PAC是部分支持由美国国立卫生研究院T32授予大学的威斯康星干细胞的培训计划（PA克拉克）。 JSK部分支持的HEADRUSH脑肿瘤研究教授，罗杰LOFF纪念的GBM研究基金，和威斯康星大学基金会/神经外科脑肿瘤研究和教育基金。 JCW和YH部分支持由NIH资助NIBIB 1R01EB009103-01。

1。 BTSC的细胞分离标志物：BTSC来自生长在无血清干细胞培养基中作为神经球的预先存在的文化。文化其中预先描述10，11。 <ol><li>准备细胞悬液从BTSC衍生的神经球。 BTSC-衍生的神经球被收集在一个15毫升锥形管，离心5分钟，在900 rpm的转速下。更快的离心剪切和/或损害的神经球。吸去上清液和的神经球的文化再悬浮在0.5毫升预热的Accutase。孵育5-10分钟，此溶液在37℃下使松开的神经球。机械破碎细胞，用10-20温柔笔划的P100移液器1.5毫升干细胞培养基，然后加入到细胞中的Accutase。 </li><li>的标志物：BTSC在1300 rpm离心5分钟，然后离心，并再悬浮在1毫升干细胞培养基。 </li><li> A 50微升等分试样从suspens删除离子和放置到一个离心管中，进行细胞计数。 50μl的台盼蓝的Eppendorf管中，并添加到该悬浮液被放置在血细胞计数器进行细胞计数。 </li><li>细胞计数后，如果需要，额外的干细胞培养基中加入的BTSC悬浮液，得到细胞密度为20000个活细胞/微升媒体。 </li></ol> 2。双向层状苏-8主站和PDMS印章成型（参见图1）的制备我们使用光学光刻和软光刻技术制作SU-8主站和PDMS印章，这是必不可少的微流体装置的装配。稍有不同的标准程序12，SU-8主是由两层组成。 3微米高的微通道浇铸在第一/底部层前面的过程中，而250微米高的播种和接收腔中的第二/上层。为了两个文化室（微之间的连接是否正确s和腔室），这两层都必须准确地对准的位置。然而，第二层的厚度和不透明度是大到足以阻止基准/光学对准器从访问的底部功能。在这里，我们设计的面具与基准标记远程定位。因此，在第一层中的这些标记物可以被有选择地屏蔽，而第二层的纺丝。其结果是，两个功能层是由在一个主站和准备用于模制在浮雕的PDMS印章。 <ol><li>设计和准备两张照片口罩。一个掩模由两个基准标记和用于第一层的微通道的阵列（长400微米，宽和10-50微米）。另一个掩模由播种和接收用于所述第二层的腔室（2毫米长和宽为600μm）。这两个掩模设计在Illustrator（Adobe公司，加利福尼亚州），激光印刷到透明薄膜（薄金属零件，科罗拉多州），用异丙醇和空气干燥清洗。 </li><li>使第一层与SU-8的光刻胶（MicroChem，马萨诸塞州）根据供应商的产品使用说明书。首先，旋涂的手柄的su-8（5），与3-μm厚的光致抗蚀剂的晶片（WRS材料，加利福尼亚），然后通过软烘烤。然后，光致抗蚀剂是紫外线曝光和固化后烘烤和显影，接着与相应的掩模紧密接触。 </li><li>旋涂用250微米厚的苏-8（2100）的第二层。为了防止厚的光致抗蚀剂的阻塞状态，从第一层的基准标记时，我们用透明胶带覆盖这些区域之前，所述第二层的旋涂。磁带，然后剥离，以显示标记的对准目的。 </li><li>紫外线暴露所述第二层与所述第二掩模。的对准标记显示，它是直接将它们对齐在使用光掩模对准第二掩模的。第二层的光致抗蚀剂，然后紫外线曝光和固化在紧密接触，然后由后烘烤和显影。/ P&gt; <li>抗粘涂层，导致主。为了帮助去除固化的PDMS，SU-8主可以通过气相沉积，使表面不沾涂层的硅烷化处理。简单地将其放置到一个干燥器中的至少一个小时，与几滴三氯（1H，1H，2H，2H-全氟辛基）硅烷的溶液在真空下。 </li><li>模具PDMS主。混合预聚物基地和的PDMS SYLGARD的184（道康宁，密歇根州）的比例为10:1彻底治疗者。将其放置在干燥器真空脱气看到，直到没有气泡。混合物倒入的面具，脱气，如果再引入新的气泡。固化在模具上的水平加热板上于90℃2小时，冷却至室温后，轻轻放开PDMS印章。因此，培养渠道刻在PDMS，并准备好组装的设备。主是可重复使用的成型，直到它被损坏或磨损。抗粘涂层，需要时，再次进行的粘性恢复。 </li></ol> 3。大会的微流体装置和细胞培养（图2） 为了培养细胞，特征刻PDMS印章附着的玻璃盖玻片上，以形成封闭的通道。入口和出口被创建加载文化/媒体。同时，清理和其他程序，以在玻璃衬底和PDMS印章是必要的，以确保细胞兼容性。 <ol><li>水库通过PDMS印章使用活检打孔器。我们选择其直径为6毫米。这些水库有两个目的服务。一是要保持额外的营养细胞的生长。另一种是移液器装载和真空抽吸的访问。 </li><li> PDMS印章在70％的乙醇浸泡30分钟，然后用清水冲洗用去离子水，再过10分钟。其目的是为了清除潜在的有机残留物，介绍了在制造过程期间，和未交联的聚二甲基硅氧烷污染。更激烈和彻底的清洗过程，如有机提取13 </sup&gt;和索氏提取14用于其他地方。然而，我们发现是BTSC文化的不必要的。 </li><li>消毒用高压釜（121℃，35分钟）的PDMS印章。这一步完成的聚二甲基硅氧烷交联反应。从这个步骤开始，直到步骤3.6中，所有的程序都是在无菌方式。 </li><li> PDMS印章然后放在玻璃盖玻片，预先涂有聚-L-赖氨酸，然后15-17的移动设备与层粘连蛋白涂覆填充与层粘连蛋白的溶液中过夜，在37℃下的信道层粘连蛋白被吸入从设备，该设备是用干细胞培养基。 </li><li>将细胞悬液从第1步到水库和渠道。 11μl的游离细胞被放置在一个播种储层。使用真空抽吸到播种室迫使细胞，如果需要。增加7微升的细胞都放在的相邻的播种水库。注意：一般的细胞密度为20000细胞/微升媒体。船尾呃五分钟，播种和接收水库充斥着媒体。 </li><li>将一个0.5毫米的前顶部蒸压的PDMS表上。自然发生的表面粘附2 PDMS件之间的密封的细胞培养和准备运输，孵化和显微成像。 </li></ol> 4。长期时间的推移成像的BTSC迁移与BioStation IM（尼康仪器公司，梅尔维尔，USA），。 结合摄像头，在一个盒子里的所有软件和孵化器，这显微系统使图像细胞培养无干扰数天。此外，其独特的电机设计物镜的移动，并保持固定在样品台访问点功能。这使得它实际的图像并行实验和跟踪在高通量检测细胞运动。 <ol><li>预平衡的Biostation IM 45分钟以稳定的温度，水分和空气的供应。此外，一些水续ained菜肴样品室是用来保持适当的湿度。 </li><li>装入的移动设备中的细胞显微镜的样品室中，和中心使用微型镊子。 </li><li>的焦点，位置点和时间点Biostation软件设置和启动时间的推移。 </li></ol> 5。代表性的成果： 目视检查和表征的癌症细胞的迁移通过使用进行划分的微流体装置的实施例在图3和图4中示出。细胞线是BTSC。我们目前的体制，细胞培养相图像，可以连续记录长为5天（图3）和频繁，每2秒（图4）。超过5天的营养补充和废物排出，文化传媒需要更换。我们的长期时间的推移，在其迁移的形态变化的基础上，确定了循环序列的6个细胞的阶段。作为出版于trated在图3中，我们描述（一）迁移前，（二）开始，（三）路径探索，（IV）巡航，（V）的目标exloration和（六）迁移后。在接收室，梭形细胞停留在阶段（i）在大型的肿瘤，是无法生长，分裂和逐步迁移。他们的做法是微通道入口，一些细胞开始阶段（ii）当他们扩大并产生粘结突出。其中之一是能够占据的入口和进行阶段（iii），这可以探索的迁移方向。一旦细胞决定的迁移方向，它的收益阶段（四）通过微巡航的稳定和高速，这是在整个微。在结束阶段（V）接收室，探索开放式的空间，然后到舞台（六）微通道，细胞的收入。微通道中，迁移能力主要是产生通过出泡活性，如在图4中示出，类似于变形虫大公升。细胞皱缩和膜变形完全记录在这里。 <img src="/files/ftp_upload/3297/3297fig1.jpg" alt="图1" /> 图1的微流体设备的制造示意图。通过修改后的软光刻技术的整个过程进行了一块3英寸的硅处理晶片。作为在第一层是由3微米高的微通道和对准标记。然后250-μm厚的su-8是旋涂，而对准标记用透明胶带覆盖，使得它们可以购买访问屏蔽由厚的光刻胶不挡视线对准。因此，腔室的功能可以作为第二层，精确对准的第一层。 PDMS印章最终成型产生的主。 <img src="/files/ftp_upload/3297/3297fig2.jpg" alt="图2" /> 图2的移动设备的组装和细胞接种过程的示意图。随信附上的PDMS和gl屁股盖玻片，3D培养空间室，水库和微通道组成的。与细胞培养的播种室充满，而接收腔室中最初填以无细胞介质。微通道，它们之间的连接提供细胞迁移的足迹从播种端至接收端。 <img src="/files/ftp_upload/3297/3297fig3.jpg" alt="图3" /> 图3。BTSC通过微转移。上部：快照的时间推移示出超过400微米的迁移从播种侧到接收侧的单个单元格（高亮显示为绿色）。整个行程大约需要2天，涉及一系列的细胞形态学变化。比例尺是40微米。下：六个迁移阶段的卡通表示。预迁移（ⅰ）：在播种室，纺锤状和可分割的细胞逐渐沿相互迁移。微通道入口附近种族相互细胞，分化细胞体和电子<html> xerting片状伪足突起。启动（二）：最高容量的迁移细胞占据了通道入口，而其竞争对手将撤退。路径的探索（三）：小极性的变形虫模式的迁移细胞的变化。这种迁移方式非常能动的，并能在各个方向上的探索路径，皱缩的小膜突起。巡航（ⅳ）：细胞的迁移路径一旦被确定，变换成一个适于变形虫模式维持一个额外的大突起标题向前。通过这种方式，细胞坚持高的蠕动，以及作为一个稳定的方向和速度。目的地探索（V）：在路径减慢，细胞和，探索接收室的任何侵略目标发展filopodias。迁移后（ⅵ）：进入接收腔室后，细胞变成星形和保持高的蠕动，但没有确定的方向。 “/&gt; 图4。快照时间推移显示出泡一个BTSC活性和膜的变形：（AB）的周期。开始（BD）。扩展;（DF）。回缩。和曲线线的箭头表示的方向和位置的膜变形，分别。快照收集每8秒。

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432, 396-401 (2004).</li><li>Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+stem+cells+derived+from+glioblastomas+cultured+in+bFGF+and+EGF+more+closely+mirror+the+phenotype+and+genotype+of+primary+tumors+than+do+serum-cultured+cell+lines.">Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines.</a> Cancer Cell. 9, 391-403 (2006).</li><li>Galli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Research. 64, 7011-7021 (2004).</li></ol>

没有利益冲突的声明。

这里提出的微流体装置和图像记录技术使细胞形态在迁移过程中的可视化表征。微流体平台与现有的传统方法相比，具有成本效益，高通量和设计灵活性的优点。所提出的微观可视化系统允许长期活细胞迁移的研究和记录。镜头机动特性使得它可以跟踪多个在一个较高的图像分辨率，而不会干扰样品的迁移路径。 在组装过程中的设备，PDMS印章非永久地结合到为方便的PDL涂层（步骤3.4）的玻璃盖玻片。本协议的成功是至关重要的，以实现良好的密封。的器件制造中，如在所述基板上的邮票或粉尘/碎屑的气泡，从引入的污染可以危及流体泄漏的接合和结果。因此，TREATI使用设备无灰尘的方式，重要的是要防止设备故障。在此之前到的PDL涂覆，玻璃盖玻片硝酸处理的PDL溶液离心以除去灰尘/碎屑。我们发现，透明胶带，酒精冲洗，水浴除尘/ PDMS印章的碎片非常有帮助的，如果一个干净的房间是无法访问的。 PDMS印章制作的玻璃盖玻片接触之后，用镊子轻轻地拍打邮票便于密封。 标志物：BTSC可以通过球体培养干细胞培养基中，类似正常的神经干细胞培养人体标本手术室丰富。以这种方式展示标志物：BTSC丰富的干细胞样特性，如表达干细胞标记物（CD133，巢），分化为多种神经系（胶质细胞和神经元），在原位免疫缺陷小鼠模型中肿瘤的发生数为100细胞18。尽管存在一些争论，提纯阳离子和维护BTSCs 1，19-21，球种标志物：BTSC更好地维护的表型和基因型的亲肿瘤22-24。 隔离的空间模拟细胞迁移的生理环境。在我们的设备，BTSC表现出强烈的迁移能力，通过一个尺寸有限的空间中，通过调节细胞的形态。我们的细胞形态学变化的特性显示模式转换中的6个的阶段序列可能模拟一个可能的新的详细说明的BTSC入侵邻近脑。在早期阶段，细胞获得了显着量的极性，并生成有效地锚定自己的微通道内的粘合剂突起。一旦细胞在微占用成立的，转换的巡航模式，并通过微全程保持这种模式。在这个阶段，BTSC保持较高的活力和一致的方向，但节省能源。在terestingly，它显示一个一个微通道的是，限制一个巡航细胞一次，这样，当一个单细胞进入到这个阶段，以及其他细胞从微通道撤退。这种机制可能确保肿瘤扩散的有效性，并防止过度的营养物质在微。在完成整个微迁移，细胞恢复其具有的多向的突起和进一步勘探入侵的目标和新的迁移路径。进行划分的微流体器件提供了一种新的体外手段，建立一个微环境为研究脑实质内的的BTSC渗透的。 这种方法可以很容易地适于癌干细胞（CSC）和洄游细胞株是来自于其他类型的肿瘤的研究。培养细胞的微流体装置，可以执行在任何现代生物学实验室]是配备的孵化器和组织培养的专业知识。配备了一个SU-8的主人，PDMS铸造和设备装配软光刻技术中的一些基本训练是可行的。此外，该平台还可以扩展其他应用程序的集成等功能模块，如梯度混合器，表面图案，流体控制和微电极。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Dulbecco改良的Eagle培养基（DMEM），高糖</td><td> Gibco / Invitrogen公司</td><td> 11965 </td><td>脑肿瘤干细胞（BTSC）文化用品</td></tr><tr><td>火腿的F12 </td><td> Gibco / Invitrogen公司</td><td> 31765 </td><td>脑肿瘤干细胞（BTSC）文化用品</td></tr><tr><td> B27的补充减去维生素A </td><td> Gibco / Invitrogen公司</td><td> 12587-010 </td><td>脑肿瘤干细胞（BTSC）文化用品</td></tr><tr><td>抗真菌抗生素（PSA）的</td><td> Gibco / Invitrogen公司</td><td> 15240 </td><td>脑肿瘤干细胞（BTSC）文化用品</td></tr><tr><td>表皮生长因子（EGF），人重组</td><td> Gibco / Invitrogen公司</td><td> PHG0313 &lt;/ TD&gt; <td>脑肿瘤干细胞（BTSC）文化用品</td></tr><tr><td>碱性成纤维细胞生长因子碱性成纤维细胞生长因子（bFGF），重组人</td><td> Gibco / Invitrogen公司</td><td> PHG0021 </td><td>脑肿瘤干细胞（BTSC）文化用品</td></tr><tr><td>肝素钠盐，从猪肠粘膜</td><td>西格玛</td><td> H1027-250KU </td><td>脑肿瘤干细胞（BTSC）文化用品</td></tr><tr><td>层粘连蛋白（天然鼠） </td><td> Gibco / Invitrogen公司</td><td> 23017-015 </td><td>脑肿瘤干细胞（BTSC）文化用品</td></tr><tr><td> Accutase </td><td>密理博</td><td> SCR005 </td><td>脑肿瘤干细胞（BTSC）文化用品</td></tr><tr><td>干细胞培养基</td><td></td><td></td><td><ul><li> ％火腿F12 30 </li><li> 70％DMEM </li><li> 1％抗生素 - 抗真菌剂</li><li>没有维生素A的2％B27 </li><li>表皮生长因子（20毫微克/毫升）和碱性成纤维细胞生长因子/肝素（20纳克/毫升碱性成纤维细胞生长因子，5毫克/毫升肝素）配合成分和使用前的无菌过滤器</li></ul></td></tr><tr><td>碱性成纤维细胞生长因子碱性成纤维细胞生长因子（bFGF）/肝素</td><td></td><td></td><td><ul><li>分装浓度为20微克/毫升</li><li> 25微克：5mg/mL的肝素加入1.25毫升25微克碱性成纤维细胞生长因子。 </li><li>对于250微克到250微克碱性成纤维细胞生长因子加入1 mL 20,10,5肝素。转移到15毫升锥形，并添加11.5毫升5毫克/毫升肝素为锥形。 </li><li>为50微升或250微升等分试样在-80℃下保存</li></ul></td></tr><tr><td>表皮生长因子（EGF） </td><td></td><td></td><td><ul><li>分装浓度为20微克/毫升</li><li>对于200微克至200微克EGF加入1 mL无菌DMEM（GIBCO 11965-118）。转移到15 mL锥形取9。 </li><li>对于500微克至200微克EGF加入1 mL无菌DMEM（GIBCO 11965-118）。转移到50 mL锥形，加入24毫升DMEM。 <li>存储为50和万亩“l”或“250微升等分试样在-80℃下</li></ul></td></tr><tr><td>肝素</td><td></td><td></td><td><ul><li>分装浓度为5毫克/毫升</li><li>称取100毫克肝素。 </li><li>加入100毫克肝素到20毫升（70％）的DMEM（30％）F12-（1％）的PSA（14毫升的DMEM，F12 6毫升，200μL的PSA） </li><li>无菌过滤器</li><li>存储为50微升或在-80℃下的250微升等分试样</li></ul></td></tr><tr><td> SU-8光刻胶</td><td> MicroChem </td><td></td><td></td></tr><tr><td>硅处理晶片</td><td> WRS材料</td><td>的3P01-5SSP-INV </td><td></td></tr><tr><td>三氯（1H，1H，2H，2H-全氟辛基）硅烷</td><td> Sigma-Aldrich公司</td><td> 448931 </td><td></td></tr><tr><td> PDMS SYLGARD 184 </td><td>道康宁</td><td></td><td></td></tr><tr><td>层粘连蛋白</td><td> BD生物科学</td><td></td><td> 50微克/毫升，在PBS缓冲液中的终浓度</td></tr><tr><td> Biostation IM </td><td>尼康仪器</td><td></td><td></td></tr></tbody></table>

evaluation of cancer stem cell migration using compartmentalizing microfluidic devices and live cell imaging

在过去的40年里，美国癌症研究上投入了超过200亿美元，导致死亡率只有5％的跌幅。改善患者的治疗效果的一个主要障碍是缺乏了解的机制与细胞迁移相关联的侵略性癌症细胞侵袭，转移和治疗性1。多形性胶质母细胞瘤（GBM），最常见的原发性恶性成人脑肿瘤2，充分体现了这个困难。尽管标准的手术，放疗和化疗，患者的中位生存期只有15个月，由于积极的GBM渗透到相邻的大脑和快速的癌症复发2。异常的细胞的的迁徙机制和肿瘤微环境的相互作用，有可能区分癌症从正常细胞3。因此，提高治疗方法的GBM需要更好地了解癌症细胞迁移机制。最近的工作表明THATA小亚群内GBM细胞，脑肿瘤干细胞（BTSC），可能是治疗和复发性负责。 BTSC迁移能力的机制才刚刚开始特点1,4。 由于在目视检查和几何操作，常规迁移试验的限制仅限于量化整个细胞群。相比之下，微流体器件允许单细胞分析的，因为兼容性与现代显微镜和控制微环境6-9。 我们提出了详细的表征的BTSC移民进行划分的微流体装置的方法。这些PDMS国产设备投的组织文化分为三个相连的车厢环境：播种室，接收室和桥接微流。我们量身定制的设备，如两院保持足够的媒体支持可行的BTSC 4-5大YS没有媒体交流。最初引入到直播室的高度移动的标志物：BTSC被隔离后，虽然桥接微流的并行接收室的迁移。这种迁移模拟癌症细胞扩散的大脑通过的间隙空间。在迁移过程中细胞形态的相位实时图像，记录了好几天。高度洄游BTSC，因此是孤立的，再培养，并作进一步分析。 进行划分的微流体可以是一个多功能的平台，研究的标志物：BTSC和其他癌症干细胞的迁移行为。通过结合梯度发电机，流体处理，微电极和其他微流体模块，这些设备也可以被用于药物筛选和疾病的诊断6。积极的游走细胞亚群的分离，使潜在的分子机制的研究。

Watch this Scientific Journal Video about Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging at JoVE.com

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

一个调查癌症干细胞迁移的微流体装置进行划分。这个新的平台创建一个可行的细胞微环境，使活细胞运动的微观可视化。高度能动的癌症细胞分离分子机制研究的积极渗透，并可能导致未来更有效的疗法。

癌症干细胞迁移的使用进行划分的微流控芯片和活细胞成像的评价

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

evaluation-cancer-stem-cell-migration-using-compartmentalizing

Research

JoVE Journal

Medicine

25.2K 次观看.  University of Wisconsin-Madison. 一个调查癌症干细胞迁移的微流体装置进行划分。这个新的平台创建一个可行的细胞微环境，使活细胞运动的微观可视化。高度能动的癌症细胞分离分子机制研究的积极渗透，并可能导致未来更有效的疗法。

视频: Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

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 ...

科研

医学

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

树脂浇铸和3维成像的小鼠门静脉或肝内胆管系统

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

共聚焦成像在肝癌细胞中观察到的异常有丝分裂细胞死亡相关

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.