Name: assessing specificity of anticancer drugs in vitro
Uploaded: 2016-03-23T14:00:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about Assessing Specificity of Anticancer Drugs In Vitro at JoVE.com

阿尔斯特夫人，西洋中部先生和姜医生都赞赏他们出色的技术援助。还赞赏是Volz的博士和提供访问他们的数字PCR设备的研究小组Dandri博士。

1.准备混合文化同时含有肿瘤细胞和非肿瘤细胞<ol><li>使用的肿瘤细胞从已建立的细胞系MPNST S462 5。使用成纤维细胞作为非肿瘤细胞6,7- </li><li>培养既肿瘤细胞和在标准DMEM培养基含有10％胎牛血清的成纤维细胞（BSA），3 2mM谷氨酰胺，0.1毫摩尔2-巯基乙醇，500U / ml青霉素，500微克/毫升链霉素和1mM丙酮酸钠，37℃和5％的CO 2 5-7在亚汇合时，收获用0.05％胰蛋白酶的细胞，并使用血细胞计数器计数。 </li><li>混合400肿瘤细胞和1600非肿瘤细胞一起在96孔培养板的每个孔在0.2ml培养基。考虑肿瘤细胞在5天治疗期的增长更快，肿瘤细胞的初始比例被设置为25％。设置4个重复每个药物浓度。孵育细胞在37℃和5％的CO 2 O / N。 </li></ol> 2.德鲁摹治疗<ol><li>使用尼罗替尼作为测试药物。制备在DMSO 0，2，4和20mM尼洛的储备溶液。通过200倍的5ml DMEM培养基稀释他们每个人的，给人为0，10，20和100微米6.7 2倍浓缩的药物溶液。 </li><li>从每个删除0.1毫升中井和加入0.1毫升含有培养基的药物。终浓度是0，5，10和50微米。继续温育在含药物培养基中的细胞5天。 </li><li>在治疗结束时，检查用相位差显微镜的附着的细胞，并拍照。吸出介质走，用0.2ml的PBS洗一次存活粘合剂细胞。 </li></ol> 3.准备的DNA数字PCR <ol><li>裂解细胞使用的HotShot 8。 <ol><li>加入50μl碱裂解溶液（25mM的氢氧化钠，0.2毫摩尔EDTA）到每个孔中。用铝箔密封井。孵育在95℃的板在烘箱中或在加热板上30分钟。查了下，MICR的Oscope以确保没有更多的完整的细胞保持在孔的表面上。万一细胞并没有完全溶解，增加加热时间或者添加洗涤剂。在-20℃冷冻过一次，还有c可增强细胞的破坏。 </li><li>加入50μl中和液（40毫摩尔Tris-HCl，pH值4.0）至各孔中。传输所有裂解物成PCR板和干在PCR循环仪中的U形形式的井在95℃下进行10至30分钟。重构用20微升蒸馏水裂解物。 </li></ol></li><li>使用下拉式透析9淡化<ol><li>使用膜过滤器与平均孔径0.025微米。 </li><li>浮在蒸馏水过滤器在一个12孔板的孔中有光泽的一面朝上。湿过滤器5分钟。注意避免过滤器和水之间产生气泡。 </li><li>吸管从步骤3.1.4各样品的重组溶胞产物小心到每个滤波器的4个区域的。覆盖板和透析30至60分钟。 </li><li>吸走水过滤器没有下过翻转过滤器，也没有扰乱滴。 </li><li>传送裂解物滴到一个新的PCR的板的孔中。干燥通过加热使DNA的液滴在95℃下进行10至30分钟，在PCR循环仪中。重构于10μl水中的溶胞产物。 </li></ol></li></ol> 4.数字PCR <ol><li>解冻冷冻组分如DNA，缓冲液和在RT测定混合物，降速10秒在任何可利用离心机的最大的力（例如，VWR微型离心机），然后保持他们在冰上。 </li><li>准备包含除DNA（ 表1）所有组件的主混合物中。考虑死体积并计算比所需量多大约10％的主混合物中。 注意:使用在市售试剂盒中使用的引物和探针。 </li><li>涡流的主混合物，降速10秒在任何可利用离心机的最大的力（例如，VWR微型离心机）并分配15微升到每个96 PCR板的井。添加所有裂解液（包含DNA）每个样品包含主混合井。转移20微升每个反应混合物的成孔上在RT盒的样本面。 </li><li>分配70微升液滴生成的油的成每个孔上的卡盒的油面。与垫圈关闭墨盒和整个设置放入一个液滴发生器。启动液滴产生。 </li><li>转移40微升液滴溶液到96孔PCR平板的孔中。密封用薄片的板和板放置到PCR仪。设定在105℃的盖温度，并在2℃/秒斜坡率。 表2中的设置进行PCR后的PCR，该板块转移到液滴的读者开始阅读。可替代地，存储在4℃的板长达24小时。 </li></ol> 5.计算肿瘤细胞的比例<ol><li>加载数字PCR数据，并执行自动analys是。 </li><li>手动检查的正和负的液滴的一维和二维分布控制各测定的结果，并确保它们很好地分离和阈系是在正确的位置上。排除的结果不符合标准，例如，正和负的液滴没有明确分开，从进一步的分析。由此产生比NF1 / RPP30复制到Excel表格中。 </li><li>计算肿瘤细胞作为比重:2×（1- NF1 / RPP30）。 </li></ol>肿瘤细胞的比例6.剂量依赖性变化<ol><li>每种药物浓度，计算从4个重复的平均值和标准偏差。绘制的手段和打击与毒品浓度的标准偏差。 </li></ol>

<ol>
	<li>Caponigro, G., Sellers, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+preclinical+testing+of+cancer+therapeutic+hypotheses.">Advances in the preclinical testing of cancer therapeutic hypotheses.</a> Nat Rev Drug Discov. 10 (3), 179-187 (2011).</li><li>McMillin, D. W., Negri, J. M., Mitsiades, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+tumour-stromal+interactions+in+modifying+drug+response:+challenges+and+opportunities.">The role of tumour-stromal interactions in modifying drug response: challenges and opportunities.</a> Nat Rev Drug Discov. 12 (3), 217-228 (2013).</li><li>Wilding, J. L., Bodmer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+lines+for+drug+discovery+and+development.">Cancer cell lines for drug discovery and development.</a> Cancer Res. 74 (9), 2377-2384 (2014).</li><li>Edwards, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+target+validation+using+patient-derived+cells.">Preclinical target validation using patient-derived cells.</a> Nat Rev Drug Discov. 14 (3), 149-150 (2015).</li><li>Frahm, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+phenotypic+characterization+of+tumor+cells+derived+from+malignant+peripheral+nerve+sheath+tumors+of+neurofibromatosis+type+1+patients.">Genetic and phenotypic characterization of tumor cells derived from malignant peripheral nerve sheath tumors of neurofibromatosis type 1 patients.</a> Neurobiol Dis. 16 (1), 85-91 (2004).</li><li>Jiang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+selectivity+of+nilotinib+on+NF1-associated+tumors+in+vitro.">Efficacy and selectivity of nilotinib on NF1-associated tumors in vitro.</a> J Neurooncol. 116 (2), 231-236 (2014).</li><li>Jiang, W., Mautner, V. F., Friedrich, R. E., Kluwe, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+assessment+of+the+anticancer+drug+response+of+plexiform+neurofibroma+tissue+using+primary+cultures.">Preclinical assessment of the anticancer drug response of plexiform neurofibroma tissue using primary cultures.</a> J Clin Neurol. 11 (2), 172-177 (2015).</li><li>Montero-Pau, J., Gomez, A., Munoz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+an+inexpensive+and+high-throughput+genomic+DNA+extraction+method+for+the+molecular+ecology+of+zooplanktonic+diapausing+eggs.">Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs.</a> Limnology and Oceanography-Methods. 6, 218-222 (2008).</li><li>Saraswat, M., Grand, R. S., Patrick, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desalting+DNA+by+drop+dialysis+increases+library+size+upon+transformation.">Desalting DNA by drop dialysis increases library size upon transformation.</a> Biosci Biotechnol Biochem. 77 (2), 402-404 (2013).</li><li>Iglesias-Bartolome, R., Martin, D., Gutkind, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+head+and+neck+cancer+oncogenome:+widespread+PI3K-mTOR+pathway+alterations+and+novel+molecular+targets.">Exploiting the head and neck cancer oncogenome: widespread PI3K-mTOR pathway alterations and novel molecular targets.</a> Cancer Discov. 3, 722-725 (2013).</li></ol>

The procedure is patent-protected (10 2013 221 452, Germany)

在用于体外评估药物特异性此genetic-和基于细胞的工具中的关键步骤是使用一个或多个肿瘤特异性的遗传特征的肿瘤细胞的比例的测定。与传统的表型特征7，遗传特性是特定的，稳定的和容易量化。例如，肿瘤特异性突变或拷贝数变异只存在于肿瘤细胞中，但不是在非肿瘤细胞。这样的遗传柱头能够可靠地和精确地定量，例如，使用数字PCR如本研究。 对于数字PCR，DNA的高纯度不是必要的。然而，脱盐并除去抑制分子是必要的，这可以通过使用合适的滤光器的下拉式透析来实现。将合并的裂解和下拉式透析是简单，快速，不需要特殊的工具，因此，可以在任何标准的实验室中进行。如果细胞不布雷亚ķ良好，另外的处理可以被认为是，如冷冻细胞在-20℃，加入洗涤剂和/或使用蛋白酶。 在本研究中，NF1基因的杂合缺失用作定量肿瘤特异性特征。同样地，也可以使用其他的基因或基因座的缺失。此外，突变也可用于定量肿瘤细胞。在这种情况下，对于突变体数字PCR测定和正常等位基因需要被广泛验证，以确保其为每个特异性。 肿瘤细胞的剂量依赖性比例为药物 - 特异性或选择性的指示。此外，它也使从混合培养的总细胞对药物（ 图4）的响应的肿瘤细胞中提取的响应。此萃取策略提供了在使用原代培养物的技术问题的解决方案（其中包含肿瘤和无正肿瘤细胞）进行药物测试。 基于细胞在本研究证明因此可能具有应用潜力的体外工具genetic-和（1）在药物发现，其中它提供了用于评估药物特异性或选择性的体外和一平台（2）个性化癌症护理它使测试药物在小学文化，因此可以辅助化疗4有助于药物的选择。此外，可以使用此工具来研究药物的药理作用机制，因为某些遗传改变或多个基因改变可以在药物治疗过程中应遵循。 在使用个别化原代培养物的一个困难是缺乏对肿瘤细胞的量化的通用遗传特征。但是，随着今天的全基因组测序提前，最频繁改变的基因和区域是已知的常见的肿瘤。例如，其中头部和颈部肿瘤，71％，23％和22％具有在TP53，FAT1突变和CDKN2A基因，60％，34％和26％具有在CDKN2A，STK11和PTEN，分别为10的基因区域缺失。因此，通过靶向测序和/或数字PCR方法筛选5至10个这样的基因或/和地区将有可能确定哪些可用于肿瘤细胞中的衍生初级培养定量的一种或多种遗传改变。另一个限制是用于建立原代培养物的切除肿瘤的量往往不足。 尽管有利特征和genetic-的有前途的应用潜力和基于细胞的体外工具，其限制，应牢记及其体外性质应该强调。例如，对肿瘤细胞的差动作用或细胞毒性药物的可能，而由于在细胞类型差异（成纤维细胞与雪旺氏细胞）或它们不同来源（来自不同个体）。在肿瘤细胞上比在非肿瘤细胞的药物中较强的作用也由一个事实，即前者增长比后者快进行说明。最重要的是，细胞在体外行为不同比在人体中的细胞，因此， 在体外评估结果经常没有或只是部分地反映真实的临床情况。因此，功效，毒性和对培养的细胞获得的药物特异性相当的生物标志物的性质，但不具有抗生素临床前测量效力的可靠性。然而，从细胞系混合培养和/或初级培养移动是在评估在体外的药物作用和特异性/选择性前进了一步。具有广泛的努力， 在体外条件可以发现这使得与患者实际响应，至少在某些药物在体外数据的合理相关性。

Therapeutic effects of anticancer drugs on tumor cells are associated with various unwished toxic effects on non-tumor cells. Toxic effects are difficult to assess in the laboratory, largely due to the lack of suitable tools1. Cell cultures containing both tumor and non-tumor cells would provide a potential in vitro resource for assessing specificity or selectivity of a drug which may shed light on clinical toxicity. However, conventional in vitro assays such as proliferation or viability assays measure effect of a drug on total cells, but cannot divide the effect into the part on tumor cells and the part on non-tumor cells. For example, a 50% reduction of total cell number in a culture composed of 50% tumor cells and 50% non-tumor cells can imply death of all tumor cells, death of all non-tumor cells or, more likely, death of parts of both at various ratios. 
Also because of this technical problem, primary cultures which contain tumor and non-tumor stromal cells are frequently unsuitable for testing drugs. On the other side, because of this cellular heterogeneity, primary cultures resemble their original tumors far better than clonal cells in established cell lines2. More importantly, primary cultures are patient-specific and may enable individualized drug testing2-4.
An essential issue is therefore the development of a method for determination of the proportion of tumor cells in mixed cultures including primary cultures. Toward this challenge, a genetic solution was conceived which is based on determination of the quantitative ratio of a tumor-specific genetic feature to a universal reference sequence. From this ratio, the proportion of tumor cells in a mixed culture can be calculated. 
This study illustrates the procedure of the genetic and cell-based tool for assessing specificity of anticancer drugs including (1) preparing a mixed culture from tumor cells of an established line and non-tumor fibroblasts, (2) treating the mixed culture with a test drug, (3) preparing DNA for digital PCR, (4) determining the ratio of an tumor-specific heterozygous deletion to a reference sequence using a dual-probe droplet digital PCR assay, (5) calculating proportions of tumor cells at each drug concentration and (6) drawing up dose-dependent change of proportion of tumor cells.

<table><tbody><tr><td>Membrane filter of 25 mm diameter</td><td>Merk-Millipore</td><td>VSWP 02500</td><td>For drop-dialysis</td></tr><tr><td>ddPCR assay for NF1</td><td>BioRad</td><td>dHsaCP1000177</td><td>FAM-labelled</td></tr><tr><td>ddPCR assay for RPP30</td><td>BioRad</td><td>dHsaCP2500350</td><td>HEX-labelled</td></tr><tr><td>QX200</td><td>BioRad</td><td>186-4001</td><td>The previous version QX was used in the study, which is not available any more. The replacement is QX200</td></tr><tr><td>Droplet oil</td><td>BioRad</td><td>186-3005</td><td></td></tr><tr><td>Cartriges&amp;nbsp;</td><td>BioRad</td><td>186-3004</td><td></td></tr><tr><td>Gaskets</td><td>BioRad</td><td>186-3009</td><td></td></tr><tr><td>Cartrige holder</td><td>BioRad</td><td>186-3051</td><td></td></tr><tr><td>Pierceble foil</td><td>BioRad</td><td>181-4040</td><td></td></tr></tbody></table>

assessing specificity of anticancer drugs in vitro

一种用于在使用同时含有肿瘤和非肿瘤细胞培养物的体外评估的抗癌药物特异性过程证明。的关键要素是使用双探针数字PCR测定和肿瘤细胞的比例的后续计算定量测定相对肿瘤特异性遗传改变的一个普遍序列。该试验是在含有和已建立的线路的肿瘤细胞的培养进行了掺入-在非肿瘤细胞。混合培养用各种浓度的试验药物进行处理。在处理后，DNA直接从在利用简单和廉价的方法的96孔板孔中的存活粘合剂细胞制备，并进行双探针数字PCR分析，用于测量肿瘤特异性遗传改变和参考普遍序列。在本示范中，NF1基因的杂合缺失用作肿瘤特异性遗传改变和一个RPP30基因作为参考基因。使用比率NF1 / RPP30，肿瘤细胞的比例计算。因为肿瘤细胞的比例的剂量依赖性变化提供了该药物的特异性的体外指示，这种遗传和基于细胞的体外测定法将可能有在药物发现的应用潜力。此外，个性化癌症护理，这genetic-和基于细胞的工具可以有助于通过使用个别肿瘤的原代培养物测试功效和候选药物特异性的手段优化辅助化疗。

使用组合的HotShot /落透析是足以令人满意的数字PCR（ 图1B）制备的DNA的质量。为2000的成纤维细胞，约得到3000±500正液滴，对应于预期4000份（在一个小区中一式两份）的75％。在这项研究中，肿瘤细胞从已建立的线路MPNST S462这是众所周知的具有NF1基因5的杂合缺失。这50％的基因剂量减少显然由双数字PCR检测（ 图1B）进行检测。与此相反，在成纤维细胞，测定该基因的两个拷贝。肿瘤和非肿瘤细胞之间的这种遗传差异使得在混合培养物的肿瘤细胞（ 图2）的比例的计算。通过测量NF1的相对基因剂量RPP30，在混合培养的肿瘤细胞的比例可以被确定在每一药物浓度（ 图3）。 <img alt="图1" src="/files/ftp_upload/53752/53752fig1.jpg" /> 图1. 数字PCR（A）说明的原则。 （B）双探针检测显示减少了肿瘤细胞MPNST S462的目标（NF1）基因相对于参考（RPP30）基因的数量。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53752/53752fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53752/53752fig2.jpg" /> 图2 肿瘤细胞的计算比例。肿瘤细胞和非肿瘤细胞以不同比例混合，并接种到96-板的各孔中。附件O / N，将细胞裂解机智后ħ的HotShot /掉落透析和脱盐，溶菌物进行这给了NF1 / RPP30的比例数字PCR。在此基础上的比例，肿瘤细胞在每孔的比例来计算。平均并计算肿瘤细胞的比例的标准偏差从4个重复在每个设置的比例。肿瘤细胞（Y轴）的计算比例分别为暗算的设立比例（X轴）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53752/53752fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53752/53752fig3.jpg" /> 图3. 肿瘤细胞比例的剂量依赖性变化。（A）剂量依赖性可见至关重要的细胞减少。 （B）中的肿瘤细胞的比例（平均为4次重复的标准差）从计算出的<html> NF1的比率: - 10μMRPP30，在0范围减小。在最高剂量50微米，一些重要的细胞被留下，很可能是由于毒性作用于所有细胞。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53752/53752fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53752/53752fig4.jpg" /> 从非肿瘤细胞的肿瘤细胞（A）的存活力和/或总细胞的增殖如图4 分离反应可使用常规的测定法来测量;可使用在本研究中表现出的程序来确定肿瘤细胞的比例。使用两个参数（B）中，混合培养对药物的总细胞的反应可以分为肿瘤细胞和非肿瘤细胞的应答的响应。.COM /文件/ ftp_upload / 53752 / 53752fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <table fo:keep-together.within-page="1"><colgroup><col /><col /><col /></colgroup><tbody><tr height="21"><td height="21" style="height:21px;width:147px;"></td><td style="width:171px;"> 1反应</td><td style="width:99px;"></td></tr><tr height="21"><td height="21" style="height:21px;width:147px;"> 2×ddPCR超混合液</td><td style="width:171px;"> 12 </td><td style="width:99px;"></td></tr><tr height="21"><td height="21" style="height:21px;width:147px;">分析组合的NF1 </td><td style="width:171px;"> 1.2 </td><td style="width:99px;"></td></tr><tr height="21"><td height="21" style="height:21px;width:147px;">测定混合物的RPP30 </td><td style="width:171px;"> 1.2 </td><td style="width:99px;"></td></tr><tr height="21"><td height="21" style="height:21px;width:147px;"> HaeIII </td><td style="width:171px;"> 0.6 </td><td style="width:99px;"></td></tr><tr height="40"><td height="40" style="height:40px;width:147px;">小计</td><td style="width:171px;"> 15 </td><td style="width:99px;">混合大师</td></tr><tr height="21"><td height="21" style="height:21px;width:147px;">脱氧核糖核酸</td><td style="width:171px;"> 9 </td><td style="width:99px;"></td></tr><tr height="40"><td height="40" style="height:40px;width:147px;">总</td><td style="width:171px;"> 24 </td><td style="width:99px;">最终的PCR混合物</td></tr></tbody></table> 表1设置双ddPCR反应 <table fo:keep-together.within-page="1"><tbody><tr><td> 步 </td><td> 行动 </td><td> 温度 </td><td> 时间 </td><td> 周期 </td></tr><tr><td> 1 </td><td> POLYM清除激活</td><td> 95℃ </td><td> 10分钟</td><td> 1 </td></tr><tr><td> 2 </td><td> DNA变性</td><td> 94℃ </td><td> 30秒</td><td rowspan="2"> 40 </td></tr><tr><td> 3 </td><td>退火/ exetension </td><td> 60℃ </td><td> 1分钟</td></tr><tr><td> 4 </td><td>聚合酶失活</td><td> 98℃ </td><td> 10分钟</td><td> 1 </td></tr><tr><td>五</td><td>保持（可选） </td><td> 4℃ </td><td></td><td></td></tr></tbody></table> 表2 PCR仪设置

Watch this Scientific Journal Video about Assessing Specificity of Anticancer Drugs In Vitro at JoVE.com

Assessing Specificity of Anticancer Drugs In Vitro

该协议的目的是评估使用同时含有肿瘤和非肿瘤细胞混合培养抗癌药物在体外的特异性。

评估抗癌药物的特异性<em&gt;体外</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

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Universidad San Sebastian

Research

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Medicine

Assessing Specificity of Anticancer Drugs In Vitro

9.4K 次观看.  University Medical Center Hamburg-Eppendorf. 该协议的目的是评估使用同时含有肿瘤和非肿瘤细胞混合培养抗癌药物在体外的特异性。 

视频: Assessing Specificity of Anticancer Drugs In Vitro

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

利用RNA测序来检测新剪接变异体在相关耐药<em&gt;体外</em&gt;癌症模型

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

科研

癌症研究

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

抗癌金属配合物的合成与细胞毒性评价MTT法

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

化学

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

球体、菌落形成测定和斑马鱼移植对抗癌香豆素 OT48 生物活性的临床前评价

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

一种基于流式细胞术的细胞表面蛋白结合测定，用于评估抗癌适配体的选择性和特异性

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

一种混合的3D组织工程<em&gt;体外</em&gt; /<em&gt;在硅片</em在具体的突变背景预测药物的有效性&gt;肺肿瘤模型

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

生物工程

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

评估抗癌药物的特异性

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利用RNA测序来检测新剪接变异体在相关耐药