我们要认识到泰森食品公司慷慨地提供受精卵。莎娜阿诺德在本议定书的拍摄。帕尔默Trenis露丝属Kirschstein国家研究服务奖（F31的美国国家癌症研究所）是由美国国立卫生研究院的支持下。部分支持这项工作是由CCSRI格兰特＃700537 JDL和NIH / NCI授予＃CA120711 - 01A1和CA120711 - 01A1对AZ。

1。准备xenografting鸡蛋（自发转移） <ol><li>刚下岗的受精鸡蛋孵化在一个旋转的孵化器，在100 ° F和60％的湿度为10天。鸡蛋旋转四次，每一个小时。 </li><li>发育10天的鸡蛋都放在一个鸡蛋机架就在自己身边。一个鹅颈灯或其他合适的光源是用蜡烛鸡蛋的蛋壳射入airsac是位于蛋的钝端的光。 </li><li>位于绒毛尿囊静脉和标记。此静脉是位于顶部的蛋壳是几个大血管交界。在此分支点血管下降，离从CAM和重视的胚胎。一个1cm见方框用铅笔在蛋壳上绘制约1厘米远离静脉分支点。包括广场周围的区域，然后用棉签在碘浸泡清洗。 </li><li>使用一个碳化硅研磨石（DREMEL部分＃84922）是通过蛋的钝端钻一个洞到空气囊装有一个旋转刀具（DREMEL）。使用同样的工具，钻了一个洞以前绘制的正方形内的鸡蛋的顶部，阻止蛋壳膜。一个与上年底的罚款钻的25号注射器针头是用来做了一个小洞，在蛋壳膜，小心不要撕破底层的CAM。 CAM是后来脱离蛋壳膜平方米面积的使用温和的吸力创建具有自动吸液管对孔放置在airsac ¼英寸聚乙烯管件与安装的援助。申请吸后，气囊应该象征方孔下方的CAM已成功删除从蛋壳。凸轮下降后，在气囊和位于广场附近的绒毛尿囊静脉孔孔是密封的一块磁带的实验室磁带。卵随后放置在100 ° F和60％的湿度预期嫁接的肿瘤细胞，成一个固定的孵化器。顺序描述见图1。 </li></ol> 2。准备用于静脉注射的鸡蛋（实验转移） <ol><li>刚下岗的受精鸡蛋孵化12天，在100 ° F和60％的湿度在一个旋转的孵化器。鸡蛋旋转四次，每一个小时。 </li><li>发育天12的鸡蛋都放在一个鸡蛋机架就在自己身边。一个鹅颈灯或其他合适的光源是用蜡烛鸡蛋的蛋壳射入airsac是位于蛋的钝端的光。 </li><li>位于绒毛尿囊静脉和标记。 1厘米是一个0.5厘米的矩形绘制蛋壳直接静脉以上的铅笔。包括广场周围的区域，然后用棉签在碘浸泡清洗。 </li><li>创建一个小窗口，在使用Dremmel钻头（＃118）在绘制的位置蛋壳。在删除该窗口，露出底层的蛋壳膜，随后呈现透明的矿物油下降。见一个图形的描绘图5A。 </li><li>此时空气囊和位于广场附近的绒毛尿囊静脉孔孔是密封的一块磁带的实验室磁带。卵随后放置在100 ° F和湿度60％，预计在注射肿瘤细胞成一个固定的孵化器。 </li></ol> 3。准备嫁接或注射的肿瘤细胞。 <ol><li> xenografting，肿瘤细胞脱离他们的培养皿中，用胰蛋白酶/ EDTA，洗净，10卷的磷酸盐缓冲液（PBS），以除去残留的媒体，胰蛋白酶，或EDTA。 </li><li>静脉注射，分离培养的细胞用非酶细胞分离解决方案，并与无血清DMEM洗。和） </li><li>细胞计数与hemacytometer为10-40万细胞/ ml的PBS重悬嫁接，或者在无血清DMEM悬浮在1万个细胞/毫升静脉注射。 </li></ol> 4。接枝的新暴露的CAM（自发转移）肿瘤细胞<ol><li>鸡蛋是从孵化器中删除和以前绘制的广场，在那里已形成新的空气袋的位置切一个小窗口。这是最容易与配备一个切割轮（＃409圆切割轮的Dremel工具）旋转工具。尖嘴钳对用于去除蛋壳的一小部分，揭露底层凸轮。 </li><li>使用棉签（费舍尔品牌＃23-400-001）凸轮磨损的5倍。对棉花，一点血应该是明显。立即损害的CAM后，细胞悬液25μl放置到受损区域。最佳的细胞数量是确定发送mpirically但可以范围从0.5X10 5到1x10 6。鸡蛋中的窗口，是用胶带封紧，小鸡离开站立10-15分钟，为了让定居的细胞。鸡蛋15分钟后返回到静止的孵化器。 </li><li>肿瘤是可以成长为5-8天，具体应用和性质的肿瘤细胞用线不同而有差异。顺序描述见图1。 </li></ol> 5。静脉注射肿瘤细胞（实验转移） <ol><li>使用30号胰岛素注射器100μL细胞悬液注入到每个胚胎的尿囊静脉，鸡蛋被退回到一个固定的孵化器，仍然有一个额外的1-7天。 </li><li>在不同的时间点，肺或较低的CAM是从每个胚胎收获和处理的肿瘤细胞的检测，如下所述。 </li></ol> 6。收获肿瘤和雏鸡组织<ol><li>准备一个夹层面积。该试剂盒检测通过使用一个高度敏感的PCR方法定量分析人类的DNA转移细胞。因此，这是非常重要的，以防止外源DNA的污染。在一个专门的区域，并进行解剖，整个解剖的工具清洗在每个动物之间。为了防止您的样品交叉污染，整个解剖过程中的3套独立的工具是:一切蛋，删除除去内脏器官的原发灶和一组一组的。此外，四个洗容器用于连续冲洗每个动物之间的工具。在这些清洗顺序是:1）双蒸水，2）双蒸水，3）含氯漂白剂，以及4）70％的乙醇。 </li><li>鸡蛋是从固定的孵化器，并置于冰上15分钟，麻醉和安乐死的动物。窗户打开，这样的肿瘤变得可见。打开窗户删除一些蛋壳更大可能是必要的的。从CAM切除原发肿瘤，称重，组织学处理。 </li><li>从小鸡的蛋壳，切成相等的半壳径向。倒出从胚胎切割外壳，并转移到一个干净的权衡船一侧乳房上来。动物解剖用新鲜，清洁工具。动物是通过胸骨切割开。一旦胚胎是开放的，一个肝片，从左边或右边的叶收集。轻轻拉动连接到肫食道和切割采钰，连接机关内部器官随后被删除。为了收集肺切肋骨打开乳房板分离和心脏被删除。在这一点上，可能减少对肺周围的四个边（最接近头顶部沿左肋，附近的隔膜和沿气管右侧底部的左边）。为了一致性，相同的肺是收集在每个动物。采伐的组织样本保存在-20 ° C，以备以后使用，可以立即处理DNA提取。 </li></ol> 7。基因组DNA提取和定量PCR分析<ol><li>基因组DNA提取使用SYBR GREEN提取物 - N放大器组织PCR试剂盒（Sigma - Aldrich公司目录＃XNATRG）的组织。提取的DNA可以被转移到一个干净的Eppendorf管中，并储存在-20 ° C，或立即使用。重要的是要在核酸自由水稀释前，在随后的PCR反应中使用的DNA 1:50。另外，DNA可以提取使用任何标准DNA提取协议（泽吉尔斯达等 ，2002）。 </li><li>人类DNA检测小鸡采用定量PCR技术对人类铝序列的具体组织。定量PCR使用ALU -特异性引物，从Exract N -放大器套件使用SYBR GREEN放大。 PCR试剂盒配备了一个2倍的反应，含有SYBR GREEN，缓冲区，盐dNTPS，Taq DNA聚合酶和Jumpstart的Taq酶抗体混合。反应混合物在终体积为15μl，引物各0.4μM。然而，对于大多数情况下，使用提取的DNA稀释1:50将提供定量数据，模板DNA的最佳数量可能需要优化经验。鸡GAPDH的引物用于PCR是在下列条件下运行，用于ALU序列作为内部控制:聚合酶激活在95 ° C为2分30个循环，在95 ° C为30秒，63 ° C第30 72 °秒，持续30℃鸡GAPDH的反应条件是相同的，用于ALU中所描述的，但是可能有必要延长40个循环的PCR。 </li><li>转移定量分析比较实验组，对照组（Schmittgen和Livak，2008;使用ΔΔCT方法执行。泽吉尔斯达等 ，2002）。或者使用人类肿瘤细胞提取使用提取N -放大器套件（泽吉尔斯达等 ，2002）的系列稀释，可以生成标准曲线。统计的意义在于评估使用学生t检验或方差分析，对照组和实验组。 </li></ol> 8。代表性的成果: <img src="/files/ftp_upload/2815/2815fig1.jpg" alt="图1"/> 图1。鸡胚转移模型。 A）四个面板图说明了该模型的的关键步骤，在横截面的外观: 1。经过10天的潜伏期。 CAM已达到接近最大尺寸。对鸡蛋的顶部位置的尿囊静脉动脉和静脉明显可见。 2。后立即鸡蛋在蛋壳上钻两个孔:到airsac和一个相邻的尿囊静脉的附着点。 3。后一种轻微的真空外观已用于疏散airsac和下降的CAM。肿瘤细胞（蓝色）适用于下降的CAM。 4。使嫁接的肿瘤细胞生长后，连同从鸡胚胎组织肿瘤的收获进行分析B）模型的序列描述: 1。孵蛋，2。这标志着尿囊静脉的位置，3。在蛋壳上钻洞，4。滴药凸轮，5。切割肿瘤嫁接细胞，6开幕。将肿瘤细胞，7。在冰上冷却鸡蛋事先收集组织，8。收获后肿瘤生长7天。 <img src="/files/ftp_upload/2815/2815fig2.jpg" alt="图2"/> 图2。CAM的肿瘤组织学。从荷瘤绒毛尿囊膜组织被固定在福尔马林，切片，与三色染色处理。一）HEp3肿瘤后7天在CAM的增长。 B和C显示放大显示肿瘤和基质地区。 D）法线的CAM。 <img src="/files/ftp_upload/2815/2815fig3.jpg" alt="图3"/> 图3。纵向分析定量转移 。高或低的转移能力（HEp3 M（高）和HEp3 M（低）分别）的头部和颈部癌HEp3的变种进行了分析，他们有能力传播使用铝PCR。十大动物进行了分析，在每个时间点为7天的时间。对这些样本进行了量化的标准曲线，并随后表示＃cells/50mg组织。 <img src="/files/ftp_upload/2815/2815fig4.jpg" alt="图4"/> 图4。在动物体内自发转移细胞的抑制与转移抑制抗体1A5的治疗。使用5X10 5到10天的胚胎CAM应用的HEp3细胞形成肿瘤。申请一半的动物治疗的肿瘤细胞与抗CD151抗体1A5后三天。从荷瘤动​​物的肺，两天后收获和实时定量ALU PCR扩增。相反实时定量PCR的chGAPDH被用来确认相当于宿主基因组DNA的数量（ΔΔCT方法被用于定量比较两组（二）存在。 <img src="/files/ftp_upload/2815/2815fig5.jpg" alt="图5"/> 图5。在转移性级联评估逮捕，生长和血管内 。从根本上说，转移到次要器官的定义是由三个方面的贡献:逮捕，生长速度，血管内率。这些可以量化的实验转移（A）和自发转移（二）结合使用。使用实验转移（a）逮捕定义为细胞数量检测注射后2小时。被捕细胞的生存和发展确定为细胞数量的变化24小时后注射。被定义为在天3-7扩散率增长。血管内确定使用（B）在intravasated细胞的数量是7天的转移负担，目前，超过该值由目前的第6天的转移性负担的增长预测中定义的自发转移。 <img src="/files/ftp_upload/2815/2815fig6.jpg" alt="图6"/> 图6。 CAM中的可视化扩张的转移 。 HEp3细胞表达GFP发育12天的静脉注射。在第1天，3和5，一个鸡蛋被牺牲的CAM部分是用荧光体视显微镜成像。创建的深绿色背景是由蛋壳，血管可见的黑线，和肿瘤细胞可见的明亮的绿色斑点。

<ol>
	<li>Ghiso, A. g. u. i. r. r. e., Kovalski, J. A., K, ., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+dormancy+induced+by+downregulation+of+urokinase+receptor+in+human+carcinoma+involves+integrin+and+MAPK+signaling.">Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signaling.</a> J Cell Biol. 147, 89-104 (1999).</li><li>Chambers, A. F., Shafir, R., Ling, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+system+for+studying+metastasis+using+the+embryonic+chick.">A model system for studying metastasis using the embryonic chick.</a> Cancer research. 42, 4018-4025 (1982).</li><li>Chambers, A. F., Wilson, S. M., Tuck, A. B., Denhardt, G. H., Cairncross, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+metastatic+properties+of+a+variety+of+mouse,+rat,+and+human+cells+in+assays+in+nude+mice+and+chick+embryos.">Comparison of metastatic properties of a variety of mouse, rat, and human cells in assays in nude mice and chick embryos.</a> In Vivo. 4, 215-219 (1990).</li><li>Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+effect+of+matrix+metalloproteinase+down-regulation+on+vascular+intravasation+and+metastasis+of+human+fibrosarcoma+cells+selected+in+vivo+for+high+rates+of+dissemination.">Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination.</a> Cancer Res. 65, 10959-10969 (2005).</li><li>Hahn-Dantona, E., Ramos-DeSimone, N., Sipley, J., Nagase, H., French, D. L., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+proMMP-9+by+a+plasmin/MMP-3+cascade+in+a+tumor+cell+model.+Regulation+by+tissue+inhibitors+of+metalloproteinases.">Activation of proMMP-9 by a plasmin/MMP-3 cascade in a tumor cell model. Regulation by tissue inhibitors of metalloproteinases.</a> Ann N Y Acad Sci. 878, 372-387 (1999).</li><li>Kim, J., Yu, W., Kovalski, K., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+specific+proteases+in+cancer+cell+intravasation+as+revealed+by+a+novel+semiquantitative+PCR-based+assay.">Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay.</a> Cell. 94, 353-362 (1998).</li><li>Leong, H. S., Steinmetz, N. F., Ablack, A., Destito, G., Zijlstra, A., Stuhlmann, H., Manchester, M., Lewis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+embryonic+and+tumor+neovasculature+using+viral+nanoparticles.">Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles.</a> Nat Protoc. 5, 1406-1417 (2010).</li><li>Lewis, J. D., Destito, G., Zijlstra, A., Gonzalez, M. J., Quigley, J. P., Manchester, M., Stuhlmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+nanoparticles+as+tools+for+intravital+vascular+imaging.">Viral nanoparticles as tools for intravital vascular imaging.</a> Nat Med. 12, 354-360 (2006).</li><li>Ossowski, L., Reich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+malignant+phenotype+of+a+human+carcinoma+conditioned+by+growth+environment.">Changes in malignant phenotype of a human carcinoma conditioned by growth environment.</a> Cell. 33, 323-333 (1983).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nat Protoc. 3, 1101-1108 (2008).</li><li>Testa, J. E., Brooks, P. C., Lin, J. M., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+expression+cloning+with+an+antimetastatic+monoclonal+antibody+identifies+a+tetraspanin+(PETA-3/CD151)+as+an+effector+of+human+tumor+cell+migration+and+metastasis.">Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis.</a> Cancer Res. 59, 3812-3820 (1999).</li><li>Zijlstra, A., Lewis, J., Degryse, B., Stuhlmann, H., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Inhibition+of+Tumor+Cell+Intravasation+and+Subsequent+Metastasis+via+Regulation+of+In+Vivo+Tumor+Cell+Motility+by+the+Tetraspanin+CD151.">The Inhibition of Tumor Cell Intravasation and Subsequent Metastasis via Regulation of In Vivo Tumor Cell Motility by the Tetraspanin CD151.</a> Cancer Cell. 13, 221-234 (2008).</li><li>Zijlstra, A., Mellor, R., Panzarella, G., Aimes, R. T., Hooper, J. D., Marchenko, N. D., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+rate-limiting+steps+in+the+metastatic+cascade+using+human-specific+real-time+polymerase+chain+reaction.">A quantitative analysis of rate-limiting steps in the metastatic cascade using human-specific real-time polymerase chain reaction.</a> Cancer Res. 62, 7083-7092 (2002).</li></ol>

鸡胚CAM已使用了几十年，作为一个模型系统，研究各方面的癌症生物学和转移性进展。原则上转移能力的评估已经在这个模型中进行的各种团体，包括休眠分析（阿吉雷Ghiso等，1999; Ossowski和帝国，1983年），组织重塑 （Deryugina等，2005; 。哈恩Dantona 等 ，1999），和转移（泽吉尔斯达等 ，2008;泽吉尔斯达等 ，2002）。它仍然是一个非常有吸引力的癌症生物学的，因为其易用性，它能够接受移植瘤模型，和其成本低（钱伯斯等人，1982年，钱伯斯等，1990） 。。使用人类特定的基于ALU -定量PCR（Kim等，1998;。 泽吉尔斯达等，2002），我们表现出的能力，分析转移性传播人类cpidermoid细胞株HEp3（图3 ）。此外使用转移抑制抗体1A5（图4，（特斯塔等 ，1999年。泽吉尔斯达等 ，2008）。）我们展示了这项技术的效用在抑制治疗的评价。 除了 ​​能够评估转移能力和治疗反应，这种模式也可以用于定量研究的转移级联（泽吉尔斯达等 ，2002）的各个组件。使用静脉注射肿瘤细胞转移的实验，这种模式允许分析癌症细胞在二级机关逮捕，并随后将其增长（图5a）。这样的增长速度，可以被用来预测在一个纵向自发转移实验的转移负担（图5b）。血管内确定使用自发转移（B）在intravasated细胞的数量是转移性负担目前7天减6天的转移性负担目前的增长预测值由定义。 随着技术的延伸报道，在这里，我们最近开发的新的战略，以可视化肿瘤细胞在体内的行为（泽吉尔斯达等2008年），它使用荧光标记的纳米粒子的目标肿瘤微环境 （丰隆等，2010; Lewis等人 ，2006年）。通过这些推进的方法，这一模式的效用可以利用可视化的复杂和动态变化，支持的生存，建立和肿瘤细胞的生长。这些技术，再加上成本相对较低，免疫缺陷性，易用性和鸡胚CAM的适应性，使这种模式成像和定量分析癌转移的宝贵。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> 1X贝科改良Eagle培养基（DMEM） </td><td> Invitrogen公司</td><td> 11995073 </td><td></td></tr><tr><td>贝科的磷酸盐缓冲生理盐水（D - PBS）（1X），液体</td><td> Invitrogen公司</td><td> 14190250 </td><td> pH值7.4 </td></tr><tr><td>胰蛋白酶，0.05％与EDTA 4Na（1X），液体</td><td> Invitrogen公司</td><td> 25300054 </td><td></td></tr><tr><td>运动员出雏</td><td>贝里山</td><td> 1550HA </td><td rowspan="2">这些孵化器是一些最容易获得的。但是，视频将表现出不同的设备。 </td></tr><tr><td>运动员孵化器</td><td>贝里山</td><td> 1502EA </td><td></td></tr><tr><td> DREMEL旋转工具</td><td> DREMEL </td><td></td><td></td></tr><tr><td> DREMEL截止车轮没有。 36 </td><td> DREMEL </td><td> 409 </td><td></td></tr><tr><td>便携式移液器援助</td><td>费舍尔</td><td> 1368115E </td><td></td></tr><tr><td>棉尖喷枪</td><td>费舍尔</td><td> 23-400-001） </td><td></td></tr><tr><td>受精卵</td><td>各种</td><td></td><td>可用于许多不同的供应商。鸡蛋应约35-55克。母鸡，规定他们应在36和55周龄之间。 </td></tr><tr><td>血球</td><td> Hausser科学，VWR </td><td> 15170-090 </td><td></td></tr><tr><td>光纤显微镜照明灯</td><td> Amscope </td><td> HL250 - AY </td><td> 150W </td></tr><tr><td> 25号针</td><td></td><td></td><td></td></tr><tr><td> SYBR绿色提取N -安培组织PCR试剂盒</td><td> Sigma - Aldrich公司</td><td> XNATRG </td><td></td></tr></tbody></table>

quantitative analysis of cancer metastasis using an avian embryo model

在转移的癌细胞从原发肿瘤的传播，侵入到周围组织，并扩散到远处器官。转移是一个复杂的过程，可能涉及到许多的组织类型，变量跨度的时间段，经常发生深刻的内部器官，使其难以进行调查和量化。此外，转移过程中的疗效的影响使其难以评估单一的肿瘤细胞行为方面的贡献，在转移级联的多个步骤。因此，经常进行转移检测在实验动物提供了一定的现实背景下，在其中学习转移。不幸的是，这些模型进一步复杂，其复杂的生理。鸡胚是一个独特的体内模型，克服了很多的局限性研究转移，由于绒毛尿囊膜（CAM），一个良好的血管多余的胚胎组织位于下面的蛋壳，是接受了肿瘤细胞的xenografting无障碍（图1）。此外，由于鸡胚是自然免疫缺陷，CAM容易支持正常和肿瘤组织植入。最重要的是，禽流感的CAM成功地支持大多数癌症细胞的特点，包括生长，侵袭，血管生成，和微环境重塑。这使得模型特别有用的调查导致癌转移的途径，并预测转移性癌的新的潜在疗法的反应。物种的具体铝PCR检测细胞播散，使人们有可能定量评估，是殖民统治只有25细胞的转移器官。使用人类的表皮细胞株（HEp3），我们使用这个模型来分析癌细胞的自发转移到远处器官，包括鸡胚肝脏和肺。此外，我们使用ALU - PCR协议表明，作为一种工具来分析和定量转移的单个元素的血管内，逮捕，外渗，和殖民的灵敏度和可重复性的检测。

Watch this Scientific Journal Video about Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model at JoVE.com

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

使用定量PCR技术，我们展示了如何建立鸡胚CAM模型可用于定量分析的人类肿瘤细胞转移到远处器官。

癌症转移，使用禽流感胚胎模型的定量分析

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

quantitative-analysis-of-cancer-metastasis-using-an-avian-embryo-model

Nanyang Technological University

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Unit 1

Cancer

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18.9K 次观看.  Vanderbilt University. 使用定量PCR技术，我们展示了如何建立鸡胚CAM模型可用于定量分析的人类肿瘤细胞转移到远处器官。

视频: Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

科研

医学

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

定量可视化和皮肤癌症使用动态热成像检测

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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 ...

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 ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

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 ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...