Name: harnessing the dna dye-triggered side population phenotype to detect and purify cancer stem cells from biological samples
Uploaded: 2017-05-10T15:00:00.000+00:00
Duration: 9 min 57 s
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合适培养基（ 例如 ，补充有10％（ v / v ）FBS，2mM L-谷氨酰胺和1×青霉素/链霉素的RPMI 1640）中培养相应的人或鼠癌细胞系和收获使用0.05％胰蛋白酶-EDTA或一些其他去除剂消化的亚融合细胞（ 即 70-90％）的细胞手术程序使用计数室和台盼蓝染色确定细胞计数并检查活力。活细胞的比例应超过85％。 注意:携带SP隔室的人类癌细胞系的实例包括MCF7 / HTB-22和SKBR3 / HTB-30（乳腺癌），A2780和SKOV-3 / HTB-77（卵巢癌）和A549（肺癌） 26 。 </li></ol></li><li> 离体肿瘤组织 <ol><li>采取新鲜的手术肿瘤标本，或者从可移植或遗传工程小鼠肿瘤模型收获肿瘤组织。取200-1,000mg组织，并通过机械解聚（ 例如 ，使用剪刀或手术刀）和酶消化（ 例如 ，使用胶原酶/分解酶/ DNA酶混合物）产生单细胞悬液。 </li><li>通过70μm截止滤网过滤样品。确定细胞计数并使用计数室和台盼检查生存力ue染色。 注意:消化鸡尾酒通常含有200μg/ mL胶原酶P，0.8U / mL分散酶和25μg/ mL DNA酶I，孵育时间通常为20-50分钟。理想地，根据针对被研究组织27,28,29优化的方案进行组织解聚。 </li></ol></li></ol> 2.测试样品<ol><li>在新鲜培养基（含营养素载体）中，将细胞浓度调整至1×10 6个细胞/ mL，因为染料挤出是一种活跃的耗能过程。最后，将1 mL样品转移到普通流式细胞仪/ FACS圆底管（标记为"测试"）。 注意:为获得最佳结果，可能需要滴定细胞浓度（例如5×10 5 -1×10 6 -2.5×10 6个细胞/ mL），但是细胞浓度通常较低产生更高的分辨率19 。只有对于需要回收最大数量的SP细胞的分选应用，建议将细胞浓度提高到1×10 7个细胞/ mL。 </li><li>向样品中加入10μM的DCV，通过轻柔涡旋或重悬至3-5倍混匀。继续进行抑制控制。 注意:最佳结果可能取决于触发染料的滴定（例如2.5; 5; 10; 20μM），但较高的染料浓度通常会产生较高的分辨率19 。 </li></ol> 3.抑制控制<ol><li>将250μL含染料的细胞悬浮液转移到新的流式细胞仪/ FACS圆底管（标记为"对照"）。加入50μM维拉帕米或20μMFTC，并通过移液混匀。 注意:维拉帕米和FTC是SP分析中最完善的抑制剂，但具有不同的外排泵特异性y:维拉帕米抑制几种ABC药物转运蛋白，包括ABCB1和ABCB5，而FTC对ABCG2具有特异性6,19。如果单个样品的SP状态未知，则建议在不同的管中使用维拉帕米和FTC。 </li></ol> 4.染色<ol><li>盖上"测试"和"对照"管，并在37℃下在黑暗中孵育90分钟，每15分钟温和搅拌。 SP细胞中的大多数染料挤出将在头45分钟内发生，但非SP细胞中的染料积累（有助于分辨）仅在75-90分钟后才达到最大值19 。 </li><li>染色完成后，将细胞洗涤在3-4mL冰冷的PBS中，并将沉淀物重悬于100μL的体积（PBS）中。将细胞保持在黑暗中并在冰上冷却，并进行附加标记染色。或者，直接进行流式细胞仪分析裂解。 </li></ol>其他标记的组合<ol><li>向DCV染色的细胞中加入所需的荧光染料缀合的抗体组，充分混合，并在4℃下在黑暗中孵育细胞20-30分钟。在3 - 4 mL冰冷的PBS中洗涤细胞，并进行死细胞鉴别。 注意:与DCV容易兼容且几乎不需要补偿的抗体格式包括PerCP或PerCP-Cy5.5，PE-Cy7，APC，Alexa Fluor 647，APC-Cy7，APC-H7，BV711，Alexa Fluor 750和BV786 。与DCV兼容但可能需要补偿的格式包括FITC，Alexa Fluor 488，PE和BV650。与DCV实际上不兼容的格式包括BV421，V450和V500。 </li><li>以特定制造商推荐的浓度将7-AAD（或PI）添加到DCV染色的细胞中，并在黑暗中孵育5-10分钟。通过70μm截止过滤器过滤样品，并进行流式细胞术分析。</li></ol>流式细胞分析注意:建议在染色过程中打开相应的流式细胞仪器，以便在样品准备就绪后立即进行一切设置。这还包括标准维护程序，如系统性能跟踪和油箱补充。 <ol><li> 获取流式细胞仪上的细胞并将其置于双变量FSC / SSC点图上（图1B）。通过比较FSC的不同信号（ 即高度，宽度，面积）来排除双重和聚合（图1C）。 <ol><li>如果已经包括死细胞标记物，则将活细胞部分（ 例如 ，7-AAD阴性）门禁（ 图1D ）。在"蓝色"和"红色"DCV发射的双变量点图上可视化单个细胞。为此，在y轴上显示"蓝色"荧光通道（ 例如 ，450/50），"红色"荧光通道（ 例如 525/50）。 </li><li>将两个通道切换到线性模式，并相应地调整检测器电压，使SP位于左下象限的图的一侧 。相反，非SP位于反映细胞周期分布（G1和G2）的图的右上象限。关闭SP并停止采集（ 图1E ）。 注意:DCV定义的SP细胞可以使用配备有紫激光激发源（ 即 405nm激光线）的流式细胞仪检测。 SP分析的一个特点是发射的荧光以线性模式测量，并在两个单独的通道中显示在双变量点图上。 DCV的"蓝色"发射在约450nm处测量（ 例如 ，使用450/40标准滤光片），并且在约510-585nm测量DCV的"红色"发射（ 例如 ，使用510/50，a五25/50或585/15过滤器） 19 。由于测定的特殊化学性质，"红色"荧光通道19中 ，SP和非SP细胞之间的分离通常较高。 </li></ol></li><li>加载禁止控制并获取数据。 SP细胞已经消失或基本上减少（ 图1F ）。如有必要，根据这些数据细化SP门。 </li><li>为了研究SP细胞的表面标志物表达，将DCV的"红色"荧光留在x轴上，并以对数模式（ 例如 ，APC）在y轴上显示相应的荧光通道。相应地调整检测器电压，并将所研究的标记物的SP细胞的分数筛选为阳性（ 图1G ）。由SP细胞共同表达特定标记物在图30的左上象限中产生相应的信号。 </li><li>记录数据和/或排序SP细胞。 </li></ol>

<ol>
	<li>Pattabiraman, D. R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+the+cancer+stem+cells+-+what+challenges+do+they+pose?.">Tackling the cancer stem cells - what challenges do they pose?.</a> Nat Rev Drug Discov. 13 (7), 497-512 (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=Heterogeneity+of+Cancer+Stem+Cells:+Rationale+for+Targeting+the+Stem+Cell+Niche.">Heterogeneity of Cancer Stem Cells: Rationale for Targeting the Stem Cell Niche.</a> Biochim Biophys Acta. 1866 (2), 276-289 (2016).</li><li>Li, Y., 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=Suppression+of+cancer+relapse+and+metastasis+by+inhibiting+cancer+stemness.">Suppression of cancer relapse and metastasis by inhibiting cancer stemness.</a> Proc Natl Acad Sci U S A. 112 (6), 1839-1844 (2015).</li><li>Greve, B., Kelsch, R., Spaniol, K., Eich, H. T., G&#246;tte, M. <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+in+cancer+stem+cell+analysis+and+separation.">Flow cytometry in cancer stem cell analysis and separation.</a> Cytometry A. 81 (4), 284-293 (2012).</li><li>Marcato, P., Dean, C. A., Giacomantonio, C. A., Lee, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase:+its+role+as+a+cancer+stem+cell+marker+comes+down+to+the+specific+isoform.">Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform.</a> Cell Cycle. 10 (9), 1378-1384 (2011).</li><li>Golebiewska, A., Brons, N. H., Bjerkvig, R., Niclou, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+side+population+assay+in+stem+cell+and+cancer+stem+cell+research.">Critical appraisal of the side population assay in stem cell and cancer stem cell research.</a> Cell Stem Cell. 8 (2), 136-147 (2011).</li><li>Hirschmann-Jax, C., 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+distinct+&amp;#34;side+population&amp;#34;+of+cells+with+high+drug+efflux+capacity+in+human+tumor+cells.">A distinct &amp;#34;side population&amp;#34; of cells with high drug efflux capacity in human tumor cells.</a> Proc Natl Acad Sci U S A. 101 (39), 14228-14233 (2004).</li><li>Zhou, 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=The+ABC+transporter+Bcrp1/ABCG2+is+expressed+in+a+wide+variety+of+stem+cells+and+is+a+molecular+determinant+of+the+side-population+phenotype.">The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.</a> Nat Med. 7 (9), 1028-1034 (2001).</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=The+side+population+of+ovarian+cancer+cells+defines+a+heterogeneous+compartment+exhibiting+stem+cell+characteristics.">The side population of ovarian cancer cells defines a heterogeneous compartment exhibiting stem cell characteristics.</a> Oncotarget. 5 (16), 7027-7039 (2014).</li><li>Szotek, P. 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=Ovarian+cancer+side+population+defines+cells+with+stem+cell-like+characteristics+and+Mullerian+Inhibiting+Substance+responsiveness.">Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness.</a> Proc Natl Acad Sci U S A. 103 (30), 11154-11159 (2006).</li><li>Brown, M. D., 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=Characterization+of+benign+and+malignant+prostate+epithelial+Hoechst+33342+side+populations.">Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations.</a> Prostate. 67 (13), 1384-1396 (2007).</li><li>Mathew, 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=ABCG2-mediated+DyeCycle+Violet+efflux+defined+side+population+in+benign+and+malignant+prostate.">ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate.</a> Cell Cycle. 8 (7), 1053-1061 (2009).</li><li>Engelmann, K., Shen, H., Finn, O. 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. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J Exp Med. 183 (4), 1797-1806 (1996).</li><li>Telford, W. G., Bradford, J., Godfrey, W., Robey, R. W., Bates, S. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Primary+Human+Colon+Tumor+Cells+from+Surgical+Tissues+and+Culturing+Them+Directly+on+Soft+Elastic+Substrates+for+Traction+Cytometry.">Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.</a> J Vis Exp. (100), e52532 (2015).</li><li>Azari, 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=Isolation+and+expansion+of+human+glioblastoma+multiforme+tumor+cells+using+the+neurosphere+assay.">Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay.</a> J Vis Exp. (56), e3633 (2011).</li><li>Hasselbach, 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=Optimization+of+high+grade+glioma+cell+culture+from+surgical+specimens+for+use+in+clinically+relevant+animal+models+and+3D+immunochemistry.">Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.</a> J Vis Exp. (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>

作者宣称没有利益冲突。还没有非作者参与手稿的准备工作。

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><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 - PI)</td><td>BioLegend</td><td>420403</td><td>Ready to use</td></tr><tr><td>Standard cell culture reagents (&lt;em&gt;e.g.&lt;/em&gt;, medium, FBS, L-glutamine, antibiotics, PBS, trypsin-EDTA, &lt;em&gt;etc.&lt;/em&gt;)</td><td>Different suppliers</td><td></td><td></td></tr><tr><td>Sample/cells of interest (&lt;em&gt;e.g.&lt;/em&gt;, 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 (FACS; 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 &amp;micro;m cut-off strainers</td><td>Different suppliers</td><td></td><td>Optional but recommended</td></tr><tr><td>Digestion enzyme mix (&lt;em&gt;e.g.&lt;/em&gt;, 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 ...

harnessing-dna-dye-triggered-side-population-phenotype-to-detect

Research

JoVE Journal

Cancer Research

7.6K 次观看.  Kantonsspital St. Gallen. 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。 

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

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

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 ...

科研

癌症研究

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

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 ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

结合条件敲击和适应球形成测定研究患者衍生胃癌干细胞的干细胞相关基因

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

医学

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

用于轻松检测侧群细胞的替代激光激发方法

Effective Detection of Hoechst Side Population Cells by Flow Cytometry

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized for the isolation of stem cells based on the dye efflux properties of ATP-binding cassette (ABC) transporters. The method was initially employed for the identification and isolation of hematopoietic stem cells (HSCs), but it has now evolved to primarily focus on the identification and isolation of cancer stem cells (CSCs). The traditional detection method of FCM uses a 355 nm laser to excite the dye to detect SP cells. Through this study, we have successfully identified alternative approaches for dye excitation that can effectively replace the detection of SP cells using a 355 nm laser. This is achieved through the utilization of high-power 375 nm or 405 nm lasers. This allows us to exercise enhanced selectivity in the detection of SP cells rather than being solely limited to the 355 nm laser flow cytometry.

作者聚焦:用于 Hoechst 染色分析侧群细胞的创新激光技术

Here, we show that high-power 375 nm and 405 nm lasers can effectively excite Hoechst 33342 and serve as a viable alternative to the 355 nm laser for side population (SP) cell detection, thereby expanding the range of available lasers in flow cytometry applications.

通过流式细胞术有效检测 Hoechst 侧细胞群

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

在侧群的分离的Myc诱导的T细胞急性淋巴细胞白血病斑马鱼

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

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

摘要

探索更多视频

此视频中的章节

从妇科和乳腺癌肿瘤获得癌症干细胞球

固体肿瘤细胞系侧面人口分析

结合条件敲击和适应球形成测定研究患者衍生胃癌干细胞的干细胞相关基因

来自患者的细胞培养和分离的CD133

癌症干细胞从人类前列腺癌样本隔离

通过流式细胞术有效检测 Hoechst 侧细胞群

通过流式细胞术有效检测 Hoechst 侧细胞群

通过流式细胞术有效检测 Hoechst 侧细胞群

研究胰腺癌干细胞特性开发新的治疗策略

在侧群的分离的Myc诱导的T细胞急性淋巴细胞白血病斑马鱼