Name: patient-derived heterogeneous xenograft model of pancreatic cancer using zebrafish larvae as hosts for comparative drug assessment
Uploaded: 2019-04-30T14:00:00
Description: Watch this Scientific Journal Video about Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment at JoVE.com

这项工作得到了国家自然科学基金81402582、上海自然科学基金12DZ2295100、14YF1400600和18ZR1404500的支持。

所有动物程序均得到复旦大学动物伦理委员会的批准和遵循,所有胰腺癌标本均从复旦大学上海癌症中心获得。道德认证得到了FUSCC道德委员会的批准,并征求了每位患者的书面知情同意。
1. 准备显微注射设备
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准备注塑板。
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<li>制备溶解在E3溶液中的1%角质的50 mL溶液(双蒸馏水中0.6克/升水族盐= 0.01 mg/L亚甲蓝)。将溶液煮沸,直到甘蔗溶解。</li>
		<li>将50 mL?...

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PDX和CDX模型都是肿瘤生物学领域的重要平台,成功的物种间移植的关键步骤是提高异种移植的存活率。 最近,一些研究表明,BCL2L1(BCL-XL)或BCL2的瞬态表达可以显著提高小鼠宿主中人类胚胎干细胞的生存能力,而不会影响细胞特性和命运23,24,25.在我们的手稿中,我们标记了含有慢病毒的细胞,这些?...

精准肿瘤学旨在为个别患者找到最有益的治疗策略。目前,提出了许多临床前模型,如体外原发培养、体外有机体培养2和小鼠中患者衍生的异种移植(PDX),在有机体培养前后被建议用于诊断和筛选/评估潜力治疗选择3.PDX模型由人类原发性癌细胞注入免疫功能受损的小鼠,是临床肿瘤学3、4中最有前途的个性化药物筛选工具之一。与体外培养的细胞系不同,PDX模型通常保持体内肿瘤环境的完整性和异质性,更好地模仿不同肿瘤患者的多样性和特异性,因此,可以预测患者的潜在医疗结果4。然而,在小鼠中生成PDX模型需要高质量的患者样本和数月时间来收集足够的细胞和模型进行多组实验,异种移植物的细胞/遗传成分可能与原来的细胞/遗传成分漂移病人的活检小鼠PDX模型的建立成功率也较低,难以在临床实践中得到广泛应用。对于携带胰腺癌等快速进展癌症的患者,他们可能无法及时从PDX实验中获得有价值的信息。
在过去几年中,斑马鱼被报道为不仅CDX(细胞衍生肿瘤异种移植)模型的潜在宿主,而且PDX模型5,6,7,8,9,10.作为脊椎动物的模型动物,斑马鱼在遗传学和生理学上都与哺乳动物有着足够的相似性,具有两个显著的优势:透明度和小尺寸11。斑马鱼的繁殖性也很高,几天之内就可以从一对12岁的成年人身上获得数百只近亲繁殖的幼虫。几项研究已经利用斑马鱼生成癌症疾病的转基因和异种移植模型13,14。与小鼠异种移植相比,斑马鱼异种移植允许在单细胞分辨率下进行跟踪。一定数量的人体组织能够产生数百种斑马鱼PDX模型(zPDX),而可能只能产生一对小鼠PDX模型15,16。此外,斑马鱼幼虫在2-5 dpf已经发展了完整的循环系统和代谢器官,如肝脏和肾脏,但不是免疫系统17,而剩余的蛋黄囊是一个天然的3D介质,理想的药物筛选,药物抗药性试验和肿瘤迁移观察6,18,19,20,21。
随着最终尝试使用zPDX作为临床使用的筛选/测试平台,在这里,我们描述了胰腺癌zPDX模型的优化方案,它允许在短时间内使用更少的细胞以更低的成本进行体内候选药物评估。与之前关于zPDX6、9、10的参考文献相比,我们引入了几种优化,使系统在临床个性化诊断中更加可行和可靠:1)对不同细胞进行预排序在原发性肿瘤组织中组并稳定原发细胞一周,然后再进行进一步的实验;2) 通过基于慢病毒的遗传修饰标记人体细胞,增强异种移植物的细胞活力;3) 优化营养补充剂(葡萄糖和谷氨酰胺)和温度的斑马鱼养殖条件;4) 以比较方式量化不同细胞类型的药物反应。我们还通过添加几种补充材料对注射液进行了更改。总之,这些改进提供了在斑马鱼宿主中快速产生一种更类似患者的异种移植物的可能性,这种异种移植可用于评估候选药物的反应。

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>sv30087.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Cliniscience</td>
			<td>Y0503</td>
			<td>Rho kinase inhibitor</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>invivogen</td>
			<td>ant-pm-1</td>
			<td>an antibiotic for primary cell cultures</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide&#160;</td>
			<td>Sigma</td>
			<td>N3376</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin streptomycin</td>
			<td>GIBCO</td>
			<td>15140122.00</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer (PBS)</td>
			<td>GIBCO</td>
			<td>C10010500CP</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS&#160;</td>
			<td>GIBCO</td>
			<td>14170112.00</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase type IV</td>
			<td>GIBCO</td>
			<td>17104019.00</td>
			<td></td>
		</tr>
		<tr>
			<td>hyaluronidase</td>
			<td>Sigma</td>
			<td>H3884</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase&#8544;</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>b-FGF</td>
			<td>GIBCO</td>
			<td>PHG0264</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>GIBCO</td>
			<td>PHG0314</td>
			<td></td>
		</tr>
		<tr>
			<td>pancreatic cancer fibroblasts inhibitor</td>
			<td>CHI Scientific</td>
			<td>FibrOUT</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#956;m sterile filter</td>
			<td>Millipore</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>concentration column</td>
			<td>Millipore</td>
			<td>Millipore UFC910008</td>
			<td>Concentrate the virus</td>
		</tr>
		<tr>
			<td>polybrene&#160;</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronic Acid Sodium Salt</td>
			<td>Sigma</td>
			<td>H7630</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>GIBCO</td>
			<td>21051024.00</td>
			<td></td>
		</tr>
		<tr>
			<td>gemcitabine</td>
			<td>Gemzan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methylcellulose</td>
			<td>Sigma</td>
			<td>M0262</td>
			<td></td>
		</tr>
		<tr>
			<td>Navitoclax(ABT-263)</td>
			<td>Selleck</td>
			<td>S1001</td>
			<td>Bcl-xL inhibitor</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>NARISHIGE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>OLYMPUS</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>LEICA</td>
			<td>SP8</td>
			<td>0.00</td>
		</tr>
	</tbody>
</table>

patient-derived heterogeneous xenograft model of pancreatic cancer using zebrafish larvae as hosts for comparative drug assessment

患者衍生的肿瘤异种移植(PDX)和细胞衍生肿瘤异种移植(CDX)是临床前评估、药物指导和基础癌症研究的重要技术。传统宿主小鼠的一代 PDX 模型非常耗时,只适用于一小部分样本。近年来,斑马鱼PDX(zPDX)已成为一种独特的宿主系统,具有体积小、效率高的特点。在这里,我们描述了一种优化的方法,用于在zPDX模型中生成双荧光标记肿瘤异种移植模型,用于比较化疗评估。肿瘤细胞和成纤维细胞在不同的培养条件下从新鲜收获或冷冻的胰腺癌组织中丰富。两个细胞组都标有表达绿色或红色荧光蛋白的慢病毒,以及抗凋亡基因BCL2L1。转染细胞被预混合,并共同注入2dpf幼虫斑马鱼,然后在32°C的改性E3培养中繁殖。用化疗药物和/或BCL2L1抑制剂治疗异种移植模型,同时研究肿瘤细胞和成纤维细胞的毒性。总之,该协议允许研究人员快速生成具有异构肿瘤微环境的大量zPDX模型,并提供更长的观察窗口和更精确的定量评估药物候选者的效率。

图 1中表示该过程的架构化大纲。简而言之,原发性癌症组织细胞在消化后种子到完整的培养基中,无论是否添加胰腺癌成纤维细胞抑制剂。癌细胞和成纤维细胞被浓缩为两个不同的群体,成纤维细胞在没有抑制剂的情况下占主导地位,而癌细胞生长在添加抑制剂后占主导地位(图2)。构建了两个慢病毒包装载体,分别表示绿色或红色荧...

Watch this Scientific Journal Video about Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment at JoVE.com

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

患者衍生的胰腺癌异质异种移植模型,使用斑马鱼幼虫作为比较药物评估的宿主

该协议描述了基于病毒的双荧光标记肿瘤异种移植模型中的优化过程,使用幼虫斑马鱼作为宿主。这种异质异种异种移植模型模仿胰腺癌在体内微环境的组织组成,并作为在个性化zPDX(斑马鱼患者衍生异种移植)模型中评估药物反应的更精确的工具。

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

今天，我们将介绍一个幼虫斑马鱼PDX模型。此模型非常重要，因为它们可以更好地模拟异种移植中类似的细胞成分，用于评估药物反应，并进一步提高结果与比较分析的一致性。这种技术有两个主要优点。首先，我们使用各种标准化学染料将细胞标记为不同的荧光，从而能够实时跟踪细胞组成和分析相的能力。其次，它还提高了斑马鱼中人类细胞的一般生存时间，其基因改造为药物检测提供了更长的观察窗口。该技术为胰腺癌患者个性化药物和临床前肿瘤药物的预评估提供了潜在的模型。该方法还可用于追踪体内斑马鱼中其他肿瘤细胞或无肿瘤细胞异种移植，并改善细胞存活时间较长。要准备注射板，请准备在E3溶液中用1%的加糖溶液中，使用50毫升溶液。煮溶液，直到加糖溶解。将整个溶液倒入10厘米培养皿中，然后将斑马鱼胚胎固定模具放在表面。当加糖溶液完全凝固时，拆下模具。要准备注射针，请使用拔针器将内尺寸为 0.9 毫米的 10 厘米玻璃毛细管拉入两根针中。使用显微镜和钳子切割针头的端，以创建开口。首先，将一对到两对成年斑马鱼放入一个交配罐中，大约在七到九个下午。第二天早上8点，收集受精卵。将受精卵转移到含有 40 毫升新鲜 E3 溶液的培养皿中。在28.5摄氏度下孵育。首先，将收集的胰腺癌样本转移到培养皿中。用PBS冲洗组织五到六次，然后用手术刀将组织切成一毫米的立方体。接下来，将切碎的组织转移到含有5毫升HBVS的50毫升管中。添加胶原酶四型，葫二丁酶，和DNAse一在最终浓度显示这里。上下移液混合物，以混合良好。在37摄氏度的20%二氧化碳培养箱中孵育混合物15至20分钟。每五分钟向上和向下移液几次混合物。在管子中加入七毫升DMEM。在110倍G和4摄氏度下离心5分钟。然后，在DMEM中抽取上能和肿瘤混合物，然后重新悬浮。将混合物镀入两个六厘米培养皿，含有三毫升的全生长介质，名为第一组和二组。在5%的二氧化碳孵化器中，在37摄氏度下孵育两组。48小时后，将胰腺癌成纤维细胞的100倍抑制剂加入第一组培养基中，去除过度生长的成纤维细胞，使癌细胞成为主要细胞类型。使用以前的条件继续孵育。产生扁病毒后，将细胞播种到密度为30%至40%的12井板中。在5%的二氧化碳培养箱中，在37摄氏度的温度下一夜之间培养细胞。第二天，以每毫升8微克的浓度更换含有多纤维素的500微升无血清介质。在37摄氏度下孵育，用5%的二氧化碳孵育4小时。然后，在介质中再添加100微升的扁病毒，并再次孵育12小时。12小时后，用两毫升完全的介质代替该介质，再孵育36小时。36 小时后，检查荧光标记。收获受感染的细胞，以一比一的比例混合，最终浓度为每毫升一百万个细胞。将细胞在110倍G下离心5分钟，并在50微升注射溶液中重新悬浮细胞混合物。麻醉斑马鱼幼虫后，将它们转移到注射板，注射板中填充了经过修饰的E3。将混合细胞悬浮液的25微升放入微胶囊针中，并将针头插入微注射操纵器。设置注射压力和时间，将50至80个细胞注射到受精后48小时的蛋黄囊中。首先，在32摄氏度下将后泽纳鱼幼虫转移到40毫升的混合溶液中。将 10 个泽诺夫特幼虫放入 12 井板的每个井中。接下来，将幼虫分成四组。使用含有 0.1% DMSO 的 E3 治疗对照组，用 Gemcitabine 和/或 Navitoclax 治疗其他组。在32摄氏度下孵育所有幼虫两天。麻醉后，对被化的幼虫进行麻醉后，将它们放在3%甲基纤维素中。使用荧光或共合显微镜从横向视图对幼虫进行成像。然后，使用图像J和图形垫软件量化红色和绿色荧光信号的强度。在这项研究中，原发性癌细胞在消化后被播种到完整的培养中，有或没有胰腺癌成纤维细胞抑制剂。癌细胞和成纤维细胞作为两个不同的种群被丰富。没有抑制剂的人口以成纤维细胞为主，而癌细胞生长在加入抑制剂后占上风。在BFL2L1中，构建了两种表达绿色或红色荧光蛋白的扁病毒包装载体。基于病毒的荧光标记代表细胞的生存状态。混合癌细胞和成纤维细胞在受精后48小时被注射到斑马鱼蛋黄囊中，然后用Gemcitabine和/或Naitoclax治疗两天。不同人群的细胞振动和泽诺格拉夫特的细胞组成随着对药物治疗的反应而变化。当处理不同类型的肿瘤时，有几个步骤可能需要反对，包括细胞消化时间，药物治疗剂量，等等。此技术可帮助研究人员使用标准 CGX 或 PDS 测定中的斑马鱼。它允许以固定比率混合和注射不同的细胞，通过将进展带与细胞生长进行比较来量化进展带。人身保护意义重大，尤其是在包装扁家病毒时。在适当的时候戴口罩和手套，尽量不要在皮肤上混在皮肤上。

Research

JoVE Journal

Cancer Research

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

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

A Melanoma Patient-Derived Xenograft Model

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

患者衍生胰腺腺腺腺腺癌有机体模型的分离和特征

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

患者衍生的中枢神经系统转移异种移植模型的建立与利用

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

斑马鱼幼虫作为评估潜在放射增敏剂或保护剂的模型

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...