Name: induction of mesenchymal-epithelial transitions in sarcoma cells
Uploaded: 2017-04-07T15:00:00.000+00:00
Duration: 11 min 42 s
Description: Watch this Scientific Journal Video about Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells at JoVE.com

JAS确认来自杜克大学癌症研究所，杜克大学的泌尿生殖系肿瘤学实验室，骨科的杜克大学部的支持。 HL是由美国国家科学基金会（NSF）中心理论生物物理的支持（NSF PHY-1427654）和NSF DMS-1361411，并作为CPRIT（癌症预防和得克萨斯州的研究所）学者在得克萨斯州癌症研究在莱斯大学。 KEW由NIH F32 CA192630 MKJ和HL从与玛丽·C·法拉克-卡森，JN Onuchic，萨米尔·M·汉什，肯内特·J·皮塔，和唐纳德·S·科费有益的讨论中受益的支持。

1.试剂的配制<ol><li>通过添加胎牛血清（FBS）和5毫升青霉素 - 链霉素（5000 U / mL）添加到500mL的DMEM中的50毫升制备DMEM用于细胞培养。此介质可以被储存在4℃下进行长达六个月。 </li><li>重悬无核酸酶水冻干引物以10μM的终浓度。在-20℃储存再悬​​浮的引物。 </li><li>制备放射免疫沉淀测定（RIPA）缓冲液（150mM NaCl的，​​0.5％脱氧胆酸钠，0.1％SDS，50毫摩尔Tris，pH8.0）中。有蛋白酶抑制剂混合物补充使用前保持在冰上缓冲在使用时。储存于4℃。 </li><li>在水和注射器过滤器制备的10mg / mL凝聚胺（海美溴铵）用0.45μm聚醚砜过滤器进行灭菌。溶液可以储存在4℃下长达六个月。 </li><li>通过在10mL PBS中溶解10毫克制备的硝基蓝四唑氯化物和1mg / mL的工作溶液。储存于4℃。</li></ol> 2. GRHL2的慢病毒转导 1天 <ol><li>板每一个6孔板的孔3×10 5个HEK293T细胞于2ml补充的DMEM。量化使用自动细胞计数器细胞。过夜培养后60％汇合，在37℃，5％CO 2 -细胞应为40。 </li></ol> 第2天 <ol start="2"><li>稀释的2μg空载体的（的pCMV-UBC-EGFP）或2的pCMV-GRHL2-UBC-EGFP 24微克，25在无血清培养基中的200μL的1.8微克pΔ8.9和0.2微克的pCMV-VSV的沿-G辅助质粒。在一个单独的管中，在稀释每次转染无血清培养基（8μL在400μL对于两个样品）的200μL基于脂质的转染试剂的4μL。添加转染试剂混合物的200μL到每个质粒溶液，孵育在室温下20分钟。 </li><li>在incuba和灰，通过真空抽吸除去培养基，并用1毫升的PBS漂洗细胞洗HEK293T细胞。与无血清培养基的800微升更换PBS。在37℃下在5％CO 2将来自步骤2.2滴加质粒混合物到每个孔中，孵育细胞2小时:20分钟后，加入200μL转染试剂。 </li><li>小心地真空抽吸除去转染培养基，并用2毫升补充的DMEM替换。返回细胞的培养箱中进行过夜培养。 注:HEK293T细胞松散粘连。媒体更换必须谨慎进行以从盘或烧瓶的底部限制除去细胞。 </li></ol> 第3天 <ol start="5"><li>刷新转染的HEK293T细胞和平板培养基每一个6孔板的孔3×10 5 RD细胞在2mL补充的DMEM。培养细胞过夜。 RD细胞是市售的人横纹肌肉瘤细胞系。 </li></ol> 第4天 </p&gt; <ol start="6"><li>从HEK293T细胞收集病毒介质。移液器从HEK293T细胞和地点的DMEM培养基到一个新的15毫升锥形。新的DMEM媒体和地方HEK293T细胞放回孵化器为第二天小心地更换。 </li><li>添加10毫克/每毫升ml聚凝胺病毒介质的2μL成两个新的50个毫升锥形管中（一个用于空载体转染和另一个用于GRHL2转染）。注射器过滤病毒介质通过从3mL注射器在柱塞和连接用0.45μm聚醚砜过滤器至尖端。 <ol><li>添加在步骤2.6中收集的病毒介质插入注射器的筒，和沉浸介质为含有聚凝胺的新50mL锥形管中。通过真空抽吸从RD细胞除去培养基并过滤病毒介质添加到RD细胞。 </li></ol></li></ol> 第5天 <ol start="8"><li>重复第4天的HEK293T细胞可以在病毒媒体已经被收集之后被丢弃。 </li></ol> 7天 </STRONG&gt; <ol start="9"><li>洗涤RD细胞用1mL PBS中，并加入0.05％胰蛋白酶的200μL每孔。孵育在37℃下5分钟，添加补充培养基的2毫升，并移动细胞悬浮液到一个新的15毫升锥形。离心细胞，在250×g下在室温下并吸出介质5分钟。在1毫升DMEM中重悬（补充有5％FBS和1％青霉素 - 链霉素）。 注:使用FBS的比例较高可能导致流式细胞仪时堵塞<ol><li>过滤器的细胞用于流式细胞术。利用吸移管通过30微米的过滤器来应用1mL的RD细胞悬液到流式细胞术管和地方试管于冰上。 </li><li>排序EGFP +细胞26进入含有0.5毫升的DMEM的1.5mL微量离心管中补充有10％FBS和1％青霉素-链霉素和置于冰上。板EGFP +排序总共1mL的补充型DMEM中的细胞进入培养箱中培养12孔板和地点的单个孔中。 ;注意:EGFP +细胞从流这在转导后流式细胞仪的产量通常很低（50,000-100,000细胞）。细胞可能需要10培养 - 在进入步骤3之前14天。 </li></ol></li></ol> 3.的miR-200S的反向转染<ol><li>在-20℃下制备使用不含核酸酶的H 2只 O.分装股票和商店的miR-200模拟物的50μM股以避免反复冷冻/解冻循环。 </li><li>添加各为50μM的miR-200模拟物（的miR-200a中，的miR-200b中，和miR-200c中）至300μL无血清培养基或50μM阴性对照miRNA至300μL无血清培养基9μL的3μL。 </li><li>添加特异性siRNA转染试剂的6μL到无血清培养基的600μL和除以该混合物的300μL成两个管，一个用于每个来自步骤3.2所述两个的miR混合物。从步骤3.2的转染试剂的300μL的混合组合每个的miR混合物的300μL。孵育在室温TE 20分钟温度。 </li><li>而孵育，制备表达在第2节中2.4毫升创建EV或GRHL2 600000个EGFP + RD细胞的细胞悬浮液每治疗无血清培养基的（250个细胞/μL）。 </li><li>在24孔板中，每添加的miR-200的混合或来自步骤3.3阴性对照混合物的孔100μL成六个孔中，然后添加各细胞悬浮液的400μL到每个的miR混合物的三个孔。 </li><li>细胞孵育在37℃下在5％CO 2过夜和改变介质到完全补充的DMEM次日。收集单元2天后，使用合适的缓冲液（见下文）分析。经历MET RD肉瘤细胞的延时成像可作为补充材料。图像每个孔使用自动化的活细胞成像器27每2小时用10X物镜。 </li></ol> 4. RNA提取，逆转录，和qPCR <ol><li>根据使用标准RNA制造商的说明提取RNA提取试剂盒24。提取的RNA可被储存在-80℃。 </li><li>量化RNA浓度。解冻并与RNA冰工作。为了定量RNA浓度，根据制造商的方案在用酶标仪分光光度计260nm处测量UV吸光度。稀释所有样品与无核酸酶水的最低浓度。 </li><li>执行的总RNA逆转录成互补DNA（cDNA）。结合不低于100ng的总RNA用PCR缓冲液，dNTP混合物（100毫米），随机六聚体引，逆转录酶和无核酸酶的水在20μL反应体积24。根据制造商的协议运行RT周期。 cDNA可在-20℃保存长期。 </li><li>稀释20μL反应以100μL80μL无核酸酶的H 2 O的</li><li>混合5μL的基于荧光的嵌入染料2X的qPCR主混合物，各10μM的引物的0.06微升，一个ND 2μL每个样品稀释RT反应的。 </li><li>根据制造商的针对基于荧光的主混合物协议的qPCR运行。 </li><li>由增量CT方法量化mRNA的相对量和标准化为GAPDH。绘制生物学重复±每个治疗组的标准偏差的平均值mRNA的表达。 注:特殊的预防措施应与RNA时，以避免RNA酶，如使用无RNase的塑料和试剂接触服用。非一次性设备应使用去除RNA酶前处理。 </li></ol> 5.免疫荧光染色<ol><li>以下在24孔格式步骤3.6，吸媒体通过真空抽吸从RD细胞。 </li><li>固定细胞，加入每孔的4％多聚甲醛（PFA）的500μL，并孵育在室温下（RT）15分钟。固定后，位置细胞在磷酸盐在4℃下过夜如果需要缓冲盐水（PBS），并存储。 </li><li>通透细胞通过加入PBS + 0.2％的Triton-X100的500μL，并在室温下孵育30分钟。通过真空抽吸，除去透化缓冲液，并用PBS洗三次。 </li><li>在500微升的5％牛血清白蛋白的（BSA）/ PBS块孵育细胞在室温下30分钟。细胞可以在4℃保存过夜，如果必要的。 </li><li>准备一抗稀释。 /移液管将200μL的5％BSA的每孔PBS到15mL锥形（12个孔= 2.4毫升）和每毫升添加5％BSA的第一抗体1μL/ PBS需要。通过真空抽吸除去阻断缓冲液并分配稀释的一抗的200μL到每个孔中。 <ol><li>孵育细胞1:在5％BSA /在RT或过夜，在4℃1000稀释的初级抗体PBS 1个小时。温育后，洗涤细胞两次，用PBS。 </li></ol></li><li>在如上述5％BSA / PBS的相同体积的制备二级抗体稀释。添加使用1远红染料缀合的二级抗体:2000稀释。另外添加1微克/毫升Hoechst染料到混合的。取出PBS并添加混合200微升至每孔。 <ol><li>孵育在室温下在黑暗中1个小时。用PBS洗涤3次，在离开PBS的细胞，并使用铝箔保护细胞免受光。细胞如果需要，可以储存于4℃。 </li></ol></li><li>图像的细胞以400X总放大倍数与激发波长594倒置落射荧光显微镜 - 650纳米。 </li></ol> 6.免疫印迹<ol><li>用冰冷的PBS洗涤从3.6细胞两次。在冰上，用1x RIPA50μL的细胞裂解缓冲液补充有1X蛋白酶抑制剂混合物。 </li><li>在4℃下15分钟岩石裂解物，裂解物收集到1.5毫升管中，并离心以高速（20,000×g）离心5分钟以澄清的样品。细胞裂解物可在必要时被存储长期在-80℃下。 </li><li>量化使用Bradford测定总蛋白和蛋白质等分的等量到新的1.5毫升离心管中。布林克样本以等体积的使用RIPA缓冲液和3倍的Laemmli负载缓冲液补充有2-巯基乙醇。 </li><li>在95℃下孵育在1×Laemmli样品上样缓冲液的样品5分钟，并使用4-12％Tris-甘氨酸凝胶在200V下45分钟运行SDS-PAGE。取决于细胞系蛋白质装载范围的量。在一些情况下，需要的总蛋白的50-100微克检测间充质细胞系E-钙粘蛋白。 </li><li>转移蛋白到硝酸纤维素膜上2小时，在50 V在1×Tris-甘氨酸转移缓冲液。 </li><li>块膜在室温下或在4℃下在基于BSA阻断缓冲液中过夜1个小时。 </li><li>稀释的初级抗体在1:1000的浓​​度在5毫升封闭缓冲液中，加入稀释至膜，并在室温下或在4℃下过夜轻轻摇动1个小时孵育。在PBS中洗涤膜3次，用0.05％Tween-20的。 </li><li>在室温下孵育1个小时的膜与红外荧光偶联secondar在1 Y抗体:20,000稀释在如上述的封闭缓冲液。 </li><li>在PBS洗涤膜两次用0.05％吐温20，然后在一个时间PBS。 </li><li>使用标准的红外荧光检测图像的膜。 </li></ol> 7.锚地独立生长试验注:对于详细软琼脂测定方案，见28。 <ol><li>温暖的无菌2X DMEM培养基至42℃，在热水浴中。在此期间，热的预灭菌的1％琼脂糖在微波中3分钟。在根据需要或直至完全熔融的时间微波另外的30秒。琼脂糖转移到一个42℃的水浴中。 </li><li>放置一个50毫升锥形管中在无菌罩与42℃的水的烧杯中，并在1混合1％琼脂糖和2x DMEM培养基:1的比例占1.5毫升混合物，每孔在6孔板中。总是准备额外的DMEM /琼脂糖以弥补移液错误，避免气泡。 </li><li>加入混合物的的井侧，确保没有气泡形成，静置在室温下30分钟。 </li><li>而底层被固化，制备各组通过抽吸培养基并用1ml的PBS洗涤一次，在步骤3转染的RD细胞。吸PBS并加入0.2毫升0.05％胰蛋白酶并在37℃下孵育5分钟。 </li><li>在0.8毫升的培养基和磨碎的细胞中和胰蛋白酶，以形成单细胞悬浮液。转移细胞悬液至15mL锥形管中。 </li><li>计数细胞并计算每孔10,000个细胞所需的细胞悬浮液的体积。 </li><li>加热0.6％琼脂糖在微波中3分钟，接着30秒一次，直到完全熔化。琼脂糖转移到42℃水浴中。 </li><li>放置一个50毫升锥形管中在无菌罩与42℃的水的烧杯中。混合0.6％琼脂糖和稀释的细胞悬浮液以1:1的比例，以制备1.5毫升每孔的混合物在6孔板中。总是准备额外的细胞悬液/琼脂糖混合以弥补移液错误为了避免气泡。 注意:这是很重要的，在42℃这里工作。如果温度太低，则混合物将电镀之前固化，温度高于42℃会影响细胞生存力。 </li><li>通过研磨细胞几次拌匀细胞混合物添加到孔中，以确保无气泡的形式，并让固化在室温下20分钟。 </li><li> 加入2毫升补充培养基到每个孔中并移动到板孵化器。 <ol><li>更改媒体每周一次，3-4周或直至多细胞集落形成。要小心，以避免因负压吸引去除媒体接触时的琼脂。或者，使用P1000，以除去液体介质的顶层。 </li><li>加入200硝基μL蓝四氮唑氯化物溶液染色软琼脂平板上（1毫克/毫升在PBS中），并在37℃过夜孵育所述板。第二天，用PBS成像更换介质。 </li><li>图像在孔在倒置显微镜上40X总放大倍数。 </li><li>在ImageJ的执行分析菌落面积和数量。积平均生物学重复的集落数±标准偏差。 </li></ol></li></ol>

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(92), e51998 (2014).</li><li>Yang, J., 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=Integrated+proteomics+and+genomics+analysis+reveals+a+novel+mesenchymal+to+epithelial+reverting+transition+in+leiomyosarcoma+through+regulation+of+slug.">Integrated proteomics and genomics analysis reveals a novel mesenchymal to epithelial reverting transition in leiomyosarcoma through regulation of slug.</a> Mol Cell Proteomics. 9 (11), 2405-2413 (2010).</li><li>Alba-Castellon, 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=Snail1+expression+is+required+for+sarcomagenesis.">Snail1 expression is required for sarcomagenesis.</a> Neoplasia. 16 (5), 413-421 (2014).</li><li>Takaishi, M., Tarutani, M., Takeda, J., Sano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+to+Epithelial+Transition+Induced+by+Reprogramming+Factors+Attenuates+the+Malignancy+of+Cancer+Cells.">Mesenchymal to Epithelial Transition Induced by Reprogramming Factors Attenuates the Malignancy of Cancer Cells.</a> PLoS One. 11 (6), e0156904 (2016).</li></ol>

作者什么都没有透露。

肉瘤是间充质细胞系的很少见，但高发性癌。尽管他们的间充质细胞系，肉瘤的一个子集似乎经历一个更上皮样状态的表型转化。这MET-像开关具有预后相关性，因为患者多上皮样的肿瘤较少侵略性24。尽管他们的临床相关性，很少有研究解决的分子机制肉瘤推动这些表型的转变。 为了检查肉瘤细胞MET样的转变，我们通过结合上皮因素GRHL2，且所述miR-200家族?...

An erratum was issued for: <a href="https://www.jove.com/video/55520/induction-of-mesenchymal-epithelial-transitions-in-sarcoma-cells">Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells</a>. An author name was updated.
One of the authors&#39; names was corrected from:
Shenghan Xu
to:
Shengnan Xu

An erratum was issued for: <a href="https://www.jove.com/video/55520/induction-of-mesenchymal-epithelial-transitions-in-sarcoma-cells">Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells</a>. An author name was updated.

Erratum: Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

可塑性是指细胞表型之间的可逆转变，并且通常分为两种类型，上皮 - 间质（EMT）的转变和间充质到上皮转变（MET）。此可塑性起着多细胞生物体，如发育和伤口愈合1的正常过程中起重要作用;然而，这些相同的途径和基因表达的方案也可导致疾病，如纤维化（评价的2，3，4）和癌转移（参考文献5，6，7审查，8）。期间转移，例如，EMT破坏细胞极性，细胞-细胞相互作用，并且促进侵袭9,10。总之，EMT贡献s至促进癌细胞的传播表型状态。此外，EMT也导致了驱动侵袭性表型的表型的其它改变，包括癌细胞代谢6的失调，药物抗性11,12的发展，增加的肿瘤引发能力13,14和宿主免疫逃避15的主机。 可塑性已经很好地研究了癌进展;然而，肉瘤还表现出表型可塑性。有趣的是，它看起来好像有些可塑性的癌相同的驱动程序也有助于肉瘤可塑性和侵略性。例如，从肉瘤患者的循环肿瘤细胞（CTC）已经显示出表达的EpCAM，是通常在上皮细胞上发现的16的细胞表面蛋白。阿迪倚重，250个软组织肉瘤样品被归类为上皮样或间充质样基于基因表达。在上皮样生物标记物标志患者比患者的间充质样生物标记物标志17预后较好。这与许多癌，在与患者多间充质样肿瘤18例相比，具有更多上皮样癌有更好的结果是一致的。 虽然一些肉瘤显示生物标记物和基因表达的途径与MET一致，此表型可塑性的分子基础仍然知之甚少。为了研究MET的机制和司机肉瘤我们开发使用两种特定的上皮细胞因子诱导MET的模型中，微小RNA（MIR）-200家庭和grainyhead样2（GRHL2）。与miR-200S是通过结合至MESSEN的3&#39; 非编码区调节基因表达的小的非编码RNA家族蒙古包RNA并防止翻译成蛋白质。与miR-200家族包括两个子组的 - 一种含的miR-141和miR-200a中，和另一种包括的miR-200b中，的miR-200c中，和miR-429。所述miR-200家族的成员富含上皮组织，和miR-200S的损失与转移癌19相关联。与miR-200家族也下调在软组织肉瘤与正常组织相比20。类似的miR-200S，GRHL2是上皮发展21重要的关键调节。所述GRHL2转录因子作用于两种方式上调上皮基因，如E-钙粘蛋白:1）在上皮细胞，GRHL2直接阻遏EMT主调节器，ZEB1 22;和2）GRHL2直接激活上皮基因23的转录。我们以前的研究已显示，在肉瘤细胞的miR-200和GRHL2的组合表达诱导MET样表型24。在这里，我们提出了一个详细的协议来创建肉瘤细胞使用的miR-200和GRHL2的异位表达MET诱导的体外模型。

<table><tbody><tr><td>Countess automated counter</td><td>Life technologies</td><td>AMQAX1000</td><td></td></tr><tr><td>Countess cell counting chamber slides</td><td>Invitrogen</td><td>C10283</td><td></td></tr><tr><td>SimpliAmp Thermal Cycler</td><td>Thermo Fisher</td><td>A24811</td><td></td></tr><tr><td>Odyssey Fc</td><td>LI-COR Inc</td><td></td><td></td></tr><tr><td>ViiA7 Real Time PCR System</td><td>Thermo Fisher</td><td>4453536</td><td></td></tr><tr><td>PCR microplate</td><td>Corning</td><td>321-29-051</td><td></td></tr><tr><td>KAPA SYBR Fast Universal qPCR Kit</td><td>KAPA Biosystems</td><td>KK4602</td><td></td></tr><tr><td>Starting Block (PBS) Blocking Buffer</td><td>Thermo Fisher</td><td>37538</td><td>BSA-based blocking buffer</td></tr><tr><td>Agarose General Purpose LE</td><td>Genesee Scientific</td><td>20-102</td><td></td></tr><tr><td>10x Tris/Glycine/SDS Buffer</td><td>Bio-Rad Laboratories Inc</td><td>161-0732</td><td>Running buffer</td></tr><tr><td>10x Tris/Glycine Buffer</td><td>Bio-Rad Laboratories Inc</td><td>161-0734</td><td>Transfer buffer</td></tr><tr><td>RIPA Buffer</td><td>Sigma Life Sciences</td><td>SLBG8489</td><td></td></tr><tr><td>Amersham Protran 0.45 &amp;mu;m nitrocellulose</td><td>GE Healthcare Lifesciences</td><td>10600012</td><td></td></tr><tr><td>Quick-RNA MiniPrep Kit</td><td>Genesee Scientific</td><td>11-358</td><td></td></tr><tr><td>Laemmli Sample Buffer (4X)</td><td>Bio-Rad Laboratories Inc</td><td>1610747</td><td></td></tr><tr><td>Mini Trans-Blot Cell</td><td>Bio-Rad Laboratories Inc</td><td>1703930</td><td></td></tr><tr><td>Mini-Protean Tetra Cell</td><td>Bio-Rad Laboratories Inc</td><td>1658005EDU</td><td></td></tr><tr><td>DPBS</td><td>Life technologies</td><td>14190-144</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life technologies</td><td>11995-065</td><td></td></tr><tr><td>DMEM</td><td>Life technologies</td><td>11995-065</td><td></td></tr><tr><td>Lipofectamine RNAi Max</td><td>Thermo Fisher</td><td>13778150</td><td></td></tr><tr><td>Lipofectamine 2000 Ragents</td><td>Thermo Fisher</td><td>11668019</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life technologies</td><td>15140-122</td><td></td></tr><tr><td>miRVana miRNA mimic negative control #1</td><td>Thermo Fisher</td><td>4464058</td><td>neg miRNA</td></tr><tr><td>hsa-miR-200 mirVana miRNA mimic</td><td>Thermo Fisher</td><td>4464066</td><td>miR200A</td></tr><tr><td>has-miR-200 mirVana miRNA mimic</td><td>Thermo Fisher</td><td>4404066</td><td>miR200B</td></tr><tr><td>has-miR-200 mirVana miRNA mimic</td><td>Thermo Fisher</td><td>4404066</td><td>miR200C</td></tr><tr><td>Opti-MEM</td><td>Life technologies</td><td>11088-021</td><td>serum-free media</td></tr><tr><td>anti-Ecadherin antibody</td><td>BD Bioscience</td><td>610182</td><td></td></tr><tr><td>anti-beta actin</td><td>Santa Cruz Biotechnology</td><td>sc-69879</td><td></td></tr><tr><td>anti-EpCam</td><td>Ab Serotec</td><td>MCA18706</td><td></td></tr><tr><td>anti-ZO1</td><td>Invitrogen</td><td>402200</td><td></td></tr><tr><td>IRDye 800W</td><td>LI-COR Inc</td><td>925-32210</td><td></td></tr><tr><td>IRDye 680</td><td>LI-COR Inc</td><td>926-32223</td><td></td></tr><tr><td>anti-mouse AlexaFluor 647</td><td>Thermo Fisher</td><td>A211241</td><td></td></tr><tr><td>anti-rabbit AlexaFluor 647</td><td>Thermo Fisher</td><td>ab150075</td><td></td></tr><tr><td>Halt Protease and Phosphatesse Inhibitor</td><td>Thermo Fisher</td><td>1861281</td><td></td></tr><tr><td>Precision Plus Protein Dual Color</td><td>Bio-Rad Laboratories Inc</td><td>161-0374</td><td></td></tr><tr><td>Partec CellTrics</td><td>Sysmex</td><td>04-004-2326</td><td>30 &amp;mu;m filter for flow</td></tr><tr><td>GAPDH-F</td><td>IDT</td><td></td><td>AGCCACATCGCTCAGACAC</td></tr><tr><td>GAPDH-R</td><td>IDT</td><td></td><td>GCCCAATACGACCAAATCC</td></tr><tr><td>Ecadherin-F</td><td>IDT</td><td></td><td>TGGAGGAATTCTTGCTTTGC</td></tr><tr><td>Ecadherin-R</td><td>IDT</td><td></td><td>CGCTCTCCTCCGAAGAAAC</td></tr><tr><td>ZEB1-F</td><td>IDT</td><td></td><td>GCATACAGAACCCAACTTGAACGTC</td></tr><tr><td>ZEB1-R</td><td>IDT</td><td></td><td>CGATTACACCCAGACTGC</td></tr><tr><td>NOTCH-F</td><td>IDT</td><td></td><td>GGCAATCCGAGGACTATGAG</td></tr><tr><td>NOTCH-R</td><td>IDT</td><td></td><td>CTCAGAACGCACTCGTTGAT</td></tr><tr><td>nitro blue tetrazolium&amp;nbsp;</td><td>Sigma</td><td>N5514</td><td></td></tr><tr><td>hexadimethrine bromide</td><td>Sigma</td><td>H9268</td><td>polybrene</td></tr><tr><td>3 mL syringe</td><td>BD Bioscience</td><td>309657</td><td></td></tr><tr><td>Sterile syringe filter</td><td>VWR</td><td>28145-505</td><td></td></tr><tr><td>5mL polypropylene round-bottom tube</td><td>352063</td><td></td><td>flow cytometry tubes</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Thermo Fisher</td><td>4368814</td><td>reverse transcription kit</td></tr><tr><td>4% paraformaldyhyde</td><td>Santa Cruz Biotechnology</td><td>sc-281612</td><td></td></tr><tr><td>Triton-X100</td><td>Sigma</td><td>93443</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A7906</td><td></td></tr></tbody></table>

induction of mesenchymal-epithelial transitions in sarcoma cells

表型可塑性是指一种现象，其中细胞中瞬时获得另一谱系的性状。在癌的进展，表型可塑性驱动入侵，传播和转移。事实上，虽然大多数表型可塑性的研究一直在上皮来源的癌的情况下，原来的肉瘤，这是起源于间质，也表现出表型可塑性，有肉瘤的一个子集进行一个类似于间充质现象上皮转化（MET）。在这里，我们分别开发了一种方法，其包括与miR-200家族和grainyhead样2（GRHL2）以模仿肉瘤患者samples.We观察GRHL2和所述miR-200家族使用细胞转导和转染顺序表达这种MET-状现象， ，以更好地理解肉瘤细胞表型，这些转变的分子基础。表达的miR-200和GRHL2肉瘤细胞显示增强的上皮characterist集成电路细胞形态和上皮和间充质标志物的改变。使用这些方法今后的研究可以被用来更好地了解肉瘤细胞MET状突起，如迁移，侵入，转移倾向，和治疗的抗性的表型的影响。

架构MET诱导细胞肉瘤对于在肉瘤细胞MET样改变感应的一般时间线示于图1。该协议开始于转导GRHL2（ 图1A），随后所述miR-200家族（ 图1B）的转染。 GRHL2或miR-200家族成员未能单独表达时冲击RD细胞的出现，但GRHL2和miR-200S的异位表达一起导致在RD细胞上皮样形态学变化。从一个纺锤状的形式，以?...

Watch this Scientific Journal Video about Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells at JoVE.com

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

我们在这里提出用于诱导基于微小RNA-200家族成员及grainyhead样2（GRHL2）的合并的异位表达肉瘤细胞间质上皮转换（MET）的细胞培养方法。这种方法适用于更好地理解可塑性的癌症侵袭性和治疗方法的生物学影响。

在肉瘤细胞间质上皮转变的诱导

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

induction-of-mesenchymal-epithelial-transitions-in-sarcoma-cells

Research

JoVE Journal

Developmental Biology

9.3K 次观看.  Duke University. 我们在这里提出用于诱导基于微小RNA-200家族成员及grainyhead样2（GRHL2）的合并的异位表达肉瘤细胞间质上皮转换（MET）的细胞培养方法。这种方法适用于更好地理解可塑性的癌症侵袭性和治疗方法的生物学影响。 

视频: Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

一个实时的基于电阻抗技术测量癌细胞入侵内皮细胞单层

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

科研

医学

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

癌症研究

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

发育生物学

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

一种酶和无血清神经干细胞培养模型EMT研究适合药物发现

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

生物工程

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

研究TGF-β信号和TGF-β引起的乳腺癌和正常细胞的上皮到中位过渡

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

生物学

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.