Name: analysis of cancer cell invasion and anti-metastatic drug screening using hydrogel micro-chamber array (hmca)-based plates
Uploaded: 2018-10-25T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates at JoVE.com

这项工作得到了佩雷斯和朱蒂丝 Weisbrodt 的遗赠的支持。

1. HMCA 板压花
注: 本协议中使用的硅油 (PDMS) 图章和 HMCA 成像板的设计和制造的完整过程在我们前面的文章15,16中详细介绍。PDMS 图章 (负形状) 用于浮雕 HMCA (正形), 由大约450个 MCs (图 1A) 组成。如图 1B所示, 每个 MCs 都有一个被截断的倒置方形金字塔的形状 (高度: 190 µm, 小底座面积:90 µm x 90 ...

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据记载, 活生物体的特点是其复杂的3D 多细胞组织与常用的2D 单层培养细胞截然不同, 强调使用细胞模型更好地模仿功能的关键需要和生物药物筛选的过程。最近, 多细胞旋转椭球体、器官型文化、organoids 和片上器官已被引入8用于标准化药物发现。然而, 3D 多细胞模型的复杂性极大地危及了其鲁棒性、并行性和数据分析, 这对检测效率至关重要。
入侵能力的?...

癌症死亡主要归因于转移细胞传播到遥远的地方。许多癌症治疗的努力重点是瞄准或预防转移性菌落的形成和系统性转移性疾病1的进展。癌细胞迁移是肿瘤转移过程中的重要一步, 因此, 对肿瘤侵袭性叶栅的研究是非常重要的, 也是寻找抗转移治疗的前提条件。
动物模型作为研究转移性疾病的工具被发现是非常昂贵的, 并不总是代表在人类的肿瘤。此外, 细胞外微环境的拓扑结构、力学和成分对癌细胞的行为有强烈的影响2。由于体内模型本身缺乏分离和控制导致癌症侵袭和转移的特定参数的能力, 因此需要对体外模型进行可控仿生。
为了转移到遥远的器官, 癌细胞必须表现出迁徙和侵入性表型特征, 这些性状可以被治疗。然而, 由于大多数体外肿瘤模型不模仿实体肿瘤3的实际特征, 因此检测生理相关表型非常具有挑战性。此外, 在肿瘤内存在的表型异质性, 决定了在单元素分辨率下分析肿瘤迁移的必要性, 以发现表现性定向疗法, 例如, 通过靶向转移起始细胞异质肿瘤中的人口4。
细胞运动和集体迁移的研究主要在同质平面表面上皮细胞的单层培养中进行。这些用于癌症细胞迁移的常规细胞模型是基于通过细胞膜和 ECM 组件5侵入的单个细胞的种群分析, 但在这种系统中, 单个细胞之间的内在差异不能研究。通过支架或无支架微结构生成3D 旋转椭球体被认为是研究肿瘤细胞生长和肿瘤侵袭6、7、8的一种优越手段。但是, 大多数3D 系统不适合高吞吐量格式, 并且在每个微井9中生成独立的单个旋转椭球体时, 通常无法实现椭球体之间的交互。最近涉及癌症迁移的研究基于微流控设备3,10,11,12, 然而, 这些复杂的微流控工具很难生产, 不能用于抗侵入性药物的高通量筛选 (HTS)。
两种主要表型, 集体和个体细胞迁移, 在肿瘤细胞中发挥作用, 克服 ECM 屏障和入侵邻近组织, 已证明13,14, 每个显示不同的形态学特征、生化、分子和遗传机制。此外, 在每个表型中观察到两种形式的迁移肿瘤细胞, 成纤维细胞样和变形虫。由于入侵表型和迁移模式, 主要是由形态学属性定义的, 因此需要对细胞模型进行基于成像的检测和检查所有形式的肿瘤侵袭和迁移细胞。
目前的方法的总体目标是提供一个简单和可重现的仿生 3D体外细胞系统, 以分析肿瘤旋转椭球体大种群的侵袭能力。在这里, 我们介绍了基于 HMCA 的6孔成像板用于肿瘤侵袭和抗转移治疗的研究。该技术能够在水凝胶微室 (MC) 结构中形成大量均匀的3D 肿瘤旋转椭球体 (450)。将各种 ECM 组件添加到椭球体阵列中, 以使细胞侵入周围环境。通过对同一个体旋转椭球体/入侵细胞的短期和长期观察, 对入侵过程进行持续监测, 并在任何时候促进形态学表征、荧光染色和特定旋转椭球体的检索。由于许多旋转椭球体共享空间和媒介, 通过个体旋转椭球体之间的可溶性分子相互作用及其对彼此的影响是可能的。使用内部 MATLAB 代码进行半自动图像分析, 从而能够更快、更高效地收集大量数据。该平台能够以高效的方式对众多旋转椭球体/入侵细胞进行准确、同时的测量, 从而实现对抗入侵药物的中等吞吐量筛选。

<table>
	<tbody>
		<tr>
			<td>6 Micro-well Glass Bottom Plates with 14 mm micro-well #1.5 cover glass</td>
			<td>Cellvis</td>
			<td>P06-14-1.5-N</td>
			<td>Commercial glass bottom plates which are used for HMCA embossing</td>
		</tr>
		<tr>
			<td>UltraPure Low Melting Point Agarose</td>
			<td>Invitrogen</td>
			<td>16520100</td>
			<td>A solution of 6% agarose is warmed up to 80&#176;C before use, a solution of 1% agarose is warmed to 37&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin EDTA solution B</td>
			<td>Biological Industries</td>
			<td>03-052-1A</td>
			<td>Warmed to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>DMEM medium, high glucose</td>
			<td>Biological Industries</td>
			<td>01-055-1A</td>
			<td>Warmed to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Special Newborn Calf Serum (NBCS)</td>
			<td>Biological Industries</td>
			<td>04-122-1A</td>
			<td>Heat Inactivated</td>
		</tr>
		<tr>
			<td>DPBS (10X), no calcium, no magnesium</td>
			<td>Biological Industries</td>
			<td>02-023-5A</td>
			<td>Kept on ice before use</td>
		</tr>
		<tr>
			<td>NaOH, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-500G</td>
			<td>Used for the preparation of 1M NaOH solution</td>
		</tr>
		<tr>
			<td>Cultrex Type I collagen from rat tail, 5mg/ml</td>
			<td>Trevigen</td>
			<td>3440-100-01</td>
			<td>Kept on ice before use</td>
		</tr>
		<tr>
			<td>Hyaluronic acid sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H5542-10MG</td>
			<td>Kept on ice before use</td>
		</tr>
		<tr>
			<td>Fisetin</td>
			<td>Sigma-Aldrich</td>
			<td>F505-100MG</td>
			<td>Added to the culture medium, invasion inhibitor</td>
		</tr>
		<tr>
			<td>DETA/NO</td>
			<td>Enzo Life Sciences</td>
			<td>alx-430-014-m005</td>
			<td>Added to the culture medium, nitric oxide donor</td>
		</tr>
		<tr>
			<td>PI</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td>Used at very low concenrtation without the need for washing</td>
		</tr>
		<tr>
			<td>Dymax 5000-EC UV flood lamp complete system with light shield &#38; Dymax 400 Watt EC power supply</td>
			<td>Dymax Corporation</td>
			<td>PN 39823</td>
			<td>Used for HMCA plate sterilization by UV</td>
		</tr>
		<tr>
			<td>Inverted IX81 microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Used for automatic image acquisition</td>
		</tr>
		<tr>
			<td>Incubator for microscope</td>
			<td>Life Imaging Services</td>
			<td></td>
			<td>Essential for time lapse experiments with image acquisition, pre adjusted to 37&#176;C, 5% CO2 and keeping a humidified atmosphere</td>
		</tr>
		<tr>
			<td>Sub-micron motorized stage type SCAN-IM</td>
			<td>Marzhauser Wetzlar GmbH</td>
			<td></td>
			<td>Used to predetermine image acquisition areas, for automatic image acquisition</td>
		</tr>
		<tr>
			<td>14-bit, ORCA II C4742-98 cooled camera</td>
			<td>Hamamatsu Photonics</td>
			<td></td>
			<td>Highly sensitive, used for imaging</td>
		</tr>
		<tr>
			<td>Fluorescent filter cube for PI detection</td>
			<td>Chroma Technology Corporation</td>
			<td></td>
			<td>Filter cube specifications: excitation filter 530-550 nm, dichroic mirror 565 nm long pass and emission filter 600-660 nm</td>
		</tr>
		<tr>
			<td>The Olympus Cell^P operating software</td>
			<td>Olympus</td>
			<td></td>
			<td>Software used to control microscope, motorized stage, camera and image acquisition</td>
		</tr>
		<tr>
			<td>Matlab R2014B analysis software</td>
			<td>Mathworks</td>
			<td></td>
			<td>Used to develop in house graphic user interface for image analysis</td>
		</tr>
		<tr>
			<td>Excel software</td>
			<td>Microsoft</td>
			<td></td>
			<td>Used for data management, calculation, plot creation and statistics</td>
		</tr>
	</tbody>
</table>

analysis of cancer cell invasion and anti-metastatic drug screening using hydrogel micro-chamber array (hmca)-based plates

癌症转移是已知的导致90% 的癌症致死率。转移是一个多级过程, 启动与渗透/侵入肿瘤细胞到邻近组织。因此, 入侵是转移的关键一步, 使入侵过程的研究和开发的抗转移药物, 具有非常重要的意义。为了满足这一需求, 需要开发 3D的体外模型, 以模拟实体肿瘤及其微环境的结构, 一方面最接近体内状态, 但同时具有可重现性、鲁棒性和适用于高产量和高含量测量。目前, 大多数入侵检测都依赖于成熟的微流控技术, 它们足够用于研究, 但不能用于高通量药物筛选。在每个井中使用具有独立单独旋转椭球体的板型设备的其他检测是材料消耗, 每个条件的样品尺寸都很低。目前的协议的目标是提供一个简单的和可重现的仿生3D 细胞系统, 以分析大种群肿瘤旋转椭球体的侵袭能力。我们开发了一种基于 HMCA 成像板的3D 入侵检测模型, 用于肿瘤侵袭和抗转移药物发现的研究。该器件可生产多种均匀的旋转椭球体 (每个条件下的高样本大小), 同时在单元素分辨率下连续、同时观察和测量旋转椭球体的介质抗转移药物的高通量筛选。这个平台是在这里提供的 HeLa 和 MCF7 旋转椭球体的例证单细胞和集体入侵的生产。比较了 ECM 成分透明质酸 (HA) 对旋转椭球体周围胶原的侵袭能力的影响。最后, 我们引入漆黄素 (入侵抑制剂) 对 HeLa 旋转椭球体和一氧化氮 (NO) (入侵激活剂) MCF7 旋转椭球体。通过内部软件对结果进行分析, 实现半自动、简单、快速的分析, 便于多参数检测。

独特的 HMCA 成像板用于3D 肿瘤旋转椭球体的侵袭性测定。整个测试, 开始与椭球体形成和结束与入侵过程和额外的操作, 在同一板块内执行。对于椭球体的形成, HeLa 细胞被加载到阵列盆地中, 并通过重力沉降到水凝胶 MCs 中。水凝胶 MCs 具有非附着性/低附着特性, 促进细胞细胞相互作用和3D 肿瘤旋转椭球体的形成。图 2A说明了在 MCs 中解决的单元格, 使?...

Watch this Scientific Journal Video about Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates at JoVE.com

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

针对入侵检测的性能, 提出了一种基于 HMCA 的成像板。该板块有助于形成三维 (3D) 肿瘤旋转椭球体和测量癌细胞侵入细胞外基质 (ECM)。通过半自动分析实现了入侵检测的定量化。

用水凝胶微室阵列 (HMCA) 板进行肿瘤细胞侵袭和抗转移药物筛选的分析

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

这种简单的可重复仿生三维细胞系统的目标是促进大量肿瘤球体的入侵能力分析，用于抗转移药物筛查。我们的水凝胶微室阵列的主要优点是，它允许生产大量的均匀球体，填充每井相同的焦平面。即使增加了细胞外基质，水凝胶微室阵列结构也保留了球体位置，从而有助于对球体和入侵过程的精确连续监测。此外，许多球体和入侵细胞的形态特征和荧光染色可以就地进行，并以单元素分辨率以具有时间效率的方式进行分析。演示水凝胶微室阵列六井成像板生产程序将玛丽亚索博列夫从我们的实验室。要准备水凝胶微室阵列板，首先预热六个聚二甲基硅氧烷（PDMS），将邮票预热至70摄氏度，并在干浴前加热至75摄氏度的干浴上放置一个六孔玻璃底板，预热至75摄氏度，几分钟。当板是温暖的，倒一个400微升预加热低熔化的阿加糖滴到六井板的每一个井的底部，并轻轻地在每滴上放置一个邮票。让设置在室温下冷却 5 到 10 分钟，随后在 4 摄氏度下冷却 20 分钟，以进行全加糖凝胶。当阿加罗斯凝固后，从每一个井轻轻剥下PDMS，使红糖凝胶与微室图案。在允许紫外灯达到最高强度后，将浮雕板放在光固化系统中三分钟，用无菌 PBS 填充紫外线消毒的宏井。然后，盖住盘子，用 Parafilm 将其包裹起来，储存四摄氏度，直到使用。要加载电池，请从每一个井上取出 PBS，并轻轻地将 50 微升的感兴趣细胞加载到每个水凝胶阵列的顶部。在允许细胞通过重力沉淀15分钟后，小心地将6至8个500微升的新鲜介质加入环和水凝胶阵列外的宏井塑料底部边缘。在37摄氏度和5%的二氧化碳中孵育细胞，用于形成球类。然后，将球体转移到冰上的生物安全罩中10分钟。当板冷却后，将移液器尖端靠在水凝胶阵列旁边的宏井塑料底部边缘，然后从阵列周围取出所有介质。然后，从阵列罐中取下介质，非常小心地将凝胶加载移液器尖端靠在阵列罐的边缘，注意不要打扰微室和球体。接下来，使用预冷冻的细移液器尖端，在阵列边缘添加两个150微升的胶原蛋白混合物，一次一个，慢慢释放混合物，以避免球形错位。第二轮胶原蛋白被添加到每井后，将球体返回孵化器一小时，以完全凝胶外基质。当基质凝固后，在凝胶顶部加入400微升预加热的1%低熔化黄糖，并在室温下孵育板，在室温下孵育5至7分钟，在4摄氏度下冷却2分钟，进行加糖凝胶化。然后，将移液器靠在大井塑料底部的边缘，轻轻地向每个井添加两毫升的完整介质。要进行入侵测定，将水凝胶微室阵列板加载到装有37摄氏度和5%二氧化碳培养箱的机动倒置显微镜舞台上，并采用4到10倍的目标预先确定每井中图像采集的位置，以便评估整个阵列区域。然后，每两到四个小时获取一次亮场图像，总共 24 到 72 小时，以跟踪细胞从球体体进入周围细胞外基质的入侵。在图像采集结束时，从图像采集软件导出延时图像，以标记图像文件格式保存到专用文件夹中。接下来，打开图形用户界面，然后单击"加载亮场"。调整图像的分割参数，实现球体及其入侵区域的精确分割，然后单击"兴趣区域分割"以在每个球体周围创建边框。如果自动分段不正确，请按"删除"或"添加"，然后手动进行相应的更正。单击"跟踪"可对每个延时图像中的球体进行编号。然后，单击"测量"以生成包含所有参数的电子表格，然后以适当的格式保存电子表格以进行数据处理和统计分析。为了研究透明质酸对自发的 HeLa 球体入侵过程的影响，在本次实验中，用胶原蛋白和透明质酸混合物溶液覆盖了两天成熟的球体，并与单独覆盖胶原蛋白溶液的两天成熟球体进行比较。经过50个小时的孵育，嵌入在胶原蛋白和透明质酸中的球体与单独嵌入在胶原蛋白中的球体相比，在周围细胞外基质中表现出较低的细胞分散。事实上，对99个球类在单球形分辨率下入侵区动力学的分析表明，在胶原蛋白中加入透明质酸会抑制球状细胞分散，导致入侵区较小。与未处理的 HeLa 球体相比，使用具有细胞和哑激素特性的生物活性类黄酮治疗 HeLa 球类化合物，可对球体周围产生抑制细胞分散。事实上，在实验结束时对488个球类的入侵区进行分析后发现，治疗后，药物的抑制作用明显，只有10微摩尔。由于水凝胶的附着性较低，这种基于微腔室的技术有助于球体的形成，即使在极少数细胞的情况下。这在宝贵的有限癌症干细胞样或患者衍生的原细胞样本中尤其具有优势。水凝胶微室成像板允许在长期实验中分析球体的确切空间位置，以在单元素水平上评估入侵过程，并提供高度的统计鲁棒性和准确性。这种方法的未来应用包括并排培养肿瘤和频闪细胞在宏观井内不同区域，以及检索单个球体进行下游分子分析。

Research

JoVE Journal

Cancer Research

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

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

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

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

在头颈部鳞状细胞癌的神经浸润模型

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

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

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 ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

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 ...