Name: harnessing the dna dye-triggered side population phenotype to detect and purify cancer stem cells from biological samples
Uploaded: 2017-05-10T15:00:00
Description: Watch this Scientific Journal Video about Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples at JoVE.com

Maximilian Boesch由奥地利科学基金会FWF（授权号J-3807）的ErwinSchrödinger奖学金支持。

该协议完全符合机构伦理审查委员会的准则。如果使用本文所述的方案调查人或动物组织，研究人员有义务获得其机构或国家相关审查委员会的批准。 注意事项安全处理生物样本的标准注意事项适用于本协议。这包括戴手套和实验室外套，并尽可能在生物安全柜内进行工作。 1.细胞的制备<ol><li> 癌细胞系 <ol><li>在37℃下，在10-30mL合适培养?...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF7+side+population+cells+with+characteristics+of+cancer+stem/progenitor+cells+express+the+tumor+antigen+MUC1.">MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1.</a> Cancer Res. 68 (7), 2419-2426 (2008).</li><li>Ho, M. M., Ng, A. V., Lam, S., Hung, J. Y. <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+human+lung+cancer+cell+lines+and+tumors+is+enriched+with+stem-like+cancer+cells.">Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells.</a> Cancer Res. 67 (10), 4827-4833 (2007).</li><li>Kato, K., 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=Endometrial+cancer+side-population+cells+show+prominent+migration+and+have+a+potential+to+differentiate+into+the+mesenchymal+cell+lineage.">Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage.</a> Am J Pathol. 176 (1), 381-392 (2010).</li><li>Bleau, A. 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=PTEN/PI3K/Akt+pathway+regulates+the+side+population+phenotype+and+ABCG2+activity+in+glioma+tumor+stem-like+cells.">PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells.</a> Cell Stem Cell. 4 (3), 226-235 (2009).</li><li>Harris, M. 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=Cancer+stem+cells+are+enriched+in+the+side+population+cells+in+a+mouse+model+of+glioma.">Cancer stem cells are enriched in the side population cells in a mouse model of glioma.</a> Cancer Res. 68 (24), 10051-10059 (2008).</li><li>Murase, 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=Side+population+cells+have+the+characteristics+of+cancer+stem-like+cells/cancer-initiating+cells+in+bone+sarcomas.">Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas.</a> Br J Cancer. 101 (8), 1425-1432 (2009).</li><li>Boesch, M., Wolf, D., Sopper, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Stem+Cell+Detection+Using+the+DyeCycle-Triggered+Side+Population+Phenotype.">Optimized Stem Cell Detection Using the DyeCycle-Triggered Side Population Phenotype.</a> Stem Cells Int. 2016, 1652389 (2016).</li><li>Golebiewska, 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=Side+population+in+human+glioblastoma+is+non-tumorigenic+and+characterizes+brain+endothelial+cells.">Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells.</a> Brain. 136 (PT 5), 1462-1475 (2013).</li><li>Boesch, 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=Drug+transporter-mediated+protection+of+cancer+stem+cells+from+ionophore+antibiotics.">Drug transporter-mediated protection of cancer stem cells from ionophore antibiotics.</a> Stem Cells Transl Med. 4 (9), 1028-1032 (2015).</li><li>Fukunaga-Kalabis, 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=Tenascin-C+promotes+melanoma+progression+by+maintaining+the+ABCB5-positive+side+population.">Tenascin-C promotes melanoma progression by maintaining the ABCB5-positive side population.</a> Oncogene. 29 (46), 6115-6124 (2010).</li><li>Dean, M., Fojo, T., Bates, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+stem+cells+and+drug+resistance.">Tumour stem cells and drug resistance.</a> Nat Rev Cancer. 5 (4), 275-284 (2005).</li><li>Goodell, M. 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E. <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+analysis+using+a+violet-excited+cell-permeable+DNA+binding+dye.">Side population analysis using a violet-excited cell-permeable DNA binding dye.</a> Stem Cells. 25 (4), 1029-1036 (2007).</li><li>Boesch, 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=High+prevalence+of+side+population+in+human+cancer+cell+lines.">High prevalence of side population in human cancer cell lines.</a> Oncoscience. 3 (3-4), 85-87 (2016).</li><li>Ali, M. Y., Anand, S. V., Tangella, K., Ramkumar, D., Saif, T. 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(83), e51088 (2014).</li><li>Boesch, 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=DyeCycle+Violet+used+for+side+population+detection+is+a+substrate+of+P-glycoprotein.">DyeCycle Violet used for side population detection is a substrate of P-glycoprotein.</a> Cytometry A. 81 (6), 517-522 (2012).</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 Res. 65 (14), 6207-6219 (2005).</li><li>Guha, P., 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=Nicotine+promotes+apoptosis+resistance+of+breast+cancer+cells+and+enrichment+of+side+population+cells+with+cancer+stem+cell-like+properties+via+a+signaling+cascade+involving+galectin-3,+alpha9+nicotinic+acetylcholine+receptor+and+STAT3.">Nicotine promotes apoptosis resistance of breast cancer cells and enrichment of side population cells with cancer stem cell-like properties via a signaling cascade involving galectin-3, alpha9 nicotinic acetylcholine receptor and STAT3.</a> Breast Cancer Res Treat. 145 (1), 5-22 (2014).</li></ol>

癌症临床管理进展也取决于针对CSCs的治疗方式的发展。允许从生物样品中可靠分离CSCs的方法将加速鉴定合适的靶标，因此在这项工作中至关重要。 SP分析是确定表达ABC药物转运蛋白的CSC群体的确定和有价值的技术，并且DCV的实施已经将其适用性扩展到标准流式细胞仪器。在机理上，SP鉴别利用ABC药物转运蛋白的活性染料流出来检测基于低荧光的CSC，从而在图的底部留下相应的非SP的特征定位。重?...

由于对该疾病的遗传和分子理解以及诸如治疗性抗体和小分子抑制剂等靶向药物的出现的引人注目的进展，原发性癌症疗法在过去十年中已经大大改善。相比之下，转移性和复发性疾病通常是不可治愈的，并且这些临床环境中的发病率和死亡率仍然很高。 CSCs在肿瘤内代表不同的亚群，并具有典型的干细胞特性，如克隆性/致瘤性，多药耐药和不对称细胞分裂1,2 。因此，CSCs不仅可以促进转移性进展和肿瘤异质性，而且在治疗期间也会持续使患者复发。因此，治疗性CSC消除是预防疾病复发并允许长期治愈癌症的重要医疗需要3 </suP&gt;。 识别脆弱性和阐明消除CSCs的策略在很大程度上取决于允许从生物样品中纯化以进行后续表达谱和/或功能调查的方法。反过来，这些方法依赖于对这些细胞特异的表面，细胞内或功能性标记。 CSC特异性表面标志物包括但不限于CD44，CD133，CD24和CD90，并且已被用于鉴定多种肿瘤实体（包括乳腺癌和结肠癌）中的CSC群体。另一种标记物醛脱氢酶（ALDH）显示细胞内定位，并且可以在功能上检测到提供酶转化产生光的各自的底物。使用此测试，CSC群体也已被鉴定在不同的肿瘤实体5 。一种补充方法，通常称为SP分析，并在此处描绘<html>方法学细节，利用ABC药物转运蛋白的活性染料流出来鉴定出少量的荧光样干细胞样细胞群6,7,8 。为了实现这一点，给定的样品在亲脂性DNA结合荧光团的存在下孵育，其通过被动扩散进入所有细胞并靶向用于结合的核和线粒体DNA。没有ABC药物转运蛋白表达的非CSCs累积染料，导致明亮的荧光，而CSCs主动挤出染料，减少荧光。药物转运蛋白活性的药理学抑制消除了功能上的SP表型，应用于控制目的。具有SP特征的CSC群体已经公开在卵巢癌9,10 ，前列腺癌11 ，&gt; 12，乳腺癌13 ，肺癌14 ，子宫内膜癌15 ，胶质瘤16,17和骨肉瘤18 。重要的是，SP测定与癌细胞系和原发性肿瘤组织相容，即使主要材料构成额外的挑战，例如对特定肿瘤细胞鉴别策略的需求（某些宿主细胞群体也可表现出SP特征） 19,20 。 两种最成功的SP赋予药物转运蛋白是ABCB1 / P-糖蛋白/ MDR1 / CD243和ABCG2 / Bcrp1 / CD338 8,9,21;然而，其他药物转运蛋白也可以是SP表型的分子决定因素（ 例如 ，ABCB5） 22 。 ABCB1可以有效地阻断维拉帕米，而ABCG2的活性可以用fumitremorgin C（FTC）6,19来明确消除。 SP分析的特殊强度是可以与其他染色（ 例如表面标志物和ALDH）组合，并且允许活细胞恢复，从而与下游功能调查相容。此外，由于CSC人群9,23中ABC药物转运蛋白的保护性高，所以SP检测广泛适用。 最初，使用Hoechst 33342作为触发染料24进行了SP检测。该染料具有优异的分辨率，但需要紫外激光激发;因此其适用性自然限于高端流式细胞仪器。 DyeCycle Violet（DCV） 25的出现开启了新的avenu用于SP分析，并将该方法扩展到缺乏紫外激光源（紫激光源足以解决DCV-SP细胞）的标准流式细胞仪器的适用性。重要的是，Hoechst 33342和DCV具有共同的泵特异性，表明染料应识别相同的细胞群体。 在这里，我们提供了基于DCV的SP分析的详细实验方案，以便在独立实验室中快速简便地再现。因此，我们认为我们的文章是CSC研究人员的资源，应该有助于这种有用的细胞生物学方法的优化和标准化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vybrant DyeCycle Violet</td>
			<td>Thermo Fisher Scientific</td>
			<td>V35003</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Verapamil hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td>Dissolve in ethanol</td>
		</tr>
		<tr>
			<td>Fumitremorgin C</td>
			<td>Sigma-Aldrich</td>
			<td>F9054</td>
			<td>Dissolve in DMSO</td>
		</tr>
		<tr>
			<td>7-AAD (or propidium iodide)</td>
			<td>BioLegend</td>
			<td>420403</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Standard cell culture reagents (e.g., medium, FBS, L-glutamine, antibiotics, PBS, trypsin-EDTA, etc.)</td>
			<td>Different suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample/cells of interest (e.g., human or murine cancer cell line, human or murine tumor tissue)</td>
			<td>Different suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometric instrument (analyzer or cell sorter)</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Violet laser source required</td>
		</tr>
		<tr>
			<td>FACS tubes (round-bottom)</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Tubes with cap recommended</td>
		</tr>
		<tr>
			<td>70 &#181;m cut-off strainers</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Optional but recommended</td>
		</tr>
		<tr>
			<td>Digestion enzyme mix (e.g., collagenase/dispase/Dnase)</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Only relevant for tissue dissociation</td>
		</tr>
		<tr>
			<td>Flurochrome-conjugated antibodies against surface markers of interest</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>ALDEFLUOR Kit</td>
			<td>STEMCELL Technologies</td>
			<td>01700</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

harnessing the dna dye-triggered side population phenotype to detect and purify cancer stem cells from biological samples

癌症是干细胞驱动的疾病，并且这些细胞的根除已成为主要的治疗目标。解密癌症干细胞（CSCs）的脆弱性和鉴定合适的分子靶标依赖于允许其在异种样品如细胞系和离体肿瘤组织中的特异性鉴别的方法。流式细胞术/ FACS是在单细胞水平上多参数解剖生物样品的强大技术，是迄今为止选择的方法，用于回收活细胞进行下游分析。表面标志物如CD44和CD133以及醛脱氢酶酶活性的检测常常用于通过FACS定义和分离来自肿瘤样品的CSCs。以方法学细节描述的补充方法利用ABC药物转运蛋白的功能性染料挤出，其识别通常称为侧群体（SP）的不同的荧光 - 细胞群体。 SP;癌细胞表现出典型的干细胞特征，并且可以使用抑制染料挤出药物转运蛋白（最常见的是ABCB1 / P-糖蛋白/ MDR1 / CD243和ABCG2 / Bcrp1 / CD338）的试剂来消除和功能确认。此外，SP测定与其他流式细胞术评估（如表面抗原染色，醛脱氢酶检测和死细胞鉴别（ 例如 ，使用7-AAD或碘化丙啶（PI））相容。因此，我们基于功能而不是表型参数，机械地描述了CSC识别，分离和子表征的有价值且广泛适用的方法。尽管最初以Hoechst 33342作为引发染料，但我们在此着重于在配备有紫色激光源的任何流式细胞仪上可分辨的最新的基于紫罗兰染料的SP表型。

提供了A2780细胞的代表性SP分析，A2780细胞是先前显示存在SP占细胞的1％以下的人卵巢癌细胞9 。细胞在37℃下在补充有10％（ v / v ）FBS，2mM L-谷氨酰胺和1×青霉素/链霉素的RPMI 1640中培养，并以0.05％胰蛋白酶-EDTA处理，以85％融合收获。将细胞在PBS中洗涤并通过70μm截止的过滤器过滤。使用计数室和台盼蓝染色测定细胞计数，并在37℃下在黑暗中用10μM...

Watch this Scientific Journal Video about Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples at JoVE.com

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

允许从生物样品中鉴定和分离干细胞群体的方法对于癌症及其以外的干细胞靶向治疗的进展至关重要。在这里，我们提供使用染料触发侧群体表型的癌症干细胞分离的详细方案。

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 ...

Research

JoVE Journal

Cancer Research

Cell-Free DNA Integrity Analysis in Urine Samples

无细胞DNA完整性分析尿样中

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

科研

癌症研究

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. ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

从妇科和乳腺癌肿瘤获得癌症干细胞球

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Analysis of Side Population in Solid Tumor Cell Lines

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 ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

癌症患者血浆样本中无细胞DNA检测

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

合并的shRNA文库筛选，以确定调节耐药性表型的因素

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples

基于简单快速的滚动圆扩增检测粗生物样品中的拓扑异构酶1活性

Isothermal amplification-based techniques such as the rolling circle amplification have been successfully employed for the detection of nucleic acids, ...