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1.克隆分析<ol><li>制备的雌激素依赖性乳腺癌细胞系的单个细胞悬浮液使用步骤MCF-7和T47D细胞概述如下<ol><li>从10cm的组织培养皿这是不超过50％的汇合与MCF-7或T-47D细胞吸出培养基（DMEM / 10％热灭活的胎牛血清/谷氨酰胺和青霉素/链霉素）。冲洗用PBS。孵育胰蛋白酶0.25％/ 2.21毫摩尔EDTA溶解在DMEM高葡萄糖，在37℃下进行1-4分钟。 </li><li>检查细胞在相差显微镜下1分钟的间隔，以确保单细胞分布。悬浮细胞用2毫升吸管吹打上下数次以破坏细胞 - 细胞接触，以实现一个几乎不变的，单细胞的状态。 </li><li>继续培养细胞中的胰蛋白酶在37℃至4分钟，如果你只有2分钟的潜伏期后观察细胞团块。如果他们仍然附着到e不要使用这些细胞克隆研究ACH胰酶消化后4分钟，因为其他错误将在菌落数收益率被引入。 注意:如果细胞结块，形成的菌落的数量将反映细胞比孵育数少的乘积。如果细胞过度胰蛋白酶，它们的克隆潜力可能会减少。 </li><li>制备1500个细胞/ ml培养基的单细胞悬浮液为24孔板，或更小，（+ 500细胞/ ml，这取决于细胞类型或通道数），由在一个主管含有所需的整个卷系列稀释所有实验中的变量。 注意:我们的目标是一个最终细胞800细胞/ cm 2（约500至1100个细胞/ cm 2的范围内）的密度。我们的目标是产生大约100±50菌落，这允许相对容易计数，防止拥挤并允许足够菌落导致显著统计差异，当菌落增加或减少实验perturba系统蒸发散。 </li></ol></li><li>在克隆形成密度使用步骤细胞培养下文概述<ol><li>孵育细胞一式四份孔24以及纤连蛋白包被的板在1500细胞的产克隆密度/孔从1500个细胞/ ml的主单细胞悬浮液管。通过制定3毫升和分配在每2个孔1毫升介质磨碎含有细胞用5毫升吸管的介质。 注:纤连蛋白包被的平板应该购买预涂覆从商业供应商。控制的质量的外涂层板，自动化过程导致不平坦的表面不适合于该测定。 </li><li>吹打向上和向下混合悬浮液5毫升吸管，吸取涨3 mL细胞悬液，并填写2井每1ml。填补只2孔中从一个移液管的任何一个时间。再次归还剩余量在吸管的细胞悬液在主管，悬浮细胞吹打向上和向下，并拟定另有3毫升填补另外2 WELLS与每1ml。 注:主管的连续混合是必要的，因为细胞会不断沉降。制订足够的体积，以填补仅2个孔是必要的，以类似的细胞数增加至每孔，因为细胞中的吸移管沉淀为好。 </li><li>工作迅速分发大量细胞，因为使细胞坐在悬浮液在室温和CO 2浓度将调节其克隆生成电位（未发表意见）。 </li><li>吹打成井菌落检测的行为在优化细胞的空间分布。这样做通过缓慢吹打含有最终细胞浓度入井的中间暂停。不要使板进一步动作之前，细胞沉淀在底部。不要摇动板，因为圆形混合将有效地离心细胞井创造高细胞密度和不可数合流菌落周边6天。 注意:不要混用，除非必要，因为这比没有混合不太理想，在所有引进的细胞悬液卷后。如果需要以混合细胞，通过移动板来回在垂直的方向上，而在于在一个平面上这样做。 </li><li>孵育细胞，在37℃，5％CO 2，而不更换培养基为6天。在一个6后以及天少量细胞不会显著影响营养或细胞因子组合物也没有原始介质的pH。 </li><li>设计该试验的时间过程如下:孵育细胞上在第1天纤连蛋白涂覆的基质替换现有介质用1ml新鲜培养基或含有新鲜培养基的FGF-2 10上如下面第6天0天染色细胞纳克/毫升1.4）。 130进行3天的任何实验扰动，如下）。 </li></ol></li><li>分子信号或粘附分子的设立实验扰动<ol><li>在第3天，添加10056;升含有扰动剂的最终预期浓度的10倍，以1毫升培养基中的孔中的溶液。不要混用。继续孵育细胞在37℃，5％CO 2的额外的3天。 注:扰动剂可以包括多种抑制剂和粘附分子，受体或其它表面蛋白，细胞内信号通路，分子，因子，辅因子或结构蛋白的抑制剂的阻断剂，这可能在支持休眠状态中起作用。 </li><li>第6天色斑殖民地，如下所示。 </li></ol></li><li>染色殖民地<ol><li> 6天后在培养用新鲜的0.1％结晶紫在2％乙醇/ 10mM的硼酸钠（pH9.0）中的溶液染色细胞。抽吸培养基并添加1毫升结晶紫溶液到各孔20分钟。 </li><li>通过与国内外开口朝下锐角变成一个冰桶洋溢着不断流动的自来水浸泡洗净他们板水在水槽。倾斜板水平角度水下后，开井下来，然后向后倾斜锐角在一个温和的流动动除去时。 </li><li>重复浸渍2次或3次，直到水在孔的底部是不再蓝色。剧烈洗涤可除去细胞或菌落不太粘附由于实验介入，显著在数量和数据的误差加。 </li><li>干板隔夜放置它们在邻近其相应的标记覆盖了台式毛巾倒挂。 </li></ol></li><li>菌落计数<ol><li>计数在各后阱6天孵育，染色和干燥生长和休眠的菌落数。在倒置相差显微镜最佳为40X放大率计数菌落。计数&gt; 30个细胞的集落作为生长和12个或更少细胞的集落，以相比生长的细胞，大EXPA非常大尺寸的外观形态nded细胞质大细胞质至细胞核的比率，在图1 7,15所示。 注:13-29细胞簇通常不计算在内，因为它们不是很频繁，直截了当休眠测定。他们如果能转移的扰动无论是增长还是在休眠细胞的增长潜力被计算在内。然后这些结果将需要与生物学意义相关联。 </li></ol></li></ol> 2.克隆形成孵化的免疫荧光研究<ol><li>在6孔板调整细胞数约为7,500-8,000细胞/孔，以对应于细胞数/表面积类似于24孔试验。 </li><li>将一个圆，无菌，纤维连接蛋白包盖玻片到每口井基地的6孔板之前除了细胞成像研究。吸管细胞在3ml体积为每孔2个孔，在一个时间，在浓度概述的，如在1.2中描述的克隆形成实验段）。 </li><li>细胞培养6天，如在1.2.4和1.2.5以上。加入扰动因素，在第3天在10倍浓度的300微升量，如在1.3中集落检测过程中的说明）。 </li><li>染色的细胞在第6天用抗体与细胞黏附分子如整合素α4，α5，α6，β1，β3，例如，粘着斑复合分子FAK，桩蛋白和纽蛋白，例如，涉及能动性蛋白质如α微管蛋白，例如，信号通路成员如磷酸化Akt，磷酸-ERK，磷酸化p38，磷酸化JNK，例如，或任何其他蛋白质即调查其在休眠作用的目标，使用标准技术进行直接或间接的免疫荧光染色。 <ol><li>用钳子一天丙酮去除幻灯片6.修复/甲醇1:1 -20 °下进行20分钟，并风干。一个替代的固定剂，如多聚甲醛，也可以使用，如果有必要的。透细胞用0.1％的Triton X-100，0.1％柠檬酸钠2分钟˚F或检测细胞内的抗原。用PBS洗涤细胞。 </li><li>对于间接免疫荧光染色，进行1小时，在室温下用5％BSA或与来自在其中产生的二次抗体的物种的10％免疫前血清块滑动。 </li><li>孵育过夜，在4℃下用初级抗体与细胞粘附分子，焦复杂分子，信号传导途径的成员或任何其它蛋白，其调查其在休眠作用稀释至在PBS中由制造商推荐特定稀释度的目标0.1％TRITON X -100。 </li><li>用PBS洗3次。孵育盖玻片用荧光团标记的抗体，在室温下搅拌2小时。作为一个例子，的Alexa Fluor 488驴抗小鼠IgG抗体可用于检测鼠单克隆初级抗体。山盖玻片细胞面朝下放在使用抗淬灭剂达皮载玻片上。密封用指甲油的周长。 </li><li>对于直接免疫荧光法，开展BSA如上述2.4.2）阻挡，孵育荧光团共轭的第一抗体过夜，在4℃。用PBS洗3次。孵化与抗淬灭剂和DAPI和密封用指甲油的幻灯片。 </li><li>盖滑动托盘铝箔和储存在4 °下成像和摄影随时随地长达数星期后。在使用放大1000倍配备有照相机任何荧光显微成像系统查看和照片细胞。 </li><li>为纤维状的肌动蛋白染色，在1％牛血清白蛋白（BSA）30分钟，并在室温下孵育在BODIPY FL-Phallacidin（绿色）或罗丹明鬼笔环肽（红色）20分钟块滑动。添加抗淬灭剂和密封以上。 </li></ol></li></ol> 3.克隆形成孵化的分子生物学研究<ol><li>蛋白质印迹<ol><li>孵育ER +乳腺癌细胞MCF-7或T47D以20,000个细胞/ 60毫米的钢板和50,000细胞/ 100毫米的钢板上fibronec克隆密度镀锡在5％CO 2板在37℃。 </li><li>孵育细胞以75,000细胞/ 100mm板为分子研究需要毫克量从裂解物蛋白质稍高的密度。最多可使用每个实验点十大板块，收集足够的蛋白质，使用Western印迹分子研究或RNA分离为北印迹。 </li><li>细胞数量基凝胶负载是一个必要的辅助在大大不同大小的种植与休眠细胞比较蛋白的表达。通过胰蛋白酶处理收集细胞，并在0.2％台盼蓝的制备裂解物进行Western印迹，代替刮掉细胞用单刃刀片的计数血球的等分试样。 </li><li>离心细胞以10,000 xg离心2分钟，通过抽吸除去培养基，加入200微升裂解缓冲液，超声处理的细胞在裂解缓冲液和测定的蛋白浓度。 </li><li>通过将蛋白质产量通过细胞的数量计算每个细胞的蛋白质的量生成的量。加载裂解物成单独的聚丙烯酰胺凝胶，表示蛋白质的代表当量细胞数两者）当量的量，和b）蛋白质的量各孔中。至少25微克蛋白质应加载到每个孔中。 注意:这将允许生长和休眠细胞，这是在大小和蛋白质含量（ 图1）显著不同之间的比较。 </li></ol></li><li>流式细胞仪<ol><li>收集从100毫米②塔板细胞通过胰蛋白酶消化，如在3.1，并通过标准荧光激活细胞分选使用伯或仲免疫荧光染色为任一细胞内或细胞外的抗原（FACS）协议分析，如在2.4中概述。 注意:通过胰蛋白酶消化从组织培养皿拆卸细胞不影响膜蛋白的浓度通过抗体标记来确定。 </li></ol></li></ol>

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E., Hameed, M., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-ordinate+loss+of+FGF-2+and+laminin+5+expression+during+neoplastic+progression+of+mammary+duct+epithelium.">Co-ordinate loss of FGF-2 and laminin 5 expression during neoplastic progression of mammary duct epithelium.</a> Human Pathology. 38 (1), 154-160 (2007).</li><li>Wieder, 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=Overexpression+of+basic+fibroblast+growth+factor+in+MCF-7+human+breast+cancer+cells:+lack+of+correlation+between+inhibition+of+cell+growth+and+MAP+kinase+activation.">Overexpression of basic fibroblast growth factor in MCF-7 human breast cancer cells: lack of correlation between inhibition of cell growth and MAP kinase activation.</a> J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Farnesyl+transferase+inhibitor+treatment+of+breast+cancer+cells+leads+to+altered+RhoA+and+RhoC+GTPase+activity+and+induces+a+dormant+phenotype.">Farnesyl transferase inhibitor treatment of breast cancer cells leads to altered RhoA and RhoC GTPase activity and induces a dormant phenotype.</a> Int J Cancer. 129 (1), 61-69 (2011).</li><li>Korah, R., Sysounthone, V., Golowa, Y., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+confers+a+more+differentiated+phenotype+in+MDA-MB-231+human+breast+cancer+cells.">Basic fibroblast growth factor confers a more differentiated phenotype in MDA-MB-231 human breast cancer cells.</a> Cancer Res. 60 (3), 733-740 (2000).</li><li>Dontu, 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=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes &amp; Dev. 17 (10), 1253-1270 (2003).</li><li>Tivari, S., Lu, H., Dasgupta, T., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+dormant+estrogen-dependent+breast+cancer+micrometastases+by+bone+marrow+stromal+injury+in+an+in+vitro+model+of+dormancy.">Reactivation of dormant estrogen-dependent breast cancer micrometastases by bone marrow stromal injury in an in vitro model of dormancy.</a> Proceedings of the 105th Annual Meeting of the American Association for Cancer Research. , (2014).</li></ol>

支持由国防部资助DAMD17-01-C-0343系和DAMD17-03-1-0524，新泽西州委员会关于癌症研究02-1140-CCR-E0和露丝·埃斯特林戈德堡纪念癌症研究（RW）

我们的模型是由休眠的骨髓中的几个关键要素。它由雌激素敏感细胞，这是可能保持休眠的 ​​骨髓长时间10的类型，它包括纤维连接蛋白，骨髓的关键结构元件，FGF-2，生长因子大量由骨髓基质合成的和大量沉积在骨髓细胞在克隆密度，其中它们之间的相互作用主要是与底层的31,32和温育的细胞外基质。而骨髓微环境是更为复杂，因为需要问特定机械问题这个简单的系统可以建?...

乳腺癌细胞转移到骨髓病前是可检测1，只要小的肿瘤发展的血管2,3。转移过程是快，但效率不高。细胞迅速进入新的血管，在数百万个4天，但行程到远处器官5几年生存。尽管如此，一些微生存在骨和可以发现以单细胞或在从初诊患者1骨髓抽吸小细胞团块。这些细胞抗蚀辅助化疗，其施用于消除他们6的根本目的。这种阻力被赋予的，基本上，通过存活通过与骨髓微7,8-相互作用信令发起的。微转移可以在大约三分之一的妇女局部乳腺癌的发现，代表了生存的独立指标时，单因素分析分析9。一些microme转移瘤是开始增长，但复发模式依赖于细胞类型。例三阴性乳腺癌往往1〜4年之间复发，这表明穷人控制了休眠状态。其他类型的细胞，包括ER / PR +细胞，可以保持休眠长达20年，复发10的稳定，连续速率。而在ER +和ER-乳腺癌细胞系和肿瘤之间休眠的 ​​基因表达签名的差异反映了不同的休眠潜力11，与骨髓基质相互作用可能代表到休眠一个显著的贡献。 休眠体内的研究是非常困难的，因为微是罕见的，是由寡不敌众造血细胞超过10倍6。因此，相关模型必须产生提供体外数据可以建议机制和产生体内检验的假设。一些休眠模式，包括MATHEmatical车型12,13， 在体外模型7,8,14,15， 体内异种移植模型16， 在体外组合和异种移植模型17,18和自发肿瘤和转移模型19，已经取得了一些见解，癌细胞休眠20 。这些模型有其自身的局限性和本身用于产生有关的分子信号和支配休眠到在多种生物相关模型中进行测试的相互作用的假设主要是有用的。 界定休眠的 ​​分子机制，以导致在周期停滞，再分化和治疗抗性和机制的微环境造成复发的ER +细胞的相互作用的总体目标，我们开发了提供选择的相关内容的体外模型基质微环境7。这种模式，而在其康波相对稀疏堂费，是足够坚固，以​​允许研究者得出影响休眠显著职能特定的分子机制。这些实验产生可在体内直接测试假设。该模型依赖于我们证明是有关休眠中的几个关键要素。它们包括在一个克隆的密度，其中它们的相互作用是主要与基质和介质的可溶性组分，纤连蛋白基质和碱性成纤维细胞生长因子的存在下，用雌激素 - 依赖的乳腺癌细胞中，细胞培养物（FGF-2）在介质中。 我们特征在于支配系统在体外机制，包括诱导细胞周期阻滞由FGF-2的21中 ，通过TGFβ22介导的，生存通过PI3激酶7,8和ERK 8和形态发生分化信令上皮表型，这依赖于RhoA的失活，整45;5β1上调和间质纤维连接蛋白生存7,15结扎（ 图1）。 FGF-2对MCF-7细胞的体外细胞周期的影响开始在浓度至少一个对数低于10毫微克/毫升21,23。基本原理是基于FGF-2的表达治乳腺导管形态，循环扩张和衰退在许多哺乳动物系统24-27的时间控制。我们证明了FGF-2的诱导分化，包括导管形态发生在3D培养28，而FGF-2的表达通常失去了与人类肿瘤29的恶变。 FGR1的表达仍然完好无损调查中的29和MCF-7细胞乳腺癌继续对所有4 FGF受体30。在休眠的 ​​上下文中，FGF-2是由出口和重沉积在骨髓基质31,32在那里它在造血干细胞33的保存功能。我们DEMonstrated的FGF-2诱导ER +乳腺癌细胞纤维连接蛋白基质，还含有丰富的骨髓，它诱导分化形态培养7休眠状态。在模型中，乳腺癌细胞的生长抑制，灭活的Rho一种贯穿RhoGap GRAF，再分化到上皮表型和重新表达整联α5β1lost与恶性进展。它们结合纤连蛋白通过整合素α5β1和激活存活信号使得它们对细胞毒药物治疗7,8,15（ 图1）的抵抗力。卢的GTP酶类的抑制先前已证明诱导休眠型34。 在这里，我们将概述的具体程序，将允许调查人员建立模型，并研究规范ER +乳腺癌细胞的休眠特异的分子和细胞机制。在这里介绍的实验来说明使用该模型的我们靶向PI3K途径（ 图1B）与Akt的抑制剂和PI3K抑制剂和Rho家族（ 图1B）与泛Rho抑制剂和作为Rho激酶（ROCK）抑制剂的所有成员。

<table><tbody><tr><td>MCF-7 cells</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>T47D cells&amp;nbsp;</td><td>ATCC</td><td>HTB-123</td><td></td></tr><tr><td>BD BioCoat Fibronectin 24 Well Clear Flat Bottom TC-Treated Multiwell Plate</td><td>Corning</td><td>354411</td><td></td></tr><tr><td>BD BioCoat Fibronectin 60 mm Culture Dishes</td><td>Corning</td><td>354403</td><td></td></tr><tr><td>BD BioCoat Fibronectin 100 mm Culture Dishes</td><td>Corning</td><td>354451</td><td></td></tr><tr><td>BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin</td><td>Corning</td><td>354088</td><td></td></tr><tr><td>6 Well tissue culture plate</td><td>CellTreat</td><td>229106</td><td></td></tr><tr><td>Dulbecco Modified Eagle Medium High Glucose 10X Powder</td><td>Corning Life Sciences</td><td>50-013-PB</td><td></td></tr><tr><td>Heat Inactivated, Fetal Bovine Serum</td><td>Serum Source International</td><td>FB02-500HI</td><td></td></tr><tr><td>0.25% Trypsin/2.21 mM EDTA</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution, 100X</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>L-Glutamine, 100x, Liquid</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>Recombinant Human FGF&amp;nbsp;basic</td><td>R&amp;amp;D Systems</td><td>234-FSE-025</td><td></td></tr><tr><td>Akt Inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate)</td><td>CalBiochem</td><td>124005</td><td></td></tr><tr><td>LY294002&amp;nbsp;</td><td>CalBiochem</td><td>19-142</td><td>Chenical PI3K inhibitor</td></tr><tr><td>C3 transferase&amp;nbsp;</td><td>Cytoskeleton</td><td>CTO3</td><td>Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1</td></tr><tr><td>Y-27632 dihydrochloride&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>129830-28-2</td><td></td></tr><tr><td>BODIPY FL-Phallacidin (green)&amp;nbsp;</td><td>Molecular Probes</td><td>B607</td><td>Fluorochrome for fibrillar actin staining</td></tr><tr><td>BODIPY FL-Rhodamine phalloidin (red)&amp;nbsp;</td><td>Molecular Probes</td><td>R415</td><td>Fluorochrome for fibrillar actin staining</td></tr><tr><td>Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent&amp;nbsp;</td><td>Molecular Probes</td><td>R37114</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI&amp;nbsp;</td><td>Molecular Probes</td><td>P-36931</td><td></td></tr></tbody></table>

an in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: a tool for molecular mechanism studies and hypothesis generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

<html>进行实验以概括的测定。实验的时间过程示于图2A中 。将细胞培养在克隆密度上-1天，FGF-2在新鲜的培养基中加入第0天和细胞进行培养，直到第6天，当它们被染色和菌落计数。任何扰动的系统都在100μl体积在10倍终浓度施用在第3天所需的。 图2B说明了生长和休眠菌落的典型外观。生长的菌落含有&gt; 30个细胞和休眠的 ​​殖民地包含12个或更少的细胞，其许多倍的大?...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

一个<em&gt;在体外</em&gt;对雌激素敏感的乳腺癌休眠模式在骨髓中:分子机理研究与假设生成的一个工具

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

8.2K 次观看.  Rutgers New Jersey Medical School. We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo. 

视频: An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

科研

癌症研究

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

医学

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 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

乳腺癌临床前模型乳腺肿瘤的矫形移植

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

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 ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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摘要

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此视频中的章节

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

一个系统来研究肿瘤休眠和转移性增长

骨肉瘤的三维骨细胞外基质模型

骨转移模型

乳腺癌临床前模型乳腺肿瘤的矫形移植

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

在乳腺癌细胞雌激素调节小分子RNA的剖析

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方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

一个

摘要

探索更多视频

此视频中的章节

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

一个系统来研究肿瘤休眠和转移性增长

骨肉瘤的三维骨细胞外基质模型

骨转移模型

乳腺癌临床前模型乳腺肿瘤的矫形移植

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

在乳腺癌细胞雌激素调节小分子RNA的剖析

一个

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型