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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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Cellular Physiology. 177, 411-425 (1998).</li><li>Brunner, G., Gabrilove, J., Rifkin, D. B., Wilson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C+release+of+basic+fibroblast+growth+factor+from+human+bone+marrow+cultures+as+a+biologically+active+complex+with+a+phosphatidylinositol-anchored+heparan+sulfate+proteoglycan.">Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan.</a> J Cell Biol. 114 (6), 1275-1283 (1991).</li><li>Wilson, E. L., Rifkin, D. B., Kelly, F., Hannocks, M. J., Gabrilove, J. 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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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MCF-7 cells</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>T47D cells&#160;</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&#160;basic</td>
			<td>R&#38;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&#160;</td>
			<td>CalBiochem</td>
			<td>19-142</td>
			<td>Chenical PI3K inhibitor</td>
		</tr>
		<tr>
			<td>C3 transferase&#160;</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&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>129830-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY FL-Phallacidin (green)&#160;</td>
			<td>Molecular Probes</td>
			<td>B607</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>BODIPY FL-Rhodamine phalloidin (red)&#160;</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&#160;</td>
			<td>Molecular Probes</td>
			<td>R37114</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI&#160;</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 ...

an-vitro-dormancy-model-estrogen-sensitive-breast-cancer-bone-marrow

Research

JoVE Journal

Medicine

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

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 ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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 ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

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一个系统来研究肿瘤休眠和转移性增长