Name: isolation and characterization of a head and neck squamous cell carcinoma subpopulation having stem cell characteristics
Uploaded: 2016-05-11T14:00:00.000+00:00
Duration: 11 min 28 s
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-CSC）是CM，结合以下内容:Dulbecco改进的Eagle培养基（DMEM）:F12（1:3，体积比），胎牛血清为5％（FCS），0.04毫克/升氢化可的松，100U / ml青霉素，0.1克/链霉素的L和20微克/升表皮生长因子（EGFR）的。 </li></ol></li><li>在层流罩，trypsinize细胞。取出介质，其中s洗terile磷酸盐缓冲盐水（PBS），并加入1 ml胰蛋白酶 - EDTA（0.5克/升）为75平方厘米的烧瓶中。孵育在37℃下3分钟。停止通过添加用于亲代细胞系培养基中的反应。计数使用细胞计数器细胞的数目。 <ol><li>为了制备用于亲代细胞系（CM-P）的是CM，有10％FCS，0.4毫克/氢化可的松L，100U / ml青霉素和0.1 g / L的链霉素）补充的DMEM。 </li></ol></li><li>稀释在步骤1.1.2获得，以获得在CM-P 10 7个细胞/ ml的细胞悬浮液。分裂中制备的2管中的细胞悬浮液:将100微升（10 6个细胞）在管"的Hoechst和维拉帕米"和4毫升（4×10 7个细胞）在管"Hoechst公司"。 </li><li>在样本"赫斯特和维拉帕米"，加入10微升5毫米盐酸维拉帕米溶液（终浓度:0.5毫米），轻轻混匀。添加1克/升的Hoechst溶液5微升（最终浓度:0.1克/升）。在样品＆＃34;赫斯特"，加入1克5微升/升赫斯特每106 个细胞溶液（200μl总）从今以后，继续使用铝箔直接光线照射的保护样本。 </li><li>孵育在水浴所有试管在37℃下为1小时30分钟。轻轻混匀，每次15分钟，以防止细胞沉淀下来。 </li><li>离心管，在250×g离心在4℃下5分钟。除去上清，悬浮颗粒每用2毫升1X PBS的。 </li><li>离心管，在250×g离心在4℃下5分钟。除去上清液。在PBS中稀释悬浮"的Hoechst和维拉帕米"粒料在500微升5毫克/升的碘化丙啶（PI）的缓冲液和4毫升5毫克/升的PI在PBS缓冲液中稀释"Hoechst的"颗粒。 </li><li>通过一个70微米的细胞滤网转移样品溶液到在流式细胞仪除去团粒，收集的单个细胞在试管样品摄取。保持冰样本，并利用铝箔直接光照保护。 </li></ol></li><li>一世侧群通过细胞分选剔除赫斯特染料的染料溶液。 <ol><li>上术分拣机的流用下列参数进行Hoechst的阴性细胞分离:紫外激光（355纳米），2个检测器，蓝色的赫斯特（450/50 BP）和红色的Hoechst（610/20 BP）滤波器的紫外激光路径上，并且2收藏家。 <ol><li>首先，分析在作为用于染色和流式细胞仪建立的阳性对照样品"Hoechst公司"。 </li><li>使用流式细胞仪软件，对"环球表"窗口中，点击"点图"（第五顶部右图），并创建全球工作表上的图表。在纵坐标，用鼠标右键，选择FSC-A（前向散射）和横坐标，SSC-A（侧散射）（ 图1A）。以同样的方式，创建一个SSC-W与SCC-H点图。在这第二点图，创造出P1区域，单击"多边形门"（第十四右上图）（ 图1B）33。 注:P1地区将包括单细胞和辨别双峰。 </li><li>可选:通过创建FSC-A与PI门上P1排除PI阳性细胞。在这个散点图中，选择PI人口负（P2）排除PI阳性死细胞。 </li><li>使用流式细胞仪软件，对"环球表"窗口中，点击"点图"，创造了全球工作表上的图表。在纵坐标，用鼠标右键，选择蓝色的Hoechst-A和横坐标，红赫斯特-A。随着人口右击，选择"显示人口"和P1。在这点图，创建一个区域（P2）选择出现作为细胞的主要群体（ 图1C）左侧的侧臂负赫司特染料侧群（SP）细胞。 </li></ol></li><li>分析样品"赫斯特"，并收集10000个事件。以确保该门的P2，其代表的SP的人口，处于有利位置，分析样品"的Hoechst和维拉帕米"（10,000个事件）观察SP人口（ 图1D）的消失。 </li><li>收集在含有1ml的CM-CSC在1.1.1.1制备15毫升管中的SP的Hoechst染料阴性细胞。 </li><li>在细胞分选结束后，离心分离机，在250×g离心5分钟将细胞悬浮液，取出上清液并用1ml的CM-CSC的悬浮颗粒。计数使用细胞计数器分选的细胞的数量和转移的细胞的适当数量的培养瓶（ 见表1）。添加CM-CSC和在37℃和5％的CO 2气氛中孵育。 </li><li>保持为最大的2次传代在相同条件下培养的细胞，直到有一个至少5×10 7个细胞（参见步骤3，"细胞培养法"）的。 </li></ol></li></ol> 2. CD44 高 / 高 ALDH在侧群分类子群体的选择<ol><li>染色50000000 SP细胞的ALDH检测试剂盒和CD44抗体。 <ol><li>预备七15毫升标记无菌试管如下:"未染色的"（管一），"CD44-APC"（管b）中，"的IgG1-APC"（管c）中，"ALDH"（管d）中，"ALDH和DEAB "（管五），"ALDH和CD44-APC"（管F），"ALDH，DEAB和CD44-APC"（管克）。保留所有试管和试剂在4℃染色过程中。 </li><li>准备缓冲液A 4.5毫升缓冲1（包含在套件中）和45微升CD44-APC（别藻蓝蛋白）抗体（稀释1:100）。用100μl缓冲液1和的IgG1-APC抗体的1微升（:100稀释1）制备的缓冲液B。制备缓冲液C用4ml缓冲液1和20μl试剂盒的试剂。 </li><li>以10 7个细胞/ ml制备预先分选的细胞（步骤1.2.5）的细胞悬浮液。添加细胞悬浮液的100微升（10 6个细胞）至铝管a，b和c和4毫升（4×10 7个细胞）至管F。暂时不要把细胞管D，E和g。 </li><li>离心含有在250 xg离心细胞5分钟的管中。除去上清液，并在管的a，b和c在100μl缓冲液1的悬浮粒料添加5微升二乙基氨基的苯​​甲酮（DEAB），的ALDH抑制剂管e和g。 <ol><li>悬浮细胞在管F用4ml试剂C，并立即转移100μl的成管d，e和克孵育在水浴所有试管在37℃下30分钟，并使用铝箔从直接曝光保护。 15分钟后，轻轻地混合细胞悬液涡旋，以防止细胞沉淀。 </li></ol></li><li>从这一刻起，保持在冰上管，并使用铝箔直接光线照射的保护。离心机在4℃以250×g离心5分钟，所有管去除上清。 </li><li>在4毫升和管b和g用100微升缓冲液A重悬管的C 100微升缓冲B.要在另一管，悬浮管F加入100微升缓冲液1孵育10分钟，在4℃;C。 </li><li>离心机在4℃以250×g离心5分钟，所有管去除上清。在250 xg离心再次用1ml缓冲液1（4毫升管f）和离心机冲洗一次，在4℃5分钟。去除上清。重悬在4毫升，并在1毫升缓冲液1的所有其它管管F沉淀。 </li><li>通过70微米的细胞过滤后将样品解决方案，在流式细胞仪除去团粒，收集的单细胞在试管适当样品吸收。 </li></ol></li><li>在CD44 高 / 高 ALDH细胞荧光人口的分离激活细胞分选。 <ol><li>执行CD44 高 / ALDH 高细胞上流式细胞分选仪的流动具有以下参数排序:用FITC过滤器（530/30）1蓝色激光路径上蓝激光（488纳米）和红激光（633纳米），1检测器探测器的APC滤波器（二十○分之六百六）和2收藏家红色激光路径上。 </li><li>使用流式细胞仪软件，点击"点普罗T"（第五右上图），如第1.2.1.2所述创建FSC-A与SSC-A点图，以检查细胞形态，并选择一个带SSC-W与SSC-H的单细胞组成的群体点图。 </li><li>使用流式细胞仪软件，对"环球表"窗口中，点击"点图"，创造了全球工作表上的图表。在纵坐标，用右键点击，以选择双染人群选择APC-A和横坐标，FITC-A。用管D和E选择ALDH 高细胞（ 图2A）创建一个门。注:阳性细胞与DEAB（ 图2B）处理管É消失。 <ol><li>在同一图中，用管b和c以选择CD44 高细胞（ 图2C和2D）创建第二栅极。阳性细胞消失含有的IgG1-APC管。分析管f和g，并创建第三个门，其中包括CD44 高 / ALDH &lt;SUP&gt;高细胞（ 图2E和2F）。 注意:如果阳性细胞存在于含的IgG1-APC（ 图2D）的管中，细胞和APC染色抗体之间的相互作用是不特定。 </li></ol></li><li>收集CD44 高 / 高 ALDH细胞进入有1ml CM-CSC的15毫升管。还低收集CD44 低 / ALDH成含有1ml的CM 19的15毫升管。注意:如步骤1.1.1.1说明准备CM-CSC。 </li><li>离心细胞悬浮液在250×g离心5分钟，去除上清，并用1ml的CM-CSC的悬浮颗粒。计数排序细胞的数目。 </li></ol></li></ol> 3.细胞培养法<ol><li>如上所述细胞的双分选后，板已排序的细胞进入一个合适的培养瓶（ 表1）与CM-CSC在37℃，5％的CO 2。经过18-24小时，检查，如果细胞已粘附和改变培养基。改变培养基每3天，直到扩张菌落大于50％汇合更大。 </li><li>使用胰蛋白酶为0.5g / L的适当体积- EDTA（ 表1），从培养瓶中trypsinize细胞。孵育细胞3至5分钟，在37℃。添加CM-CSC（ 表1）的适当体积以停止胰蛋白酶的作用。 </li><li>计数得到的细胞和板4×10 5个细胞在与CM-CSC 175平方厘米培养瓶中的数量。在37℃和5％的CO 2。在这一集中，24小时的倍增时间细胞就会7天70％汇合。 </li><li>它们经历了3代之前使用的CSCs 在体外 或体内实验。 </li></ol> 4.确认肿瘤潜力和CSC特性<ol><li>肿瘤球形成以确认CD44 高 / ALDH的肿瘤潜力高细胞。 <ol><li>在3.2中描述Trypsinize细胞。 1×10 6个细胞添加到15毫升管中并离心，在250×g离心5分钟。除去上清液并重新悬浮在DMEM中沉淀:F12（3:1）培养基无FCS，20纳克/毫升的rhEGF的，肝素和1x B27的4毫克/升。孵育在6孔低锚固板培养瓶中的细胞，在37℃和5％ 的 CO 2。 注意:使用的DMEM:F12（3:1）含有FCS 5％，20纳克/毫升的rhEGF的，4毫克/肝素L的和1x B27，如果细胞不不含FCS生长介质。 </li><li>观察播种（ 图3A）之后的肿瘤球形成具有从4至10天的光学显微镜。 注:肿瘤球体直径应大于35微米。 CD44 高 / ALDH 高细胞将显示数量和大小方面具有更快和更增强的肿瘤球形成相比CD44 低 / ALDH 低 。 </li></ol></li><li> 体内致瘤性的评估后，在NOD-SCID小鼠CD44 高 / 高 ALDH细胞皮下注射。 <ol><li>排序后，在三个不同浓度（10 4个细胞/ ml，10 5个细胞/ ml，和10 6个细胞/ ml）再悬浮细胞于PBS中。注入皮下100微升的10 4个细胞/ ml（10 3个细胞）的6只小鼠的右胁区域。执行相同的10 5个细胞/ ml（10 4个细胞）和10 6细胞/ ml（10 5个细胞）。 注:下稀释比10 3个细胞可以进行测试。 </li><li>注入在左翼区域的CD44 低 / ALDH 低细胞的相同浓度。长达10周监测注射的小鼠看到肿瘤进展19。 </li></ol></li></ol>

<ol>
	<li>Baumann, M., Krause, M., Hill, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+role+of+cancer+stem+cells+in+radioresistance.">Exploring the role of cancer stem cells in radioresistance.</a> Nat Rev Cancer. 8 (7), 545-554 (2008).</li><li>Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proc Natl Acad Sci USA. 100 (7), 3983-3988 (2003).</li><li>Singh, S. 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=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432 (7015), 396-401 (2004).</li><li>Collins, A. T., Berry, P. A., Hyde, C., Stower, M. J., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+prostate+cancer+stem+cells.">Prospective identification of tumorigenic prostate cancer stem cells.</a> Cancer Res. 65 (23), 10946-10951 (2005).</li><li>Eramo, 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=Identification+and+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15 (3), 504-514 (2008).</li><li>Dalerba, 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=Phenotypic+characterization+of+human+colorectal+cancer+stem+cells.">Phenotypic characterization of human colorectal cancer stem cells.</a> Proc Natl Acad Sci USA. 104 (24), 10158-10163 (2007).</li><li>Hermann, P. 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=Distinct+populations+of+cancer+stem+cells+determine+tumor+growth+and+metastatic+activity+in+human+pancreatic+cancer.">Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.</a> Cell Stem Cell. 1 (3), 313-323 (2007).</li><li>Yang, Z. F., 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=Significance+of+CD90+cancer+stem+cells+in+human+liver+cancer.">Significance of CD90 cancer stem cells in human liver cancer.</a> Cancer Cell. 13 (2), 153-166 (2008).</li><li>Fang, 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=A+tumorigenic+subpopulation+with+stem+cell+properties+in+melanomas.">A tumorigenic subpopulation with stem cell properties in melanomas.</a> Cancer Res. 65 (20), 9328-9337 (2005).</li><li>Prince, M. E., 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=Identification+of+a+subpopulation+of+cells+with+cancer+stem+cell+properties+in+head+and+neck+squamous+cell+carcinoma.">Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.</a> Proc Natl Acad Sci USA. 104 (3), 973-978 (2007).</li><li>Clarke, M. F., 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+--+Perspectives+on+current+status+and+future+directions:+AACR+Workshop+on+cancer+stem+cells.">Cancer stem cells -- Perspectives on current status and future directions: AACR Workshop on cancer stem cells.</a> Cancer Res. 66 (19), 9339-9344 (2006).</li><li>Soltanian, S., Matin, M. M. <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+and+cancer+therapy.">Cancer stem cells and cancer therapy.</a> Tumor Biol. 32 (3), 425-440 (2011).</li><li>Molyneux, 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=BRCA1+basal-like+breast+cancers+originate+from+luminal+epithelial+progenitors+and+not+from+basal+stem+cells.">BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells.</a> Cell Stem Cell. 7 (3), 403-417 (2010).</li><li>Vermeulen, 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=Single-cell+cloning+of+colon+cancer+stem+cells+reveals+a+multi-lineage+differentiation+capacity.">Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity.</a> Proc Natl Acad Sci USA. 105 (36), 13427-13432 (2008).</li><li>Ratajczak, M. Z. <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+--+Normal+stem+cells+'Jedi'+that+went+over+to+the+'dark+side.'.">Cancer stem cells -- Normal stem cells 'Jedi' that went over to the 'dark side.'.</a> Folia Histochem Cytobiol. 43 (4), 175-181 (2005).</li><li>Bao, 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=Glioma+stem+cells+promote+radioresistance+by+preferential+activation+of+the+DNA+damage+response.">Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.</a> Nature. 444 (7120), 756-760 (2006).</li><li>Liu, 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=Analysis+of+gene+expression+and+chemoresistance+of+CD133++cancer+stem+cells+in+glioblastoma.">Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma.</a> Mol Cancer. 5, 67 (2006).</li><li>Moncharmont, 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=Targeting+a+cornerstone+of+radiation+resistance:+Cancer+stem+cell.">Targeting a cornerstone of radiation resistance: Cancer stem cell.</a> Cancer Lett. 322 (2), 139-147 (2012).</li><li>Bertrand, 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=Targeting+Head+and+Neck+Cancer+Stem+Cells+to+Overcome+Resistance+to+Photon+and+Carbon+Ion+Radiation.">Targeting Head and Neck Cancer Stem Cells to Overcome Resistance to Photon and Carbon Ion Radiation.</a> Stem Cell Rev. 10 (1), 114-126 (2013).</li><li>Das, B., Tsuchida, R., Malkin, D., Koren, G., Baruchel, S., Yeger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+tumor+stemness+by+increasing+the+invasive+and+tumorigenic+side+population+fraction.">Hypoxia enhances tumor stemness by increasing the invasive and tumorigenic side population fraction.</a> Stem Cells. 26 (7), 1818-1830 (2008).</li><li>Diehn, 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=Association+of+reactive+oxygen+species+levels+and+radioresistance+in+cancer+stem+cells.">Association of reactive oxygen species levels and radioresistance in cancer stem cells.</a> Nature. 458 (7239), 780-783 (2009).</li><li>Chen, Z. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+stem+cell+concept+in+progression+of+head+and+neck+cancer.">The cancer stem cell concept in progression of head and neck cancer.</a> J Oncol. 2009, 894064 (2009).</li><li>Zhang, P., Zhang, Y., Mao, L., Zhang, Z., Chen, W. <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+oral+squamous+cell+carcinoma+possesses+tumor+stem+cell+phenotypes.">Side population in oral squamous cell carcinoma possesses tumor stem cell phenotypes.</a> Cancer Lett. 277 (2), 227-234 (2009).</li><li>Zhang, Q., 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+subpopulation+of+CD133(+)+cancer+stem-like+cells+characterized+in+human+oral+squamous+cell+carcinoma+confer+resistance+to+chemotherapy.">A subpopulation of CD133(+) cancer stem-like cells characterized in human oral squamous cell carcinoma confer resistance to chemotherapy.</a> Cancer Lett. 289 (2), 151-160 (2010).</li><li>Sun, S., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Head+neck+squamous+cell+carcinoma+c-Met&#8314;+cells+display+cancer+stem+cell+properties+and+are+responsible+for+cisplatin-resistance+and+metastasis.">Head neck squamous cell carcinoma c-Met&#8314; cells display cancer stem cell properties and are responsible for cisplatin-resistance and metastasis.</a> Int J Cancer. 129 (10), 2337-2348 (2011).</li><li>Chen, Y. 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=Aldehyde+dehydrogenase+1+is+a+putative+marker+for+cancer+stem+cells+in+head+and+neck+squamous+cancer.">Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer.</a> Biochem Biophys Res Commun. 385 (3), 307-313 (2009).</li><li>Lim, Y. 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=Cancer+stem+cell+traits+in+squamospheres+derived+from+primary+head+and+neck+squamous+cell+carcinomas.">Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas.</a> Oral Oncol. 47 (2), 83-91 (2011).</li><li>Yu, Q., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface-localized+matrix+metalloproteinase-9+proteolytically+activates+TGF-beta+and+promotes+tumor+invasion+and+angiogenesis.">Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis.</a> Genes Dev. 14 (2), 163-176 (2000).</li><li>Krishnamurthy, 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=Endothelial+cell-initiated+signaling+promotes+the+survival+and+self-renewal+of+cancer+stem+cells.">Endothelial cell-initiated signaling promotes the survival and self-renewal of cancer stem cells.</a> Cancer Res. 70 (23), 9969-9978 (2010).</li><li>Chikamatsu, K., Takahashi, G., Sakakura, K., Ferrone, S., Masuyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoregulatory+properties+of+CD44++cancer+stem-like+cells+in+squamous+cell+carcinoma+of+the+head+and+neck.">Immunoregulatory properties of CD44+ cancer stem-like cells in squamous cell carcinoma of the head and neck.</a> Head Neck. 33 (2), 208-215 (2011).</li><li>Chen, Y. 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=Cucurbitacin+I+suppressed+stem-like+property+and+enhanced+radiation-induced+apoptosis+in+head+and+neck+squamous+carcinoma--derived+CD44(+)ALDH1(+)+cells.">Cucurbitacin I suppressed stem-like property and enhanced radiation-induced apoptosis in head and neck squamous carcinoma--derived CD44(+)ALDH1(+) cells.</a> Mol Cancer Ther. 9 (11), 2879-2892 (2010).</li><li>Clay, M. R., 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=Single-marker+identification+of+head+and+neck+squamous+cell+carcinoma+cancer+stem+cells+with+aldehyde+dehydrogenase.">Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase.</a> Head Neck. 32 (9), 1195-1201 (2010).</li><li>Meinelt, E., 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> Technical Bulletin: Standardizing Application Setup Across Multiple Flow Cytometers Using BD FACSDiva Version 6 Software. , (2012).</li><li>Zhou, L., Wei, X., Cheng, L., Tian, J., Jiang, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133,+one+of+the+markers+of+cancer+stem+cells+in+Hep-2+cell+line.">CD133, one of the markers of cancer stem cells in Hep-2 cell line.</a> Laryngoscope. 117 (3), 455-460 (2007).</li><li>Fukusumi, T., 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=CD10+as+a+novel+marker+of+therapeutic+resistance+and+cancer+stem+cells+in+head+and+neck+squamous+cell+carcinoma.">CD10 as a novel marker of therapeutic resistance and cancer stem cells in head and neck squamous cell carcinoma.</a> Br J Cancer. 111 (3), 506-514 (2014).</li><li>Shen, C., Xiang, M., Nie, C., Hu, H., Ma, Y., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44+as+a+molecular+marker+to+screen+cancer+stem+cells+in+hypopharyngeal+cancer.">CD44 as a molecular marker to screen cancer stem cells in hypopharyngeal cancer.</a> Acta Otolaryngol. 133 (11), 1219-1226 (2013).</li><li>Kanojia, 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=Proteomic+profiling+of+cancer+stem+cells+derived+from+primary+tumors+of+HER2/Neu+transgenic+mice.">Proteomic profiling of cancer stem cells derived from primary tumors of HER2/Neu transgenic mice.</a> Proteomics. 12 (22), 3407-3415 (2012).</li><li>Higgins, D. 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=Brain+tumor+stem+cell+multipotency+correlates+with+nanog+expression+and+extent+of+passaging+in+human+glioblastoma+xenografts.">Brain tumor stem cell multipotency correlates with nanog expression and extent of passaging in human glioblastoma xenografts.</a> Oncotarget. 4 (5), 792-801 (2013).</li></ol>

The authors have nothing to disclose.

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 100x</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&amp;nbsp;</td><td>CKX31</td><td></td></tr><tr><td>SQ20B cell line</td><td>Gift from the John Little&amp;rsquo;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 ...

isolation-characterization-head-neck-squamous-cell-carcinoma

Research

JoVE Journal

Medicine

8.5K 次观看.  Université de Lyon. 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).

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

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

巨蟹座的建立源于人类传统的骨肉瘤细胞培养

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

科研

癌症研究

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

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 ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

从3维球体培养中分离出类似干细胞的细胞

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

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

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

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 ...

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 ...

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.

分离和表征头颈部鳞状细胞癌亚群具有干细胞特性

摘要

探索更多视频

此视频中的章节