这项工作是由中国社会科学院基础科学和医学格兰特的支持; （MB）;澳大利亚计划资助一个国家健康与医学研究理事会（KLS，338516）;以及由维多利亚州政府的运作基础设施支持计划（澳大利亚）。

细胞的准备工作1。，媒体和试剂<ol><li>含甲基纤维素 - 中的制备<ol><li>溶解1.5克甲基纤维素在250毫升的烧瓶用100ml无菌的Dulbecco改良的Eagle培养基（DMEM）（无添加剂）的。 </li><li>加热，在微波5分钟，在低的溶液;小心避免沸腾了。 </li><li>一旦甲基纤维素粉末是半溶解后，加一个干净的磁力搅拌器和媒体采取的体积至125ml。 </li><li>混合，反转，并在室温下搅拌1.5小时，随后在4℃下搅拌过夜。 </li><li>平分的解决方案分为四个50ml试管和离心1.5小时在2,300×g的。 </li><li>收集清，高粘度上清液（股票约90-95％），分装到无菌的50ml试管，并储存在4℃可保存3个月。 </li></ol></li><li>文化与细胞的标记<ol><li>保持在单层卵巢癌细胞系中的TiSSUE培养箱中，在37℃，5％CO 2在适当的生长培养基（DMEM为KGN单元17和MCBD110：M199为OVCA429和OVCA433细胞系18），补充有10％胎牛血清（FBS），2mM的L -谷氨酰胺和1％青霉素 - 链霉素。 </li><li>保持LP9人类间皮细胞在M199：火腿含有F12培养基15％FBS，10毫微克/毫升的表皮生长因子（EGF）和400毫微克/毫升氢化可的松，在培养箱中在37℃，5％CO 2。 </li><li>种子细胞中的所需烧瓶大小和生长至约75％汇合。用0.05％胰蛋白酶/ EDTA，旋在186×g离心5分钟，并用PBS洗涤细胞沉淀物两次收获的细胞用胰蛋白酶消化。 </li><li>使用血细胞计数器计数细胞。 </li></ol></li><li>可选的荧光细胞标记方法<ol><li>重悬癌症细胞以1×10 6个细胞/ 1毫升预热的PBS/0.1％BSA中的溶液的最终浓度。 </li><li>加入2μl的5毫米的股票手机风帆王牌CSFE溶液（或保持长期其它优选的细胞标记）的细胞，以10μM的终浓度和孵育在37℃水浴中10-15分钟。 </li><li>淬火用1ml冰冷的培养基中含有10％FBS和再沉淀通过离心分离该反应。重悬在10毫升的适当预热的生长培养基（见步骤1.2.1）。 </li><li>种子细胞成75 厘米过滤器覆盖的烧瓶中，并培养于37℃，5％CO 2。 </li><li>检查在荧光显微镜下观察细胞，确认荧光标记的电平足够进行检测。一旦细胞达到约75％汇合，收获和计数按照步骤1.2.3和1.2.4以上。 </li></ol></li></ol>球体的2。在生成甲基纤维素<ol><li>计算从第1卵巢癌细胞所需的球体产生的（共300000细胞的每个都需要完整的96孔板实验）的体积。无TE：如果使用不同的细胞系比这里提出，细胞密度为均匀的球体产生，可能需要对每行进行优化。 </li><li>计算稀释细胞甲基媒体，首先减去15毫升要求（步骤2.1）的细胞体积。 </li><li>加3的甲基股票溶液（使20％的甲基纤维素最终）至体积的培养基适合于每个细胞系，等于15毫升减去细胞体积和轻轻翻转充分混合。 </li><li>新增300,000细胞含有甲基纤维素中，轻轻颠倒充分混匀。 </li><li>吸移管150微升的细胞/甲基纤维素/媒体组合（总共3000个细胞/孔的）到一个96孔的凹底培养板中，培养在37℃下的每一个孔的，5％CO 2中培养1-4天，或直到均匀球体组成（1球体/孔通常观察到的）。 </li></ol> 3，RTCA细胞侵袭实验 NOTE：阻抗读数被表达为细胞指数（CI），一个无量纲参数，它是在表示单元的状态测量电池阻抗的相对变化。为了便于分析，数据导入到电子表格中，例如Microsoft Excel。 <ol><li>板准备<ol><li>工作中的4个孔组的时间，加50μl的基质（稀释于无血清培养基中1:10），一个16 - 孔RTCA CIM板的上部腔室的每个孔中，以确保总表面积被覆盖。立即取出30微升矩阵，但慢慢地，摆脱任何多余的液体。 </li><li>培养板在37℃下，在组织培养箱中在使用前4小时。 </li><li>加适合于每个细胞系的上部腔室和160微升的媒体有或无血清（按您的实验设计）的下部腔室30微升无血清培养基（SFM）中。 </li><li>通过点击下部腔室入上腔体和equilibr装配在CIM平板吃在37℃培养箱中培养1小时在组织培养的孵化器。 </li></ol></li><li>编程RTCA仪器<ol><li>打开RTCA程序，并选择“布局”选项卡。突出显示所有实验井。 </li><li>右键单击井，并选择“打开井”;然后根据需要填写的实验条件和单元名称。 </li><li>要添加步骤或子步骤的程序，选择“添加了一步”的“时间表”标签并点击右键。第一个步骤是预编程的背景扫描，它会自动并入程序一次&#39;添加步骤“被选择。 </li><li>输入所需的程序细节的RTCA方案，该方案将建立LP9单层的过程中录制的读数步骤2。通过选择“间隔”选项卡，进入&#39;15分钟&#39;添加阻抗读数之间的时间间隔。增加实验时间通过选择“持续时间”选项卡中的ð进入12-24小时之间的时间。 </li><li>要添加后续步骤中，选择“添加了一步”的“时间表”选项卡，右键点击并输入的额外步骤的详细信息，如上。在RTCA程序步骤3将直接仪球体入侵期间采取读数;输入&#39;下&#39;的&#39;时间&#39;标签下间隔“选项卡，&#39;48小时&#39;&#39;5分钟。 </li><li>选择&#39;板&#39;在菜单栏上，并保存。 </li></ol></li><li>球体入侵LP9单层和计量的产生<ol><li>从上面的步骤3.2将CIM板RTCA仪器和开放的程序。在添加单元格，选择“执行”菜单栏中，然后“开始”。背景扫描将自动执行（步骤1中的RTCA软件的说明）。 </li><li>从仪器下面的后台扫描和地方组织培养罩取下CIM板。 </li><li>板50,0悬浮00 LP9细胞在160微升的SFM到每个CIM板的上部腔室良好。 </li><li>将板在RTCA仪器，并采取读数，每15分钟，同时LP9单层建立过夜（RTCA该计划的步骤2）。 </li><li>下步&#39;在甲基纤维素球体的一代“2收成产生癌症的球体，上面。 <ol><li>在无菌条件下，切1毫米销1毫升移液管尖的顶部，并用它来轻轻地检索每个的内容很好。转移到无菌管中。 </li><li>离心机的球状体，在120×g离心8分钟，并且通过温和抽吸除去含有甲基纤维素培养基中。 </li><li>用PBS两次洗球体，在离心120×g离心8分钟每次洗涤后沉淀的球体。 </li><li>对于每个实验的井，在160微升培养基中的无FBS重悬，共10球状体。 </li><li>通过选择菜单栏中的“执行”，并检查与暂停的RTCA实验＃8216;暂停“。从仪器上取下CIM板，并将其放置在组织培养罩。吸断从每个孔中的介质，并替换为椭球体的含介质（10球在160微升）或LP9只对照孔新鲜培养基。 </li></ol></li><li>返回板的RTCA仪器。选择菜单栏中“执行”，并选中“中止一步”。该程序会自动移动到下一个步骤。要继续实验，然后从菜单栏中“执行”，并勾选“开始/继续”。在RTCA程序步骤3将启动。 </li></ol></li><li>数据分析<ol><li>以确定球体细胞侵袭的程度，减为LP9单层仅由为在每个时间点的卵巢癌细胞系中获得的单个读数得到的值。 </li><li>绘制&#39;球体入侵&#39;，归一化的所有值在该球体被添加到板的时间点，由s廷该时间点为&#39;0&#39;。 </li></ol></li></ol>

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入侵的卵巢癌细胞与多种细胞类型的腹膜的表面相互作用，包括成纤维细胞，脂肪细胞和间皮细胞和癌细胞和腹膜细胞之间的双向通信被认为是在驱动转移过程2中发挥关键作用。一旦掌握，本文描述的协议是很容易地适应这些相互作用的腹膜微环境中的研究， 例如 ，通过添加外源性细胞因子，生长因子，或特异性抑制剂到CIM板孔或遗传操作的上部或下部腔室的的癌症或腹膜细胞系。此外，这种方法可与新鲜收获的来自恶性腹水和/或原发性腹膜细胞来获得直接见解的调节信号和哪些支配腹腔内建立一个转移灶的细胞相互作用原发性卵巢肿瘤细胞中可以使用<s了&gt; 2。 使用RTCA仪器来测量单个细胞或球粒的侵入能力提供了比传统的单个端点检测和隔时图像分析的几个优点。在RTCA仪器测量细胞侵袭在定义的时间间隔连续地测定法，它可以是几小时或几天的过程。这使得改变的决心入侵的速度随着时间的推移，它克服了单一的端点检测的局限性。例如，检查从在一个单一的端点（ 图2）， 例如 ，3小时的不同肿瘤细胞系的侵袭数据，有可能导致错误的结论KGN和OVCA433细胞远比OVCA429细胞更具侵入性的。该技术的另一个优点是，RTCA仪器测量的变化的电阻抗。这意味着，检测可以在不与外源试剂标记细胞，这可能对细胞啉意想不到的效果运行otype或功能。此当 ​​学习从恶性腹水，与它必须保持操作到最低限度，以避免引入的分子和表型的改变，是不反光的他们的体内性质2衍生的原发性卵巢癌细胞变得尤为重要。此外，使用RTCA仪器能够定量数据的收集实时，这不仅避免了耗时随时间推移显微镜相关分析也可以快速确定关键的实验窗口分析优化。比较各种因素对球体侵袭的影响时，这是特别有用的，因为不同的因素可能会发挥其最大效果在不同的时间点，或随时间大大不同的访问。 虽然这个协议是经得起改变（ 例如 ，使用不同的细胞外基质基质，不同的肿瘤细胞株，或不同目标的CELls）的，如果这样做，必须注意的是不同实验条件下，必须首先进行优化是非常重要的。例如，前起球体入侵研究中，癌细胞/ CIM板的最佳数目以及应首先由细胞数目滴定在单层培养物测定法测定。球状​​体，每孔使用的最佳数量，然后在此基础上分析。例如，在当前的方法中，球体包括约3,000个细胞每一次时产生。如果初始细胞数滴定表明，30,000细胞单层是最优的检测入侵分析，然后10球粒每CIM板孔加入。此外，进行共培养实验时，它研究的“目标”细胞单层的行为分开，以确定这些细胞是否是天生侵入性，因为这可能会混淆的结果是很重要的。在展示的实例中，癌症球体被镀上一个LP9细胞monola的顶揭掉，有和没有FBS加入到底部以及化学引诱物。并联，LP9细胞单独培养，每个处理条件下进行。 同样，当学习细胞和球状体的侵入能力，但同样重要的是检查它们的迁徙能力以及为了区分这两种行为（ 即侵入需要一个ECM屏障的蛋白水解消化而迁移不）。要做到这一点，省略ECM屏障（步骤3.1.1 - 3.1.3），并进行分析与到位的ECM屏障平行。后者的“入侵”的措施可以得到纠正，以昔日的“迁移”的措施，如果需要的话。一旦细胞已侵入底腔，改变其附着，铺展，并且在膜的下侧的增殖可以有助于改变：最后，需要注意的是从电阻抗的测量中获得的信息在范围上受到限制是非常重要的阻抗REA钟声。因此，在与球体细胞活力，粘附，迁移和形态等分析相结合，以全面评估球体细胞行为时使用这种方法是最翔实。例如，理想情况下，为了充分理解球体 - 间皮的相互作用，分离的共培养也应周期性地根据标准相显微镜成像，使得球体的形态和卫生质量评估可以与入侵定量RTCA测定平行进行。如果执行可选的标记步骤，然后单个癌细胞的行为，可以在荧光显微镜来确定，因为他们从球体分解，并与未标记的间皮细胞相互作用。的荧光标记的肿瘤细胞时共培养与第二细胞类型的附加的好处是，它是那么可以确认，只在癌细胞已经通过细胞与细胞外基质屏障侵入。

卵巢癌具有最高的病死率所有妇科癌症1。在世界范围内，有〜230000箱子和超过10万人死亡，每年1由于疾病。大多数患者（&gt; 75％）被诊断后的癌症已经转移时，治疗是进一步提高对化疗的抵抗复杂和预后较差2。患者的Ⅲ〜Ⅳ期转移性疾病有只有30-40％，3年生存率。因此，目前迫切需要更好地了解细胞行为相关的卵巢癌转移，才能有效地治疗晚期疾病。 卵巢癌转移的当前模型提出在转移过程，其中每一个出现在一个独特的微环境对肿瘤行为影响的几个步骤。转移性卵巢癌的细胞最初是从原发肿瘤部位流下并在nonadher生存腹腔液为单细胞或三维的多细胞结构内的耳鼻喉科状态，称为多细胞聚集体或球状体2,4,5。不形成悬浮球体细胞更容易受到失巢凋亡2,4,5。球状体也表现出增加抗化疗药物，有助于残留病灶和随后的复发癌2,4,5。在卵巢癌转移的第二个关键步骤是聚集来自自由浮动集群腹腔壁及大网膜5,6内建立继发肿瘤的过渡。细胞-细胞和细胞-基质相互作用有牵连的聚集形成，生存，并坚持外基质（ECM）通过的4-11腹膜间皮面料制作。迄今，这些过程知之甚少。 确定涉及卵巢癌的传播机制，球体可以被根儿ated从使用非贴壁培养的方法，保持细胞悬浮液通过添加甲基纤维素的培养基12,13或由在低附着板2,14培养细胞建立细胞株。当以这种方式培养，卵巢癌细胞组装成多细胞聚集体或球状体而表现出类似的细胞，分子和生化特性的那些肿瘤的聚集的体内 2,14找到。形成后，球体可随后从非粘附介质收获和再接种到各种固体表面进行进一步的功能研究。这代表一个比预先测定单层培养，其不准确建模的卵巢癌细胞中的非粘附的环境，如腹腔内转移流体的行为。 肿瘤球体的侵袭能力可以在传统的端点检测在Boyden小室2进行评估</suP&gt;，但这种方法可能会引起误解，因为卵巢癌细胞入侵是一个动态的过程随着时间的推移。最近的实时方法已经被使用皮细胞间隙的时间推移视频显微镜，入侵卵巢癌的球体15,16设计;然而，时间间隔分析数据是耗时的，并且可以是主观的。本文中，用于评估在实时的卵巢癌的球状体的侵入行为一个快速，定量方法进行说明。首先，建立一个球体，间皮细胞模型表示的卵巢癌转移的阶段时的多细胞球状体附着并侵入腹膜衬里以形成继发性肿瘤。随后，方法为实时细胞分析仪（RTCA，见材料表公司资讯）被改编进行成长为球体，并在此模型中测试了不同的卵巢癌细胞侵袭能力的实时定量测量。 在RTCA中沪西仪器，细胞的反应是连续监测了一个测定的过程中，而不需要外源的标签，通过测量变化的电阻抗。专门设计的培养板有位于微孔膜的下面以一个2腔以及（&#39;CIM“板）的上部和下部腔室之间的界面涂金微电极。电极在下部腔室中的位置的变化使电阻抗的测量作为细胞通过一个ECM或ECM /蜂窝屏障侵入。 2室每个CIM板孔之间的界面膜首先被涂覆有感兴趣的细胞外基质成分。在椭球间皮细胞模型系统，该接口被涂上一层基质胶（见材料表公司信息），以模仿复杂的ECM的腹膜间皮衬底层，其次是人皮的汇合单层&#39;目标的细胞。最后，预成型的卵巢癌的球体是广告DED。卵巢癌的球体细胞必须积极通过侵入靶细胞层和基质到达底部腔和改变电阻抗读数。是对自己的靶细胞也评估，以确保它们不通过矩阵层侵入，并适合使用在该测定。

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assessment of ovarian cancer spheroid attachment and invasion of mesothelial cells in real time

卵巢癌转移由脱落到腹腔液和分散到腹膜内远端站点。单层培养物没有准确地模拟癌细胞的行为中的非粘附性的环境中，如肿瘤细胞本身聚集成由所附和侵入腹膜衬里以形成继发性肿瘤向转移过程的多细胞结构。建模的卵巢癌转移，多细胞聚集体，或球状体这一重要的阶段，可以从非粘性的条件下保持既定卵巢癌细胞系来产生。以模仿由肿瘤细胞在体内遇到的腹膜微环境，一个球体，间皮共培养模型建立在其中预先形成的球状体被镀上一个人腹膜间皮细胞单层的顶端，形成有外基质屏障。方法然后使用实时细胞分析仪进行定量实时研制成长为球状体不同卵巢癌细胞系的侵袭能力的时间测量。这种方法允许入侵过的时间，这比传统的端点检测和比较费力实时显微图像分析几个优点长时间的连续测量。总之，这种方法能够快速，测定其调节卵巢癌的细胞球体通过一段时间的间皮和基质屏障入侵之间的互动因素。

卵巢癌的球状体可以从多种类型的细胞系中通过它们在悬浮液中培养的或由恶性腹水2,14收获原发肿瘤细胞的产生。此处2上皮性卵巢癌细胞系，OVCA433和OVCA429，和卵巢粒层细胞瘤细 ​​胞系，KGN，已被用来生成球状体（ 图1A）。在该方法中，细胞培养在U型底的井，而悬浮于其中已取得的粘性通过添加甲基纤维素的介质。在这些条件下，许多卵巢癌细胞表现出聚集形成球状体13的固有能力。以下过夜培养悬浮在甲基纤维素/媒体，所有三种细胞系形成的直径约400-500微米（ 图1A）紧凑球体结构。同时，RTCA CIM板的制备中，首先通过涂布在多孔膜的界面与矩阵，然后汇合人类MES的单层othelial细胞（ 图1B）。一旦形成，球体，然后转移到所制备的CIM板。收获肿瘤球状体镀上LP9单层的顶部，并如下所述进行评估。平行培养按照标准相显微镜图像（或荧光显微镜下，如果可选的细胞标记的步骤之后）定期在RTCA数据的解释（ 图1C），以帮助拍摄。 它是信息性评估侵袭癌细胞株两者的基础水平，以及趋化因子诱导的侵袭。通常，基底侵袭是通过加入SFM到上部和下部腔室测定。完全培养基（10％FBS），它包含许多潜在的趋化因子，是用在当前的例子中，研究“趋化因子诱导的入侵（ 图2）。单独LP9间皮细胞的平行研究证实，这些细胞是在最低限度侵袭性根据任一基底或“趋化因子”诱导条件，从而在2天数分别为一良好的细胞类型共培养测定法使用具有卵巢癌的细胞（ 图2A和2B）。与此相反，使用任何的三种细胞系的产生卵巢癌球体呈现侵入朝向趋化因子（FBS）（ 图2A-2C）的能力。使用RTCA实时工具比传统的终点测定法的主要优点是，改变细​​胞/球体行为可以被量化随着时间的推移。在2天测定中，KGN，OVCA429和OVCA433细胞系都表现出侵入朝向趋化因子（通过增加细胞指数表示），但侵入的低基础水平（ 图2C）的能力。该细胞系表现出入侵发生率相似，由曲线（ 图2C）的平行斜坡所示;然而，OVCA429曲线表现出较高的上部渐近线，这表明相比于其它细胞系侵袭的更高的最大电平。此外，该细胞系中表现出他们的时间的差异发病入侵，与KGN细胞迅速入侵细胞和基质屏障和OVCA433和OVCA429细胞服用3到5倍更长的侵入，分别为（ 图2D）。重要的是，在入侵发生的行为，并不能预测其对入侵的整体能力（ 图2C和2D）的。这表明，肿瘤细胞株的不同内在功能，但也可能预示着不同的因素可以调节球体的早期和晚期侵入性的行为。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51655/51655fig1highres.jpg" width="500" /> 图1球体产生的实验和模型建立A：我：B下生长为单层（ 底部 ）的匹配细胞系的球体在甲基纤维素/媒体（ 上 ）形成相衬显微镜和法师：示意图显示了RTCA 2腔的CIM板以及设立，其中预先形成的卵巢癌球体被镀上一个LP9间皮层/基质屏障在上部腔室和媒体±FBS的趋化因子的单层的顶端被加入到下室。 。KGN球体上相差显微镜（ 左 ）或荧光显微镜（ 右 ）下的LP9单层的顶部图片：2室度量增加电阻抗为多个小区通过障碍下部室C侵入的界面下方的电极。比例尺= 100微米。 <a href="https://www.jove.com/files/ftp_upload/51655/51655fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 。图2实时卵巢癌球体入侵数据A - B：由有和无FBS的CIM板的底腔井进行了RTCA侵袭实验的代表性结果。 KGN细胞的侵袭相比LP9间皮细胞。结果是从一式三份孔在24小时的时间点（A）和在整个2天试验期（B）显示为平均值±标准差细胞指数C - D：KGN，OVCA429的侵袭能力的比较，和OVCA433细胞描绘在24小时内（C）和超过2.5小时的时间段（D）更详细的时装系列。

Watch this Scientific Journal Video about Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time at JoVE.com

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

卵巢癌细胞侵入的腹膜间皮衬里是一个动态过程，随着时间的推移。利用实时分析器，卵巢癌细胞中的球状体，间皮细胞共培养模型的侵袭能力可以量化在延长的时间段，提供见解因子调控的转移过程。

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

assessment-ovarian-cancer-spheroid-attachment-invasion-mesothelial

Research

JoVE Journal

Medicine

17.2K 次观看.  MIMR-PHI Institute of Medical Research. 卵巢癌细胞侵入的腹膜间皮衬里是一个动态过程，随着时间的推移。利用实时分析器，卵巢癌细胞中的球状体，间皮细胞共培养模型的侵袭能力可以量化在延长的时间段，提供见解因子调控的转移过程。 

视频: Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

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 ...

科研

医学

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

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 ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

球体的检测，以测量TGF -β诱导的入侵

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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 ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

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, ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...