细胞培养基 + 挖空血清置换 + 胰岛素-转铁蛋白-硒): 1x 青霉素/链霉素, 10 人/毫升碱性成纤维细胞生长因子 (bFGF), 20 ng/毫升人表皮生长因子 (EGF), 0.4% 牛血清白蛋白 (BSA), 1x 胰岛素-转铁蛋白-硒, 1% 挖空 (高) 血清置换 (不冻融)。</li>
	<li>过滤-通过0.4 µm 瓶顶过滤系统的补充剂消毒整个介质。</li>
	<li>根据推荐的常规培养条件, 分离癌细胞株, 从传统的细胞培养烧瓶中洗涤 3x, 用1x 磷酸盐缓冲盐水 (PBS), 加入适量的0.25% 胰蛋白酶 (1 毫升每5厘米2) 为5分钟, 在单元格上创建一个薄层。将胰蛋白酶与含有血清的培养基的数量5x 中和, 然后进行计数细胞。例如, 使用25毫升培养基5毫升的胰蛋白酶。</li>
	<li>将细胞溶液的浓度调整为 62.5 x 104细胞/毫升, 以将8µL 球形培养基中的500个细胞的总种子, 放入1536井组织培养处理的板材中。</li>
	<li>在一个生物安全柜中, 用一个用蠕动泵为基础的消毒的小的不锈钢尖盒来种子细胞。在不同的细胞线之间, 冲洗的卡带与 1x PBS 和70% 乙醇的导管十年代。</li>
	<li>或者, 使用在高效过滤室内的液体处理器, 使用灭菌的、小的不锈钢尖盒和蠕动泵或附加的注射器泵, 根据所需的每个细胞线的数量来播种细胞。</li>
	<li>使用透气的粘接板密封密封板, 手动或使用板封口机。</li>
	<li>将盘子放在一个纺纱孵化器设置在 10 rpm 和37°c 与5% 二氧化碳 (CO2) 和95% 相对湿度。允许球体形成 3 d。</li>
</ol>2. 培养1536年井2D 癌症线
注: 1536 井2D 癌症线应培养24小时之前, 添加的化合物到3D 板块。
<ol><li>通过将以下试剂添加到500毫升适当的生长培养基中, 制备2D 癌细胞培养基: 1x 青霉素/链霉素和10% 胎牛血清 (血清)。</li>
	<li>过滤-通过0.4 µm 瓶顶过滤系统的补充剂消毒整个介质。</li>
	<li>根据推荐的常规培养条件, 分离癌细胞株, 从传统的细胞培养烧瓶中冲洗3x 的 1x PBS, 并添加适量的0.25% 胰蛋白酶 (1 毫升每5厘米2), 以5分钟在单元格上创建一个薄层。将胰蛋白酶与含有血清的培养基的数量5x 中和, 然后进行计数细胞。例如, 使用25毫升培养基5毫升的胰蛋白酶。</li>
	<li>将细胞溶液的浓度调整为 62.5 x 104细胞/毫升, 以便在8µL 的完全培养基中, 将500个细胞的总粒数种到 1536-良好的组织培养处理板中。</li>
	<li>在一个生物安全柜中, 用一个用蠕动泵为基础的消毒的小的不锈钢尖盒来种子细胞。在不同的细胞线之间, 冲洗导管的卡带与1x 无菌 PBS 和70% 乙醇十年代。</li>
	<li>或者, 使用在高效过滤的机器人室内的液体处理器, 使用灭菌的、小的不锈钢尖盒和蠕动泵或所附的注射器泵, 根据每个单元格的板量来种子细胞线需要。</li>
	<li>使用透气的粘接板密封密封板, 手动或使用板封口机。</li>
	<li>将板块放在一个纺纱孵化器设置在 10 rpm 和37°c 24 小时之前, 复合添加, 与 5% CO2和95% 相对湿度。</li>
</ol>3. 复合加法
<ol><li>在100% 二甲基亚砜 (亚砜) 上制备8点3倍的 MAPK 抑制剂序列稀释, 其浓度为10毫米。蛋白酶体抑制剂佐米被用来作为一个完整的细胞杀死的积极控制, 以确定的动态范围的检测。</li>
	<li>快速旋转所有的化验板和复合板在 100 x g 收集任何冷凝。除去每个盘子上的粘合剂密封, 并将一个盘子放在一个音响分配器上, 以增加每一个混合物的 8 nL, 代表最终浓度为0.1% 亚砜每井和一个最高的复合剂量10µM。</li>
	<li>在化合物的添加完成后, 放置一个定制的, 不锈钢细胞培养盖子, 防止在板上的蒸发, 并将板块放入一个旋转孵化器在37°c 为 5 d, 与 5% CO2和95% 相对湿度。</li>
</ol>4. 检测试剂添加和原始数据的获取
<ol><li>预热适量的细胞裂解试剂, 含有荧光素, 以检测三磷酸腺苷 (ATP) 的变化, 从而改变细胞在室温或37°c 水浴中的增殖。使用蠕动泵在每个井中添加3µL 的检测试剂, 并在室温下孵化出至少15分钟的平板。</li>
	<li>捕捉紫外线板上的发光。</li>
</ol>5. 数据处理
<ol><li>从仪器中提取原始数据, 并将包含测试化合物的所有字段正常化为仅含有亚砜的所有井的平均值作为中性控制。从这个值计算每个化合物的生长抑制百分比。这是在 Microsoft Excel 中使用公式函数完成的, 其中f (x) = {(示例值相对光单位 (RLUs)/(平均亚砜值 RLUs) * 100)}。</li>
	<li>通过图形程序将步骤5.1 中规范化数据的值与复合浓度进行绘制, 生成剂量响应曲线和抑制 IC50s。 注: 我们使用了一个内部 NIBR 软件工具分析了数据。为了完成这一功能, 我们选择了非参数曲线分析工具。</li>
</ol>

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</ol>

作者是诺华的雇员, 一些员工也是股东。

本文介绍了如何在1536年的大型复合筛板上生产肿瘤球体的详细协议。这些方法最初是根据国家癌症研究所的工作改编的, 那里的肿瘤球体生长在6井和96井板块的低吞吐量化验中, 询问有关遗传依赖性和复合敏感性13的问题,14,15. 本议定书的一个关键和独特的特点是, 球体是由传统的2D 组织培养的细胞组成的 1536年-板块使用定义的媒体称为 SCM + 高 + 它。一旦细胞被播种, 他们被密封的透气粘合板密封, 以防止任何蒸发超过3维球体形成的时间。球体还可以使用这种方法16培养更长的时间。在 3 d 的球体形成后, 添加了5维化合物, 然后用裂解基增殖试剂测定细胞生长的变化。对控制井进行规范化处理, 并对 2D与3D 培养的细胞间的 IC50s 进行了计算, 并将这些细胞的变化与对照处理后的细胞进行了比较。
由于1536井板是常规的组织处理板, 而不是水凝胶涂层或细胞驱板, 因此该协议的限制是使用所定义的介质, 这是成功执行该协议的关键因素。高血清置换最初的测试, 以成功地帮助在胚胎干细胞的生长在 3D17 , 并在本议定书已经适应了许多肿瘤球体线13,15的增长,18,19. 值得注意的是, 虽然媒体条件对2D 细胞的生长与3D 球体的增长是不同的, 但我们以前已经证明, 药物敏感性的差异效应是由生长形式介导的, 而不是简单的区别在媒介构成9。
为了确保该协议的成功, 作为一个故障排除步骤, 建议首先进行一系列的测试开发实验, 以优化每个细胞线, 然后再开始大规模的屏幕。在这个例子中, 所有的细胞系检测首先评估, 以确定它们是否可以播种在相同的起始密度为500细胞每井。然后, 他们用亚砜唯一的板块来检验标准偏差 (SD) 和方差系数 (CVs) 在生长期的持续时间。即使有了这种检测方法的发展, 某些线的增长速度也比其他的要快, 这将有利于进一步优化播种密度和这些更具挑战性的线的实验持续时间。最后, 这里提出的数据表明, 有一种趋势, 而不是一个普遍的现象, 对于某些细胞系, 如非 NSCLC 线 Calu-6 是唯一更敏感的化合物靶的 MAPK 路径在3D 相比, 在2D 增长。正在进行更多的实验, 以确定哪些生物机制可能有助于调节敏感度的显著差异。
在超高温超导环境中筛选球体的能力允许在药物发现过程中早期使用3D 区域性, 并且可以识别仅在3D 环境中活动的新化学物质。由于患者缺乏疗效, 许多临床考生从未进入市场, 尽管在体外显示生长抑制和体内回归, 但小组已经开始质疑实施3D 检测是否能在发现过程中及早弥合这个缺口。在使用96井或384井方法时, 在3D 培养细胞中存在许多不同的板块类型。在这里, 我们提出了1536井方法, 产生多个病灶的个别球体。在未来, 这些发现可以与类似的屏幕使用正在开发中的新技术, 如1536井板, 可以种植细胞作为悬挂滴, 或涂有水凝胶/胶的板块, 是圆底,这将允许形成一个球体每井。这些条件将引入额外的层次的复杂性, 用于筛选的文化条件, 使其更具生理学意义, 并有可能代表提高翻译从板凳到床边的小说疗法。这些技术还将使涉及缺氧或扩散梯度的科学问题, 这需要使用更大的球体。
这里提出的方法重点是筛选这些球体对一个小分子图书馆的化合物。未来, 这些方法可以扩大到筛选新的生物制剂和 biotherapeutics, 如双特异抗体或抗体-药物结合, 其中3D 的文化可能提供更多的生理相关的抗体-抗原相互作用。此外, 使用3D 球体可能对发展与疾病有关的化验, 以了解新出现的免疫肿瘤学空间21中的目标和干预措施至关重要。

在过去的十年中, 越来越多的研究已经实施了3D 细胞培养模型, 以了解不完全概括的概念, 在2D 的塑料上生长的细胞。这些概念的例子包括正常上皮细胞极性1的交替, 在那里细胞的顶端和基底层的空间方向丢失, 以及细胞外基质在调节生存和细胞命运方面的作用。特别是肿瘤学研究已经使用3D 模型来了解癌细胞的基本生物学和2D 和3D 细胞培养系统之间的差异3,4。更复杂的细胞培养技术的发展以及它们对多井格式的进一步适应使得在3D 环境中寻找新药。与2D 条件下生长的细胞相比, 3D 模型的肿瘤范围复杂, 从分层细胞系统5到单细胞型球体不同大小, 到复杂的多细胞型球体6,7,8. 因此, 发现完全诱导这些3D 模型系统中的细胞死亡的新型化合物或生物制品, 对药物发展运动有很大的兴趣。这些化验的终点通常与在2D 文化中进行的实验的端点相同, 以评估细胞增殖的变化, 但是当在生理上相关的环境中进行时, 它们可能揭示目标基因或通路的真正依赖性水平。审问。
如上所述, 已经开发了各种模型系统来研究3D 培养系统中的药物反应, 但是大多数使用96或384井微量滴定板, 并且不容易适应经常使用的高通量筛选 (高温超导) 格式。药物发现筛选运动。这类系统包括使用悬浮液滴技术、球形培养、带磁性微粒的脉动细胞来诱导漂浮, 以及含有天然或合成凝胶的培养物, 如胶原蛋白、胶或聚乙二醇 (PEG)2.在这里, 我们提出了一个以前开发的技术的详细方法, 以生产3D 球状文化从已建立的癌细胞系1536井板格式。在本协议中, 使用高度定义的介质来防止附着正常附着的细胞线9。这个系统有局限性 (也就是说, 它不能完全重述一个复杂的癌症模型系统), 但尽管如此, 这些化验能够对大量小分子集合进行高通量筛选, 并对药物反应进行横向比较。在2D 和3D 之间的文化对各种细胞系和化合物。
所选的细胞系用于演示本文中的方法, 所有与 MAPK 信号通路相关的基因的海港突变, 一种在癌症中高度失调的通路, 并且有许多疗法可用。许多线有激活致癌突变在柯尔多大鼠肉瘤病毒也称为 KRAS, 神经母细胞瘤 RAS 或 NRAS, 哈维大鼠肉瘤病毒癌基因或 HRAS, 和相关的激酶迅速加速纤维肉瘤, 也称为RAFs。最近的文献表明, 在3D 条件9,10的情况下, 这种通路的不同节点抑制剂在细胞系的子集中具有独特的功效。一项研究发现, 当有活性 RAS 的癌细胞在3D 培养时, 它们显示出对 MAPK 抑制剂的敏感性增强, 而且这种方法可以识别传统2D 中可能遗漏的通路和靶向抑制剂。设置。本研究的目的是介绍用于培养这些细胞系的方法, 并进一步证明对这些抑制剂的差异反应, 只有在使用3D 细胞培养系统时才能观察到。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM Dulbecco`s Modified Eagle Media (DMEM)/F12</td><td>ATCC</td><td>30-2006</td><td>Purchased as a prepared solution. This reciepe was found to be preferred compared to other vendors.</td></tr><tr><td>DMEM Dulbecco`s Modified Eagle Media (DMEM)/F12</td><td>Lonza</td><td>12-604F</td><td>Purchased as a prepared solution.</td></tr><tr><td>Roswell Park Memorial Institute (RPMI)</td><td>Lonza</td><td>12-115Q</td><td>Purchased as a prepared solution.</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Seradigm</td><td>1500-500</td><td>Purchased as a prepared solution.</td></tr><tr><td>1x 0.25% Trypsin with Ethylenediaminetetraacetic acid (EDTA)</td><td>Hyclone</td><td>SH30042.01</td><td>Purchased as a prepared solution.</td></tr><tr><td>1x Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td>Purchased as a prepared solution.</td></tr><tr><td>10 ng/mL Human basic Fibrobast Growth Factor (bFGF)</td><td>Sigma</td><td>F0291</td><td>Resuspend in sterile water.</td></tr><tr><td>20 ng/mL Human Epidermal Growth Factor (EGF)</td><td>Sigma</td><td>E9644</td><td>Resuspend in sterile water.</td></tr><tr><td>0.4% Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A9418</td><td>Resuspend in sterile PBS and filter.</td></tr><tr><td>1x Insulin Transferrin Selenium (ITS)</td><td>Gibco</td><td>51500056</td><td></td></tr><tr><td>1% KnockOut (KO) Serum Replacement</td><td>Gibco</td><td>10828-028</td><td>Add fresh right before use.</td></tr><tr><td>100% Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>276855-100ML</td><td></td></tr><tr><td>CellTiter-Glo</td><td>Promega</td><td>G7573</td><td>Purchased as a prepared solution.</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Small Combi Cassette</td><td>ThermoFisher</td><td>24073295</td><td></td></tr><tr><td>Small Multiflow Cassette</td><td>BioTek</td><td>294085</td><td></td></tr><tr><td>1536-well sterile TC plates (white)</td><td>Corning</td><td>3727</td><td></td></tr><tr><td>1536-well sterile TC plates (black)</td><td>Corning</td><td>3893</td><td></td></tr><tr><td>Scivax Plates</td><td>MBL International</td><td>NCP-LH384-10</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adhesives seals</td><td>ThermoFisher</td><td>AB0718</td><td></td></tr><tr><td>Spinning Incubator</td><td>LiCONiC</td><td></td><td></td></tr><tr><td>Stainless Steel Lids</td><td>The Genomics Institute of Novartis Research Foundation (GNF)</td><td></td><td></td></tr><tr><td>ATS Acoustic Dispensor</td><td>EDS Biosystems</td><td></td><td></td></tr><tr><td>Echo Acoustic Dispensor</td><td>Labcyte</td><td></td><td></td></tr><tr><td>Luminometer</td><td>Envision-Perkin Elmer</td><td></td><td></td></tr><tr><td>Peristaltic Pump- Multidrop Combi</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Liquid Handler-Multiflow FX</td><td>BioTek</td><td></td><td></td></tr></tbody></table>

generation of high-throughput three-dimensional tumor spheroids for drug screening

癌细胞通常在塑料表面的两个维度 (2D) 培养。然而, 这种技术缺乏肿瘤肿块在体内暴露的真实环境。实体肿瘤生长不像附着在塑料上的一张纸, 而是作为一个三维 (3D) 空间中的克隆细胞的集合, 与邻居相互作用, 并具有不同的空间特性, 如正常细胞极性的破坏。这些相互作用导致 3 d-培养细胞获得形态学和细胞特征, 这是更相关的体内肿瘤。此外, 肿瘤肿块与其他细胞类型 (如基质和免疫细胞) 直接接触, 以及其他所有细胞类型的细胞外基质。沉积的基质由大分子如胶原蛋白和纤连蛋白组成。
为了增加从长凳到床边的肿瘤学研究结果的翻译, 许多团体开始研究3D 模型系统在药物开发策略中的应用。这些系统被认为在生理上是相关的, 因为它们试图重述肿瘤的复杂和异构环境。然而, 这些系统可能相当复杂, 尽管可以在96井格式中增长, 而有些现在甚至在 384, 但它们对于大规模的增长和筛选提供了很少的选择。这一观察到的差距导致了这里详细描述的方法的发展, 以培养肿瘤球体在高吞吐量能力在 1536年-井板。这些方法是对高度复杂的基于矩阵的系统的一种折衷, 它们很难筛选, 而且是常规的2D 检测。各种癌细胞系窝藏不同的基因突变被成功筛选, 检查复合功效通过使用一个精心策划的化合物库, 针对丝裂原活化蛋白激酶或 MAPK 通路。然后将球状培养反应与2D 生长的细胞反应进行比较, 并报告差异活动。这些方法为在高通量3D 设置中测试复合活动提供了唯一的协议。

在2D 的培养条件下, 各种已知生长良好的癌细胞系使用此处概述的方法进行了测试。图 1中有来自各种 MAPK 突变癌细胞系 (Calu-6:KRAS、NCI-H1299:NRAS、SK-MEL-30:NRAS 和 KNS-62:HRAS) 的代表性图像。这些图像表明, 虽然细胞系有不同的形貌, 每一个形成了3D 结构在1536井的化验板块。图 2表明, 当用于大规模筛选时, 可以观察2D 和3D 之间的差异复合敏感度 。图上的每个点代表一个化合物的抑制浓度, 其中有50% 细胞杀死或化合物的 IC50 反应在两个设置。为了证明这种效果不完全由不同的媒介, Calu-6 细胞培养在同一媒体使用2D 组织培养板或 3D Scivax 生物膜板, 防止附着。对于同一介质中的两种化合物, 在补充图 1中, 增加了3D 生长条件的灵敏度或较低的 IC50。像 Calu-6 这样的线在3D 条件下生长时, 对化合物的敏感性增强, 而像 SK-MEL-30 这样的线在3D 与2D 相比更不敏感。表1包含有关此屏幕中使用的所有单元格线的其他背景信息。
典型剂量-响应曲线的例子在图 3中被可视化。这个数字显示了两种化合物的差异效果, RAF265 (图 3A) 和 RAF709 (图 3B), 这抑制了皇家空军激酶的活动。这两种化合物在3D 条件下更有效 Calu-6, 这是一个非小细胞肺癌线窝藏 Q61K, 活化突变的蛋白质 KRAS。同时, 这两种化合物在3D 球体和2D 的 HCT116 结肠癌系中同样有效, 其中含有 KRAS 的 G13D-activating 突变。为了证明增加的灵敏度的影响是介导的增长条件3D 对 2D, 而不是简单地由不同的媒介条件, Calu-6 细胞生长使用在两种条件下的血清。如补充图 1所示, 当2D 和3D 条件都使用了 "血清" 时, 3D 区域性仍然显示出对产生较低 IC50 的化合物的灵敏度增加。
最后, 3D 检测还允许观察到的矛盾生长活化现象, 这是已知的发生时, 有不完全抑制皇家空军脂肪酸在高度活化的 RAS 突变体线, 而不是在二聚体无关的途径激活发现在 BRAF V600E 突变11,12。在图 4中, BRAF 抑制剂 Dabrafenib 在 KNS-62、HRAS 突变的小细胞肺癌线以及非典型 BRAF 突变体胰细胞系 BxPC-3 的治疗中, 可以看到这种活化。这种生长是在药物的低浓度激活, 而生长只被压抑在高浓度的药物 (图 4B)。这种活化在 KRAS 突变体线 Calu-6 中是出人意料的, 在 V600E BRAF 突变线 A375 中是不被预期的。我们观察到使用其他3D 增长模型 (未显示数据) 的 Calu-6 的悖论激活。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57476/57476fig1.jpg"> 图 1: 在 3D 1536-井板中生长的癌细胞的代表性图像.在 1536年, 用试剂分配器和一个小卡带将细胞镀了 3 d。在使用4X 透镜的倒置显微镜下获得的明亮场图像, 展示了 MAPK 突变细胞系 Calu-6:KRAS、NCI-H1299:NRAS、SK-MEL-30:NRAS 和 KNS-62:HRAS 的各种3D 形态。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57476/57476fig2.jpg"> 图 2: 一个散点图, 比较 2D-与3 维复合效果之间的细胞线.球体, 这是以前产生的 3 d, 然后再用化合物治疗 5 d 之前, 复合功效确定。每个圆圈代表一个唯一的复合响应。用裂解细胞增殖试剂检测细胞增殖的变化, 用四参数的逻辑回归分析方法计算 IC50s, 并将其称为 "太阳神"。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57476/57476fig3.jpg"> 图 3: 比较2D 与 3 d 复合功效的剂量反应曲线.剂量-反应曲线与亚砜控制相比, 显示了复合效果。两个皇家空军抑制剂, RAF265 和 RAF709 处理的 KRAS 突变体 Calu-6 和 HCT116 细胞的曲线在这里显示为细胞生长变化的百分比 (% 生长抑制) 相比, 亚砜控制井。在2D 条件下生长的细胞的数量以红色显示, 而生长为3D 球体的单元格呈蓝色。数据周围的实线表示每个数据集的95% 置信区间。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57476/57476fig4.jpg"> 图 4: 某些3D 区域性捕获了悖论性增长激活现象.剂量-反应曲线显示复合功效 (% 生长抑制) 相比, 亚砜控制。KRAS 突变体 Calu-6、BRAF V600E 突变 A375、HRAS 突变 KNS-62、非典型 BRAF 突变 BxPC-3 细胞的曲线, 用悖论活化剂 Dabrafenib 处理。在2D 条件下生长的细胞的数量以红色显示, 而生长为3D 球体的单元格呈蓝色。数据周围的实线表示每个数据集的95% 置信区间。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/57476/57476figS1.jpg"> 补充图 1: 剂量-反应曲线比较 2D-对 3 d 复合效果的血清中含有培养基.剂量-反应曲线与亚砜控制相比, 显示了复合效果。两个皇家空军抑制剂, RAF265 和 RAF709 治疗的 KRAS 突变体 Calu-6 细胞的曲线显示, 在细胞生长变化的百分比 (% 生长抑制) 相比, 亚砜控制井。在2D 条件下生长的细胞的数量以红色显示, 而生长为3D 球体的单元格呈蓝色。两种情况都使用含有含血清的介质。误差线表示来自2个单独数据集的标准偏差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476figS1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>单元格行名</td>
			<td>血统</td>
			<td>BRAF</td>
			<td>Ras</td>
		</tr>
<tr>
<td>A-375</td>
			<td>黑色素 瘤</td>
			<td>V600E</td>
			<td>WT</td>
		</tr>
<tr>
<td>BxPC-3</td>
			<td>胰腺</td>
			<td>V487-P492 > A</td>
			<td>WT</td>
		</tr>
<tr>
<td>Calu6</td>
			<td>非小细胞肺癌</td>
			<td>WT</td>
			<td>KRAS Q61K</td>
		</tr>
<tr>
<td>116</td>
			<td>大肠</td>
			<td>WT</td>
			<td>KRAS G13C</td>
		</tr>
<tr>
<td>IPC-298</td>
			<td>黑色素 瘤</td>
			<td>WT</td>
			<td>NRAS Q61L</td>
		</tr>
<tr>
<td>KNS-62</td>
			<td>鳞状肺</td>
			<td>WT</td>
			<td>HRAS Q61L</td>
		</tr>
<tr>
<td>NCI-H1299</td>
			<td>肺</td>
			<td>WT</td>
			<td>NRAS G12D</td>
		</tr>
<tr>
<td>PANC-1</td>
			<td>胰腺</td>
			<td>WT</td>
			<td>KRAS G12D</td>
		</tr>
<tr>
<td>SKMEL-30</td>
			<td>黑色素 瘤</td>
			<td>WT</td>
			<td>NRAS Q61K</td>
		</tr>
</tbody></table>表 1: 单元格线信息。

Watch this Scientific Journal Video about Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening at JoVE.com

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

在各种疾病征兆中, 越来越多的生理相关模型被开发并实施到药物发现计划中。这里描述的新的模型系统展示了三维肿瘤球体如何在高通量1536井板系统中进行培养和筛选, 以寻找新的肿瘤药物。

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

generation-high-throughput-three-dimensional-tumor-spheroids-for-drug

Nanyang Technological University

Research

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Cancer Research

8.6K 次观看.  Novartis Institutes for Biomedical Research. 在各种疾病征兆中, 越来越多的生理相关模型被开发并实施到药物发现计划中。这里描述的新的模型系统展示了三维肿瘤球体如何在高通量1536井板系统中进行培养和筛选, 以寻找新的肿瘤药物。

视频: Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

三维肿瘤球体的纵向形态和生理监测

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

科研

癌症研究

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

癌症细胞球体机器人生产与药物测试的双水相系统

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

生物工程

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

用于肿瘤球体半高通量分析的快速光学清除

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

生成用于药物评估研究的 3D 肿瘤微球

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

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 ...

医学

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

肿瘤细胞球体的高通量图像分析:一个用户友好的软件应用来测量球体的大小自动准确

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...