作者没有确认。

1. TTFields 体外应用系统-底板和Dish维护<ol><li>冲洗流动的自来水下菜和菜盖。然后用去离子水和风干面朝下冲洗。 </li><li>如果化疗剂/药物已被添加到培养皿中事先的实验，填充用5％的光洗涤剂溶液各培养皿并留下过夜。 <ol><li>彻底冲洗流动的自来水下避免培养皿内的洗涤剂的任何痕迹的菜肴。冲洗菜肴和菜肴用去离子水和风干面朝下覆盖。 </li></ol></li><li>将干净且干燥的菜肴，其覆盖到杀菌袋。密封和放置行李在高压釜中，用菜朝下。高压釜30分钟，在不超过121℃高。 </li><li>将反应釜上的干燥程序，部分打开高压釜门，擦盘子30分钟。 </li><li>擦拭用70％乙醇轻轻浸湿的布在底板上。 </li> &lt;/醇&gt; 2.实验设置注意:在这个协议中使用的所有的设备和材料在材料清单中有描述。所有下面的步骤应在层流柜内，同时保持无菌条件下进行。 <ol><li>制备清洁和无菌TTFields菜肴和盖，如部分1中所描述擦拭底板与用70％乙醇轻轻润湿的布。 </li><li>通过按下轻轻放在培养皿中并通过约5mm，直至盘锁的轮辋上在底板的三个销顺时针旋转安装TTFields菜肴盖到基板。放置一个无菌的22毫米的盖玻片（处理过的塑料或玻璃）上的每个培养皿的底部。 </li><li>准备在完全生长培养基（细胞悬浮液的Dulbecco改良的Eagle为U型87 MG和F98培养基; RPMI为A2780和OVCAR-3;如上述补充 材料清单）。 注:CEll浓度取决于细胞类型和实验持续时间，实验结束时不应超过80％-90％汇合的细胞生长。 注意:人体细胞系可能代表生物危害，应以适当的安全措施处理。 </li><li>将200μL细胞悬浮液（通常在200μL中为200μL，在72小时实验中取决于倍增时间），作为每个盖玻片中心的下降并用盖盖覆盖。 </li><li>在CO 2培养箱中37℃孵育，直至细胞粘附;在这个阶段可以过夜孵化。 </li><li>使用200或1,000μL移液管从液滴中吸出液体。 </li><li>轻轻移液2 mL完全生长培养基，以填补每一道菜。使用无菌尖端吸移并轻轻敲击盖玻片边缘，以释放偶尔被夹在滑块下面的气泡。 </li><li>用盖子盖上碗碟，放在里面IDE在37℃的CO 2培养箱直到TTFields治疗的开始。 </li></ol> 3. TTFields应用<ol><li>传送与附加TTFields菜底板到冷藏CO 2培养箱中培养。 注意: 在体外 TTFields应用会产生热量;因此，冷藏培养箱需要补偿的餐具的变暖。更高TTFields强度会产生更多的热量，将需要较低的培养箱环境温度（ 见表1）。当培养箱温度TTFields应用是在18-30℃的范围内的临床相关TTFields强度来实现的。 </li><li>扁平电缆母连接器连接到所述底板。接通TTFields发电机。 </li><li>启动体外应用系统软件的TTFields和选择实验设置。 <ol><li>定义一个新的实验。键入exp的名字eriment并在计算机屏幕上的软件用户界面的实验所有者。调整为每道菜或底板上的频率和目标温度。 </li></ol></li><li>开始使用该软件的应用TTFields。 </li><li>验证所有菜肴都正确连接，并且显示屏上出现淡蓝色。如果一个菜是用红色框圈（意思是说没有正确连接），轻轻按下了下去，慢慢地来回转动，直到触点恢复，盘中出现淡蓝色。 </li><li>发表长达24小时运行在体外应用系统的TTFields。 </li><li>如果实验达到终点，通过点击该软件的最终试验按钮停止实验并继续执行步骤5.如果不是，请点击暂停试验。 </li><li>断开底板的扁平电缆连接器。删除与从培养箱餐具的底板成层流柜。 </li><li>在所有的菜更换介质ES每24小时和基板返回到冷藏培养箱中。底板连接到使用扁平电缆的发电机。通过点击Continue按钮继续实验。 </li></ol> 4.控制样品 注:生长控制的细胞中相似的条件TTFields处理的细胞，不含TTFields应用。 <ol><li>板的对照样品具有相同的悬浮液，并在用于电镀TTFields处理的样品类似的表面。 </li><li>放置控制菜在CO 2培养箱中在37℃。 </li><li>替换在餐具的介质每24小时以匹配TTFields处理的培养皿中的条件。 </li></ol> 5.实验结束<ol><li>点击结束实验后，等待该软件从系统中检索的所有记录，并记录保存到计算机。 </li><li>使用报表按钮查看温度，电流和电阻日志˚F每个基板，以验证所有的菜均按照实验计划进行处理。 注意:实验结束后，所有报告和日志将保存到计算机，并可随时进行检查。 </li><li>关闭TTFields生成器。 </li><li>断开扁平电缆与基板的连接，并从培养箱中取出。 </li><li>要取下陶瓷盘，请按下并逆时针转盘，将其从基板上解锁。 </li><li>将菜肴放入层流柜中，无菌取出盖玻片。将其转移到含有新鲜培养基或磷酸盐缓冲盐水（PBS）的无菌培养皿中进一步检查和评价。 </li></ol>评估TTFields的效果注意:TTFields的效果可以通过几种方式确定。使用一种或多种以下方法将处理的细胞与未处理的对照细胞进行比较: <ol><li>细胞计数<ol><li>从每个包含盖玻片培养皿中取出培养基/ PBS。 </li><li> 0.5mL的0.25％胰蛋白酶/ EDTA加入各培养皿并孵育至多10分钟（ 即，直到细胞开始从盖玻片表面脱离）在37℃下在CO 2培养箱中培养。 </li><li>加入0.5毫升的完全生长培养基中和胰蛋白酶，并通过向下轻轻吹打并重新悬浮细胞。 </li><li>计数使用任何标准细胞计数技术，该技术是具有低细胞浓度计数兼容的细胞（ 即，大约20,000细胞/ mL）。 </li></ol></li><li>克隆分析<ol><li>板从每个皿到新的无菌皮氏培养皿或含有2毫升的新鲜完全生长培养基的6孔板中的细胞相似的数目（每皿100-500细胞）。 </li><li>孵育在CO 2培养箱中在37℃下进行1-3周（取决于各细胞系的性质），直到由〜50个形成细胞的集落。评估细胞通过光学显微镜每集落数。 </li><li>除去培养基并用PBS冲洗。 </li><li>除去PBS并加入冰冷的甲醇（1mL）中。孵育在-20℃下10分钟或更长的时间。 </li><li>除去甲醇。 </li><li>添加结晶紫溶液（0.1％w / v的在水中的25％V / V甲醇）并孵育20分钟。 注意:水晶紫是一个潜在的致癌物质;使用手套，用结晶紫工作时。 </li><li>除去结晶紫，用去离子水洗涤3次，并风干。 </li><li>计数形成在每个培养皿的菌落。 </li></ol></li></ol>

<ol>
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波拉特Yaara，Giladi摩西，施奈德曼S·罗莎，BLAT罗尼，Shteingauz安娜，泽维Einav，明斯特Mijal，沃洛申大里，凯南·诺亚，塔尔奥娜和KirsonD·艾隆D的在Novocure有限公司，以色列支付员工。温伯格URI是Novocure有限公司，卢塞恩的工资的员工。约拉姆是帕提在Novocure有限公司，NY股东。

TTFields是基于适当调整交变电场1,2，8，9，10，17的连续应用的新兴抗肿瘤方式。最大化抗肿瘤功效为所有的治疗方式所希望的结果。因此，"战斗"为癌细胞生长抑制的每增加百分之可能对长期的临床结果为患者显著的效果。这是因为应用TTFields的所需持续性和所得到的累积效果。最大化TTFields应用可以以多种方式来实现:（1）增加的电场强度1，7，（2）延长治疗时间5，（3）找到最有效combinati与其他治疗模式18，19，和（4）限定的最佳频率1，2，4，6，7。在肿瘤位点最大化的电场强度通过优化在患者皮肤上的阵列的位置来实现;这允许传送的最大电场强度，以基于患者20的个体解剖肿瘤。延长的治疗持续时间主要依赖于病人遵守治疗（每天至少18个小时）17。发现与其它疗法的正确组合和确定最佳频率在很大程度上依赖于在体外结果，作为用于TTFields治疗结果没有确认标记是当前可用的。在这项工作中，我们提出了实验p需要rocedures确定最佳TTFields频率使用TTFields 体外应用系统癌性细胞系。这里描述的方法可以潜在地用于筛选其他癌症治疗方式（ 例如，化学治疗剂或辐射）与TTFields的组合，并确定用于每个特定联合治疗用于TTFields给药的最佳频率。 与以前的出版物线，这里示出的结果表明，对于这两种神经胶质瘤细胞和卵巢癌细胞的治疗的最佳频率是200kHz 1,7。在这项工作中，我们展示的第一次，最佳TTFields频率，以降低克隆形成电势与通向最大的细胞毒性作用的频率有关。在这项工作中所用的方法来量化TTFields的效应（ 即，细胞毒性和克隆生成）一只需要许多可能的标准终点分析中的两个来评估治疗结果。额外的治疗结果测试包括:（1）在显微镜上固定，染色和安装细胞在其上电镀的盖玻片，以观察细胞内结构; （2）从TTFields皿本身或将盖玻片转移到新的一次性盘上之后进行蛋白质和RNA提取物的测定;和（3）用于流式细胞术分析染色的胰蛋白酶化细胞。 仔细的实验​​规划将影响TTFields交付后的治疗结果。关键步骤包括确保整个实验中的细胞增殖不会导致过度生长并使用适当的电场强度，因为当应用于敏感细胞系时，过高的强度将导致太少的细胞用于所需的测定法来确定最佳频率。相反，TTFields以非常低的强度应用上较不敏感的细胞系将导致可以由固有变化所掩盖小的效果。治疗日志应检查关于温度稳定性，电电流和电阻为每整个实验每道菜有价值的信息。在治疗开始时更换有故障的菜肴和不符合理想的治疗参数，将最大限度地减少重复之间的变异菜排除数据。 总之，TTFields是已经证实在临床设置8，9，10的疗效和安全性的新出现的抗癌治疗方式。利用这里所描述的协议， 在体外环境测试TTFields可以允许在临床上治疗TTFields参数的优化和拓宽可我们行动的基本机制的理解。

肿瘤治疗领域（TTFields）是胶质母细胞瘤和潜在的其它癌症类型的治疗中抗有丝分裂方式。字段被通过低强度的连续应用（1-3伏/厘米），中频（100-500千赫）递送，交变电场的肿瘤1，2的区域。 在体外和体内应用TTFields显示出抑制各种癌细胞系的两个生长和肿瘤的几种动物肿瘤模型1，2，3，4，5，6，7的进展。在实体瘤患者，包括胶质母细胞瘤和非小细胞肺癌试点临床试验和大规模的随机研究，有demonst评估连续TTFields应用的安全性和有效性8,9,10。发现TTFields的功效是:（1）频率依赖性，具有特定的最佳频率，导致来自不同来源的细胞系的细胞计数的最大降低1,2,4,5,6,7; （2）电场强度依赖性，具有约1V / cm的活性的最小阈值和更强的较高强度1，2，7，11; （3）治疗持续时间较长时增强5 ;和（4）当垂直于每个oth施加2个方向TTField时更高ER，相比于从单个方向施加1电场。基于上述发现，TTFields可以应用于患者使用2台定位于患者的皮肤换能器阵列，以最大化在肿瘤床12，13中的电场强度长的持续时间。 研究TTFields对体外癌细胞的影响目前提供，以确定最佳频率，以适用于特定肿瘤类型的唯一途径。为最佳频率测试需要一种装置，其允许不同频率的在100-500千赫的范围内的应用和在高达3 V / cm的根的强度均方（RMS）的细胞培养物。作为TTFields应用产生热量，该应用系统需要耗散过量的热，同时保持在温度严格控制的能力。 一些设备WER多年来开发E要允许TTFields应用到细胞培养物1，2，5，14，15，16。在所有这些装置中，所用的电极，以避免涉及使用导电电极，例如在电极表面的电子交换和有毒的金属离子释放到介质1的警告进行绝缘。测试的各种TTFields应用系统之间的主要区别在于所使用的，与由与绝缘体2，14，15，16的薄膜或具有高介电常数的材料绝缘的金属导线或者电极（ 例如电极绝缘的类型，铌镁酸铅titanatE（PMN-PT））6。而绝缘导线电极提供TTFields应用相对简单且成本有效的解决方案，它们通常由以实现有效的电场强度高于1伏/厘米阈值和通过可用于细胞接种的表面所需的高电压的限制，作为电极之间的距离相对较小。基于使用高介电常数材料绝缘电极系统需要特殊的设计和制造能力，但它们不需要高电压，并能提供用于在电极之间细胞生长的更大的面积。 在这项工作中所用的TTFields 体外应用系统属于后一类的系统中，与所述芯单元是培养皿（TTFields菜，参见图1）构成的高介电常数的陶瓷（ 即，PMN-PT）。两对电极是在所述外垂直威名印刷一个TTFields菜ls以允许电场的从2个方向中的应用。的电极连接到一个正弦波形发生器和一个放大器，它允许在50-500千赫的频率范围内应用TTFields。为了散发过多的热量，所述TTFields培养皿保持在冷藏孵化器内，与介质温度控制使用的菜温度和调整由系统所施加的电压的恒定监测执行。在实践中，培养箱设定为更低的温度将导致更高的电场强度，作为系统中，直到盘内的目标温度达到增加电压。皿内的温度和孵化器温度之间的差异可能会导致一些蒸发，这取决于温度梯度;因此，培养基中需要每24个小时进行更换以保持足够的生长条件。 下面的协议描述的实验方法来优化TTFields频率癌细胞的应用程序，以便在细胞中的最大降低计数，并且在存活细胞的电势的降低，形成的菌落来实现。

<table><tbody><tr><td>inovitro system and software</td><td>Novocure</td><td>ITG1000 and IBP1000</td><td>Each unit contains 1 TTFields generator, 1 base plate, 8 TTFields dishes with covers and 1 flat cable.&amp;nbsp;</td></tr><tr><td>Sterilization bags</td><td>Westfield medical</td><td>24882</td><td></td></tr><tr><td>Plastic cover slides</td><td>Thermo Scientific (NUNC)</td><td>174977</td><td>Pre treated and sterilized</td></tr><tr><td>Glass cover slides</td><td>Thermo Scientific (Menzel-Gl&amp;auml;ser)</td><td>CB00220RA1</td><td>Sterilize if necessary</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium</td><td>Biological Industries (Israel)</td><td>01-055-1A</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>21875-034</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biological Industries (Israel)</td><td>04-007-1A</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>Gibco</td><td>25030-029</td><td></td></tr><tr><td>Pen/Strep (10,000 U/mL Penicillin, 10,000 &amp;micro;g/mL Streptomycin)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Sodium Pyruvate solution 100 mM&amp;nbsp;</td><td>Biological Industries (Israel)</td><td>03-042-1B</td><td></td></tr><tr><td>Hepes buffer 1 M</td><td>Biological Industries (Israel)</td><td>03-025-1B</td><td></td></tr><tr><td>Insuline solution from bovine pancreas</td><td>Sigma-Aldrich</td><td>10516-5ML</td><td></td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Biological Industries (Israel)</td><td>03-050-1B</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Methanol</td><td>Merck</td><td>1.06009.2511</td><td>Cool to -20 &amp;deg;C in the freezer before use</td></tr><tr><td>Crystal violet</td><td>Sigma-Aldrich</td><td>120M1445</td><td>Harmful. Prepare 0.1% w/v crystal violet solution in 25% Methanol 75% water.</td></tr><tr><td>Light detergent</td><td>Alcononx</td><td>242985</td><td>Prepare 5% solution in water, or according to manufacurer&#039;s instrutions.</td></tr><tr><td>PBS</td><td>Biological Industries (Israel)</td><td>02-023-1A</td><td>Without calcium and magnesium</td></tr><tr><td>A2780</td><td>ECACC</td><td>93112519</td><td>Grow in RPMI 1640 supplemented with FBS (10%), pen/strep (100&amp;nbsp;U/mL / 100&amp;nbsp;&amp;micro;g/mL), sodium pyruvate (1&amp;nbsp;mM) and Hepes buffer (12&amp;nbsp;mM).</td></tr><tr><td>F98</td><td>ATCC</td><td>CRL-2397</td><td>Grow in Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium supplemented with FBS (10%), pen/strep (100&amp;nbsp;U/mL / 100&amp;nbsp;&amp;micro;g/mL), sodium pyruvate (1&amp;nbsp;mM) and glutamine (2&amp;nbsp;mM).</td></tr><tr><td>Ovcar-3</td><td>ATCC</td><td>HTB-161</td><td>Grow in RPMI 1640 supplemented with FBS (20%), pen/strep (100&amp;nbsp;U/mL / 100&amp;nbsp;&amp;micro;g/mL), sodium pyruvate (1&amp;nbsp;mM), Hepes buffer (12&amp;nbsp;mM) and insulin (10&amp;nbsp;&amp;micro;g/mL).</td></tr><tr><td>U-87 MG</td><td>ATCC</td><td>HTB-14</td><td>Grow in Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium supplemented with FBS (10%), pen/strep (100&amp;nbsp;U/mL / 100&amp;nbsp;&amp;micro;g/mL), sodium pyruvate (1 mM) and glutamine (2&amp;nbsp;mM).</td></tr><tr><td>refrigirated CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>CARON</td><td>7404-10-3</td><td></td></tr><tr><td>Laminar flow cabinet</td><td>ADS Laminair</td><td>Bio12 and VSM12</td><td></td></tr></tbody></table>

determining the optimal inhibitory frequency for cancerous cells using tumor treating fields (ttfields)

肿瘤治疗领域（TTFields）是通过低强度的连续的，非侵入性的应用（1-3 V / cm）的递送的有效的治疗方式，在交替的几百kHz的频率范围内的电场。 TTFields的组织培养研究中进行体外应用系统，该系统允许改变的频率和强度，以陶瓷的培养皿具有高介电常数（ɛ&gt; 5000）的电场的应用程序中使用TTFields。在陶瓷培养皿底部铺于盖玻片癌细胞系经受在各种频率在两个正交方向输送，以促进治疗结果的测试中，如细胞计数和集落形成测定法TTFields。本报告中呈现的结果表明，该TTFields相对于两个细胞计数和集落形成测定法的最佳频率对于两个卵巢癌和神经胶质瘤细胞200千赫。

<html> TTFields应用扫描不同频率后的结果可以基于不同的测定法进行量化，例如细胞计数，比色测定，克隆形成测定法，以及使用放置在特殊高壁TTField内的Boyden腔室侵入细胞数量变化的检查细胞培养皿。基于预定的细胞倍增时间的仔细的实验​​规划将允许细胞在治疗期间达到有丝分裂事件的最大数量，从而使治疗结果最大化。 图2显示了A2780（ 即人卵巢癌细胞; 图2A ） 7和F-98（ 即大鼠神经胶质瘤细胞）的平均细胞计数（ 即细胞数）和克隆形成测定法的典型频率扫描结果。"&gt;图2B）1两个定向TTFields处理（频率范围:100-500千赫，RMS:1.7 V / cm，并且温度培养箱:18℃）的结果表明施加在所有的在细胞计数一个显著减少频率，在200千赫兹的最大减少用于测试（单向ANOVA与多重比较）的所有细胞系的最适频率导致在克隆形成潜力的最高减少是两个A2780和F98细胞（ 图2B和C）相同的。细胞数目，并在每个频率下的集落生成效应之间的Pearson相关系数为0.967（p值= 0.002）和0.755（p值= 0.083），用于分别和A2780 F98。 图3表明用于OVCAR-3的平均细胞计数的频率扫描的结果（ 即，人卵巢癌细胞; 图3A）和U-87中号G（ 即，人神经胶质瘤细胞; 图3B）细胞以不同强度TTFields的两个方向上处理（RMS:1.7，1.3和1.0伏/厘米，分别和培养箱温度:18℃，24℃，和28° C，分别地）。结果表明，虽然仍存在与1.3-V /厘米TTFields处理（培养箱温度:24℃）后在OVCAR-3细胞计数显著减少，效果相比，当细胞进行处理所获得的结果是相对小的与1.7伏/厘米（培养箱温度:18℃）。 U型87 MG细胞用1.0-V /厘米TTFields处理（培养箱温度:28℃）也证明了在细胞数量的减少当与1.7伏/厘米视为类似的趋势，但效果并不显著在较低强度。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55820/55820fig1.jpg" /> FIGURe 1 。 TTFields菜肴安装在底板单元上，并连接到扁平TTFields控制电缆。 <a href="http://ecsource.jove.com/files/ftp_upload/55820/55820fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55820/55820fig2.jpg" /> 图2 。频率扫描确定（A）A2780和（B）F98细胞的最佳TTFields抑制频率。用不同频率（1.7V / cm和100-500kHz）的TTField处理细胞72小时。使用细胞计数和克隆形成测定法估计TTFields治疗的效果。箭头表示最佳频率。结果表示每个测试频率至少6次重复的平均值±SD。 （ C ）克隆形成的代表性图像<html>以下在各种频率TTFields治疗A2780细胞的urvival。 <a href="http://ecsource.jove.com/files/ftp_upload/55820/55820fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55820/55820fig3.jpg" /> 图3。 用于（A）OVCAR-3和（B）的最佳TTFields抑制频率的确定频率的扫描的U 87 MG细胞。将细胞处理72小时具有不同频率的（100-500 kHz）和强度的TTFields（OVCAR-3:1.3和1.7伏/厘米; U型87 MG:1.0和1.7伏/厘米）。采用细胞计数估计TTFields治疗的效果。箭头指示的最佳频率。结果基于在测试的每个频率至少6次重复的平均值表示±标准差。PG"目标=‘_空白’&gt;点击此处查看该图的放大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 孵化器的环境温度 </td><td> 预计TTFields强度 </td></tr><tr><td> （C） </td><td> （V /厘米RMS） </td></tr><tr><td> 18 </td><td> 1.62 </td></tr><tr><td> 19 </td><td> 1.55 </td></tr><tr><td> 20 </td><td> 1.48 </td></tr><tr><td> 21 </td><td> 1.41 </td></tr><tr><td> 22 </td><td> 1.33 </td></tr><tr><td> 23 </td><td> 1.26 </td></tr><tr><td> 24 </td><td> 1.19 </td></tr><tr><td> 25 </td><td> 1.12 </td></tr><tr><td> 26 </td><td> 1.04 </td></tr><tr><td> 27 </td><td> 0.97 </td></tr><tr><td> 28 </td><td> 0.9 </td></tr><tr><td> 29 </td><td> 0.83 </td></tr><tr> &lt;TD&gt; 30 </td><td> 0.76 </td></tr></tbody></table> 表1.孵化环境温度和预期TTFields培养皿内TTFields强度。

Watch this Scientific Journal Video about Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields TTFields at JoVE.com

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields TTFields

肿瘤治疗领域（TTFields）是通过低强度，中频，交流电场的连续的，非侵入性的应用递送的有效的抗肿瘤的治疗模式。 TTFields应用到使用TTFields 体外应用系统的细胞系允许导致在细胞计数的最高还原的最佳频率的确定。

确定癌细胞使用肿瘤治疗领域的最佳抑制频率（TTFields）

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), ...

determining-optimal-inhibitory-frequency-for-cancerous-cells-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields)

14.8K 次观看.  Novocure Ltd., Haifa, Israel. 肿瘤治疗领域（TTFields）是通过低强度，中频，交流电场的连续的，非侵入性的应用递送的有效的抗肿瘤的治疗模式。 TTFields应用到使用TTFields 体外应用系统的细胞系允许导致在细胞计数的最高还原的最佳频率的确定。 

视频: Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields TTFields

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

科研

免疫与感染

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

肿瘤治疗胶质母细胞瘤的临床应用

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

癌症研究

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.
In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

协议的射频评估与互动金纳米粒子和生物系统的非侵入性癌症热疗治疗

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

医学

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

肿瘤治疗领域的治疗与联合贝伐单抗治疗复发性胶质母细胞瘤的治疗

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

离子通道治疗实体瘤的药理学靶向

Screening Ion Channels in Cancer Cells

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.

作者聚焦:探索离子通道在癌症中的作用:表征和可能的治疗方法 

The pharmacological targeting of ion channels is a promising approach to treating solid tumors. Detailed protocols are provided for characterizing ion channel function in cancer cells and assaying the effects of ion channel modulators on cancer viability.

筛选癌细胞中的离子通道

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development ...

Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In addition, this method can be used in combination with specialized agents known as sonosensitizers to increase the extent of preferential damage exerted by ultrasound against neoplastic cells, an approach referred to as sonodynamic therapy (SDT). The methodology for generating and applying low frequency ultrasound in a preclinical in vitro setting is presented to demonstrate that reproducible cell destruction can be attained in order to examine and compare the effects of sonication on neoplastic and normal cells. This offers a means by which to reliably sonicate neoplastic cells at a level of consistency required for preclinical therapeutic assessment. In addition, the effects of cholesterol-depleting and cytoskeletal-directed agents on potentiating ultrasonic sensitivity in neoplastic cells are discussed in order to elaborate on mechanisms of action conducive to sonochemotherapeutic approaches.

Selective damage of human leukemia cells can be achieved through a novel approach of applying low frequency ultrasound both with and without chemotherapeutic pretreatment of leukemic and normal hematopoietic cells.

代脉冲式低频超声定量分析，以确定未处理的声波的敏感性和治疗肿瘤细胞

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

在预处理肿瘤细胞的同种异体共培养环境中分析人 T 细胞活性

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

确定癌细胞使用肿瘤治疗领域的最佳抑制频率（TTFields）

摘要

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此视频中的章节

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

肿瘤治疗胶质母细胞瘤的临床应用

协议的射频评估与互动金纳米粒子和生物系统的非侵入性癌症热疗治疗

肿瘤治疗领域的治疗与联合贝伐单抗治疗复发性胶质母细胞瘤的治疗

筛选癌细胞中的离子通道

筛选癌细胞中的离子通道

筛选癌细胞中的离子通道

代脉冲式低频超声定量分析，以确定未处理的声波的敏感性和治疗肿瘤细胞

在预处理肿瘤细胞的同种异体共培养环境中分析人 T 细胞活性

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植