Name: analysis of side population in solid tumor cell lines
Uploaded: 2021-02-23T13:00:00
Description: Watch this Scientific Journal Video about Analysis of Side Population in Solid Tumor Cell Lines at JoVE.com

本工作由中国自然科学基金委员会81572599、81773124、81972787资助：天津市自然科学基金（中国） 19JCYBJC27300：天津市人民医院 - 南开大学合作研究资助2016rmnk005;中央大学基础研究基金、南开大学63191153。

1. 细胞准备
<ol><li>细胞消化和中和<ol>
<li>种子肿瘤细胞（如MDA-MB-231细胞）在6井板，并在37°C孵化器中培养他们提供5%的二氧化碳。</li>
		<li>当细胞密度达到90%左右时，收获细胞。彻底吸气培养介质，用3mL磷酸盐缓冲盐水（PBS）清洗细胞2倍。 注意：为了检查信号通路抑制剂（例如FRA1抑制剂）或活化剂（例如STAT3活化剂）对肿瘤细胞干细胞特性的影响，肿瘤细胞在6个井板中播种，并在收...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+cells+in+human+cancers.">Side population cells in human cancers.</a> Cancer Letters. 268 (1), 1-9 (2008).</li><li>Patrawala, 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=Side+population+is+enriched+in+tumorigenic,+stem-like+cancer+cells,+whereas+ABCG2++and+ABCG2-+cancer+cells+are+similarly+tumorigenic.">Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic.</a> Cancer Research. 65 (14), 6207-6219 (2005).</li><li>Yamada, M., 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=Nasal+Colivelin+treatment+ameliorates+memory+impairment+related+to+Alzheimer's+disease.">Nasal Colivelin treatment ameliorates memory impairment related to Alzheimer's disease.</a> Neuropsychopharmacology. 33 (8), 2020-2032 (2008).</li><li>Marotta, L. 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=The+JAK2/STAT3+signaling+pathway+is+required+for+growth+of+CD44(+)CD24(-)+stem+cell-like+breast+cancer+cells+in+human+tumors.">The JAK2/STAT3 signaling pathway is required for growth of CD44(+)CD24(-) stem cell-like breast cancer cells in human tumors.</a> Journal of Clinical Investigation. 121 (7), 2723-2735 (2011).</li><li>Zhang, C. H., 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=From+Lead+to+Drug+Candidate:+Optimization+of+3-(Phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine+Derivatives+as+Agents+for+the+Treatment+of+Triple+Negative+Breast+Cancer.">From Lead to Drug Candidate: Optimization of 3-(Phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine Derivatives as Agents for the Treatment of Triple Negative Breast Cancer.</a> Journal of Medicnal Chemistry. 59 (21), 9788-9805 (2016).</li><li>Tam, W. 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=Protein+kinase+C+alpha+is+a+central+signaling+node+and+therapeutic+target+for+breast+cancer+stem+cells.">Protein kinase C alpha is a central signaling node and therapeutic target for breast cancer stem cells.</a> Cancer Cell. 24 (3), 347-364 (2013).</li><li>Ibrahim, S. F., Diercks, A. H., Petersen, T. W., van den Engh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analyses+as+a+critical+parameter+in+defining+the+side+population+(SP)+phenotype.">Kinetic analyses as a critical parameter in defining the side population (SP) phenotype.</a> Experimental Cell Research. 313 (9), 1921-1926 (2007).</li><li>Petriz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+the+side+population+(SP).">Flow cytometry of the side population (SP).</a> Current Protocol in Cytometry. , 23 (2013).</li><li>Hiraga, T., Ito, S., Nakamura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+in+MDA-MB-231+human+breast+cancer+cells+exhibits+cancer+stem+cell-like+properties+without+higher+bone-metastatic+potential.">Side population in MDA-MB-231 human breast cancer cells exhibits cancer stem cell-like properties without higher bone-metastatic potential.</a> Oncology Reports. 25 (1), 289-296 (2011).</li><li>Shen, W., 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=ICAM3+mediates+inflammatory+signaling+to+promote+cancer+cell+stemness.">ICAM3 mediates inflammatory signaling to promote cancer cell stemness.</a> Cancer Letters. 422, 29-43 (2018).</li><li>Koh, S. Y., Moon, J. Y., Unno, T., Cho, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baicalein+Suppresses+Stem+Cell-Like+Characteristics+in+Radio-+and+Chemoresistant+MDA-MB-231+Human+Breast+Cancer+Cells+through+Up-Regulation+of+IFIT2.">Baicalein Suppresses Stem Cell-Like Characteristics in Radio- and Chemoresistant MDA-MB-231 Human Breast Cancer Cells through Up-Regulation of IFIT2.</a> Nutrients. 11 (3), 624 (2019).</li><li>Lee, H., Park, S., Kim, J. 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对于 SP 分析，需要牢记几个要点。首先是为每个细胞系选择适当的阻滞剂，如维拉帕米尔或雷塞平，因为 SP 细胞的"门"位置根据添加阻止器后大量 SP 细胞消失的位置确定。对于MDA-MB-231细胞系，雷瑟平工作得很好。然而，对于其他细胞系，不同的阻滞剂可能效果更好。
第二个是霍赫斯特33342的浓度。有代表性的数据显示，随着霍赫斯特33342的染色浓度降低，SP细胞的百分比增?...

癌症干细胞（CSC）是具有自我更新能力和多分化潜力的细胞子集，在肿瘤生长、转移和复发1、2中起着至关重要的作用。目前，CSC已确定存在于各种恶性肿瘤中，包括肺癌、脑癌、胰腺癌、前列腺癌、乳腺癌和肝癌3、4、5、6、7、8、9。这些肿瘤中CSC的鉴定主要基于表面标记蛋白的存在，如CD44、CD24、CD133和Sca-19、10的高和/或低表达，但迄今尚未报告能够区分CSC与非CSC的独特标记。目前，用于识别和净化肿瘤组织或肿瘤细胞系中的 CSC 的几种技术。这些技术是根据 CSC 的特定属性设计的。其中，侧群（SP）细胞的测定和排序是两种常用的方法。
SP细胞最初是由古德尔等人11日发现的，当时它们特征是小鼠骨髓细胞中的造血干细胞。当小鼠骨髓细胞被贴上荧光染料Hhchst 33342的标签时，一小群Hchst 33342模糊染色细胞出现在流动细胞测定测量的二维点图中。Hoechst 33342 是一种 DNA 结合染料，至少有两种绑定模式，导致不同的光谱特征。同时以两个波长查看荧光发射时，可以显示多个种群12。在他们的测定中，Hoechst 33342 在 350 nm 时兴奋不已，荧光是使用 450/20 nm 带通 （BP） 滤光片和 675 nm 边缘滤光长通 （EFLP）11来测量的。与整个骨髓细胞群相比，这组细胞富含称为SP细胞11的造血干细胞。SP细胞能够快速驱逐霍赫斯特33342。这种染料的流向与ATP绑定盒式磁带（ABC）运输机13有关，可以抑制一些代理，如富米特雷莫金C14，维拉帕米尔和雷塞平15，16。之后，在各种组织、器官、肿瘤组织和肿瘤细胞系17、18、19中检测出不同比例的SP细胞。这些SP细胞具有干细胞的许多特征17，19。
本手稿描述了霍奇斯特33342对培养肿瘤细胞的标记和染色，以及通过流动细胞学对SP细胞的分析。此外，还显示了使用此方法优化的 Hoechst 33342 浓度和特定肿瘤细胞系的适当阻滞剂选择。最后，证明了干细胞促进或抑制信号对肿瘤细胞SP比例的影响。实验实例表明，对SP的分析可用于探索各种信号的影响，如基因表达、小抑制剂、活化剂、细胞因子和化疗因子，对肿瘤茎的作用。与其他分离和纯化 CSC 的方法（如 CD44+/CD24） 种群排序、醛脱氢酶 （ALDH） 分析和肿瘤球体形成检测）相比，该方法更容易操作，且具有成本效益。

<table>
	<tbody>
		<tr>
			<td>6 well cell culture plate</td>
			<td>CORNING</td>
			<td>3516</td>
			<td>9.5 cm2 (approx.)</td>
		</tr>
		<tr>
			<td>Colivelin</td>
			<td>MCE</td>
			<td>HY-P1061A</td>
			<td>Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-Pro-Ala-Gly-Ala-Ser-Arg-Leu-Leu-Leu-Leu-Thr-Gly-Glu-Ile-Asp-Leu-Pro</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biological Industries (BIOIND)</td>
			<td>04-001-1ACS</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>BD LSRFortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer software</td>
			<td>BD Biosciences</td>
			<td>FACSDiva</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry analysis software</td>
			<td>BD Biosciences</td>
			<td>FlowJo</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>bisBenzimide H 33342 trihydrochloride</td>
		</tr>
		<tr>
			<td>Polystyrene round bottom test tube</td>
			<td>CORNING</td>
			<td>352054</td>
			<td>12 x 75 mm, 5mL</td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td>3,8-Diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide</td>
		</tr>
		<tr>
			<td>Reserpine</td>
			<td>Sigma-Aldrich</td>
			<td>83580</td>
			<td>(3&#946;, 16&#946;, 17&#945;, 18&#946;, 20&#945;)-11,17-Dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester</td>
		</tr>
		<tr>
			<td>SKLB816</td>
			<td>Provided by Dr. Shengyong Yang, Sichuan University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Verapamil hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td>5-[N-(3,4-Dimethoxyphenylethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile hydrochloride</td>
		</tr>
	</tbody>
</table>

analysis of side population in solid tumor cell lines

癌症干细胞（CSC）是肿瘤生长、转移和复发的重要原因。CSC的分离和鉴定对肿瘤研究具有重要意义。目前，几种技术用于从肿瘤组织和肿瘤细胞系中识别和净化CSC。侧群 （SP） 细胞的分离和分析是两种常用的方法。这些方法依赖于 CSC 快速排出荧光染料（如 Hoechst 33342）的能力。染料的流出与 ATP 绑定盒式 （ABC） 运输机相关联，可由 ABC 运输器抑制剂抑制。介绍了用Hchst 33342染色培养肿瘤细胞的方法，并通过流动细胞测量分析其SP细胞的比例。此检测方便、快速且经济高效。此测定中生成的数据有助于更好地了解基因或其他细胞外和细胞内信号对肿瘤细胞干细胞特性的影响。

根据这种方法进行了四次实验性SP分析。在第一个，我们检测到SP细胞在MDA-MB-231中的比例，这是人类乳腺癌细胞系的三重阴性，在正常条件下。在细胞计数后，Hoechst 33342 被添加到一个包含 1 x 106细胞的管中，最终浓度为 3 μg/mL。Reserpine 和 Hoechst 33342 分别添加到另一根管中，最终浓度分别为 40μM 和 3μg/mL。PI 被添加到两个管中。FSC-A（X轴）与PI-A（Y轴）的点图显示了三个种群：1） 代表死细...

Watch this Scientific Journal Video about Analysis of Side Population in Solid Tumor Cell Lines at JoVE.com

Analysis of Side Population in Solid Tumor Cell Lines

提出了一种方便、快速、经济的方法，测量固体肿瘤细胞系中侧群细胞的比例。

固体肿瘤细胞系侧面人口分析

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

侧群细胞分析是从肿瘤组织和肿瘤细胞系中分离和识别癌症干细胞的常用方法之一。此检测方便、快速且经济高效。这种方法有助于更好地了解基因或其他细胞外和细胞内信号对肿瘤细胞干细胞特性的影响。许多因素会影响SP细胞在分析中的百分比，因此在正式实验之前探索适当的条件至关重要。在六井板中播种肿瘤细胞，并在37摄氏度的孵化器中孵化，提供5%的二氧化碳。当细胞密度达到约 90% 时，吸气培养介质，用三毫升 PBS 洗两次细胞。然后，在每口井中加入500微升的0.25%的三氯辛-EDTA，在37摄氏度下孵化板一至三分钟。轻轻敲击板分离细胞，在每口井中加入三毫升PBS，并加2%FBS，将细胞上下吹动三到五次，以分散细胞团。检查显微镜下的细胞悬浮。如果存在细胞团，则通过 70 微米过滤器通过悬架。将细胞转移到15毫升离心管，并在5分钟内以200倍g的离心机将其离心。取出超自然物，用三毫升PBS补充2%FBS，将细胞上下吹管三到五次混合。使用血细胞计在显微镜下计数细胞，并在PBS中稀释它们，辅以2%FPS，最终浓度为每毫升6个细胞的1倍10倍。准备两个五毫升，聚苯乙烯，圆底试管，并添加一毫升的细胞悬架到每个管。将一根管子标记为试管，另一根将另一根管子标记为阻滞管。通过稀释到适当的浓度来准备阻滞剂的解决方案。将其添加到阻滞管中并混合良好，然后在 37 摄氏度下孵育管 30 分钟。每10分钟摇动一次管子。通过稀释到适当的浓度，准备霍赫斯特 33342 的解决方案。将其分别添加到试管和阻滞剂控制管中，混合良好，然后在 37 摄氏度下孵育管 60 分钟。每10分钟摇动一次管子。孵化后，在200倍和4摄氏度下将细胞离心5分钟，然后小心地吸气超自然。用一毫升冰冷的PBS补充2%FBS，然后将细胞上下吹拌，以补充每个管中的细胞。在每个管子中加入一微升每毫升碘化物丙酸或PI，然后混合。单击流细胞仪软件中的参数选项卡。首先，分别选择 FSC、SSC、霍奇斯特蓝、霍赫斯特红和 PI 的参数。其次，选择 PI 参数的对数比例。最后，分别选择 FSC、SSC、霍奇斯特蓝、霍赫斯特红和 PI 参数的面积和宽度比例。先从试管中运行电池，然后使用相同的电压和门从阻滞剂控制管中运行电池。从每个样本中收集 20 到 10 万个事件，以分析 SP 细胞的百分比。该方法用于检测MDA-MB-231人类乳腺癌细胞系中SP细胞的比例。FSC-A与PI-A的点图显示了死细胞、细胞碎片和PI负细胞的主要种群，并进行了进一步分析。单个细胞群从 FSC-A 与 FSC-W 的点图中封闭，使用 SSC-A 与 SSC-W 的点图来分析 SP 细胞的比例，在未经处理的细胞中，SP 细胞的比例约为 0.9%，在用 reserpine 处理的细胞中约为 0.09%。在MDA-MB-435细胞上测试了霍奇斯特33342的不同染色浓度，确定每毫升两微克最适合SP分析。低浓度使得SP细胞与其他种群难以区分，而高浓度则导致SP细胞消失。对维拉帕米尔和树泽平进行了测试，以确定MDA-MB-435细胞的适当阻滞剂。大约 0.4% 的细胞在 verapamil 治疗后排出染料，但经过再塞平治疗后，该比率下降到约 0.1%，这表明树泽平是该细胞系更合适的阻滞剂。该协议还用于确定使用STAT3活化剂结肠素治疗的A549人肺腺癌细胞中的SP细胞比例，以及用FRA1抑制剂SKLB816治疗的T47D人类乳腺癌细胞中的SP细胞比例。该检测为信号通路对癌症干细胞特征的调节作用提供了线索，并有助于发现新的机制，最终指导肿瘤的靶向治疗。

Research

JoVE Journal

Cancer Research

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

利用DNA染色体引发的侧群体表型，从生物样品中检测和纯化癌细胞

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

科研

癌症研究

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

肺癌细胞系中癌症干细胞的放射敏感性

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

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 ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

循环肿瘤细胞系：基础和转化研究的创新工具

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...