Name: evaluating the effectiveness of cancer drug sensitization in vitro and in vivo
Uploaded: 2015-02-06T14:00:00.000+00:00
Duration: 9 min 19 s
Description: Watch this Scientific Journal Video about Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo at JoVE.com

这项工作成为可能，补助JK从卓越中心安大略省和安大略研究基金。 我们想拍摄期间感谢Siddika Pardhan和彼得·弗格森博士的技术援助。

注意:下面的协议被设计为与贴壁细胞使用。修改都需要该方法适用于在悬浮液中生长的非贴壁细胞。在协议中所描述的CAM实验被设计用于与细胞表达的荧光标记物（ 例如 ，GFP，RFP， 等等 ）的使用。九日龄鸡胚上需要的实验方案为所述CAM实验的第2天。 1.准备和肿瘤细胞与反义寡核苷酸转染（艾滋病服务） <ol><li>从包含感兴趣的细胞T75烧瓶（S）吸中，洗1XPBS，并加2ml 0.25％胰蛋白酶/ EDTA的。加入10 ml完全生长培养基到每个烧瓶中，并在溶液中转移细胞到50ml管中。计数细胞并调节音量，以使最终浓度为1.0×10每毫升5个细胞。 </li><li>标签两组T25烧瓶:一组将包含8瓶，标记1-8，并为USED为实时代谢测定实验 ;和第二组也将包含8瓶和将被用于鸡胚胎的CAM实验 。在溶液中加2ml细胞到每个烧瓶中，并发生在培养箱中（37℃，5％CO 2的O / N）。 </li><li>第二天早上（第0天，时间0），标号35毫升管如下:对照（非靶向ASO或siRNA），&#39;靶的兴趣"（即 ，靶向ASO或siRNA），和脂质体2000（或类似的转染试剂）。稀释股票ASO解到适当的浓度与在相应的管无血清培养基中。 <ol><li>添加所需的转染试剂的量，以适当的管中并孵育在无血清培养基中5分钟。转染试剂溶液添加到ASO管并孵育20分钟。 </li></ol></li><li>加入250微升的控制的ASO转染试剂溶液向每个烧瓶1-4中各组中，与250微升"的目标利率的"ASO转染试剂溶液每每组5-8瓶的。孵育4小时（37ᵒC，5％的CO 2）。 <ol><li>在培养过程中，开展2.1节所述的步骤。培养后，加入3 ml完全培养基的每个烧瓶中所述CAM实验组中，并返回到培养箱O / N（37ᵒC，5％的CO 2）。根据第2.2节的描述过程中的代谢实验组。处理CAM实验根据第4节的说明进行设置。 </li></ol></li></ol> 2.生物传感器芯片制备及细胞种植<ol><li>在无菌环境中，仔细清洗6的生物传感器芯片用400μlPBS中。消毒芯片用400μl的70％乙醇20分钟。用400微升PBS洗三次。 <ol><li>将里面的无菌组织文化性的芯片Ë菜肴以防止污染。将三个芯片在标有"控制"一盘菜，三个在标有"目标的景点一盘。 </li></ol></li><li>标签2 50ml试管为"控制"和"目标的景点。用PBS洗涤细胞，并添加0.25％胰蛋白酶/ EDTA的适当体积。等到单层分离并从每个烧瓶中收集细胞。存款解决方案到相应的管。 <ol><li>计数细胞并调节音量，以使最终浓度为6.0×10 5细胞/ ml。离心机和重悬细胞，如果初始浓度太低。 </li></ol></li><li>加入250微升的细胞溶液以每生物传感器芯片中的相应组（总共每片1.25×10 5个细胞）的。在一个时间执行此任务一组，以减少混淆的芯片的危险。放置装有芯片湿度腔室内部以防止蒸发无菌菜肴，并放置湿度茶MBER在孵化器O / N（37ᵒC，5％的CO 2）。 </li><li>第二天早上（第1天），小心地从每个芯片吸出培养基，并用250微升生长培养基补充有0.2％胎牛血清（FBS）和1×青霉素和链霉素（P / S）取代。孵育4小时（37ᵒC，5％的CO 2）。 </li></ol> 3.代谢测量系统准备和方案准备<ol><li>在孵化，准备必要的药品解决方案，并为即将到来的检测代谢测量系统。 注:新陈代谢测量系统共包含六个模块。每个模块可容纳每个实验一筹。 <ol><li>从各"控制"和"目标的兴趣&#39;基团以接收车辆的测定法的持续时间中的一个芯片;:细分测定成以下的实验组从每组两个芯片来接收药物治疗所界定的时间周期的一个中SSAY，随后车辆在清除期。 </li><li>用于药物致敏研究，修改长度，定时和浓度的药物治疗，以适合所考虑的特定的药物。 注:对于顺铂，被凭经验用A549细胞中确定的下列步骤，并且可以根据细胞系而改变。完成所有阶段以4微升/分钟默认流量。 6小时生长培养基+ 0.2％FBS和1×P / S的 6μM顺铂媒体24小时+ 0.2％FBS 1倍P / S 生长培养基+ 0.2％FBS和1×P / S中的24小时 18小时生长培养基+ 0.2％FBS和1×P / S的 4小时0.1％的Triton-X的 注:从这些步骤的偏差，需要的计算来确定的基础上，期望的流速和试验持续时间中，药物溶液的所需体积。下面的章节是基于以上所述的步骤。 </li></ol></li><li>标签和填补14 50ml试管用50毫升生长培养基+ 0.2％FBS的ð1倍P / S。放置6在一个代谢测量系统管架，在另一个6，并在第三个试管架还包含管与顺铂的其余两个。密封第一两个机架具有空气可渗透膜以防止蒸发。 </li><li>准备6μM顺铂培养基+ 0.2％FBS与1倍P / S的解决方案（适当的量）。免除顺铂方案所需的量分成四个标有50ml试管。 <ol><li>将管在第三代谢测定管机架的其余四个槽。确保将管放置在对应于所需biomodules适当的槽。封用的空气可渗透膜的管中。 </li></ol></li><li>准备和标记6 50ml试管并用各20ml的培养基中0.1％的Triton-X溶液填充它们。所述的Triton-X溶液将溶解任何残留的细胞，并提供一个"零"值的每个芯片。将管在第四代谢测量单元管架和密封机智h的空气permable膜，以防止过度蒸发。 </li><li>加载芯片到biomodules并开始实验。 </li></ol> 4. CAM实验药物治疗和注射注:该步骤和程序，准备鸡胚的CAM实验在别处描述19 <ol><li>开始处理的CAM实验 48小时转染后（第2天）。子分割"控制"和"目标利益团体分成两个瓶中的车辆，以及两个顺铂。吸从烧瓶的培养基，并补充有是3毫升的新鲜培养基，或6μM顺铂。孵育6小时（37ᵒC，5％的CO 2）。 </li><li>吸出培养基，然后洗净，trypsinize细胞;收集的胰蛋白酶的细胞中溶液分为四个适当标记的15ml试管。 <ol><li>离心机和洗涤细胞三次，用PBS。重新悬浮在5ml PBS中的的细胞沉淀ð计数细胞。 </li><li>调节音量，以使最终的细胞浓度为1.0×10 6细胞/ ml。转移的细胞溶液倒入标5毫升管注射，并置于冰上。倒置试管之前轻轻注射，以确保细胞是充分混合。 </li></ol></li><li>设置共24鸡胚（9日龄）注射（每组6只）一边。执行使用连接到一个柔性管连接到1ml注射器的玻璃针注射。执行注射者必须失明的治疗组。 <ol><li>使用立体显微镜，注入1.0×10 5个细胞中的100微升的体积为所述CAM的静脉循环。使用缓慢，脉动技术，细胞的最大音量注入。轻轻地轻拍注射部位用软擦拭，以阻止血液的流动和吸收任何泄漏。标签每个胚胎适当的容器在每次注射。 </li></ol></li><li>确认存在和DISTRI通过使用荧光显微镜可视化它们下列荧光细胞在CAM的脉管bution注射。请注意其中的肿瘤细胞数量出现低或分布不佳任何胚胎。 </li><li>将鸡胚胎在湿度室中，并将它们存储在培养箱（37℃，60％湿度）。等待7-9天历数转移灶之前。 </li></ol> 5，转移灶枚举<ol><li>在7-9天的潜伏期，监控胚胎并丢弃任何死的。保证室内湿度在孵化期间保持湿润，在任何时候。 </li><li>用荧光立体显微镜在20X放大倍率，扫描胚胎的CAM的整个上表面以规则的，格子状图案。 </li><li>算转移灶的数目在每个视场，则相符的数量为每胚胎的最终总。这样做对各组中的6胚胎将提供一个平均数ö每处理F转移灶。 </li></ol>

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由于与临床试验相关的固有的成本和风险，有必要开发更好的和更严格的临床前测试方法，以充分评估新的抗癌治疗方案。当前常用的技术都表现出弱点，这可能限制它们的潜在有希望的治疗靶标/剂之间进行区分的能力，以及那些可能不太有效。我们设计了一个协议，以评估新的抗癌方法，解决了许多传统方法的不足之处。 所描述的协议的一个特别重要的特征是，以实时测?...

获得抗性靶向和/或细胞毒性癌症治疗的一个重要的临床问题，可导致治疗失败，复发，并增加患者的死亡率1。给定异质性的高水平，在大多数肿瘤中，它是一个具有足够高的细胞数的肿瘤将包含细胞抗单一或组合疗法靶向在其上的那些细胞赖以生存2,3-分子途径的子集的数学确定性。这样的肿瘤细胞可以在治疗过程中可靠地选择，从而导致疾病复发。新疗法，能同时针对不同的癌细胞存活机制之前或治疗介导的选择之后发展，因此是临床上重要的。 肿瘤基因组高水平的基因组不稳定和突变是区分癌细胞非肿瘤宿主细胞4根本特征。因此，一个usefu升的策略来增加DNA损伤化疗的效果，并防止耐药性的发展是积极抑制DNA修复中的肿瘤细胞5。这是调查和各种新的DNA修复的目标的一个活跃的领域正在探索在临床前设置。一些小分子或它们的靶的反义基抑制剂已经被开发并正在接受测试6-8。该目标是确定最有希望的临床前候选并评估其安全性和有效性的临床试验。 临床试验的成本高，失败的风险（为各种原因，包括亚最佳的临床前评估）是难以克服的障碍中的新疗法9发展进步。使用适当的和严格的临床前模型，以充分评估新的治疗靶点和候选药物可能会降低的临床试验10高故障率</suP&gt;。 一些常用的临床前的方法来评估的新的抗癌疗法的功效是:a）测量能力，减少肿瘤细胞增殖在体外 （细胞增殖测定）中的肿瘤细胞的能力，二）治疗诱导减少的形式的组织培养的菌落（菌落形成测定法）中的肿瘤细胞的代谢活性，三）治疗诱导的减少在体外 （氧化还原染料的转换）中，d）治疗诱导的诱导体外肿瘤细胞死亡（凋亡，坏死，自噬相关联在体内有丝分裂及其他）11，和e）治疗诱导的减少生长人和小鼠异种移植物的12-14或消融。 列出的体外方法的一个主要弱点是，他们没有提供候选疗法的效果持续的实时评估。相反，他们只在选定的，广泛分隔时间点提供信息在治疗的过程中。这样的测量已经减少容量以准确地反映了肿瘤细胞应答的大小和定时。 体内小鼠异种移植模型也受限于成本高，时间来完成的长度，和亚最佳剂量和治疗时间（调度）的风险。此外，有证据表明，啮齿动物异种移植物模型是临床疗效在人类有限的预测相比， 在体外评估的原代人肿瘤细胞的应答，并建立了人肿瘤细胞系对候选治疗性干预15,16。 我们设计了一种新的组合协议来评估新的药物组合临床前，在解决了上面列出的更多的一般的步骤的弱点的方式。代替扩散，集落形成，或氧化还原染料转化测定中，我们利用了代谢测定单元来分析细胞粘附，呼吸，并实时酸化在整个治疗期间17。同时，我们通过使用鸡胚绒毛尿囊膜（CAM）模型的侵袭和转移18,19的研究体内治疗相结合的效果。我们使用这些方法来评价反义寡核苷酸（ASO）靶向BRCA2能增强常用化疗药物顺铂的有效性的能力。

<table><tbody><tr><td>A549 cells</td><td>ATCC</td><td>CCL-185</td><td>http://www.atcc.org/products/all/CCL-185.aspx</td></tr><tr><td>AeraSeal film</td><td>Carl Roth GmbH</td><td></td><td></td></tr><tr><td>AMEM</td><td>Wisent Bioproducts</td><td>210-011-QK</td><td></td></tr><tr><td>Antisense oligodeoxynucleotides</td><td>Avecia</td><td></td><td>BRCA2 target sequence: 5&#039; - UAAGGAACGUCAAGAGAUAC - 3&#039; (bases 7241-7259 )</td></tr><tr><td>Axio Zoom V16 Microscope</td><td>Carl Zeiss</td><td></td><td>http://www.zeiss.ca/microscopy/en_ca/products/stereo-zoom-microscopes/axio-zoom-v16.html</td></tr><tr><td>Bionas Biosensor Chips</td><td>Bionas GmbH and Micronas GmbH</td><td>BIS8001D</td><td></td></tr><tr><td>Bionas Discovery 2500 System&amp;nbsp;</td><td>Bionas GmbH</td><td></td><td>http://www.bionas-discovery.com/prodservices/instruments/system2500/</td></tr><tr><td>Cisplatin</td><td>Sigma Aldrich</td><td>479306</td><td></td></tr><tr><td>Fertilized chicken eggs</td><td>Sourced locally</td><td></td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Gibco - Life Technologies</td><td></td><td></td></tr><tr><td>Lipofectamine 2000&amp;nbsp;</td><td>Invitrogen - Life Technologies&amp;nbsp;</td><td>12566014</td><td>http://www.lifetechnologies.com/ca/en/home/life-science/protein-expression-and-analysis/transfection-selection/lipofectamine-2000.html</td></tr><tr><td>PBS</td><td>Wisent Bioproducts</td><td>311-010-CL</td><td></td></tr><tr><td>Trypsin (0.25%)/EDTA</td><td>Wisent Bioproducts</td><td>325-043-CL</td><td></td></tr></tbody></table>

evaluating the effectiveness of cancer drug sensitization in vitro and in vivo

由于异质性和固有的人类癌症，单一药剂的治疗或组合治疗方案靶向相同途径突变的水平高，很可能会失败。必须强调在通路是负责对疗法的固有和/或适应性阻力的抑制。调查的一个活跃的领域是开发和DNA修复抑制剂促进的作用，和防止抗，常用的化疗和放疗的测试。我们使用了一种新的协议，以评估BRCA2抑制的有效性，以敏感的肿瘤细胞对DNA损伤药物顺铂的装置。肿瘤细胞代谢（酸化和呼吸）实时一段72小时的划定上的分一秒基础治疗效果进行监测。在组合中，我们使用的是鸡胚胎绒毛尿囊膜（CAM）外渗及浸润模型进行转移频率的评估。此协议解决了一些常用的体外和体内方法来评价新的癌症治疗方案的弱点。它可以在除了常见的方法，例如细胞增殖试验，细胞死亡测定法中使用，并且在体内小鼠异种移植物的研究，以更紧密地辨别之间候选目标和剂，并仅选择最有前途的候选者的进一步发展。

结果与数字改编许可，从发表的作品22。 BRCA2诱导抑制呼吸的下降顺铂治疗的肿瘤细胞 与BRCA2靶向ASO和顺铂表现在呼吸早期的和不可逆转的下降，相比于它接收到的控制ASO与顺铂单独处理细胞A549人类肺癌细胞; 24小时顺铂治疗的BRCA2 ASO和控制ASO呼吸作用的差值处理的细胞后为39％（图1A）。重要的是，这减少呼吸开始在细胞粘附?...

Watch this Scientific Journal Video about Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo at JoVE.com

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 ...

evaluating-effectiveness-cancer-drug-sensitization-vitro

Research

JoVE Journal

Medicine

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

8.3K 次观看.  Western University. 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. 

视频: Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

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 ...

科研

癌症研究

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

球体、菌落形成测定和斑马鱼移植对抗癌香豆素 OT48 生物活性的临床前评价

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

体外肿瘤细胞恢复对 chimeric 抗原受体 t 细胞抗肿瘤功能的预测评价

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

生物工程

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 ...

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 ...

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

光控生物活性化合物的体外和体内评价 - 癌症光药理学的潜在候选药物

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

化学

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

评估癌症药物过敏的有效性

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