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Ricerca sul cancro

乳腺癌临床前模型乳腺肿瘤的矫形移植

Published: May 18, 2020
doi:
10.3791/61173
, , , , ,
1Department of Molecular and Cellular Biology,Baylor College of Medicine, 2Lester and Sue Smith Breast Center,Baylor College of Medicine, 3Dan L. Duncan Cancer Center,Baylor College of Medicine

Summary

患者衍生异种移植（PDX）模型和可移植的基因工程小鼠模型忠实地概括了人类疾病，是基础和转化乳腺癌研究的首选模型。在这里，一种方法被描述为正位移植乳房肿瘤片段到乳腺脂肪垫研究肿瘤生物学和评估药物反应。

Abstract

临床前模型，忠实地回顾肿瘤异质性和治疗反应是关键转化乳腺癌研究。不朽的细胞系很容易生长和基因改造，以研究分子机制，但来自细胞培养的选择性压力往往导致遗传和表观遗传的变化随着时间的推移。患者衍生异种移植（PDX）模型忠实地概括了人类乳腺肿瘤的异质性和药物反应。PDX模型在矫形移植后表现出相对较短的延迟，有助于乳腺肿瘤生物学和药物反应研究。可移植的基因工程小鼠模型允许研究乳腺肿瘤免疫力。目前的协议描述了将乳腺肿瘤片段矫止移植到乳腺脂肪垫中的方法，然后是药物治疗。这些临床前模型为研究乳腺肿瘤生物学、药物反应、生物标志物发现和耐药机制提供了有价值的方法。

Introduction

大多数乳腺癌死亡可归因于对常规疗法1，2具有抗药性的复发性疾病。乳腺癌的肿瘤间和肿瘤内异质性有助于治疗抵抗。此外，肿瘤异质性会影响准确的预后，并挑战疾病管理3，4。识别反应的预测生物标志物将显著改善乳腺癌患者的临床结果。尽管大多数乳腺癌类型是免疫学上”冷”的肿瘤，可能对免疫治疗没有反应，但免疫检查点抑制剂在临床试验2，5中已显示出希望。例如，第三阶段的试验显示，无病生存（DFS）和初步证据表明，atezolizumab（对PD-L1的单克隆抗体）与纳布-帕利塔塞尔相结合，可以提供整体生存效益相比，纳布-帕利塔塞尔单独在肿瘤中≥1%PD-L1染色6。开发使乳腺肿瘤对免疫疗法敏感的疗法将彻底改变治疗方案。

临床前模型，忠实地回顾人类乳腺癌异质性和药物反应是研究肿瘤生物学和确定潜在的生物标志物有针对性的治疗的关键。不朽的细胞系被广泛用于乳腺癌研究，因为这些细胞系很容易生长和基因改造，以研究分子机制。然而，由于来自体外长期细胞培养的选择性压力，遗传漂移可能会随着时间的推移而发生，乳腺癌细胞系可能携带不同于原发性乳腺肿瘤7、8、9畸变的细胞系特异性基因组改变。

患者衍生的异种移植（PDX）肿瘤块能够概括人类疾病的异质性，在组织学和免疫造血学上类似于原发肿瘤10、11、12、13、14、15、16、17、18、19、20、21、22 ，23，24，25，26，27，28，29。重要的是，PDX模型在多种移植中表型稳定，从组学、转录学、蛋白质组和基因组分析10、11、12、13、14、15、16、17、18、19、20、21、22中可以证明这一点，23，24，25，26，27，28，29。PDX 模型显示的治疗反应与临床观察到的 10、11、12、13、14、15、16、17、18、19、20、21、22、23、24 等治疗反应相当， 25，26，27，28，29。已建立了雌激素受体阳性（ER+）、黄体酮受体阳性（PR+）、表皮生长因子2阳性（ERBB2+、HER2+）和三阴性乳腺癌（TNBC）PDX模型的PDX模型，为检测内分泌、化疗和靶向治疗提供了一个极好的平台。然而，PDX模型目前的主要警告之一是小鼠缺乏功能免疫系统。

基因工程小鼠模型（GEMM），如Trp53同源空、cMyc、Wnt1、PyMT或Her2过度表达模型，允许在完整的免疫系统背景下研究自发肿瘤的启动、进展和转移。然而，肿瘤延迟时间长，这使得它很难进行临床前试验与多臂30，31。然而，GEMM可以移植到同步宿主，产生足够数量的肿瘤，密切回顾人类肿瘤32，33，34，35，36，37，38，39，40，41，42，43，44，45，46，47，48，49，50，51，52，53，54，55。例如，从p53-空BALB/c小鼠的乳腺上皮被移植到同步野生型受体小鼠的清除脂肪垫中，形成原发性肿瘤，可以进一步移植到同步宿主56，57。p53-空肿瘤回顾了人类肿瘤的不同亚型。

PDX模型和可移植GEM的结合为研究乳腺癌生物学、药物反应和抗肿瘤免疫提供了宝贵的临床前工具。在目前的协议中，描述了PDX和GEMM肿瘤片段在小鼠乳腺脂肪垫中的矫形移植方法。这些模型适合串行段落，通常保留稳定的表型。为了减轻遗传漂移或失去异质性的风险，随着时间的推移，多个组织片段被冷冻保存在每一个通道，以备随后移植的情况下，生物或形态的变化观察到时间29，58。

Protocol

使用动物的所有协议都已得到机构动物护理和使用委员会（IACUC）的审查和批准。肿瘤片段，约1×2毫米3 的大小，是从贝勒医学院的患者衍生异种移植和高级在Viva模型核心获得的可冷冻库存。 1. 准备冷冻保存的乳腺肿瘤片段进行移植 将带有肿瘤片段的冷冻液从液氮转移到 37 °C 的水浴中。 解冻时偶尔轻轻轻拂一下，搅拌低温。 组织解冻后，将?…

Representative Results

图1显示了矫形移植的设备（图1A）和关键程序（图1B）。图2显示了移植PDX肿瘤（MC1）的特征。MC1模型的肿瘤片段（1毫米3）被移植到SCID/米色小鼠的#4脂肪垫中。一个月后，肿瘤平均大小达到350毫米3左右。肿瘤体积每周监测两次，为期一个月?…

Discussion

为了减少动物肿瘤生长的变化，将肿瘤组织切成1毫米3 的片段进行移植至关重要。生长软组织的模型更难处理，肿瘤片段需要稍微大一点（1~2毫米3）。当将组织放入乳腺脂肪垫口袋时，请注意不要将组织分割成多个块，因为这会导致多个小肿瘤或形状奇怪的肿瘤。

此外，使用新鲜肿瘤移植动物，将用于药物治疗研究。从冷冻保存中植入组织将产生更可变的接…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了美国国家卫生研究院（R37CA228304和R01HL146642至西陈，CA148761至杰弗里·罗森），美国国防部（W81XWH-19-1-0524至西陈， W81XWH-19-1-0035 至徐向东、美国癌症协会（RSG-18-181-01-TBE 至西辰）和德克萨斯州癌症预防与研究所（RR150009 CPRIT 癌症研究学者奖授予陈希） 贝勒医学院的患者衍生异种移植和高级体内模型核心（来自RP170691 CPRIT核心设施奖和NCI-CA125123 P30癌症中心支持补助金）。

Materials

1 mg/mL Buprenorphine-SR ZooPharm (via BCM veterinarians) Sterile
26G syringe BD 148232E Sterile
Betadine Scrub Fisher 19-027132
Cotton Swabs VWR International Laboratory 89031-272 Sterile
DMEM Fisher MT 10-013-CM Sterile
Electric shaver Oster 78005-050
Glass beads sterilizer (Germinator) Roboz Surgical Store DS-401
Lubricant ophthalmic ointment Akorn Animal Health 17478-062-35
Micro Dissecting Forceps; Serrated, Angular (regular forceps) Roboz Surgical Store RS-5139 Sterile
Micro Dissecting Spring Scissors (fat pad cutter) Roboz Surgical Store RS-5658BT Sterile
Micro Forceps (tissue placing forceps) Roboz Surgical Store RS-5069 Sterile
Petri Dish Fisher 08-757- 100D Sterile
Sterile drape Sai Infusion Technology PSS-SD1 Sterile
Surgery scissors Roboz Surgical Store RS-5960 Sterile
Tissue Forceps (claw forceps) Roboz Surgical Store RS-5158 Sterile
Wound clip applier BD Autoclip Wound System 01-804 Sterile
Wound clip remover BD Autoclip Wound System 01-804-15 Sterile
Wound clips BD Autoclip Wound System 01-804-5 Sterile
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Tags

Orthotopic TransplantationBreast TumorPreclinical ModelBreast CancerTumor BiologyDrug ResponseDrug ResistanceMammary Fat PadPatient-derived XenograftSubcutaneous ImplantationCryopreservationThawingTumor DissectionDMEM
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Lv, X., Dobrolecki, L. E., Ding, Y., Rosen, J. M., Lewis, M. T., Chen, X. Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer. J. Vis. Exp. (159), e61173, doi:10.3791/61173 (2020).
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