Name: generation of high-throughput three-dimensional tumor spheroids for drug screening
Uploaded: 2018-09-05T15:30:00
Description: Watch this Scientific Journal Video about Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening at JoVE.com

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?...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+three-dimensional+(3D)+models+in+cancer+research:+an+update.">In vitro three-dimensional (3D) models in cancer research: an update.</a> Molecular Carcinogenesis. 52 (3), 167-182 (2013).</li><li>Mathews Griner, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+pharmacological+profiling+of+3D+tumor+models+of+cancer+cells.">Large-scale pharmacological profiling of 3D tumor models of cancer cells.</a> Cell Death &amp; Disease. 7 (12), e2492 (2016).</li><li>Polo, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsiveness+to+PI3K+and+MEK+inhibitors+in+breast+cancer.+Use+of+a+3D+culture+system+to+study+pathways+related+to+hormone+independence+in+mice.">Responsiveness to PI3K and MEK inhibitors in breast cancer. Use of a 3D culture system to study pathways related to hormone independence in mice.</a> PLoS One. 5 (5), e10786 (2010).</li><li>Hatzivassiliou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAF+inhibitors+prime+wild-type+RAF+to+activate+the+MAPK+pathway+and+enhance+growth.">RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.</a> Nature. 464 (7287), 431-435 (2010).</li><li>Bhargava, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registered+report:+RAF+inhibitors+prime+wild-type+RAF+to+activate+the+MAPK+pathway+and+enhance+growth.">Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.</a> Elife. 5, e09976 (2016).</li><li>Mathews, L. A., Hurt, E. M., Zhang, X., Farrar, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+CpG+promoter+methylation+in+invasive+prostate+cancer+cells.">Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells.</a> Molecular Cancer. 9, 267 (2010).</li><li>Mathews, L. A., Crea, F., Farrar, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+gene+regulation+in+stem+cells+and+correlation+to+cancer.">Epigenetic gene regulation in stem cells and correlation to cancer.</a> Differentiation. 78 (1), 1-17 (2009).</li><li>Mathews, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+DNA+repair+genes+in+invasive+human+pancreatic+cancer+cells.">Increased expression of DNA repair genes in invasive human pancreatic cancer cells.</a> Pancreas. 40 (5), 730-739 (2011).</li><li>Mathews, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+1536-well+quantitative+high-throughput+screen+to+identify+compounds+targeting+cancer+stem+cells.">A 1536-well quantitative high-throughput screen to identify compounds targeting cancer stem cells.</a> Journal of Biomolecular Screening. 17 (9), 1231-1242 (2012).</li><li>Horii, T., Nagao, Y., Tokunaga, T., Imai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+murine+primordial+germ+cells+and+embryonic+germ+cells.">Serum-free culture of murine primordial germ cells and embryonic germ cells.</a> Theriogenology. 59 (5-6), 1257-1264 (2003).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+SOX9+by+the+NF-kappaB+signaling+pathway+in+pancreatic+cancer+stem+cells.">Epigenetic regulation of SOX9 by the NF-kappaB signaling pathway in pancreatic cancer stem cells.</a> Stem Cells. 31 (8), 1454-1466 (2013).</li><li>Mathews, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+1536-well+quantitative+high-throughput+screen+to+identify+compounds+targeting+cancer+stem+cells.">A 1536-well quantitative high-throughput screen to identify compounds targeting cancer stem cells.</a> Journal of Biomolecular Screening. 17 (9), 1231-1242 (2012).</li><li>Mathews Griner, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+combinatorial+screening+identifies+drugs+that+cooperate+with+ibrutinib+to+kill+activated+B-cell-like+diffuse+large+B-cell+lymphoma+cells.">High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (6), 2349-2354 (2014).</li><li>Herter, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+three-dimensional+heterotypic+spheroid+model+for+the+assessment+of+the+activity+of+cancer+immunotherapy+agents.">A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents.</a> Cancer Immunology, Immunotherapy. 66 (1), 129-140 (2017).</li></ol>

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?...

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>Reagents</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>Consumables</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>Equipment</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?...

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 ...

Research

JoVE Journal

Cancer Research

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

肿瘤组织中高通量芯片测序的染色质状态全基因组图谱集成平台

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

科研

癌症研究

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

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

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, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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

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 ...

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

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 ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

小肠神经内分泌肿瘤类球体的建立与特征

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

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 ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

研究结肠癌干细胞的球体三维模型

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

使用患者源性肿瘤类器官的高通量 体外 检测

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...