Name: looking for driver pathways of acquired resistance to targeted therapy: drug resistant subclone generation and sensitivity restoring by gene knock-down
Uploaded: 2017-12-11T14:00:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down at JoV

作者希望感谢维罗妮卡 Zanoni 博士编辑手稿。

注: 本议定书已作了具体调整, 以分析对她阳性胃癌细胞株曲妥珠单抗的抗性机制。在材料表中报告所需的所有实验室仪器。
1. 有针对性的抗 Subclones 治疗的产生
注意: 这是最关键和难以标准化的步骤在协议中, 由于不同的细胞线可能表现出不同的敏感性对靶向治疗抑制剂独立于其肿瘤 histotype 或药物浓度使用.
<ol><li>培养 NCI-N87 细胞系在 RPMI 培养基中补充胎小牛血清 (10%) 和谷氨酰胺 (2 毫米, 1%), 作为单层在37°c, 并种子它到25厘米2培养瓶。</li>
	<li>添加到每瓶10µg/毫升曲妥珠单抗的培养基作为起始剂量。将细胞暴露到起始剂量, 直到它们恢复生长。 注: 靶向治疗抑制剂的起始剂量应低于其血浆峰值浓度的10倍。血浆峰值浓度是在患者血液中检测到的药物浓度的最高水平, 通常遵循多剂量标准疗法。这个值可以从药代动力学的研究中检索出来。</li>
	<li>用台盼蓝排除法监测细胞生长;当细胞生长恢复时 (通常在三周的时间内), 将细胞暴露在更高剂量浓度的曲妥珠单抗。</li>
	<li>将细胞暴露在逐渐增加的曲妥珠单抗剂量 (从100µg/毫升到400µg/毫升), 直到细胞持续生长 (大约2周), 尽管存在着高浓度的药物 (至少 2.5-4 倍高于血浆峰值)。</li>
	<li>
执行克隆分析, 以评估目标治疗抑制剂在培养基中浓度的每一次变化对靶向治疗的抗药性水平, 并定义相对电阻 ic50索引 (RR ic50), 如下所示。
	<ol>
<li>种子500细胞在24多井培养板材。 注: 每个条件设置必须准备5口井。</li>
		<li>暴露细胞以增加目标治疗抑制剂 (曲妥珠单抗) 浓度从100µg/毫升多达400µg/毫升。</li>
		<li>在 CO2孵化器中孵化出15天37摄氏度的细胞。</li>
		<li>孵化后, 修复和染色样品如下。<ol>
<li>去除培养基和冲洗细胞与10毫升 1x PBS。去除 1x PBS 和添加 2-3 毫升的固定溶液 (乙酸: 甲醇, 1:7, (卷/卷))。取出固定液, 加入0.5% 晶紫溶液。室温孵育2小时</li>
			<li>用吸管吸吸并浸泡在自来水中, 冲洗掉任何结晶紫溶液残渣, 小心地去除水晶紫溶液。在室温下风干2天。</li>
		</ol>
</li>
		<li>使用倒置显微镜 (4X 放大倍数) 计数超过50个细胞的菌落数。 注: 为此需要两名独立观察员。</li>
		<li>将 RR ic50计算为目标抗治疗亚克隆细胞的 IC 50 值与原始/天真细胞的值之间的比值.</li>
	</ol>
</li>
</ol>2. 候选靶向治疗抗性基因 (R 基因) 的验证协议
注: 在基因表达的特征后, 与目标治疗的后天抗性和任何候选基因作为抵抗的驱动因素相关的签名将被调查通过: a) rt-pcr, 和 b) 西方印迹分析。
<ol><li>
实时 rt-pcr
	<ol>
<li>用酸性胍硫氰酸盐-苯酚-氯仿混合物提取细胞系中的总 RNA (请参阅材料表)。</li>
		<li>使用随机水溶液引物进行反向转录 (RT) 反应, 并设置热循环仪如下: 在25摄氏度启动5分钟, rt 46 摄氏度20分钟, rt 钝化95摄氏度为1分钟。</li>
		<li>使用 400 ng 总 RNA 的20µL 反应。准备样品的反应混合物如下: 7 µL RNase 免费 H2O, 2 µL cDNA (10 ng/µL), 1 µL 20x 荧光标记目标特定探针, 10 µL 2x 混合包含缓冲, dNTPs 和 DNA 聚合酶 (见材料表)。</li>
		<li>将反应混合物的20µL 添加到每个井板上, 然后运行以下程序: 保持阶段在50°c 为2分钟, 在95°c 为10分钟 (1 个周期);然后40个周期在95°c 为十五年代和在60°c 为1分钟。</li>
	</ol>
</li>
	<li>
西部污点
	<ol>
<li>通过加入2毫升0.05% 胰蛋白酶/EDTA 溶液来恢复细胞悬浮, 并允许胰蛋白酶工作 2-3 分钟。</li>
		<li>添加2个含有10% 胎牛血清的培养基, 以中和胰蛋白酶 (例如, 每1毫升胰蛋白酶的2毫升培养基) 和离心机在 1200 x g 处为5分钟. 弃上清和洗涤细胞与冷 1x PBS。</li>
		<li>离心机在 1200 x g 5 分钟, 并丢弃上清。并用重悬细胞颗粒与溶解缓冲和孵育在一个摇杆在4°c 30 分钟。<ol>
<li>溶解缓冲量取决于单元格数 (例如, 100 µL 为 1 x 106单元格);每次都要准备新鲜的溶解缓冲液。1毫升的裂解缓冲准备 (在冰上留下): 使用975µL M 每哺乳动物裂解缓冲液, 15 µL 蛋白酶和磷酸酶抑制剂, 10 µL 100 µM PMSF 抑制剂。</li>
		</ol>
</li>
		<li>用细胞裂解离心法回收上清蛋白, 在 1.3万 x g 处4摄氏度, 15 分钟。</li>
		<li>使用一项蛋白质测定法确定蛋白质浓度。</li>
		<li>添加 4x LaemmLi 样本缓冲区到蛋白质样品 (至少 20-30 µg 蛋白质) 和热量在100°c 3 分钟。</li>
		<li>使用预制 4-20% 凝胶和负载 20-30 µg 的 LaemmLi 蛋白溶液和 5-10 µL 蛋白质标准每样。</li>
		<li>用三/GLICYNE/SDS 1x 缓冲器填充西部印迹环, 并在110伏处运行凝胶, 30-60 分钟 (当时间停止检查染料已达到凝胶的末端, 否则再运行几分钟)</li>
		<li>Electroblot 使用半干燥系统将蛋白质转移到 PVDF 膜 (参见材料表)。</li>
		<li>在室温下的振动筛中浸泡适当的蛋白质溶液 (牛奶/BSA 5%), 以1小时的温度阻挡膜。</li>
		<li>在5% 牛奶溶液中用 anti-IQGAP1 原抗体制作1:400 溶液 (参见材料表)。</li>
		<li>一夜之间将细胞膜放在一个冷室的振动筛上。</li>
		<li>第二天, 放弃抗体溶液, 浸泡在 t-tbs 1x 溶液 (1x t-tb: 1x 三盐缓冲 Tween20 0.1%) 在室温下的振动筛7分钟。</li>
		<li>丢弃 T-tb 1x 解决方案并更换。重复3次。</li>
		<li>为标准的西方印迹检测添加抗体, 制作1:10,000 次小鼠抗体溶液 (参见材料表) 并将膜放在振动筛上;在室温下离开2小时。</li>
		<li>丢弃抗体溶液, 在室温下的振动筛中浸泡7分钟的 T-TBS 1x 溶液。</li>
		<li>丢弃 T-tb 1x 解决方案并更换。重复3次。</li>
		<li>在化学发光溶液中浸泡5分钟的膜, 并将膜成像在化学发光成像仪上。</li>
	</ol>
</li>
</ol>3. 候选 R 基因击倒法恢复靶向治疗抑制剂敏感性
<ol><li>
细胞制剂
	<ol>
<li>用 trypsinization 制备单细胞悬浮液。在细胞被镀的当天进行转染。<ol>
<li>用 PBS 冲洗 NCI-N87 细胞, 在37°c 处孵育0.05% 胰蛋白酶/EDTA 溶液, 5-10 分钟. 添加2容量的 RPMI 培养基, 含10% 胎牛血清, 以中和胰蛋白酶 (例如, 2 毫升培养基为每1毫升胰蛋白酶)。</li>
			<li>用台盼蓝染色 hemocytometer 细胞计数。种子 2.5 x 105单元 (60% 汇合) 到 T25 cm2烧瓶为每个条件。 注: 适当数量的播种细胞是由细胞系的加倍时间和实验的持续时间决定的。目的是在实验的整个持续时间内实现目标基因的击倒。九 T25 cm2烧瓶是理想的转染条件和克隆检测 (3 个烧瓶的控制, 3 烧瓶负转染控制, 3 烧瓶基因靶向转染)。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
反向转染
	<ol>
<li>为每个 T25 cm2烧瓶准备基因靶和阴性对照转染组合, 如下所示。<ol>
<li>稀释12.5 µL 的每个基因靶向 siRNA 寡核苷酸或负控制 siRNA 寡核苷酸在极小的必要的转染培养基 (比率 1:55; 请参阅材料表)。</li>
			<li>添加阳离子脂质体转染试剂 (1:1 与基因靶向 siRNA 寡核苷酸的比值; 请参阅材料表)。室温孵育20分钟。 注: 转染混合料总容积为700µL。</li>
			<li>将转染混合的700µL 添加到每个 T25 cm2烧瓶中。孵育细胞在37°c 1-3 天。</li>
		</ol>
</li>
		<li>通过 rt-pcr 和印迹分析验证基因击倒。通过菌落形成实验, 对所假定的 R 基因击倒对靶向治疗剂敏感性恢复的影响进行双重检查 (重复步骤 1.5)。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

虽然个性化和有针对性的治疗已经引起了越来越多的热情, 这些所谓的 "聪明" 疗法面临着与传统化疗药物相同的主要障碍,即, 快速和不可预知的药物耐药性的获取。为了提高靶向治疗的效果, 必须通过更好地了解导致药物耐药性的机制来解决抗药性问题。在这里, 我们提出了一个广泛的可访问的体外方法来研究肿瘤细胞系模型中靶向治疗药物的后天抗性机制。
由?...

在分子和细胞生物学的关键发现之后, 一些新的合成抗癌分子被开发来选择性地靶向致癌信号通路在广泛的肿瘤类型。特别是, 两类靶向治疗剂具有独特的性质在肿瘤学, 即合成化合物和重组单克隆抗体, 已知已取得临床成功, 并显著改变癌症护理最近数十年1,2,3。
然而, 尽管他们对治疗的初步反应令人印象深刻, 但大多数癌症患者对所有靶向治疗剂, 无论是单克隆抗体还是激酶抑制剂都有抗药性。因此, 耐药性是经典抗癌药物的主要障碍4, 对于新出现的靶向治疗 (5,6) 仍然是一个巨大的挑战。
抗靶向治疗可能是内在的 (即, 主) 或后天 (即, 次要)。内在的阻力描述了一个从头缺乏对治疗的反应, 而继发性抵抗发生后的反应, 药物治疗的时间7。后者是由肿瘤细胞的小群在原始肿瘤的大部分或依偎在远端隐蔽的解剖龛, 表现出多达90% 抵抗一个或多个靶向治疗剂。针对靶向治疗的抗性发展的分子机制主要是由于目标基因突变和其他支持生存信号通路的冗余激活, 其理解仍然远未完成8。
在这项工作中, 我们提出了一种时间和经济高效的方法来调查体外机制的后天抗性治疗药物, 从产生耐药细胞 subclones 的描述沉默用于恢复对抑制剂敏感度的程序, 是用于测试和验证工作假说的关键工具。特别是, 我们的方法被用来调查, 在胃癌, 抗性机制曲妥珠单抗(例如, 赫赛汀), 一个人性化的单克隆抗体靶向细胞外领域的 HER2蛋白9。曲妥珠单抗 + 化疗被广泛接受为标准一线治疗 HER2-positive 转移性乳腺癌。多亏最近的临床前研究表明, anti-HER2 疗法在体外和体内HER2-positive 胃癌模型10,11中都有显著的活性, 分子药物靶向 HER2 已在临床试验中广泛检查, 其中一些仍在进行中, 对胃食管癌患者12,13,14,15。
这些研究强调了对曲妥珠单抗16 的耐药性患者数量的增加, 类似于其他靶向治疗抑制剂的观察结果。特别地, 曲妥珠单抗的反应率为 47%, 曲妥珠单抗 + 化疗患者的总存活率为2.7 月以上, 仅供化疗患者治疗的时间为17。这表明, 虽然主要曲妥珠单抗抵抗是普遍的, 次要曲妥珠单抗抵抗是不可避免的。因此, 迫切需要澄清导致胃癌 HER2-targeted 治疗阻力的机制, 鉴于这种肿瘤的肿瘤遗传异质性, 这是更成问题的问题18。
此外, 胃癌耐曲妥珠单抗的机制已经证明具有挑战性, 部分原因是难以获得可靠的临床前模型代表这种情况。大岛et . 报告了一个体内选择方法的曲妥珠单抗抗胃癌细胞系, 包括重复培养的小残留腹膜转移治疗后,曲妥珠单抗19。然而, 需要一个围栏, 特定的动物处理专门知识, 并批准动物伦理委员会作出贡献, 使其成为一个耗时和成本昂贵的方法。我们在这项工作中描述的方法是简单和广泛适用的, 可以很容易地扩展到对其他治疗药物在不同的肿瘤 histotypes20的抗性机制的研究。

<table><tbody><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596026</td><td>TRIzol Reagent is a reagent for isolating high-quality total RNA or simultaneously isolating RNA, DNA, and protein from a variety of biological samples</td></tr><tr><td>ISCRIPT CDNA SYNTHESIS KIT</td><td>Bio-Rad</td><td>1708891</td><td>The iScript cDNA synthesis kit is a sensitive and easy-to-use first-strand cDNA synthesis kit for gene expression analysis using real-time qPCR.</td></tr><tr><td>TaqMan gene expression assay</td><td>Life Technologies</td><td>4331182</td><td>Applied Biosystems TaqMan Gene Expression Assays consist of a pair of unlabeled PCR primers and a TaqMan probe with an Applied Biosystems FAM or VIC dye label on the 5&amp;rsquo; end and minor groove binder (MGB) and nonfluorescent quencher (NFQ) on the 3&amp;rsquo; end.</td></tr><tr><td>M-PER Mammalian Protein Extraction Reagent</td><td>Thermo Scientific</td><td>78501</td><td>Thermo Scientific M-PER Mammalian Protein Extraction Reagent is designed to provide highly efficient total soluble protein extraction from cultured mammalian cells.</td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>Thermo Scientific</td><td>78440</td><td>Thermo Scientific Halt Protease and Phosphatase Inhibitor Cocktail (100X) provides the convenience of a single solution with full protein sample protection for cell and tissue lysates.</td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td>The Thermo Scientific Pierce BCA Protein Assay Kit is a two-component, high-precision, detergent-compatible assay reagent set to measure (A562nm) total protein concentration compared to a protein standard.</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747</td><td>Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample.</td></tr><tr><td>4&amp;ndash;20% Mini-PROTEAN TGX Precast Protein Gels</td><td>Bio-Rad</td><td>456-1094</td><td>Long-life TGX (Tris-Glycine eXtended) Gels have a novel formulation and can be used for both standard denaturing protein separations as well as native electrophoresis.</td></tr><tr><td>Precision Plus Protein WesternC Blotting Standards</td><td>Bio-Rad</td><td>1610376</td><td>Precision Plus Protein Prestained Standards are available in Dual Color, All Blue, Kaleidoscope, and Dual Xtra formats. All four have the same gel migration patterns, with 3 high-intensity reference bands (25, 50, and 75 kD).</td></tr><tr><td>10x Tris/Glycine/SDS</td><td>Bio-Rad</td><td>1610732</td><td>10x Tris/glycine/SDS is a premixed running buffer for separating protein samples by SDS-PAGE.</td></tr><tr><td>Trans-Blot Turbo Mini PVDF Transfer Packs</td><td>Bio-Rad</td><td>1704156</td><td>Trans-Blot Turbo Mini PVDF Transfer Packs for fast, efficient transfer of proteins from mini gels using the Trans-Blot Turbo Transfer System. Each vacuum sealed, ready-to-use transfer pack contains two buffer-saturated ion reservoir stacks and a prewetted PVDF membrane.</td></tr><tr><td>Precision Protein StrepTactin-HRP Conjugate</td><td>Bio-Rad</td><td>1610381</td><td>StrepTactin-Horseradish Peroxidase (HRP) Conjugate for chemiluminescent or colorimetric detection of Precision Plus Protein Unstained Protein Standards or Precision Plus Protein WesternC Protein Standards on western blots.</td></tr><tr><td>Immun-Star WesternC solution</td><td>Bio-Rad</td><td>5572</td><td>Immun-Star WesternC solution, a method of protein detection , detects mid-femtogram amounts of protein.</td></tr><tr><td>Universal Negative Control</td><td>Invitrogen</td><td>12935300</td><td>Negative Control siRNA has no significant sequence similarity to mouse, rat, or human gene sequences. The control has also been tested in cell-based screens and proven to have no significant effect on cell proliferation, viability, or morphology.</td></tr><tr><td>opti-MEM Glutamax Medium</td><td>Thermo Fisher Scientific</td><td>51985026</td><td>Opti-MEM Medium is an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in growth rate or morphology.</td></tr><tr><td>Silencer Select Pre-Designed &amp;amp; Validated siRNA</td><td>Ambion</td><td>4390824</td><td>RNA interference (RNAi) is the best way to effectively knock down gene expression to study protein function in a wide range of cell types.</td></tr><tr><td>Lipofectamine RNAiMAX Transfection Reagent</td><td>Invitrogen</td><td>13778075</td><td>Lipofectamine RNAiMAX Transfection Reagent offers an advanced, efficient solution for siRNA delivery. No other siRNA specific transfection reagent provides such easy and efficient siRNA delivery in a wide variety of cell lines including common cell types, stem cells and primary cells, as well as traditionally hard-to-transfect cell types.</td></tr><tr><td>Mouse anti-IQGAP1</td><td>Invitrigen</td><td>33-8900</td><td>working concentration: 1:400 in 5% milk solution</td></tr><tr><td>goat anti-mouse IgG-HRP: sc-2005</td><td>Santa Cruz</td><td>sc-2005</td><td>working concentration: 1:10000 in 5% milk solution</td></tr><tr><td>PMSF</td><td>Sigma Aldrich</td><td>P 7626</td><td>this is an inhibitor of serine proteases and acetylcholinesterase</td></tr><tr><td>Thermocycler for RT-PCR</td><td>MJ Research</td><td>MJ Research PTC-200 Thermal Cycler</td><td>it allows temperature homogeneity and a ramping speed of up to 3&amp;deg;C/sec. The protocol can be performed also with other thermalcyclers</td></tr><tr><td>Real Time RT-PCR instrument</td><td>Applied Biosystems</td><td>7500 RT-PCR system</td><td>This is a five-color platform that uses fluorescence-based polymerase chain reaction (PCR) reagents to provide a relative quantification using comparative C&lt;sub&gt;T&lt;/sub&gt; assay type. The protocol can be performed also with other thermalcyclers</td></tr><tr><td>Microvolume Spectrophotometers</td><td>Thermo Scientific</td><td>Nanodrop</td><td>It provides an accurate and fast acid nucleid measurement using little volume of sample</td></tr><tr><td>Blotting system</td><td>Bio-Rad</td><td>Trans-Blot Turbo system</td><td>It perfoms a semy-dry transfer in a few minutes</td></tr><tr><td>Image acquisition system and gel documentation</td><td>Bio-Rad</td><td>Chemidoc image system</td><td>It is easy to use for chemiluminescent detection</td></tr></tbody></table>

looking for driver pathways of acquired resistance to targeted therapy: drug resistant subclone generation and sensitivity restoring by gene knock-down

过去的两年, 在医学肿瘤学中, 从细胞毒药物到靶向治疗的转变。虽然靶向治疗剂显示了更深刻的临床疗效和最小化的副作用比传统治疗, 耐药性已成为主要限制他们的利益。在临床实践中, 主要采用两种策略: i. 基因处理方法对后天抗性药物的基因型进行建模, 二) 体外/在体内选择耐药性模型。在目前的工作中, 我们提出了一个统一的框架, 用于调查负责对靶向治疗剂产生抗药性的基本机制, 从生成耐药细胞 subclones 开始, 到描述沉默的程序, 用于恢复对抑制剂的敏感性。这种简单的时间和成本效益的方法是广泛适用的, 可以很容易地扩展到研究其他靶向治疗药物在不同的肿瘤 histotypes 的抗性机制。

图 1概述了实验过程的框架。三株胃癌细胞线表达高水平的 HER2 和敏感的曲妥珠单抗 (IC 50 < 峰值血浆水平的药物,图 2A, 左图) 是种植培养基中含有靶向治疗剂。10倍的剂量低于峰值血浆浓度的曲妥珠单抗 (10 µg/毫升) 被设置为起始剂量, 并逐渐增加到400µg/毫升在 8-12 月的时间。之后, 我们获得了三 subclones 的特点是稳定的?...

Watch this Scientific Journal Video about Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down at JoV

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

这是一个时间和经济高效的体外协议, 研究对靶向治疗剂的后天抗性机制, 这是癌症管理中极不满足的医疗需求。

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 ...

looking-for-driver-pathways-acquired-resistance-to-targeted-therapy

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

7.1K 次观看.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. 这是一个时间和经济高效的体外协议, 研究对靶向治疗剂的后天抗性机制, 这是癌症管理中极不满足的医疗需求。

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

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 ...

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

用抗体阵列对信号转导通路的审问对酪氨酸激酶抑制剂的抗性评价

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

建立过度表达 dr3 的细胞系, 评估抗有丝分裂治疗的凋亡反应

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

建立和表征三个抗阿法替尼的肺腺癌PC-9细胞系与增加剂量的阿法替尼开发

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

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 ...

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 ...

医学

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.

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

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

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 ...

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 ...

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

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利用RNA测序来检测新剪接变异体在相关耐药