Name: chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds
Uploaded: 2015-06-06T15:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds at JoVE.com

We thank Marleen De Meulemeester for demonstrating the assay technique in the video film. B. I. R. is a Postdoctoral Research Fellow of the Research Foundation &#8211; Flanders (FWO &#8211; Vlaanderen). L.M.M. is a recipient of an Emmanuel van der Schueren grant from the Flemish League against Cancer (Vlaamse Liga tegen Kanker).

<img alt="图1" src="/files/ftp_upload/52792/52792fig1.jpg" /> 图的不同检测步骤1.示意图。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1.准备预培养心的碎片（的PHF） <ol><li>孵育受精鸡卵在37℃9天。通过允许在4天后续准备的PHF（ 例如，在星期四）日期和最后的对抗肿瘤细胞聚集完成这个孵化（ 例如，在鸟巢星期一）。 </li><li>消毒壳与在水中的70％（体积/体积）乙醇。进行使用的无菌溶液和材料所有进一步操作在组织培养橱。打开外壳使用时钝钳胚胎极。 </li><li>拉出胚胎由HOL丁俊晖与眼球摘除勺子（ 图2）颈部。放置在玻璃培养皿的胚胎用直径为5厘米含有5毫升林格氏盐溶液。 <ol><li>打开腹胸部皮肤被撕开使用锋利的镊子双手肌肤，由同一类型的操纵去除胸骨，并用显微虹膜切除术剪刀将其切断大血管解剖出来的心脏。执行与下一个校准目镜网格macroscope所有进一步的操作。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/52792/52792fig2.jpg" /> 图2.抬起出胚胎用勺子去核卵细胞中。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="4"><li>的心脏转移到一个玻璃培养皿一个直径为5厘米含有用5％胎牛血清5毫升MEM-安瑞嘉3培养基。通过采用切除显微剪刀虹膜切除术心脏的上部颅第三卸下心房和相关的船只。从解剖心室心包有一对锋利的钳子。 </li><li>请使用显微剪刀虹膜切除术心室矢状半切，并清除血液通过用锋利的钳轻轻晃动。 </li><li>转移脑室到另一个玻璃培养皿的直径为5厘米含有用5％胎牛血清加入5ml新鲜MEM-安瑞嘉3培养基中。用显微虹膜切除术剪刀剪心室成约为0.4毫米直径件。一个心脏可以产生约100片段。 注:一个心脏可以产生约100片段</li><li>旋转培养皿轻轻地来驱动所有心室碎片朝菜的中心。删除所有语料库槲用不锈钢针用Sep从心室片段arating它们和驱动它们朝向培养皿的周边。 </li><li>的心脏的片段，用玻璃巴斯德吸管转移到含有培养基（MEM-安瑞嘉3加10％体积/体积的胎牛血清）的2-至3ml 50毫升锥形瓶中。 注意:确切培养基体积取决于个体烧瓶的可变底部凸部:其底部的中心应涵盖的仅液体的薄膜。 </li><li>气的烧瓶（多个），用5％的CO 2的空气中经由塞子的混 ​​合物，并温育于旋转摇床烧瓶（多个），在37℃下以70转/分钟（rpm）离心24小时。此悬浮培养步骤的理由是为了获得适合于与肿瘤细胞聚集随后对抗球形心脏组织碎片。 </li><li>的心脏的片段和它们的培养基转移至培养皿。丢弃语料库槲，坏死（深）的片段，并用针心脏碎片集团。 </li><li>如在步骤1.9中所述另一个60小时孵育在含有6毫升培养基的另外50毫升的锥形烧瓶中剩余的心脏片段。 注:在此潜伏期，碎片将成为球形。它们包括成肌细胞的芯和一薄层成纤维细胞在外围的。 </li><li>选择球状片段表现出成纤维细胞的薄，均匀的层（形成半透明胶囊）中，并由macroscope和针的装置的直径为0.4毫米。一鸡心脏将产生约20合适的PHF。转移这些的PHF，其中许多将收缩节奏在37℃下，于含有新鲜培养基另一个培养皿。这些都是的PHF准备与测试细胞对抗。 </li></ol> 2.编写和测试球状细胞聚集对抗<ol><li>制备半固体琼脂培养基:通过煮沸三次溶解在15毫升林格氏盐溶液100mg的琼脂。冷静该悬浮液至40℃并加入7.5林格氏盐溶液毫升/蛋白（1:1）和7.5毫升胎牛血清。 </li><li>制备6-毫升含在50ml锥形瓶中1×10 5个测试细胞/在其适当的培养基ml的悬浮液。为此3天对抗文化开始计划之前。孵育在旋转摇床烧瓶在37℃在70rpm下培养3天。气的烧瓶用5％或10％（体积/体积） 的 CO 2在空气中的混合物中，这取决于所用的培养基的类型。 </li><li>查看骨料下配有校准的目镜网格macroscope。选择（用针）球状细胞聚集体的直径为0.2毫米。 </li><li>使用巴斯德吸移管向在培养基中的最小量的传送8所选的PHF（直径= 0.4毫米），含半固体琼脂培养基中13的胚胎手表玻璃。单独移动的PHF用针，以形成一个圆。吸过量的培养基用一小片滤纸（ 见图3）。 </li></ol><img alt="图3" src="/files/ftp_upload/52792/52792fig3.jpg" /> 图3.吸气各地的PHF多余的液体 。八的PHF被放置在半固体琼脂中的胚胎表玻璃，和流体过量时有小的三角片运滤纸除去。箭头指示的8个独立的PHF。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li>带上十大评选球状细胞聚集（直径= 0.2 MM）到使用巴斯德吸管圈。吸过量的培养基。移动一个骨料相向与针的PHF的，直到它们使彼此接触。用一小片滤纸抽吸过量的培养基。 </li><li>密封该手表玻璃用石蜡的盖，和INCU软化在37℃下进行4 - 24小时，根据测试细胞对的PHF的粘合性能。 </li><li>浸入面临对用预温（37℃）培养液中，并用巴氏吸管转移的每个单独的一对进含有1.5毫升培养培养基的5毫升锥形瓶中。 </li><li>孵育在旋转摇床，在37℃下调整至120转的烧瓶中。气用5％或10％（体积/体积）的CO 2的烧瓶中的空气，这取决于所用的试验电池（ 见图4）培养基的类型。 <ol><li>为了避免媒体的浓度，通过两个补充5毫升锥形瓶中填充用2ml林格氏盐溶液通过使它们润湿气体。刷新培养基每8天。 </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/52792/52792fig4.jpg" /> 图4.小锥形瓶上gyrotory SHAK顶部。呃用1.5ml培养基的烧瓶被密封用装有气体中硅氧烷瓶塞-和出口针，并移动以120rpm在37℃。该气体通过入口管（1），以较大的Erlenmeyer烧瓶（2）充满无菌林格氏盐溶液主导，并进一步分散（3和4）含有与个别相对的对培养基小瓶（5和6）。最后，废气被收集在一个更大的烧瓶用无菌水器（7），从那里可以逸出到空气中（8）。该图显示了两套上自制的平台板顶部文化电池。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.组织学的面对文化<ol><li>准备布安 - 奥朗德的固定液。 <ol><li>溶解将2.5g乙酸铜（中性）的100ml单蒸馏水和慢慢加入4.0克苦味酸。 </li><li>过滤通过纸过滤器的溶液。加入10 mL福尔马林和1ml乙酸。拌9份的该溶液与1份的单蒸馏水饱和氯化汞溶液中。 注意:该解决方案包含多种有害物质。福尔马林是吸入有毒，与皮肤接触和吞食。乙酸具有腐蚀性的口腔和肠道消化后。苦味酸是过敏和爆炸时迅速加热或敲击。氯化汞是高度腐蚀性粘膜和nefrotoxic。穿戴防护服和手套准备布安 - 奥朗德的解决方案，它在处理火灾通风良好的地方偏僻。另一种固定方法是基于在磷酸缓冲盐水的4％甲醛。 </li></ol></li><li>经过数天或数周的潜伏期如下固定个体培养物。首先，将培养物转移到林格氏盐几秒钟溶液以除去血清蛋白。接着，沉浸在培养皿中，直径为3厘米的装有3毫升布安-奥朗德的固定溶液约2小时。 </li><li>孵育它们在水中2小时以除去尽可能多的固定溶液尽可能之前冲洗培养在3ml去矿物水洗涤三次。最后，转移到3毫升70％（体积/体积）乙醇在水中。保持在该溶液中O / N培养物，但是这可以延长天数。 </li><li>通过包含每个2小时100毫升的96％（体积/体积）乙醇在水中，100％乙醇或100％的异丙醇，和100％的二甲苯罐依次转印脱水。 <ol><li>各培养转移到一个独立的玻璃盖玻片（用二甲苯的最小量），放置在酒瓶胶囊石蜡包埋底部的盖玻片，并覆盖固定材料与液体石蜡在56℃。孵育在56℃下放置24小时，然后冷却该嵌入材料至RT。 注意:由于苯衍生物二甲苯可吸入后有毒，应通风良好的区域仅在处理。 </li></ol></li><li>取出酒瓶胶囊和盖玻片，并切出其中包含的固定文化蜡块。 </li><li>请用切片机14的整个相对的一对8微米厚的石蜡切片，并收集三个交替显微镜玻片已预先用组织粘合剂溶液作为粘着剂的所有部分。避免蛋清作为粘着剂，因为它可能会干扰免疫。 注意:在显微镜玻片的预处理是通过提供1小滴的组织粘合剂溶液和3小滴软化水用巴斯德移液管，并且将它们混合，以覆盖滑动完成。 </li><li>在装有100毫升100％二甲苯10分钟一罐浸泡两次取下第一张幻灯片的石蜡。 </li><li>再水化的载玻片浸入10秒中罐子含有100ml的下列溶液:二甲苯/乙醇（1:1），100％的乙醇，96％（体积/体积）的水乙醇，70％在水（v / v）的乙醇，最后软化水。 </li><li>溶解通过浸入氯化汞晶体中含有100ml的0.5％（重量/体积）中的碘96％（体积/体积）的水乙醇进行2分钟的罐子。 </li><li>清除在含有100毫升5％（重量/体积）硫代硫酸钠在水中10秒的罐部分。然后在蒸馏水中彻底冲洗的幻灯片。 </li><li>孵育在含有100ml哈里斯&#39;苏木2分钟一个罐子的幻灯片，在0.1M的HCl简要淹没，然后洗在流动的自来水10分钟。 </li><li>孵育在含有100ml曙红0.1％（重量/体积）的水进行1分钟的罐子。 </li><li>通过短暂浸没在含有100ml以下一系列溶液的罐脱水:软化水，70％（体积/体积）乙醇，96％（体积/体积）乙醇，100％乙醇洗涤两次，二甲苯 - 乙醇（1:1） ，最后在100％二甲苯。 </li><li>安装幻灯片通过提供2个独立的介质液滴在载玻片安装介质，让这些扩展通过将一个盖玻片在它们上面，并让它在室温下硬化24小时。 </li></ol> 4.免疫组化<ol><li>制备的Tris缓冲盐水（TBS）。溶解将6.0g三（羟甲基）氨基甲烷（Tris碱）45.0克氯化钠在4.5升的去离子水中。带来至pH 7.6，用1M的HCl（约42毫升）。添加蒸馏水至5 L. </li><li>制备的Tris-HCl缓冲液中。溶解60克Tris碱的在800毫升软化水中。带来至pH 7.6用6M盐酸（约65毫升）。加蒸馏水至1升稀释10倍的蒸馏水后使用。 </li><li>准备的Tris-BSA 0.1％的缓冲。溶解12.1克Tris碱的45.0克NaCl的4.5升的去离子水中。带来至pH 8.2，用1M的HCl（约42毫升）。加将5.0g牛血清白蛋白（BSA）和6.5克的NaN 3。添加软化水，使5 L. 注意:叠氮化钠是一种高度有毒产品。与酸接触释放剧毒气体。戴上手套，避免摄入一切手段。叠氮化钠与形式铜，铅和其他金属非常敏感的爆炸性化合物。冲洗水槽用大量的水冲洗。另一种防腐剂是0.01％硫柳汞。 </li><li>按照项目3.2至3.10。 </li><li>浸在TBS或三-0.1％BSA的缓冲幻灯片，擦去周围用纸巾部分多余的液体。 </li><li>在TBS或在5％（重量/体积）牛血清白蛋白的Tris-0.1％应用150微升正常山羊血清以1:20稀释（重量/体积）BSA缓冲液在高湿度室中30分钟（一个封闭的盒子用于共-incubation用开水冲收件人确保内高湿度）的幻灯片。然后去除多余的液体用纸巾。 </li><li>应用150微升初级抗血清稀释在Tris-0.1％（重量/体积）牛血清白蛋白在高湿度室中补充有1％（体积/体积）正常山羊血清，至少2小时。确定初级抗血清的最佳稀释度empiri地作为备用。 注意:免疫组织化学可用于检测鸡心脏抗原但是，使用特异性抗体，鸡心脏抗原所描述的技术可以应用到其他抗原（如绿色荧光蛋白）15为好。 </li><li>在Tris-0.1％洗两次幻灯片w / v的BSA在摇表5-10分钟。 </li><li>用纸巾为至少1小时在高湿度室中除去多余的流体，并应用150微升的山羊抗兔抗血清在过量（ 例如 ，在TBS中1:20）。 </li><li>用TBS在摇表5-10分钟洗两次。 </li><li>用纸巾去除多余的液体。所适用的过氧化物酶 - 抗过氧化物酶复合物稀释1:250的TBS在高湿度室中补充有1％（体积/体积）正常山羊血清为至少1小时。该复合物的稀释取决于批量。 </li><li>用TBS洗涤载玻片并转移到的Tris-HCl缓冲液pH 7.6。 </li><li>转移滑入三 -HCl缓冲液含有0.25克/升二氨基联苯胺和0.01％（体积/体积） 的 H 2 O 2的几分钟。停止孵化，当鸡心脏被染色。定期检查，在显微镜下。 </li><li>转移幻灯片的Tris-HCl中10分钟，然后以蒸馏水。 </li><li>按照项目3.13 3.14和。 注意:对于固定过程中容易被破坏抗原，培养的cryosectioning可以为免疫组化分析的替代。这在以下方案步骤描述: </li><li>用3毫升林格氏盐溶液洗活培养物以除去血清蛋白。 </li><li>加入一滴包埋剂的预冷（-16°C）标本冷冻切片机的持有人。从玻璃盖玻片边缘转移培养的组织，以这种下降的顶部之前它被完全冻结。 </li><li>酷文化到-16℃，切6微米厚的冰冻切片，并收集他们在明胶包覆载玻片上。 </li><li>固定在丙酮中的幻灯片，在4℃下储存于4℃之前10分钟。 </li><li>按照项目4.5至4.15。 </li></ol> 5.评价试验结果注意:在本测定中，侵入被定义为逐行占领PHF由面对测试电池。所有连续部分从一个面对培养的微观分析允许测试细胞聚集和在三维空间中的PHF之间的相互作用的重建。 <ol><li>观察交互并根据以下标准等级的不同的模式（参见图5）。 0级:只有PHF观察。没有面对可观察细胞。 I级:该面对测试细胞附着到PHF，但不占用的心脏组织，甚至没有最外的成纤维细胞的细胞层。 等级IIa族:所述PHF的职业是仅限于外成纤维细胞样细胞和成肌策LL层。 级防爆:本PHF已包围的细胞聚集体，但有占领的迹象。 III级:面对的细胞已经占领了住房公积金，但超过一半的心脏组织完好的原始金额留下更多的。 IV级:该面对细胞已超过一半的PHF的原体积的占用更多。 注:等级I和II与无创性细胞群观察到，而三年级，四是典型的入侵。为了评估不同的时间框架内发展，组织学分析应适用于文化面临不同的固定培养时间之后。 </li></ol><img alt="图5" src="/files/ftp_upload/52792/52792fig5.jpg" /> 图相互作用5.实施例面临癌细胞和的PHF之间 。面对培养物的组织切片染色或者用苏木精 - 曙红（左小图）或immunohistochemically随鸡对心脏（右图）的抗体。互动牌号在协议文本中定义。 （H:心脏组织，T:肿瘤细胞）。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 6.毒性评估后的药物治疗<ol><li>转移在最小体积到24孔组织培养multidishes用巴斯德吸移管的所面临的文化。 </li><li>通过加入500μl的不含药物的培养基中洗涤培养物，和6小时后刷新。 </li><li>每天检查所有孔从使用倒置显微镜（物镜40×）的外植体的细胞生长。除以增长超过培养由外植培养物的总数，得到的相面对培养物存活的索引的数目。比较药物处理的培养物与溶剂处理的那些的结果。 注:使用免疫组织化学Ki67表达的（细胞prolif累加器）和TUNEL法的（凋亡）的建议作为互补技术的植试验评估毒性。 </li></ol>面对文化7.成长性评价<ol><li>使培养物的黑与白的照片只是固定前，用配有数字photocamera显微镜（物镜50X） </li><li>在毫米测量较大（a）和小（B）直径考虑到放大率的培养物。 </li><li>通过式16计算出近似体积（V）在毫米3 </li><li> V = 0.4 xaxb 2 </li><li>通过在对峙的开始比较与PHF和细胞聚集体的组合体积这个最终体积计算的培养物的生长。 注意:因为孤的PHF倾向于培养过程中减少其体积，相面对成对的生长是依赖于肿瘤细胞。 </li></ol>

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The authors declare that they have no competing financial interests.

期间的PHF的制备，所述片段可以不留在悬浮液中，但附着于血管壁;这可以通过增加培养基的体积来克服。如果的PHF的数目太低，其大小过大，降低培养基的体积。测试电池的故障聚集，可能是由于在该温度或微生物感染的波动。可替代地，一个无法聚集可能是该细胞的固有特性。期间附着聚集到PHF的，附着力差可以由延伸在半固体琼脂培养基的顶部的潜伏期或通过吸收滤纸装置除去周围的培养多...

侵袭是恶性肿瘤的标志。此活动不仅导致周围组织的破坏，但也牵连转移形成。由于癌症患者从浸润和转移，以及高效抗侵入性治疗死仍然很少，实验室化验模仿的肿瘤细胞的侵袭已经开发出来。小鸡心脏检测的目标是提供一个相关的器官培养的方法来研究肿瘤浸润在三个维度。该分析方法可侵入性和非侵入性的细胞之间的区别，并允许研究试验化合物对肿瘤的侵袭的影响。 使用该测定的背后的基本原理是实际的概念，即肿瘤是生态系统，其中的肿瘤细胞不断地与他们的基质（宿主细胞和细胞外基质）相互作用，并通过这些分子间的相互作用的入侵是微调1。所以，在测定肿瘤细胞所面临的LIVI纳克胚胎鸡心脏片段2，它不仅作为由肿瘤细胞侵袭基板，还可以作为不同类型的基质细胞和矩阵元素的来源。小鸡心脏包含肌细胞，成纤维细胞和内皮细胞，以及细胞外基质由层粘连蛋白，纤连蛋白和不同类型的胶原蛋白的。以这种方式，三维器官培养技术涵盖牵连侵袭患者肿瘤的许多细胞和分子的相互作用。 小鸡心脏测定的主要优点是对基质的实施效果。这方面比体外其他入侵检测是基于肿瘤细胞侵入到非生物凝胶基底膜3或间质基质4分子构成更加完整。在器官培养实验发现肿瘤细胞和正常的生活宿主组织之间对抗的概念已经推出几个作者包括沃尔夫和施奈德在法国5，Easty和Easty在英国6和施莱希德国7。以上所引用的方法的鸡心脏侵袭测定两种技术优点是，片段的体积很容易被标准化，并且它们保持收缩，这允许功能完整性监视器官培养期间。此外，禽类胚胎是优选的，因为它们可以容易地从蛋的无菌内容解剖。该测定具有通过提供周围的肿瘤细胞的复合基质概念相似小鸡尿囊绒膜膜试验8。 该测定法已成功应用于从同一人肿瘤侵入性和非侵入性的细胞的变体之间进行区分，例如，在MCF-7（乳房）9和HCT-8（结肠）10细胞系的家庭。该技术可用于检测潜在的抗侵入性化合物以及<suP&gt; 11,12。如进一步说明的，它可以用于建立有机小分子的结构 - 活性关系。该试验确实，但是，没有考虑到免疫细胞对癌症侵袭的贡献。应当强调，该技术不能被认为是因为高数操作的，试验运行的有限数目（最多30培养物）和长的周转时间（约1个月），为高通量分析系统。

<table><tbody><tr><td>Ringer&#039;s salt solution</td><td>Braun</td><td>CEO123</td><td></td></tr><tr><td>Bacto-agar</td><td>Becton Dickinson</td><td>214010</td><td></td></tr><tr><td>Tris-(hydroxymethyl)-aminomethaan Analar</td><td>VWR</td><td>103157P</td><td></td></tr><tr><td>BSA : albumin from bovine serum, cohn V fract.</td><td>Sigma</td><td>A4503-500G</td><td></td></tr><tr><td>MEM-Rega 3</td><td>Life Technologies&amp;nbsp;</td><td>19993013</td><td></td></tr><tr><td>Gyrotory shakers</td><td>New Brunswick Scientific</td><td>G10 and G33</td><td></td></tr><tr><td>Erlenmeyer flasks 50 ml</td><td>Novolab</td><td>92717</td><td></td></tr><tr><td>Glass Petri dishes</td><td>Novolab</td><td>68516</td><td></td></tr><tr><td>Iridectomy scissors</td><td>Rumex</td><td>11-0625</td><td></td></tr><tr><td>Macroscope with calibrated ocular grid</td><td>Wild</td><td>157702</td><td></td></tr><tr><td>Paraffin wax</td><td>International Medical products</td><td>8599956</td><td></td></tr><tr><td>Eosin Y</td><td>Sigma</td><td>230251-25g</td><td></td></tr><tr><td>Harris&#039; hematoxylin</td><td>Sigma</td><td>HHS32-1L</td><td></td></tr><tr><td>Coverslipping resin (Tissue-Tek)</td><td>Sakura</td><td>4494</td><td></td></tr><tr><td>Paraffin melting apparatus</td><td>GFL</td><td>1052</td><td></td></tr><tr><td>Microtome for paraffin sectioning</td><td>Reichert</td><td></td><td></td></tr><tr><td>Stppers for Erlemeyer flasks with gas in- and outlets</td><td>Novolab</td><td>2602421 and 260243</td><td></td></tr><tr><td>Diaminobenzidine</td><td>Sigma</td><td>D8001</td><td></td></tr><tr><td>REAX rocking table</td><td>Heidolph</td><td>54131</td><td></td></tr><tr><td>24-well tissue dishes</td><td>VWR</td><td>NUNC142475</td><td></td></tr><tr><td>Ophtalmological enucleation spoon</td><td>Rumex</td><td>16-060</td><td></td></tr><tr><td>Sharp forceps</td><td>Rumex</td><td>4-111T</td><td></td></tr><tr><td>Blunt forceps</td><td>Nickel-Electro LTD</td><td>7112</td><td></td></tr><tr><td>Erlenmeyer flasks 5 ml</td><td>Novolab</td><td>211901</td><td></td></tr><tr><td>Microscope slides washed and degreased</td><td>International Medical products</td><td>8613908</td><td></td></tr></tbody></table>

chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds

小鸡心脏检测的目标是提供一个相关的器官培养的方法来研究肿瘤浸润在三个维度。该分析方法可侵入性和非侵入性的细胞之间的区别，并允许试验化合物对肿瘤侵袭的影响研究。癌细胞 - 无论是作为聚集体或单个细胞 - 都面临着小鸡胚胎心脏的片段。在悬浮器官培养几天或几周后，将面对培养物固定并包埋在石蜡中用于组织学分析。癌细胞和正常组织之间的三维相互作用，然后从用苏木精 - 曙红或免疫组化染色的表位在心脏组织或对抗癌细胞后的连续切片重建。该测定是用最近的概念，癌浸润是癌细胞和它们相邻的基质主元件（肌成纤维细胞，endoth之间的分子相互作用的结果是一致的elial细​​胞，细胞外基质成分等）。在这里，这种基质环境提供给癌细胞作为活体组织片段。配套方面来测定的相关性是多方面的。侵袭测定中是按照癌症侵袭的标准:在时间渐进占领和更换和宿主组织的空间，以及侵袭和非侵袭性的对抗细胞体内通常与该测定的结果。此外，细胞在体内的侵入图案，由病理学家所定义，是反映在组织学图像中测定。定量结构活性关系（QSAR）分析与众多潜在抗侵入有机同类化合物所得到的结果所允许的在临床中使用的类黄酮和查耳酮，和已知的抗转移药的结构-活性关系（ 例如，微管抑制剂的研究）抑制侵袭测定中也是如此。 HoweveR，该法没有考虑到癌症侵袭账户免疫学的贡献。

在图5中呈现的组织切片作为显示一些成功试验的最终结果。培养物的声音组织学表明活细胞，并允许解释肿瘤细胞和正常组织之间的相互作用。此外，从正常组织无免疫反应可以观察到，这证实使用如小鸡胚胎的正确年龄，免疫排斥反应系统的开发之前。有没有细菌可见，这表明在培养期间不存在（毛）的污染。最后部分的圆形周边印证文化悬浮没有（临时）坚持锥形瓶中墙上的迹...

Watch this Scientific Journal Video about Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds at JoVE.com

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

在这里，我们提出了一个协议入侵肿瘤细胞研究到生活的正常组织碎片在三个方面。这种器官培养技术主要应用于体外测试潜在抗侵袭药物。

鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

chick-heart-invasion-assay-for-testing-invasiveness-cancer-cells

Research

JoVE Journal

Medicine

9.0K 次观看.  University of Ghent. 在这里，我们提出了一个协议入侵肿瘤细胞研究到生活的正常组织碎片在三个方面。这种器官培养技术主要应用于体外测试潜在抗侵袭药物。 

视频: Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

科研

癌症研究

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

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

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

一个实时的基于电阻抗技术测量癌细胞入侵内皮细胞单层

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

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 ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

遗传学

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

使用快速和定量的细胞内胆碱膜模型发现转移调节器

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

生物学

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

癌细胞球体检测，以评估入侵的3D设置

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性

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