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
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 ?...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapeutic+resistance:+surviving+stressful+situations.">Chemotherapeutic resistance: surviving stressful situations.</a> Cancer Res. 71 (15), 5062-5066 (2011).</li><li>Izar, B., Rotow, J., Gainor, J., Clark, J., Chabner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics,+clinical+indications,+and+resistance+mechanisms+in+molecular+targeted+therapies+in+cancer.">Pharmacokinetics, clinical indications, and resistance mechanisms in molecular targeted therapies in cancer.</a> Pharmacol Rev. 65, 1351-1395 (2013).</li><li>Ellis, L. M., Hicklin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+Targeted+Therapies:+Refining+Anticancer+Therapy+in+the+Era+of+Molecular+Oncology.">Resistance to Targeted Therapies: Refining Anticancer Therapy in the Era of Molecular Oncology.</a> Clin Cancer Res. 15 (24), 7471-7478 (2009).</li><li>Asi&#263;, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mechanisms+of+primary+resistance+differ+from+dominant+mechanisms+of+secondary+resistance+to+targeted+therapies.">Dominant mechanisms of primary resistance differ from dominant mechanisms of secondary resistance to targeted therapies.</a> Crit Rev Oncol Hematol. 97, 178-196 (2016).</li><li>Arienti, C., 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+evidence+of+multiple+mechanisms+underlying+trastuzumab+resistance+in+gastric+cancer.">Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer.</a> Oncotarget. 7 (14), 18424-18439 (2016).</li><li>Tanner, 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=Amplification+of+HER-2+in+gastric+carcinoma:+association+with+Topoisomerase+IIalpha+gene+amplification,+intestinal+type,+poor+prognosis+and+sensitivity+to+trastuzumab.">Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab.</a> Ann Oncol. 16 (2), 273-278 (2005).</li><li>Bang, Y. 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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=Genomic+landscape+and+genetic+heterogeneity+in+gastric+adenocarcinoma+revealed+by+whole-genome+sequencing.">Genomic landscape and genetic heterogeneity in gastric adenocarcinoma revealed by whole-genome sequencing.</a> Nat Commun. 5, 5477 (2014).</li><li>Oshima, Y., 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=Lapatinib+sensitivities+of+two+novel+trastuzumab-resistant+HER2+gene-amplified+gastric+cancer+cell+lines.">Lapatinib sensitivities of two novel trastuzumab-resistant HER2 gene-amplified gastric cancer cell lines.</a> Gastric Cancer. 17 (3), 450-462 (2014).</li><li>Pignatta, 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=Prolonged+exposure+to+(R)-bicalutamide+generates+a+LNCaP+subclone+with+alteration+of+mitochondrial+genome.">Prolonged exposure to (R)-bicalutamide generates a LNCaP subclone with alteration of mitochondrial genome.</a> Mol Cell Endocrinol. 382 (1), 314-324 (2014).</li></ol>

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&#8217; end and minor groove binder (MGB) and nonfluorescent quencher (NFQ) on the 3&#8217; 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&#8211;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 &#38; 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&#176;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 CT 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。

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Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

抗有丝分裂药物多西紫杉醇的前列腺癌细胞模型的建立

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

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

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 ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

使用锚定多路聚合酶链反应的致癌基因融合检测,随后进行下一代测序

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

研究利普托梅宁格疾病直接靶向治疗的穆林·奥马亚异种格拉夫特模型

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

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

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 ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

组织中蛋白质和RNA表达的空间分析：一种微调虚拟显微切割的方法

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...