我们感谢他们提出深思熟虑的意见和有益的建议，分子肿瘤研究所和 Editage 提供的援助与英语语言编辑成员。这项工作是为私立大学 （2012年-2016 年） 从教育部、 文化、 体育、 科学和技术的日本战略研究基金会支持由文部科学省支持程序。钱 Ohmori 收到勃林格殷格翰 （殷格翰，德国） 资金 （2015年-2016 年） 的研究。

1.建立吉非替尼耐药细胞
<ol>
	<li>3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 溴 (MTT) 法测定确定初始吉非替尼治疗浓度。
	<ol>
		<li>细胞生长介质中的 PC-9 (rpmi-1640 培养基用 10%胎牛血清、 1%抗生素 (青霉素: 100ml U 和链霉素: 100 微克/毫升))，在一个 5%CO2的孵化器在 37 ° c。</li>
		<li>调整单元格到 1.0 × 105细胞/毫升使用自动化的单元反击 （见材料表） 和种子 50 µ L 的这在每口井的 96 孔板使用培养基;最后浓度的细胞应该是 5.0 × 103细胞/井。</li>
		<li>孵育板一夜之间在 37 ° c。</li>
		<li>添加不同浓度的吉非替尼 （在 2 X 终浓度) 50 µ L: 0 0.002 0.006、 0.02、 0.06、 0.2、 0.6、 2，6，、 20 µ M 到这些井这种每年最终卷好是 100 μ L。</li>
		<li>孵育板为 72 h 在 37 ° c。</li>
		<li>添加染料溶液 15 µ L （见表的材料） （使用最后 13%浓度的染料溶液），每口井和孵育 4 h 在 37 ° c。</li>
		<li>添加 100 μ L 的增溶作用/停止解决方案 （见表的材料） 为溶解并结合产生的染料溶液 （见表的材料），每年得很好，和孵化它一夜之间在 37 ° c。</li>
		<li>测量的光学密度在 570 毫微米 (OD570) 使用酶标仪申领 IC50值通过生成细胞增殖曲线。</li>
		<li>使用统计软件 （见表的材料），以图形方式显示数据，并计算 IC50值 (图 2)。</li>
		<li>准备 6-12 复制和重复实验至少三次。</li>
	</ol>
	</li>
	<li>连续逐步剂量-升级的吉非替尼治疗的 PC-9 细胞。
	<ol>
		<li>PC-9 细胞培养 （在分合流条件 1 毫升） p100 菜用 10 毫升的生长介质在 5%CO2孵化器在 37 ° c。</li>
		<li>添加到 p100 菜吉非替尼的 IC50值的十分之一。<ol>
			<li>当培养基中的细胞成为分合流时，添加 10 毫升的新鲜培养基使用 1 毫升吸管 10-30%浓度较高的吉非替尼在相同的培养条件下细胞 1-2 毫升。</li>
		</ol>
		</li>
		<li>吉非替尼浓度逐步增加 10-30%与每个分裂，直到它到达 1 µ M;这可能需要大约 6-12 个月。 注意: 当吉非替尼的浓度达到 IC50值时，细胞增殖变得要慢得多，和因此，2-4 毫升的细胞可能会添加到 10 毫升的新鲜培养基浓度较高的吉非替尼;减少的吉非替尼治疗浓度的增加可能会解决问题。此外，它将理想储存在-80 ° C，剩余的单元格并重新使用它们在以前的浓度。由于 PC-9 细胞悬浮液中，它是不必要胰蛋白酶用于传代;然而，在过程中吉非替尼治疗浓度的增加，细胞可附加到培养皿的底部。在这种情况下，细胞刮刀可以用于分离的贴壁细胞。</li>
		<li>生长介质与 1 µ M 的吉非替尼吉非替尼耐药的 PC-9 细胞培养。</li>
		<li>执行 mtt 法检测以确认吉非替尼耐细胞13;这些吉非替尼耐药细胞被命名为 PC 9MET1000 （指定为 MET1000 细胞），因为他们表现出增加的蛋白质和 RNA 水平的大都会 （图 3A、图 4A、 B）。</li>
	</ol>
	</li>
</ol>
2.建立双电阻对吉非替尼和 PHA665752
<ol>
	<li>确定 PHA665752 mtt 法检测的初始浓度。
	<ol>
		<li>MET1000 培养细胞在 p100 菜用 10 毫升的生长介质和 1 µ M 吉非替尼治疗中 5%CO2孵化器在 37 ° c。</li>
		<li>到 96 孔板在 5.0 × 103细胞/井与 50 µ L 的生长介质在每口井的种子细胞。调整终浓度为细胞到 1.0 × 105细胞/毫升使用自动化的单元反击 （见表的材料）。</li>
		<li>孵育板一夜之间在 37 ° c。</li>
		<li>添加 50 µ L 的 PHA665752，大都会-使用 tki 药物治疗 （在 2 X 最终浓度)，对不同浓度的细胞: 0 0.002 0.006、 0.02、 0.06、 0.2、 0.6、 2，6，、 20 µ M，包括 2 µ M 吉非替尼治疗 （在 2 X 最终浓度）;在每口井的最终数量应该是 100 μ L。</li>
		<li>孵育板为 72 h 在 37 ° c。</li>
		<li>添加染料溶液 15 µ L （见表的材料），以每个好，并孵育 4 h 在 37 ° c。</li>
		<li>为溶解并结合产生的染料溶液添加 100 μ L 的增溶作用停止解决方案 （见表的材料），每个都很好，并再一次孵化一夜之间在 37 ° c。</li>
		<li>测量的光学密度在 570 毫微米 (OD570) 使用酶标仪申领 IC50值通过生成细胞增殖曲线。</li>
		<li>使用统计软件 （见表的材料），以图形方式显示数据，并计算 IC50值的 PHA665752 在吉非替尼 (图 3B)。</li>
		<li>准备 6-12 复制和重复实验至少三次。</li>
	</ol>
	</li>
	<li>建立双电阻在 MET1000 细胞对吉非替尼和 PHA665752。
	<ol>
		<li>在 p100 菜用 10 毫升生长的培养基，细胞培养 MET1000 及 1 µ M 吉非替尼治疗中 5%CO2孵化器在 37 ° c。</li>
		<li>添加到生长介质中，吉非替尼具有相同的文化条件 1 µ M PHA665752 IC50值的十分之一。</li>
		<li>逐步增加，PHA665752 浓度由 10-30%，每个分裂直到它到达 1 µ M。这可能需要大约 6-12 个月。 注意: 当 PHA665752 的浓度达到 IC50值时，细胞增殖变得要慢得多，和因此，2-4 毫升的细胞可能使用添加至 10 毫升新鲜生长的培养基 1 毫升吸管浓度较高的 PHA665752;减少的 PHA665752 浓度的增加可能会解决问题。此外，它将理想储存在-80 ° C，剩余的单元格并重新使用它们在以前的浓度。这是没有必要使用胰蛋白酶传代;然而，在 PHA665752 浓度的增加，过程中的细胞可能会附加到培养皿的底部。在这种情况下，细胞刮刀可以用来分离细胞。</li>
		<li>最后，文化 PC 9 细胞耐药吉非替尼和 PHA665752 在生长培养基与 1 µ M 每个吉非替尼和 PHA665752 在一个 5%CO2的孵化器在 37 ° c。</li>
		<li>执行 mtt 法检测以确认细胞是双耐药吉非替尼和 PHA66575213，而且他们是不敏感的 PHA665752 在吉非替尼;这些双电阻细胞被命名为 PC 9 博士 （指定为博士细胞） (图 5)。</li>
	</ol>
	</li>
</ol>

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V., 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=Genotypic+and+histological+evolution+of+lung+cancers+acquiring+resistance+to+EGFR+inhibitors.">Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors.</a> Sci Transl Med. 3 (75), 75ra26 (2011).</li><li>Engelman, J. 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作者有没有利益冲突的披露。

这里描述的协议可以用于获得双抗性使用 2 步剂量递增法体外癌细胞中建立。许多研究论文报道，PC-9 细胞抗药性 EGFR-使用 tki 药物治疗 （吉非替尼或厄洛替尼） 通过 T790M EGFR 突变15，16。然而，我们没有检测到 T790M 突变基因外显子 20 的我们的 PC 9MET1000 细胞通过直接测序法 (图 4)。此外，大都会放大观察作为信号转导通路?...

致癌性突变中表皮生长因子受体 (EGFR) 发现在非小细胞肺癌 (NSCLC) 患者的一个子集，重要预测生物标记物此疾病1，2。这些最常用激活 EGFR 突变发生作为帧中删除在 19 号外显子 (delE746-A750) 或替换亮氨酸与精氨酸的外显子 (存在 L858R) 21 号 858 密码在 EGFR 酪氨酸激酶域3，4点突变。窝藏 EGFR 突变的非小细胞肺癌患者的临床有效治疗是治疗 EGFR 酪氨酸激酶抑制剂 （测出）。然而，这种疗法被受到限制抗 EGFR 测出吉非替尼和埃罗替尼等必然采集。最常见的获得性耐药发生，通过二次 T790M 突变基因外显子 20 的 EGFR，大约 60%的患者获得 EGFR-使用 tki 药物治疗 （吉非替尼或厄洛替尼） 电阻检测到。其他伴 EGFR 测出获得性耐药的分子机制并激活的旁路信号引起的大都会放大，转型小细胞肺癌，与上皮-间充质转变5，6。受体肝细胞生长因子，由 MET 基因，编码是 dysregulatedin 许多肿瘤，据报是重要的一个候选靶向疗法7，8。
战略，以克服 EGFR 测出获得性的耐药正进行临床评价。临床前和临床研究表明，第三代 EGFR 抑制剂如 osimertinib 是 T790M 突变9患者有效。此外，可通过联合治疗 EGFR 和大都会测出6，10抑制 EGFR 突变癌症与大都会放大的增长。评估患者的大都会和 EGFR 抑制剂组合与获得性耐药吉非替尼和埃罗替尼的临床试验正在进行现在是11。
研究对化疗药物产生获得性耐药的分子机制，最初响应抑制剂的细胞系是与这些抑制剂继续治疗。两种方法可用来建立获得性的耐药细胞系。一是逐步的剂量递增，和另一种是接触浓度高。逐步升级方法已被更常用，因为它具有很高的成功率。单元格第一次接触低浓度的抑制剂，近十分之一的半-极大抑制浓度 (IC50) 值，和浓度然后逐渐增加，由 10-30%直到达到靶浓度。高质量浓度暴露法涉及细胞暴露于最后一剂到培养基中，是超过 IC50值，所以几乎彻底杀死亲本细胞抑制剂。此高质量浓度暴露方法具有更低成功率比逐步升级方法。
在以往的研究中，EGFR-使用 tki 药物治疗与 MET-使用 tki 药物治疗联合的治疗被证明是有效的体外系统。获得性的耐药 EFGR-使用 tki 药物治疗，吉非替尼通过逐步升级为 12 个月，MET-放大，成立，因此，吉非替尼耐药细胞线称为 PC 9MET1000 生成的12。
该协议是两步剂量升级程序获得双抗 EGFR-使用 tki 药物治疗，吉非替尼和大都会-使用 tki 药物治疗，PHA665752 的 EGFR 突变的非小细胞肺癌 PC 9 细胞 (图 1)。这种方法建立获得性耐药细胞系中是相对较容易执行，成功率高。描述的协议可以修改为其他分子靶向药物的细胞毒性药物以及抑制剂的应用。

<table><tbody><tr><td>RPMI-1640</td><td>Wako</td><td>189-02025</td><td>with L-Glutamine and Phenol Red</td></tr><tr><td>CellTiter 96</td><td>Promega</td><td>G4100</td><td>Non-Radioactive Cell Proliferation Assay</td></tr><tr><td>FBS</td><td>gibco</td><td>26140-079</td><td></td></tr><tr><td>Pen Strep</td><td>gibco</td><td>15140-163</td><td></td></tr><tr><td>gefitinib</td><td>Selleck</td><td>S1025</td><td>stocked 10 mM/EMSO at -20&amp;ordm;C</td></tr><tr><td>PHA665752</td><td>Selleck</td><td>S1070</td><td>stocked 10 mM/EMSO at -20&amp;ordm;C</td></tr><tr><td>96-well plate</td><td>IWAKI</td><td>860-096</td><td>flat bottom with lid, low evaporation type</td></tr><tr><td>TC10 Automated Cell Counter</td><td>BIO RAD</td><td>#1450010</td><td></td></tr><tr><td>CellTiter 96 Non-Radioactive Cell Proliferation Assay</td><td>Promega</td><td>G4100</td><td>Dye Solution and Solubilization/Stop Solution</td></tr><tr><td>Powerscan HT microplate reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>GraphPad Prism v.5.0 software</td><td>GraphPad Inc.</td><td></td><td></td></tr><tr><td>PC-9</td><td>Riken BioResource Center</td><td>RCB4455</td><td></td></tr><tr><td>P100 dish</td><td>FALCON</td><td>353003</td><td></td></tr></tbody></table>

establishing dual resistance to egfr-tki and met-tki in lung adenocarcinoma cells in vitro with a 2-step dose-escalation procedure

耐药是在癌症治疗中的一项重大挑战。耐药株在体外生成是发现新的机制，以克服这一挑战的必要条件。在这里，一种建立双电阻对表皮生长因子受体 (EGFR) 的 2 步剂量升级方法-酪氨酸激酶抑制剂 （使用 tki 药物治疗）、 吉非替尼和大都会-使用 tki 药物治疗，PHA665752，描述。这种方法基于简单逐步剂量递增抑制剂诱导的获得性抗性细胞系。产生耐药株的备用方法涉及细胞暴露于高浓度抑制剂中的一步。逐步的剂量递增方法具有更高的成功诱导可能性获得性抗性比这种方法。激活 EGFR 突变是响应与 EGFR-使用 tki 药物治疗，治疗的生物标志物应用的一线治疗非小细胞肺癌 (NSCLC)，那怀有这些突变。然而，尽管作出有效反应的报告，EGFR-使用 tki 药物治疗使用是有限因为肿瘤不可避免地获得耐药性。EGFR-使用 tki 药物治疗抵抗背后的主要机制包括看门人网站、 在 EGFR 外显子 20 T790M 和大都会旁路信号二次突变。因此，为这一问题的潜在解决方案将结合 EGFR-使用 tki 药物治疗和大都会-使用 tki 药物治疗。这个综合的治疗已被证明是有效的体外研究模型。获得性吉非替尼耐通过 MET 放大而成立的逐步剂量递增的吉非替尼治疗 12 个月，命名为 PC 9MET1000 细胞株在先前的研究中生成。进一步探讨获得性的 MET-使用 tki 药物治疗和 EGFR-使用 tki 药物治疗的机制抵抗，大都会-使用 tki 药物治疗，PHA665752，管理，给这些细胞逐步剂量递增吉非替尼在为 12 个月。本议定书也成功地适用于组合疗法建立获得性的耐药其他抑制剂分子的数目。

这手稿 》 中提出了议定书 》 概述如图 1，包括对吉非替尼和 PHA665752 的抗性双感应 PC 9 单元格中通过 2 步剂量升级过程所示。图 2显示了细胞增殖的家长 PC 9 随着浓度的增加吉非替尼的跌幅。这表明，PC-9 细胞变得敏感对吉非替尼剂量升级过程完成之后。图 3显示 PC 9MET1000 细胞表现出电阻对吉非替尼，但?...

Watch this Scientific Journal Video about Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure at JoVE.com

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

在体外建立双抗 EGFR-使用 tki 药物治疗和癌症细胞中大都会使用 tki 药物治疗方法。此方法是用于开发陈列尽管大都会放大 EGFR-使用 tki 药物治疗治疗疾病进展的 EGFR 突变，患者的治疗方法。它也可以修改为针对其他分子的抑制剂。

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 ...

establishing-dual-resistance-to-egfr-tki-met-tki-lung-adenocarcinoma

Research

JoVE Journal

Cancer Research

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

8.7K 次观看.  Showa University. 在体外建立双抗 EGFR-使用 tki 药物治疗和癌症细胞中大都会使用 tki 药物治疗方法。此方法是用于开发陈列尽管大都会放大 EGFR-使用 tki 药物治疗治疗疾病进展的 EGFR 突变，患者的治疗方法。它也可以修改为针对其他分子的抑制剂。

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

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

开发和维护的临床前患者来源的肿瘤异种移植模型为新的抗癌疗法的调查

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

科研

癌症研究

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

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

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 ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

利用18f-葡萄糖 PET/CT 成像和定量组织学测量肺癌小鼠模型葡萄糖代谢的动态变化

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

合成肺腺癌细胞原位移植研究 pd-l1 表达

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

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 ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

使用结构DNA改变分析和患者衍生异种移植测试癌症的靶向疗法

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

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

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

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

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

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

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

生物工程

多细胞球体的肺内接种以模拟原位 NSCLC

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model.
Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease&#39;s progression and facilitate the development of potential anticancer drugs.

作者聚焦：建立小鼠非小细胞肺癌模型以开发抗癌药物纳米制剂

This study presents an orthotopic non-small cell lung cancer (NSCLC) model based on intrapulmonary inoculation of multicellular spheroids of fluorescent A549-iRFP cells. The model recapitulates clinical NSCLC stages and responds to cisplatin, according to dynamic in vivo monitoring of long-wavelength fluorescence.

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models ...

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

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此视频中的章节

开发和维护的临床前患者来源的肿瘤异种移植模型为新的抗癌疗法的调查

抗有丝分裂药物多西紫杉醇的前列腺癌细胞模型的建立

利用¹⁸f-葡萄糖 PET/CT 成像和定量组织学测量肺癌小鼠模型葡萄糖代谢的动态变化

合成肺腺癌细胞原位移植研究 pd-l1 表达

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

使用结构DNA改变分析和患者衍生异种移植测试癌症的靶向疗法

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

一种混合的3D组织工程

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建