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Krebsforschung

间变性甲状腺癌和头颈部鳞状细胞癌患者来源异种移植模型的建立与表征

Published: June 02, 2023
doi:
10.3791/64623
, , , , , , , , ,
1Precision Medicine Research Center, Sichuan Provincial Key Laboratory of Precision Medicine, National Clinical Research Center for Geriatrics, West China Hospital,Sichuan University, 2Department of Thyroid Surgery, West China Hospital,Sichuan University, 3Sichuan Kangcheng Biotech Co., 4Sichuan Cancer Hospital

Summary

由于PDX模型正在迅速成为转化肿瘤学领域的标准，因此本协议建立并表征了间变性甲状腺癌（ATC）和头颈部鳞状细胞癌（HNSCC）的患者来源异种移植（PDX）模型。

Abstract

患者来源的异种移植（PDX）模型忠实地保留了原发肿瘤的组织学和遗传学特征，并保持了其异质性。基于PDX模型的药效学结果与临床实践高度相关。间变性甲状腺癌（ATC）是甲状腺癌最恶性的亚型，侵袭性强，预后差，治疗有限。虽然ATC的发病率仅占甲状腺癌的2%-5%，但其死亡率高达15%-50%。头颈部鳞状细胞癌（HNSCC）是最常见的头颈部恶性肿瘤之一，全球每年有超过60万例新病例。本文提出了详细的协议来建立ATC和HNSCC的PDX模型。本工作分析了影响模型构建成功率的关键因素，比较了PDX模型与原发肿瘤的组织病理学特征。此外，通过评估成功构建的PDX模型中代表性临床使用药物的 体内 治疗效果，验证了该模型的临床相关性。

Introduction

PDX模型是一种动物模型，其中人类肿瘤组织被移植到免疫缺陷小鼠中并在小鼠提供的环境中生长1。传统的肿瘤细胞系模型存在一些缺点，例如缺乏异质性，无法保留肿瘤微环境，在重复体外传代过程中易受遗传变异的影响，以及临床应用不良2，3。基因工程动物模型的主要缺点是人类肿瘤基因组特征的潜在丧失，新的未知突变的引入，以及难以确定小鼠肿瘤与人类肿瘤之间的同源程度4。此外，基因工程动物模型的制备成本高、耗时且效率相对低下4.

PDX模型在反映肿瘤异质性方面比其他肿瘤模型具有许多优势。从组织病理学的角度来看，虽然小鼠对应物随着时间的推移取代了人类基质，但PDX模型很好地保留了原发肿瘤的形态结构。此外，PDX模型保留了原发肿瘤至少四代的代谢组学特性，更好地反映了肿瘤细胞与其微环境之间复杂的相互关系，使其在模拟人肿瘤组织的生长、转移、血管生成和免疫抑制方面独树一帜5，6，7.在细胞和分子水平上，PDX模型准确反映了人类肿瘤的肿瘤间和肿瘤内异质性，以及原始癌症的表型和分子特征，包括基因表达模式、突变状态、拷贝数以及DNA甲基化和蛋白质组学8，9。不同传代的PDX模型对药物治疗具有相同的敏感性，表明PDX模型的基因表达高度稳定10，11。研究表明，PDX模型对药物的反应与患者对该药物的临床反应之间存在极好的相关性12，13。因此，PDX模型已成为一种强大的临床前和转化研究模型，特别是在药物筛选和临床预后预测方面。

甲状腺癌是内分泌系统常见的恶性肿瘤，是一种人类恶性肿瘤，近年来发病率迅速上升14。间变性甲状腺癌（ATC）是最恶性的甲状腺癌，患者的中位生存期仅为4.8个月15。虽然中国每年只有少数甲状腺癌患者被诊断为ATC，但死亡率接近100%16，17，18。ATC通常生长迅速并侵入颈部的邻近组织以及颈部淋巴结，约一半的患者有远处转移19，20。头颈部鳞状细胞癌（HNSCC）是世界上第六大常见癌症，也是癌症死亡的主要原因之一，估计每年有60万人患有HNSCC21，22，23。HNSCC包括大量肿瘤，包括鼻子，鼻窦，口腔，扁桃体，咽部和喉部的肿瘤24。ATC和HNSCC是两种主要的头颈部恶性肿瘤。为了促进新型治疗药物和个性化治疗的开发，有必要开发稳健和先进的临床前动物模型，例如ATC和HNSCC的PDX模型。

本文介绍了建立ATC和HNSCC皮下PDX模型的详细方法，分析了模型构建中影响肿瘤取样率的关键因素，并比较了PDX模型与原发肿瘤的组织病理学特征。同时，在这项工作中，使用成功构建的PDX模型进行了 体内 药效学测试，以验证其临床相关性。

Protocol

所有动物实验均按照四川大学华西医院机构动物护理和使用委员会批准的实验动物护理评估与认证协会指南和协议进行。本研究使用4-6周龄的NOD-SCID免疫缺陷小鼠（两性）和4-6周龄的雌性Balb / c裸鼠。这些动物是从商业来源获得的（见 材料表）。华西医院伦理委员会授权对人类受试者进行研究（方案编号2020353）。每位患者都提供了书面知情同意书。 1. 实验准…

Representative Results

共移植甲状腺癌标本18例，成功构建甲状腺癌PDX模型5例（肿瘤取率为27.8%），其中未分化甲状腺癌4例，间变性甲状腺癌1例。分析模型构建成功率与年龄、性别、肿瘤直径、肿瘤分级、分化等相关性。虽然4级肿瘤样本的模型成功率高于低分级样本，未分化肿瘤样本的成功率也高于高分化样本，但相关性分析结果表明，这些因素与PDX模型的成功率无关（表1).接种了17个HNSCC样本，并成功?…

Discussion

本研究成功建立了ATC和HNSCC的皮下PDX模型。在PDX模型构建过程中，有很多方面需要注意。当肿瘤组织与患者分离时，应尽快放入冰盒中送实验室接种。肿瘤到达实验室后，操作人员必须注意保持无菌区域并练习无菌程序。对于穿刺活检样品，由于肿瘤组织特别小，接种后将样品与基质凝胶混合会更有利于模型的建立。原发肿瘤组织也应尽可能保存、固定和冷冻，以备将来研究之用。在接种过程中?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

本工作得到了四川省科技支撑计划（批准号：2019JDRC0019和2021ZYD0097）、四川大学华西医院优秀学科1.3.5项目（批准号ZYJC18026）、四川大学华西医院优秀学科1.3.5项目-临床研究孵化项目（批准号：2020HXFH023）、中央高校基本科研业务费专项（SCU2022D025）的支持。 成都市科技局国际合作项目（批准号：2022-GH02-00023-HZ）、四川大学创新星火项目（批准号：2019SCUH0015）、电子科技大学华西医院医工融合人才培养基金（批准号：HXDZ22012）。

Materials

2.4 mm x 2.0 mm trocar Shenzhen Huayang Biotechnology Co., Ltd 18-9065
Balb/c nude mice Beijing Vital River Laboratory Animal Technology Co., Ltd. 401
Biosafety cabinet Suzhou Antai BSC-1300IIA2
Blade Shenzhen Huayang Biotechnology Co., Ltd 18-0823
Centrifuge tube  Corning 430791/430829
Cryopreservation tube Chengdu Dianrui Experimental Instrument Co., Ltd /
Custodiol HTK-Solution Custodiol 2103417
Dimethyl sulfoxide(DMSO) SIGMA-ALORICH D5879-500mL
Electronic balance METTLER ME104
Electronic digital caliper Chengdu Chengliang Tool Group Co., Ltd 0-220
fetal bovine serum(FBS) VivaCell C04001-500
IBM SPSS Statistics 26 IBM
Ketamine Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd  100761663
Lenvatinib ApexBio A2174
NOD SCID immunodeficient mice Beijing Vital River Laboratory Animal Technology Co., Ltd. 406
Pen-Strep Solution Biological Industries 03-03101BCS
Petri dish WHB WHB-60/WHB-100
Saline  Sichuan Kelun W220051705
Scissor Shenzhen Huayang Biotechnology Co., Ltd 18-0110
Tweezer Shenzhen Huayang Biotechnology Co., Ltd 18-1241
Vet ointment Pfizer Inc. P10015353
Xylazine Dunhua Shengda Animal Medicine Co., Ltd 070031777
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Tags

Keywords: Patient-derived XenograftPDXAnaplastic Thyroid CarcinomaATCHead And Neck Squamous Cell CarcinomaHNSCCTumor HeterogeneityMolecular DiversityPersonalized TreatmentAnti-cancer Drug Sensitivity ScreeningTumor TransplantationSubcutaneousCryopreservationTumor Growth Curve
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Wu, M., Liu, Y., Zhao, Y., Zhang, Y., Huang, L., Du, Q., Zhang, T., Zhong, Z., Luo, H., Xiao, K. Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma. J. Vis. Exp. (196), e64623, doi:10.3791/64623 (2023).
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