建立胃癌患者衍生异种移植模型和原细胞系
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 40 μm Cell Strainer | Biologix, Shandong, China | 15-1040 | |
| Biological Microscope | OLYMPUS, Tokyo, Japan | OLYMPUS CKX41 | |
| Centrifuge | Eppendorf, Mittelsachsen, Germany. | 5427R | |
| CO2 Incubator | Thermo Fisher Scientific, Carlsbad, California, USA | HERACELL 150i | |
| DPBS | Basalmedia Technology, Shanghai, China | L40601 | |
| Electro-Thermostatic Water Cabinet | Yiheng, Shanghai, China | DK-8AXX | |
| Fetal bovine serum | Wisent Biotechnology, Vancouver, Canada | 86150040 | |
| Isoflurane | Baxter, China | CN2L9100 | |
| Live Tissue Kit Cryo Kit | Celliver Biotechnology, Shanghai, China | LT2601 | |
| Live Tissue Thaw Kit | Celliver Biotechnology, Shanghai, China | LT2602 | |
| NSG | Biocytogen, Beijing, China | B-CM-002-4-5W | |
| Penicilin&streptomycin | Thermo Fisher Scientific, Carlsbad, California, USA | 15140122 | |
| Red blood cell lysis buffer | Solarbio, Beijing, China | R1010 | |
| RPMI-1640 medium | Thermo Fisher Scientific, Carlsbad, California, USA | 8118367 | |
| Surgical Suture Needles with Thread | LingQiao, Ningbo, China | 3/8 arc 4×10 | |
| Tissue-processed molds and auxiliary blades | Celliver Biotechnology, Shanghai, China | LT2603 | |
| Trypsin-EDTA | Thermo Fisher Scientific, Carlsbad, California, USA | 2003779 | |
| Type 1 collagenase | Thermo Fisher Scientific, Carlsbad, California, USA | 17100017 |
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