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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">membrane filter of 25 mm diameter</td>
			<td style="width:71px;">Merk-Millipore</td>
			<td style="width:119px;">VSWP 02500</td>
			<td style="width:167px;">for drop-dialysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ddPCR assay for NF1</td>
			<td style="width:71px;">BioRad</td>
			<td>dHsaCP1000177</td>
			<td>FAM-labelled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ddPCR assay for RPP30</td>
			<td style="width:71px;">BioRad</td>
			<td>dHsaCP2500350</td>
			<td>HEX-labelled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QX200</td>
			<td style="width:71px;">BioRad</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;width:216px;">Droplet oil</td>
			<td style="width:71px;">BioRad</td>
			<td>186-3005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cartriges&#160;</td>
			<td style="width:71px;">BioRad</td>
			<td>186-3004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gaskets</td>
			<td style="width:71px;">BioRad</td>
			<td>186-3009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cartrige holder</td>
			<td style="width:71px;">BioRad</td>
			<td>186-3051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pierceble foil</td>
			<td style="width:71px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

assessing-specificity-of-anticancer-drugs-in-vitro

Nanyang Technological University

Research

JoVE Journal

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

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

在利用老鼠的气管收缩的体外测量

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

科研

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

光动力疗法治疗骨肉瘤细胞的细胞毒功效<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

评估传染性海绵状脑病物种界限与<em&gt;在体外</em&gt;朊蛋白转化测定法

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

眼内药物的药代动力学研究采用兔眼

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀:一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

降血脂药物对胆固醇颗粒形态的差异化作用

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

评估下肢缺血的Murine模型的治疗血管生成

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...