Name: implementation of in vitro drug resistance assays: maximizing the potential for uncovering clinically relevant resistance mechanisms
Uploaded: 2015-12-09T14:00:00.000+00:00
Duration: 8 min 46 s
Description: Watch this Scientific Journal Video about Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms at JoVE.com

The authors would like to acknowledge our colleagues at H3 Biomedicine for their feedback during the manuscript preparation.

1.评估GI 50的目标化合物（S） <ol><li>产生生长曲线（多个）感兴趣以评估相应的接种密度的细胞系。绘制在不同时间点在播种之后的细胞数（天2，4，6和8），相对于第0天这将提供所关注的细胞系的相对生长速率，应用于评估最初的接种密度，使得在96孔板未达到7天内汇合。 </li><li>一旦生长曲线已经确定，在细胞非透明，透明底96-孔板中加入100μl细胞培养基的种子适当数目来评估的GI 50。种子基于使用和1.1所确定的细胞系的细胞数。通常情况下，种子3×10 3细胞快速生长细胞系倍增时间24小时的〜， 例如 ，HCT116。 <ol><li>制备测定板（天-1）。为测定板，种子细胞在100微升培养基我期望的密度N阱中的阴影蓝色（ 图1）。威尔斯在白色高亮只包含媒体。 </li><li>准备一个控制板（天-1）。为控制盘，种子细胞的相同的密度在步骤1.2.1于100μl培养基中的单独的96孔板中。这节0"读数将在解释化合物的细胞生长抑制/细胞毒性性质帮助被分析（ 图2）。 </li></ol></li><li>第二天（0天），阅读控制板。平衡发光细胞存活力试剂（CELLTITER-格洛衬底根据制造商的说明用缓冲液混合）至RT，并通过反转内容以获得均匀的溶液轻轻混匀。添加80微升试剂到100微升细胞/媒体组合和摇内容30分钟以诱导细胞裂解。 <ol><li>记录发光用发光设定为0.1-1.0秒，检测560nm处的波长的曝光。 </li></ol></li><li>添加试验化合物（化合物景点）到测定板（第0天）。得到1:4连续稀释在DMSO中的化合物的200倍，在最终浓度为总共10个浓度（含有化合物和只有一个-DMSO，化合物板9稀释）。作为初始起点，目的是为0.03微米的200倍最低剂量和顶部剂量2000微米（最终体积200微升）。 </li><li>添加系列稀释的化合物中，使化合物的培养基混合在10倍终浓度（最终体积100微升，中间板）。存储化合物板在-20℃下对测定的3天使用。添加10微升化合物的培养基混合的细胞以一式三份，使得最高剂量是10μM的（例如，行B，C和D，第0天）（ 图1）。孵育测定板3天，在37℃。丢弃使用后的中间板（多个）。 </li><li>在第3天，使用在1.4中制备的化合物平板制备400μl的1×化合物的培养基混合。倒置试验板取出媒体抹干对汽车claved纸巾2-3倍，以去除残留的媒体。添加化合物/媒体（100μl/孔）到孔中的阴影部分和蓝色加100微升媒体至周边孔以防止蒸发（ 图1）。再孵育测定板在37℃另外3天。 </li><li>在第6天，通过如步骤1.3中所述进行发光读数评估存活细胞的相对数。 </li><li>使用0天读数来评估化合物的静态/有毒的性质。有毒物质诱导细胞凋亡而抑制细胞生长的药物诱导细胞周期阻滞。如果化合物是有毒的（一天6读数低于日0阅读），考虑选择GI 50和GI 50×5浓度性检测。然而，如果化合物诱导瘀（等于或高于D0读），考虑GI 100和GI 100×5为电阻测定（ 图2）。 </li></ol> 2.设置耐药性测定<ol><li>如果工作机智公顷细胞抑制剂，种子细胞在30-40％150毫米2组织培养皿（体积= 30毫升）中的电阻测定中汇合。如果具有毒性剂工作，种子在70-80％汇合。 </li><li>为此怀有一个完整的错配修复（MMR）机制的细胞系，从步骤孵育细胞2.1 O / N在37℃下用致癌物N-乙基-N-亚硝基脲（ENU）。处理细胞用30微升的50毫克/毫升ENU（终浓度50微克/毫升）的储备溶液，以提高基因组不稳定性。对于有缺陷的MMR（ 表2和3），用ENU或其它致癌物处理可能不是必需的细胞系。 <ol><li>对于其它细胞系，确定MMR -缺乏通过使用微卫星不稳定性12 NCI标准，或基于使用微卫星不稳定性测定或MMR基因13-15的基因组/表观仿形公布表征。 </li></ol></li><li>处理的细胞与测试化合物（多个）在浓缩的兴趣在步骤1.8确定。对于引起细胞死亡在一个典型的三天生存试验10高毒性的化合物，开始用低剂量的治疗（即，GI 50）和逐步增加浓度（GI 50的倍数）化合物的每2-3周，直到健壮性观察。补充媒体和复合每3天。 </li><li>一旦选择已经启动，改变媒体/复合混合每隔3-4天，直到耐药克隆出现。电阻定义出现时处理的细胞表现出在药物治疗相对于急性期治疗DMSO处理的对照细胞的更大的增长/可行性。 </li></ol> 3.隔离单细胞克隆<ol><li>用相差显微镜（放大率40X）对细胞的存活簇检查培养皿。 </li><li>马克满意的克隆在用标记笔在培养皿底部。挑选克隆，平均规模（大落可能从多个细胞起源），并从其他殖民地以及隔离。选择使用在步骤3.3概述了两种方法中的一种克隆。 </li><li> 方法1:使用吸移管（图3） <ol><li>除去生长培养基，用1X PBS冲洗，以消除任何漂浮细胞。 </li><li>使用黑色标记为先导，以"捡"克隆与枪头（连接到移液器，P200最好）。 </li><li>转移克隆入48孔板用200μl新鲜介质（具有半化合物浓度，以允许细胞的最佳回收）。 </li><li>让细胞加入200微升新鲜培养基/理想的化合物浓度，然后才能恢复2-3天。 </li><li>继续改变媒体/复合每隔3-4天，并不断扩大的克隆。 </li></ol></li><li> 方法二:使用克隆光盘（图3） <ol><li>如步骤3.2中所述标记的克隆。 </li><li>放置3毫米克隆光碟含有5ml的0.25％吨10cm的组织培养皿rypsin-EDTA 2分钟。 </li><li>含盘吸媒体性克隆和重叠使用一次性无菌镊子胰蛋白酶浸泡克隆光盘克隆。 </li><li>离开1-2分钟，这取决于克隆如何容易地抬离板，在37℃培养箱。 </li><li>拿起用无菌镊子克隆光盘和转移到48孔板用200微升新鲜培养基和化合物的浓度的一半。 </li><li>移液器上下轻轻地从克隆光盘撞出细胞，并培育O / N，37°C（离开井克隆光盘）。 </li><li>第二天早上，从48孔板取出克隆光盘和补充井用200微升新鲜培养基/化合物（半理想的化合物浓度）。 </li><li>三天后，补充媒体与化合物的理想浓度。 </li><li>继续改变媒体/复合每隔3-4天，并不断扩大的克隆。 </li></ol></li></ol> 4.评估的R等级分离克隆的esistance <ol><li>一旦:10-20菌落被扩展，生成的GI 50的曲线如在步骤1中所述，以评估抗性程度。 </li><li>始终包括在选择过程用DMSO处理的对照人群。它很可能是抗性的频谱将是大的（一些示出的部分，而其他显示全电阻）。收集来自每个类作为电阻可以在两个组之间变化的机制的一些克隆。 </li></ol> 5.新一代测序<ol><li>在15毫升降速2亿个细胞（控制和耐药克隆）（500 XG 5分钟），锥形瓶为基因组DNA和RNA集合（因此2×2万美元）。 </li><li>用1×PBS洗涤两次，并冻结-80°C的颗粒，直到准备隔离。 </li><li>使用商业提取试剂盒根据制造商的协议以分离RNA或基因组DNA。 </li><li>提交样品用于新一代测序使用供应商的协议10。 </li></ol> 6.生物信息学分析样品（全外显子组测序） <ol><li>预处理根据最佳实践的DNA序列管线16序列数据。 <ol><li>该地图所有读取以参考使用BWA 17人类基因组GRCh37。转换未压缩的SAM格式的比对，以压缩BAM格式Samtools 18。排序比对与Samtools坐标。添加使用皮卡德阅读群体。 （对于具体的BWA，Samtools和皮卡尔命令，见补充文本1，线1-4） </li><li>标记所有重复读取使用MarkDuplicates命令从皮卡工具集19。该文件的索引与Samtools。 （附文1，第5-6行） </li><li>为了尽量减少错配碱基在所有读取，重新调整在局部地区窝藏使用插入缺失realigner 20个小插入或缺失的读取。 （附文1，第7-8行） </li><li>为了进一步提高accuracY变体调用，用碱质量分数校准工具凭经验重新校准基地的质量分数。基础质量校准工具不仅要正确的初始质量分数，还要考虑到的几个特点，包括阅读组，机器周期，基地的位置，二核苷酸上下文（前+电流基地）账户共变。生成重新校准BAM与皮卡德PrintReads。 （有关此步骤的具体命令，见补充文本1，行9-10） </li><li>重复步骤6.1.1至6.1.4（附文1，行1-10）的测序每个样品。 </li></ol></li><li>确定每个克隆的单核苷酸变异（SNV）采用配对变量调用tool19，21-23。对于测序每个克隆，运行配对变体使用亲本克隆为匹配的"正常"呼叫。 （附文1，第11行） </li><li>过滤复发性，高品质的变体和用于注释制备具有变异注释工具 24。 Deprioritize VAriants并非适用于所有抗性克隆。 （参见附文2 R脚本） </li><li>注释与注释工具25，26变种。许多变种注释工具有一个附带的Web应用程序允许数据被上传，并通过远程服务器进行处理。 </li><li>使用功能影响预测工具27-29优先预测具有高功能性影响的那些变体。像变种注释，有几个工具可以通过Web界面功能影响的预测。 </li></ol>

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本文的出版费用由H3生物医学支付。

选择细胞系（S）的:遗传状态，基因组不稳定性特征 毫无疑问，在成功地揭示临床相关耐药机制的一个最重要的因素是初始小区选线。两个因素应予以考虑。首先，目的是选择相同谱系/亚型窝藏该疾病的定义遗传性状的细胞系（多个）（例如，BRAF V600E在黑素瘤）。公开可用的转录和变异数据，这两种细胞系30-32和主/转移瘤，适用于各种适应症33,34的讯问将有利于选择过程。虽然鉴定细胞系临床相关的遗传改变是理想的，在某些情况下，这是不可能的，由于缺乏可用的细胞系或可行由于因素如困难筛选过程的和长度。 35。基于所述COSMIC数据库，几个候选细胞系缺乏存在两种常见的突变基因，MMR，MSH2或MLH1酮（ 表2和3）。或者，如果所关注的细胞系中不MMR，与物理或DNA的反应性化学诱变剂的急性治疗存在轴承缺陷如烷基化剂N-乙基-N- -nitrosourea（ENU）可用于提高基因组不稳定性。虽然这两种方法可能显著s ^霍顿获得抗性克隆和后续测序时，严格的功能测试应该在候选基因作为更大数量的非功能性来进行，乘客的突变是可能出现。 SNVs可以排名责令最大化机会识别功能相关的基因突变。首先，选择那些反复发作的独立克隆的突变会增加这些突变性的驱动程序的可能性。在事件经常突变的尚未鉴定的，着眼于适合的药物作用机制SNVs（例如，药物靶标或药物靶的已知下游效应）可能是有意义的。最终，金标准是始终候选SNVs异位cDNA表达在药物敏感的亲代细胞的抗性的活性的实验评价。 DNA VS RNA测序 一旦抗性克隆已经生成，DNA和/或RNA可以根据需要进行测序。 DNA测序，无论是外显子组或全基因组测序，将允许鉴定的种系和体细胞的变体，如单核苷酸多态性，插入缺失和拷贝数变体。而更经济友好外显子测序着重从已知的编码区上生成读，全基因组测序将产生的测序数据为整个基因组，其可有利于在非编码元件如增强或miRNA的36鉴定的突变。然而，由于基因表达数据不是DNA测序期间衡量，这是很难预测哪些突变很可能是一个功能的驱动程序。在这方面，核糖核酸的测序，尽管成本更高，提供这种优势。即只对表达的RNA种类进行突变呼叫的事实增强了可能性感兴趣的突变可以是功能性的驱动程序。除了允许突变更集中的调查，为后续功能测试，RNA测序也提供了能够鉴定基因表达的改变，剪接和新的嵌合RNA种类，包括基因融合，可能也有助于电阻的作为强有力的驱动额外的好处。 生物信息学管道 提供用于说明目的例如命令，但更详细的文档和教程可以从Broad研究所16，应该开始NGS分析前仔细阅读。下面的命令是针对已经预先安装了所有的工具和参考的数据在一个系统上的UNIX shell环境。这些命令也假定FASTQ文件含配对末端序列从两个样品读取，命名为"父母"和"抗性"，已收到来自厂商和放置在"数据"目录。在大多数情况下，这些命令应适应或使用其他命令行参数的特定应用进行优化uments（ 例如 ，添加"-t 8"的BWA命令允许在多线程操作在8个CPU核心）。读组（其分配比对生物样品），必须经常被添加到BAM文件，即使有每咣文件只有一个样本中，为了遵守文件格式要求某些工具。阅读组参数RGID，RGSM，RGPL，RGPU和RGLB可以是任意的字符串描述样品名称，测序平台和库策略。 体外 VS 体内试验 虽然确定了体外选择几个耐药机制已被证实是临床相关的，存在一种可能性，即机制可能不服务于临床电阻相关或主要机制。其原因之一可以包括用于微环境中行驶阻力来治疗中起重要作用，一个组件，不含在实验方案/设置ð迄今iscussed。实际上，一些研究表明，抗癌剂，其能够杀死肿瘤细胞被失效当肿瘤细胞在基质细胞暗示阻力由基质37,38赋予固有机制的存在下培养。鉴定这类基质诱导获得性抗性机制，可以考虑进行体外共培养或体内肿瘤耐药性测定。由于前者测定法是相当复杂的，许多都使出产生耐药肿瘤异种移植物，解决了基质中驱动阻力的潜在作用。这样的研究已经发现相对于体外选择抗性的，两个相同的5和39的独特的机制，这意味着该基质可能确实起到在后者的作用。然而，必须牢记的时间长度可能需要产生这种抗性肿瘤和后续基因组分析-complexitie的复杂性的原因可能会在肿瘤内的分子和细胞异质性。 目标识别 除了揭示耐药机制，这NGS系的基因组谱的方法也可以应用于以确定化学探针的细胞靶。在历史上，多种无偏方法已经被用于识别操作的细胞机制的低分子量的化学品具有生物活性，包括亲和纯化加上定量蛋白质组学，酵母基因组的方法，筛选RNA干扰，和计算推理办法40和目标。作为一个扩展使用NGS-基于基因组或转录组表型耐药细胞群，鉴定独特的经常性的单核苷酸变异（SNVs）或表达变化的分析，使电阻可提供见解化合物的功能性细胞靶点药物耐药机制的阐明。 Ť他是基于如下思想的观察抗性机制的一个子集可以涉及经常突变的基因编码的小分子的直接蛋白质靶。最近，几个报告证实了该方法的效用，特别是与其它方法包括大型癌细胞的敏感性分析相结合，以揭示小分子的细胞目标探测9,10。

肿瘤基因组强劲的测序技术和改进的数据分析工具的具体分子特征，导致关键基因改变的特定类型癌症1,2的发现。靶向疗法旨在这些遗传病变，如HER2，BCR-ABL，EGFR和ALK的发展，已经显著生活质量的提高患者1,2-。然而，尽管这种方法的特异性，所述临床反应最单一疗法一直亚最佳为电阻最终出现。近日，显著的进展已经取得理解性的分子基础，以有针对性的治疗。有趣的是，这是越来越明显的阻力的一个突出机制涉及持久性目标/通路活性。作为一个典型的例子，雄激素受体（AR）-directed enzalutamide前列腺癌的治疗导致激活的AR本身的突变，保持富集AR-信号OUTPUT里的抑制剂3-5的存在。这方面的知识，导致积极运动，以1）开发的第三代拮抗剂，可以继续在抑制WT和突变AR功能enzalutamide抗性前列腺3和2）确定的AR信令可能被靶向用于治疗性干预的潜在下行节点。同样，耐其它类抑制剂如靶向EGFR，BRAF和ABL往往会导致该激活原有上瘾激酶途径1突变。 与电阻不可避免地出现在大多数患者的认识，发展的办法，尽快将这些机制，光线将允许有效的后续治疗发展。正被广泛使用的一种方法是，分析的临床难治的肿瘤基因组学相对于初治或敏感的肿瘤，以确定在遗传病变富集/枯竭可能适合与d地毯的发现。尽管它的诺言，还有这种方法的两个主要责任妨碍快速发现。首先，获得及时获得肿瘤材料基因组审讯可以作为一个显著障碍从治疗转向治愈。其次，遗传病变的抗性设置无数的去卷积可以是具有挑战性的，因为肿瘤可呈现显著肿瘤内异质6,7。 鉴于这些挑战，人们日益依赖的耐药机制临床前发现。这种方法可以允许鉴定突出的耐药机制的临床试验1可以指导临床替代管理策略在承担治疗前这些机制的一个或下发性的患者之前。 一个这样的临床前发现的工具，正在被广泛使用的是采用无偏功能RNAi筛选。对于考试PLE，惠特克和他的同事应用基因组规模的RNAi筛选，以确定NF1损失通过持续MAPK信号通路的激活8介导的抗RAF和MEK抑制剂。这些结果被认为是临床上相关的作为丧失功能的突变NF1 BRAF中-mutant肿瘤细胞，以抑制RAF和在黑素瘤肿瘤抗vemurafenib活化8内在抗性进行观察。然而，尽管这种方法的成功，许多临床相关指标经常没有确定，大概是由于丧失功能的偏压这种方法。 相反，较少偏见工具的抗性机制的临床前的发现包括通过长时间暴露于的加上NGS基于基因组或转录组仿形兴趣化合物抗性细胞系的产生。这种方法已成功地由几个组实施，以确定自发复发性单核苷酸变异或表达的改变，使电阻5,9,10。例如，在AR中的经常F876L突变最近发现在体外 3-5抗性克隆，并在之前识别该突变的异种移植肿瘤在体内 5在诊所4。最近，Bhang和他的同事（2015）中使用ClonTracer两种临床相关模型来显示，多数长时间暴露于药物过程中出现的是一个预先存在的亚群这表明大多数功能相关的突变的部分抗性克隆的11很可能已经预先存在在选择11成为选择。 相对于前面讨论肿瘤的基因组分析，为"同质"性克隆用于分析由少异质性这一做法的好处有利于潜在的司机更准确的遗传剖析。 Furtherm矿石，令人兴奋的，除了用于发现耐药机制的潜力，这种方法也可以应用于以确定行动的细胞机制的生物活性的小分子和目标的量此信息是未知的10。给出明确的优势，这种方法的多种用途，在这里，我们提出了一个协议，详细说明成功实施这样的临床前屏幕，最大限度地为临床意义的发现的潜力。

<table><tbody><tr><td>48-well plates</td><td>Fisher Scientific</td><td>07-200-86&amp;nbsp;</td><td>Expanding clones</td></tr><tr><td>96-well plates</td><td>Fisher Scientific</td><td>07-200-588</td><td>Generating GI&lt;sub&gt;50&lt;/sub&gt; curves</td></tr><tr><td>CellTiter-Glo</td><td>Promega</td><td>G7572</td><td>Viability testing</td></tr><tr><td>Cloning discs (3 mm)</td><td>Sigma</td><td>Z374431</td><td>Picking clones</td></tr><tr><td>Sterile forceps</td><td>Unomedical</td><td>DF8088S</td><td>Picking clones</td></tr><tr><td>RNeasy Plus RNA extraction kit</td><td>Qiagen</td><td>74134</td><td>Isolating RNA</td></tr><tr><td>Blood and Tissue DNeasy extraction kit</td><td>Qiagen</td><td>69581</td><td>Isolating gDNA</td></tr><tr><td>GATK</td><td>The Broad Institute</td><td></td><td>Indel realigner</td></tr><tr><td>MuTect</td><td>The Broad Institute</td><td></td><td>Paired variant calling tool</td></tr><tr><td>Oncotator</td><td>The Broad Institute</td><td></td><td>Variant annotation tool</td></tr><tr><td>MutationAccessor</td><td>The Broad Institute</td><td></td><td>Functional impact prediction tool</td></tr></tbody></table>

implementation of in vitro drug resistance assays: maximizing the potential for uncovering clinically relevant resistance mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

为了最大限度地发现耐药的主要功能驱动的潜力，选择单细胞克隆扩张，表型检测和排序。 如图4A所示，HCT116细胞治疗后长时间用细胞毒性化合物＃1导致抗性克隆的，持续的治疗（虚线黑圆点）时生长自发出现。这些克隆使用方法＃1突出显示在图3中，随后扩大了表型分析回升。 如图4B所示，抗性克隆1-3都表现出显著阻力为化合物＃1和其附近的模拟化合物＃2（大于生存能力/生长），而所有克隆显示敏感性无关的细胞毒性化合物万珂。以下确认表型抗性的，基因组DNA分离并提交全基因组测序分析。生物信息学工具施加缩小在这些结构的变体是:1）经常性和2）具有功能性影响（图5A）的潜力。结构变体满足这两个标准由独立的测序工具进行了证实。 如图5B所示，使用采用Sanger测序法全基因组测序，确认鉴定出杂合错义突变（上面板，WT序列;下面板，突变序列）。以下序列证实，原本用于测定电阻的亲代细胞系被基因工程化以表达突变的cDNA在功能上证实该突变的作用。 如图6所示，而WT cDNA的过度表达未能赋予阻力，迫使突变体的cDNA显著赋予的表型抗性的表达对于化合物＃1，在确认这个结构变异的功能作用为电阻的驱动力。用于此实验的所有试剂在表1中概述了</s<html>仲&gt;。 <img alt="图1" src="/files/ftp_upload/52879/52879fig1.jpg" /> 图1.布局分析和控制板。蓝荫，复合处理井。白翳，唯一媒介。化合物连续稀释1:4和施用以一式三份（BD或EG）。 2化合物可以每板被应用。 <img alt="图2" src="/files/ftp_upload/52879/52879fig2.jpg" /> 图2.代表活力曲线。红色虚线表示0天的读数。X轴表示剂量的化合物的向右递增和y轴表示相对于DMSO对照的孔的生存能力。 GI使用，实现100％的增长抑制100 =剂量; GI 50 =用于减少生存能力DMSO对照的50％的剂量。 _upload / 52879 / 52879fig3.jpg"/&gt; 图3.两种方法用于拾取抗性克隆用于扩展。进场1-使用移液器拿起和良好定义的克隆转移至48孔板。方法2-使用胰蛋白酶的浸泡克隆光盘解除克隆并转移到48孔板。 <img alt="图4" src="/files/ftp_upload/52879/52879fig4.jpg" /> 图4.确认实现对HCT116克隆到化合物在体外 ＃1的电阻。（A）的化合物＃1的耐HCT116克隆出现以下连续三周的选择。的控制和抗性克隆（B）的存活力后用各种化合物72小时处理试验。复方＃2是化合物＃1的密切模拟。万珂用作控制细胞毒性剂。数据显示为三个生物学重复平均值±标准偏差。 图5.识别一个独特的，经常性的突变（单核苷酸变体，SNVs）在基因中的A化合物＃1耐HCT116克隆。（A）的工作流程，以确定存在于所有抗性克隆SNVs和预测具有较高的功能性影响通过MutationAssessor。 （二）确认的突变基因的一个由桑格测序。 <img alt="图6" src="/files/ftp_upload/52879/52879fig6.jpg" /> 图6.重新表达突变基因一个赋予耐复合体外 ＃1。改造的HCT116细胞的存活后，与化合物＃1 72小时的治疗试验。 HCT116-WT或突变的细胞系的稳定表达野生型或A基因突变的cDNA，分别。数据显示为平均值±标准差Ø˚F三次生物学重复。 <table><tbody><tr><td>样品名称</td><td>氨基酸</td><td colspan="2">原代</td><td>接合性</td></tr><tr><td> C-33-A </td><td> p.E768fs * 44 </td><td colspan="2">宫颈</td><td>杂</td></tr><tr><td> C-33-A </td><td> p.S860 * </td><td colspan="2">宫颈</td><td>杂</td></tr><tr><td> CML-T1 </td><td>第？ </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>纯合子</td></tr><tr><td> CP66-MEL </td><td> p.C822F </td><td colspan="2">皮肤</td><td>杂</td></tr><tr><td> CTV-1 </td><td> P.0？ </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>纯合子</td></tr><tr><td> EFO-27 </td><td> p.Q130fs * 2 </td><td colspan="2">子房</td><td>杂</td></tr><tr><td> EFO-27 </td><td>第？ </td><td colspan="2">子房</td><td>杂</td></tr><tr> &lt;TD&gt; HCC2218 </td><td> p.E467K </td><td colspan="2">乳房</td><td>杂</td></tr><tr><td> J-RT3-T3-5 </td><td> p.R711 * </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>纯合子</td></tr><tr><td>将LNCaP </td><td>第？ </td><td colspan="2">前列腺</td><td>纯合子</td></tr><tr><td>的LoVo </td><td>第？ </td><td colspan="2"> large_intestine </td><td>纯合子</td></tr><tr><td> MOLT-13 </td><td> p.R711 * </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>纯合子</td></tr><tr><td> NALM-6 </td><td>第？ </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>纯合子</td></tr><tr><td> NCI-H630 </td><td> p.R680 * </td><td colspan="2"> large_intestine </td><td>杂</td></tr><tr><td> SKUT-1 </td><td> p.L787fs * 11 </td><td colspan="2">子宫内膜</td><td>纯合子</td></tr><tr><td> SKUT-1B </td><td> PL787fs * 11 </td><td colspan="2">子宫内膜</td><td>纯合子</td></tr><tr><td> SUP-T1 </td><td>第？ </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>纯合子</td></tr></tbody></table> 表1. MSH2 -mutated细胞系。细胞系（样品名），氨基酸取代，起源谱系和接合性表示。 <table><tbody><tr><td>样品名称</td><td>氨基酸</td><td colspan="2">原代</td><td>接合性</td></tr><tr><td> CCRF-CEM </td><td> p.R100 * </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>杂</td></tr><tr><td> CCRF-CEM </td><td>第？ </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>杂</td></tr><tr><td> CW-2 </td><td> p.Y130fs * 6 </td><td colspan="2"> large_intestine </td><td>纯合子</td></tr><tr><td> DU-145 </td><td>第？ </TD&gt; <td colspan="2">前列腺</td><td>纯合子</td></tr><tr><td> GR-ST </td><td>第？ </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>纯合子</td></tr><tr><td> HCT-116 </td><td> p.S252 * </td><td colspan="2"> large_intestine </td><td>纯合子</td></tr><tr><td> IGROV-1 </td><td> p.S505fs * 3 </td><td colspan="2">子房</td><td>纯合子</td></tr><tr><td> MN-60 </td><td>第？ </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>纯合子</td></tr><tr><td> NCI-SNU-1 </td><td> p.R226 * </td><td colspan="2">胃</td><td>纯合子</td></tr><tr><td> P30，乐施会</td><td>第？ </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </td><td>纯合子</td></tr><tr><td> PR-MEL </td><td>第？ </td><td colspan="2">皮肤</td><td>纯合子</td></tr><tr><td> REH </td><td>第？ </td><td colspan="2"> haematopoietic_and_lymphoid_tissue </TD&gt; <td>纯合子</td></tr><tr><td> SK-OV-3 </td><td> P.0？ </td><td colspan="2">子房</td><td>纯合子</td></tr><tr><td> SNU-1544 </td><td> p.S2L </td><td colspan="2"> large_intestine </td><td>杂</td></tr><tr><td> SNU-1746 </td><td> p.E523K </td><td colspan="2"> large_intestine </td><td>纯合子</td></tr><tr><td> SNU-324 </td><td> p.C233R </td><td colspan="2">胰腺</td><td>杂</td></tr><tr><td> SNU-324 </td><td> p.V384D </td><td colspan="2">胰腺</td><td>杂</td></tr><tr><td> SNU-478 </td><td> p.V384D </td><td colspan="2">胰腺</td><td>杂</td></tr></tbody></table> 表2. MLH1 -mutated细胞系。细胞系（样品名），氨基酸取代，起源谱系和接合性表示。

Watch this Scientific Journal Video about Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms at JoVE.com

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

为靶向治疗耐药性十分普遍，有必要确定耐药机制 - 前或后临床发作 - 是指导临床的替代管理策略的关键。在这里，我们提出了一个协议，以在体外测序耐药行夫妇的推导，以加快发现这些机制。

实施<em&gt;体外</em&gt;耐药性检测:最大化的潜力，揭开临床有关的耐药机制

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

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Research

JoVE Journal

Medicine

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

10.5K 次观看.  H3 Biomedicine. 为靶向治疗耐药性十分普遍，有必要确定耐药机制 - 前或后临床发作 - 是指导临床的替代管理策略的关键。在这里，我们提出了一个协议，以在体外测序耐药行夫妇的推导，以加快发现这些机制。 

视频: Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

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

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

An in vitro method for establishing dual resistance to an EGFR-TKI and a MET-TKI in cancer cells is described. This method is useful for developing treatments for patients with EGFR-mutations, who exhibit disease progression despite EGFR-TKI treatment with MET-amplification. It can also be modified for inhibitors targeting other molecules.

建立双抗 EGFR-使用 tki 药物治疗和满足使用 tki 药物治疗在肺腺癌细胞体外与 2 步剂量升级程序

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

协议的鲁棒抗除草剂试验在不同杂草

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

环境科学

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

寻找获得性抗靶治疗的驱动途径: 耐药亚克隆生成与基因击倒敏感性恢复

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

遗传学

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

患者源性肿瘤外植体作为预测患者耐药性的"实时"临床前平台

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

合并的shRNA文库筛选，以确定调节耐药性表型的因素

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

对于资源有限设置一个负担得起的HIV-1耐药性监测方法

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

医学

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 ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

对于耐药性突变对激酶抑制剂的筛选和验证方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

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利用RNA测序来检测新剪接变异体在相关耐药