Name: establishment of gastric cancer patient-derived xenograft models and primary cell lines
Uploaded: 2019-07-19T14:00:00
Description: Watch this Scientific Journal Video about Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines at JoVE.com

这项工作得到了国家自然科学基金(81572392)的支持;国家重点研究与开发计划(2016YFC1201704);广东省科技创新青年人才专项扶持计划(2016TQ03R614)。
我们特别感谢广州萨金生物科技有限公司在编制数字方面提供的援助。

这项研究得到了中山大学癌症中心(中国广州SYSUCC)的制度伦理审查委员会的批准。动物研究得到了中山大学动物护理与使用委员会的批准。注:所有实验均符合相关法律和机构准则,包括《防止血液传播病原体的职业接触指南》。
1. 样品制备
<ol><li>直接从手术中获得胃癌组织(P0 = 通道0)。肿瘤标本应大于0.5厘米3。</li>
	<li>制备3-4 mL的库存溶液:例如,RPMI-1640介质(1x)辅以10%?...

<ol>
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T., 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=Multimodal+treatment+in+locally+advanced+gastric+cancer.">Multimodal treatment in locally advanced gastric cancer.</a> Updates in Surgery. 70 (2), 173-179 (2018).</li><li>Graziosi, L., Marino, E., Donini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+Treatment+of+Locally+Advanced+Gastric+Cancer:+Will+the+West+Meet+the+East?.">Multimodal Treatment of Locally Advanced Gastric Cancer: Will the West Meet the East?.</a> Annals of Surgical Oncology. 26 (3), 918 (2019).</li><li>Choi, Y. Y., Noh, S. H., Cheong, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Gastric+Cancer+Treatment:+From+the+Golden+Age+of+Surgery+to+an+Era+of+Precision+Medicine.">Evolution of Gastric Cancer Treatment: From the Golden Age of Surgery to an Era of Precision Medicine.</a> Yonsei Medical Journal. 56 (5), 1177-1185 (2015).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCGA+divides+gastric+cancer+into+four+molecular+subtypes:+implications+for+individualized+therapeutics.">TCGA divides gastric cancer into four molecular subtypes: implications for individualized therapeutics.</a> Chinese Journal of Cancer Research. 33 (10), 469-470 (2014).</li><li>Tirino, G., 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=What's+New+in+Gastric+Cancer:+The+Therapeutic+Implications+of+Molecular+Classifications+and+Future+Perspectives.">What's New in Gastric Cancer: The Therapeutic Implications of Molecular Classifications and Future Perspectives.</a> International Journal of Molecular Sciences. 19 (9), (2018).</li><li>Roschke, A. 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=Karyotypic+complexity+of+the+NCI-60+drug-screening+panel.">Karyotypic complexity of the NCI-60 drug-screening panel.</a> Cancer Research. 63 (24), 8634-8647 (2003).</li><li>Wilding, J. L., Bodmer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+lines+for+drug+discovery+and+development.">Cancer cell lines for drug discovery and development.</a> Cancer Research. 74 (9), 2377-2384 (2014).</li><li>Siolas, D., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+tumor+xenografts:+transforming+clinical+samples+into+mouse+models.">Patient-derived tumor xenografts: transforming clinical samples into mouse models.</a> Cancer Research. 73 (17), 5315-5319 (2013).</li><li>Xu, 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=Patient-derived+xenograft+mouse+models:+A+high+fidelity+tool+for+individualized+medicine.">Patient-derived xenograft mouse models: A high fidelity tool for individualized medicine.</a> Oncology Letters. 17 (1), 3-10 (2019).</li><li>Lai, 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=Current+status+and+perspectives+of+patient-derived+xenograft+models+in+cancer+research.">Current status and perspectives of patient-derived xenograft models in cancer research.</a> Journal OF Hematology &amp; Oncology. 10 (1), 106 (2017).</li><li>Kawaguchi, T., 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=Current+Update+of+Patient-Derived+Xenograft+Model+for+Translational+Breast+Cancer+Research.">Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research.</a> Journal of Mammary Gland Biology and Neoplasia. 22 (2), 131-139 (2017).</li><li>Cassidy, J. W., Caldas, C., Bruna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+Tumor+Heterogeneity+in+Patient-Derived+Tumor+Xenografts.">Maintaining Tumor Heterogeneity in Patient-Derived Tumor Xenografts.</a> Cancer Research. 75 (15), 2963-2968 (2015).</li><li>Liu, X., Meltzer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+Cancer+in+the+Era+of+Precision+Medicine.">Gastric Cancer in the Era of Precision Medicine.</a> Cellular and Molecular Gastroenterology and Hepatology. 3 (3), 348-358 (2017).</li><li>Shultz, L. D., 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=.">.</a> Human cancer growth and therapy in immunodeficient mouse models. (7), 694-708 (2014).</li><li>McDermott, S. P., 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=Comparison+of+human+cord+blood+engraftment+between+immunocompromised+mouse+strains.">Comparison of human cord blood engraftment between immunocompromised mouse strains.</a> Blood. 116 (2), 193-200 (2010).</li><li>Ito, 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=NOD/SCID/gamma+(c)+(null)+mouse:+an+excellent+recipient+mouse+model+for+engraftment+of+human+cells.">NOD/SCID/gamma (c) (null) mouse: an excellent recipient mouse model for engraftment of human cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Wege, A. 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胃癌是一种具有攻击性的疾病,治疗选择有限;因此,胃癌模型已成为一个关键资源,使功能研究直接翻译到诊所4,8,17。在这里,我们描述了建立胃癌PDX模型和原细胞系的方法和协议。重要的是,胃癌标本的形态和生物学特征大多保留在PDX模型中。
在建立PDX模型的协议中,需要强调一些要点,以提高移植率。NSG(NOD-SCI...

胃癌是全球第五大常见癌症,也是癌症死亡的第三大原因。2018年,全球新诊断出100万多例胃癌病例,估计有78.3万人死于此病。在东北亚国家,胃癌的发病率和死亡率仍然很高。尽管在癌症治疗领域取得了显著进展,但晚期胃癌患者的预后仍然很差,五年生存率约为25%4,5,6 7,.因此,迫切需要制定治疗胃癌的新策略
胃癌的治疗具有挑战性,因为它具有很高的异质性8,9。因此,如何解决肿瘤异质性的挑战,实现精密医学是癌症研究的核心。体外和体内模型在阐明胃癌的异质机制和生物学中起着至关重要的作用。然而,虽然有许多胃癌细胞系和许多传统的转基因小鼠模型用于临床前研究,这些模型的缺点限制了其应用10。由于这些模型在文化中的特征已经改变,它们不再模拟肿瘤异质性,它们的反应也未能预测人类的反应。这些问题严重限制了确定癌症患者对靶向药物作出反应的子组的可能性。原发性肿瘤的短期培养为研究抗癌药理特性提供了一种相对快速和个性化的方法,这可能是个性化癌症治疗的标志。
患者衍生的异种移植物(PDXs)是药物反应分析12的替代临床前模型的首选。此外,PDX模型为研究癌症的起始和进展提供了一个强大的工具13,14。PDX模型保留了癌细胞的组织外观,保留了肿瘤内异质性,并更好地反映肿瘤微环境的相关人体成分15、16。然而,广泛使用的PDX模型的局限性是建立和连续传播人类实体肿瘤的成功率较低。在这项研究中,描述了建立PDX模型和主细胞系的相当成功的方法。

<table>
	<tbody>
		<tr>
			<td>40 &#956;m Cell Strainer</td>
			<td>Biologix, Shandong, China</td>
			<td>15-1040</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Microscope</td>
			<td>OLYMPUS, Tokyo, Japan</td>
			<td>OLYMPUS CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, Mittelsachsen, Germany.</td>
			<td>5427R</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Fisher Scientific, Carlsbad, California, USA</td>
			<td>HERACELL 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Basalmedia Technology, Shanghai, China</td>
			<td>L40601</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-Thermostatic Water Cabinet</td>
			<td>Yiheng, Shanghai, China</td>
			<td>DK-8AXX</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent Biotechnology, Vancouver, Canada</td>
			<td>86150040</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter, China</td>
			<td>CN2L9100</td>
			<td></td>
		</tr>
		<tr>
			<td>Live Tissue Kit Cryo Kit</td>
			<td>Celliver Biotechnology, Shanghai, China</td>
			<td>LT2601</td>
			<td></td>
		</tr>
		<tr>
			<td>Live Tissue Thaw Kit</td>
			<td>Celliver Biotechnology, Shanghai, China</td>
			<td>LT2602</td>
			<td></td>
		</tr>
		<tr>
			<td>NSG</td>
			<td>Biocytogen, Beijing, China</td>
			<td>B-CM-002-4-5W</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin&#38;streptomycin</td>
			<td>Thermo Fisher Scientific, Carlsbad, California, USA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Solarbio, Beijing, China</td>
			<td>R1010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Thermo Fisher Scientific, Carlsbad, California, USA</td>
			<td>8118367</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Suture Needles with Thread</td>
			<td>LingQiao, Ningbo, China</td>
			<td>3/8 arc 4&#215;10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-processed molds and auxiliary blades</td>
			<td>Celliver Biotechnology, Shanghai, China</td>
			<td>LT2603</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific, Carlsbad, California, USA</td>
			<td>2003779</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 1 collagenase</td>
			<td>Thermo Fisher Scientific, Carlsbad, California, USA</td>
			<td>17100017</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of gastric cancer patient-derived xenograft models and primary cell lines

使用临床前模型来增进我们对肿瘤生物学的理解,并研究治疗剂的疗效是癌症研究的关键。虽然有许多既定的胃癌细胞系和许多传统的转基因小鼠模型用于临床前研究,但这些体外和体内模型的缺点限制了其应用。由于这些模型在文化中的特征已经改变,它们不再模拟肿瘤异质性,它们的反应也未能预测人类的反应。因此,正在开发更好地代表肿瘤异质性的替代模型。患者衍生的异种移植(PDX)模型保留了癌细胞的组织外观,保留了肿瘤内异质性,并更好地反映肿瘤微环境的相关人体成分。然而,开发PDX模型通常需要4-8个月,这比许多胃患者的预期存活时间更长。因此,建立原发性癌细胞系可能是药物反应研究的有效补充方法。目前的协议描述了从外科胃癌样本建立PDX模型和原发性癌细胞系的方法。这些方法为药物开发和癌症生物学研究提供了有用的工具。

在这里,肿瘤组织从手术被保存在库存溶液,直到下一步。在4小时内,肿瘤组织被切成小块,植入NSG小鼠的背侧,这些小鼠使用松草浸泡的棉花进行麻醉。大于1cm3的肿瘤可以被切除,植入新小鼠(图1),或按照协议小心切片并保存在液氮中。在这项研究中,第一代肿瘤的生长速度比后世的要慢,需要3周或更长时间才能达到适当的大小。第一代皮下肿瘤形成成功率大于80%。我们在?...

Watch this Scientific Journal Video about Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines at JoVE.com

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

目前的协议描述了从外科胃癌样本中建立患者衍生异种移植(PDX)模型和原发性癌细胞系的方法。这些方法为药物开发和癌症生物学研究提供了有用的工具。

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. ...

患者衍生的异种移植模型和原发细胞系正在成为药物开发和肿瘤生物学研究的启动和进展不可或缺的一部分。患者衍生的异种移植模型保留了癌细胞的组织外观，保留了肿瘤内异质性，并更好地反映了肿瘤微环境的相关人类成分。他的模型是更准确地表现人类癌症，然后传统的癌细胞系，并有可能改善临床前评估新的抗癌疗法。此外，这些模型可能有助于比较癌症患者不同子组之间的分子特征或肿瘤特征。建议将NSG小鼠作为免疫缺陷小鼠模型。由于小鼠的严重免疫缺陷，所有实验中必须保持不育。在无菌条件下，使用钳子和剪刀仔细解剖肿瘤组织，并把它们修剪成几个小块。麻醉鼠标后，使用无菌剪刀在两个侧翼上做一厘米的切口。使用无菌钳子破坏皮下组织。然后，夹住肿瘤组织的一块，并放入深层位点。用手术缝合针通过皮下缝合关闭植入区域。用碘消毒伤口。在此之后，轻轻地将小鼠放在空笼中，同时保持胸骨的卧发性。密切注意老鼠，因为他们应该醒来，并开始步行约三到四分钟后。将接受手术的老鼠放在一个新的笼子里，与那些没有接受手术的老鼠分开。通过植入点皮评估肿瘤大小，并每周两次使用Vernier卡钳测量肿瘤大小。对小鼠实施安乐死后，使用无菌钳子和剪刀慢慢将肿瘤从小鼠中分离开。在10厘米的培养皿中清洗DPBS中的肿瘤组织。你钳和剪刀解剖和去除坏死区，脂肪组织，血块和结缔组织。接下来，使用模具将肿瘤组织切割到最大厚度为一毫米。在10厘米的培养皿中用DPBS清洗肿瘤片。要开始玻璃化过程，使用钳子将切片转移到管V1中，并在4摄氏度下孵育4分钟。滚动并反转管子，在四摄氏度下孵育4分钟。接下来，将 V1 溶液倒入 10 厘米的培养皿中，并使用钳子将切片转移到管 V2 中。在4摄氏度下孵育4分钟。滚动并反转管子，并在四摄氏度下孵育四分钟。在此之后，将 V2 溶液和切片倒入 10 厘米的盘中。将切片转移到V3管中，在4摄氏度下孵育5分钟。滚动并反转管子，在四摄氏度下再孵育五分钟。确保所有切片都沉入管底。如果几个切片仍然漂浮，滚动和反转管短暂和孵育在四摄氏度，直到所有的切片完全下沉。然后，将 V3 溶液倒入 10 厘米的盘中。将组织支架切割到适当的长度，并把它们放在无菌纱布上。将切片转移到支架上。用纱布包裹支架。使用钳子，将支架放在液氮中，让其孵育五分钟。在此之后，用组织信息标记低温小瓶。将带组织切片的支架转移到小瓶中，小瓶将储存在液氮中。首先，从被切除的标本或收获的PDX组织中重新检测胃癌样本。将组织放在冰上，并转移到10厘米的无菌培养皿中。使用钳子和剪刀去除坏死区、脂肪组织、血块和结缔组织。在10厘米的培养皿中用含有青霉素和链霉素的DPBS清洗肿瘤组织一次。接下来，将肿瘤切成盘子盖上的碎片。每件的最大厚度应为一毫米。将碎片转移到一个50毫升的离心管中，管中含有7毫升1型胶原蛋白酶和三辛酶溶液。短暂地涡旋混合物。在37摄氏度的水浴中孵育30至40分钟，确保每5分钟旋转一次混合物。在此之后，加入等量的RPMI-1640介质，辅以10%FBS，彻底涡旋混合物。通过40微升过滤器缓慢过滤，将混合物转移到新的50毫升离心管中。在室温下以 113 和 163 倍 g 的速度离心滤金物 5 至 7 分钟。小心地取出此上一液。用五毫升 PBS 清洗颗粒，并小心地取出上一液。如果颗粒是红色的，则含有红细胞。轻轻用500微升红细胞解液缓冲液重新暂停颗粒，并在室温下孵育5分钟。然后，添加五毫升的PBS。在室温下以113和163倍g的速度离心5至7分钟。小心地去除上一提液，用培养基重新暂停颗粒，将混合物转移到无菌的10厘米培养皿中。在37摄氏度下用5%的二氧化碳孵育培养基，确保每两到三天用含血清的培养基代替培养基。在这项研究中，建立了胃癌PDX模型和原发细胞系。第一代肿瘤的生长速度比后世的肿瘤慢，需要三周或更长时间才能达到适当的大小。第一代皮下肿瘤形成成功率超过80% PDX模型的癌细胞通过H&E染色从PDX模型中得到证实。从低温保存肿瘤组织肿瘤形成的成功率是大约95%肿瘤细胞很容易识别的形态差异。原细胞由两名病理学家在显微镜下独立验证。为了进一步确认，H&E染色用于观察固定后癌细胞形态。原发细胞系的成功分离率约为40%这些模型已被证明对临床结果具有预测性，并用于临床前药物评估，通过标记识别、生物学研究和个性化医学策略。

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

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

病人衍生软组织肉瘤原代培养的建立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

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

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

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 ...

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 ...

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 ...

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

使用急性淋巴细胞白血病患者来源的异种移植小鼠模型评估嵌合抗原受体T细胞相关毒性

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...