Name: isolation and characterization of a head and neck squamous cell carcinoma subpopulation having stem cell characteristics
Uploaded: 2016-05-11T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics at JoVE.com

We thank Thibault Andrieu and Sebastien Dussurgey from the Flow Cytometry Platform of UFR BioSciences Gerland-Lyon-Sud (UMS3444/US8) for their advice and help during our sorting. This work was achieved within the scientific framework of ETOILE and Labex-PRIMES (ANR-11LABX-0063).

根据对动物保健当地指南进行所有动物的程序。这项研究的所有细节是由CECCAPP，法国伦理委员会批准。 1.由赫斯特染料流出试验选择侧群（SP）的<ol><li>染色50000000细胞用Hoechst 33342染料。 <ol><li>准备两个15毫升锥底无菌试管：一管标记为“赫斯特”，一个标有“赫斯特和维拉帕米”。制备的10ml无菌水中的5mM盐酸维拉帕米溶液。制备用于干细胞的培养基（CM）。 <ol><li>以制备用于CSC（CM-C...

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这个协议描述的CSCs的从特定细胞系适用于其它HNSCC细胞系中成功地分离出一种可靠的方法。孤立的头颈部肿瘤干细胞是那么适合在免疫缺陷小鼠19 在体外和功能验证进一步分子表征移植。然而，一些修改可以根据侧群或存在于亲本细胞系的CD44 高 / ALDH 高百分比进行测试。例如，如果细胞在侧人口的百分比太低在特定细胞系中，CD44 高 / ALDH 高排序，?...

头颈部鳞状细胞癌（头颈部鳞癌）是全球常见的恶性肿瘤之一，尽管在目前的治疗进展，晚期患者预后较差。患者的总的5年存活率为30％左右，尽管的治疗方法包括手术，化疗放疗和靶向疗法的组合。最近的研究属性局部复发和远处转移癌干细胞样细胞的抗癌如下1疗法的生存（CSCS）。。有越来越多的证据支持细胞呈递的各种实体肿瘤，包括乳腺癌，脑癌，前列腺癌，肺癌，结肠癌，胰腺癌，肝和皮肤2-干细胞特性（未分化的状态，自我更新和分化的能力，和端粒酶活性）的存在10。但是，肿瘤干细胞的起源仍不清楚11,12。它们可能会导致从正常干细胞3,13或dedifferen的恶性转化该收购的CSC样的功能14,15肿瘤细胞的tiation。因此，了解有关肿瘤干细胞独特的途径将提供深入了解早期诊断和治疗性头颈部鳞癌。 已经提出了肿瘤干细胞也具有该躲避标准化疗和放疗抗性表型，导致肿瘤复发相比大块肿瘤细胞16-19的和被本地化为低氧龛20。多种因素已被提出来解释的CSCs的这些电阻，如倾向静止，增强DNA修复，上调细胞周期控制机制，和自由基清除21。此外，一些癌基因的分子途径可以具体在的CSCs 17激活。为了改善的CSCs进一步靶向疗法的知识，我们需要确定和的CSCs的隔离可靠的方法，因为干细胞相关的标记物中的异质各种癌症22。 在头颈部鳞癌，干细胞样肿瘤起始细胞已经从初级的肿瘤患者通过排序表达不同的生物标志物CSC（如药物转运蛋白的表达23，CD44 高 ，CD24 CD133 低 高的c-Met +表型10,24细胞中分离出来， 25，或ALDH活性高 26）或原发性培养的肿瘤病人，形成具有CSC性质squamospheres。尽管如此，squamospheres的第二代后显着降低，从而使作进一步鉴定一个小样本研究27。因此， 在体外测定从良好建立的细胞系开始是一个更简单的解决方案，以提高的CSCs的知识来设计的实验。 本文的目的是提出使用多参数流式细胞仪分析来隔离HNSCC细胞系的CSCs的方法第二细胞分选。 CD44在相关的几个CSCs的属性，包括ALDH活性的表达，侧群（SP）的表型，球体形成的能力和致瘤性被用来分离和鉴定的CSC的该亚群。 CD44，细胞表面糖蛋白，是参与细胞粘附和迁移。 CD44在许多实体瘤中高度表达的CSCs 28，包括头部和颈部癌症模型29-31。此外，CD44 高细胞可在体内产生肿瘤的异质性，而CD44 低细胞不能10。在SP测定是基于细胞的差动电位通过ATP结合盒（ABC）家族的CSC膜内过表达转运蛋白的向外排的Hoechst的染料22。此法包括使用ABC转运抑制剂如控制样品中维拉帕米。醛脱氢酶（ALDH）是参与转换视黄醇与沤胞内酶早期干细胞分化过程中25,26酸inoic。显示出在头颈部鳞癌26日高点 ALDH活性秀干细胞样细胞的行为和ALDH 高细胞的极少数细胞能够在体内 26,32产生肿瘤。 这些标记和属性的组合物成功地用于由贝特朗（E T）人研究在体外和这些的CSCs的体内光子和碳离子辐射19的电阻。其结果清楚地表明，不同的细胞标记和属性的组合是用在HNSCC的CSCs种群比单标记的方法有用的研究更多的选择性。

<table>
	<tbody>
		<tr>
			<td>Fetal Calf Serum Gold</td>
			<td>GE Healthcare</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone water soluble</td>
			<td>Sigma-Aldrich</td>
			<td>H0396-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100 X</td>
			<td>Dominique Dutscher</td>
			<td>L0022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>61965-026</td>
			<td></td>
		</tr>
		<tr>
			<td>F12 Nut Mix (1X) + GlutaMAX-I</td>
			<td>Gibco</td>
			<td>31765-027</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Promega</td>
			<td>G5021</td>
			<td>The solution must be prepared just before use because it is very unstable</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>StemcellTM Technologies</td>
			<td>7980</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), minus vitamin A</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma-Aldrich</td>
			<td>14533</td>
			<td>Corrosive, acute toxicity (oral, dermal, inhalation) category 4</td>
		</tr>
		<tr>
			<td>Verapamil hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>V-4629</td>
			<td>Acute toxicity (oral, dermal, inhalation) category 3</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td>Acute toxicity (oral, dermal, inhalation) category 4</td>
		</tr>
		<tr>
			<td>ALDEFLUOR Kit</td>
			<td>Stem Cell</td>
			<td>1700</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44-APC, human antibody</td>
			<td>Miltenyi Biotech</td>
			<td>130-095-177</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG1-APC, human antibody</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-189</td>
			<td></td>
		</tr>
		<tr>
			<td>Z1 coulter particle</td>
			<td>Beckman Coulter</td>
			<td>6605698</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Olympus&#160;</td>
			<td>CKX31</td>
			<td></td>
		</tr>
		<tr>
			<td>SQ20B cell line</td>
			<td>Gift from the John Little&#8217;s Laboratory</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>FaDu cell line</td>
			<td>ATCC</td>
			<td>HTB-43</td>
			<td></td>
		</tr>
		<tr>
			<td>Low anchorage plates</td>
			<td>Thermo Fischer Scientific</td>
			<td>145383</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva software v8.0.1</td>
			<td>BD Biosciences</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of a head and neck squamous cell carcinoma subpopulation having stem cell characteristics

尽管在头颈部鳞状细胞癌（头颈部鳞癌）进展的认识的进步，五年存活率因局部复发和远处转移仍然很低。一个假设来解释这一复发是呈现固有化疗和放射抗性癌干细胞样细胞（CSCs的）的存在。为了开发新的治疗策略，有必要有验证的靶向治疗的有效性的实验模型，因此具有用于肿瘤干细胞的鉴定和分离的可靠方法。为此，我们提出了一个协议，用于的CSCs的从人HNSCC细胞系依赖于通过荧光激活细胞进行的两个连续细胞分类法分选（FACS）的组合的隔离。第一种是基于的CSCs的属性以过表达的ATP结合盒（ABC）转运蛋白，从而排除除其他外，重要的DNA染料例如Hoechst的33342的细胞进行排序与​​第是方法被标识为一个“侧群”（SP）。作为SP细胞代表低百分比的亲代细胞的（&lt;5％），越来越相是必要的，以便在第二细胞分选之前增加其数量。下一步允许该拥有另外两个头颈部鳞癌干细胞的特性即细胞表面标志物CD44（CD44 高 ）和乙醛脱氢酶的过表达（ALDH 高 ）的高表达细胞的选择。因为使用单个标记的具有许多局限性和缺陷对于CSCs的，SP的组合的分离，CD44和ALDH标记将提供来隔离为需要的活细胞进一步分析和功能分析的CSCs的有用工具。的CSCs的干细胞样特征终于在体外通过tumorispheres的形成和β连环蛋白的表达进行验证。

从头颈部鳞癌细胞系肿瘤干细胞的分离必要的，因为肿瘤干细胞的亲本细胞系的比例很低的两个连续排序。第一分类是基于肿瘤干细胞的排除由于药物转运蛋白赫司特染料的能力。这导致获得的排序的总的细胞群体（ 图1）的1-5％。在赫斯特染色阴性细胞分选，通过查看FSC-A与SSC-A散点图（ 图1A）检查大小和排序细胞的肉芽。然后，通过使用SSC-W与S...

Watch this Scientific Journal Video about Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics at JoVE.com

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

分离和表征头颈部鳞状细胞癌亚群具有干细胞特性

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...

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Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

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