在免疫缺陷小鼠中生成肝脏正交人子宫黑色素瘤异种移植平台
Summary
正交人肝转移性黑色素瘤异种移植小鼠模型是利用手术正位植入技术与患者衍生的肿瘤块和针注射技术与培养人类uveal黑色素瘤细胞系创建。
Abstract
近几十年来，与传统的人类细胞相比，皮下植入的患者衍生异种移植肿瘤或培养的人类细胞系越来越被公认为研究免疫缺陷小鼠中人类癌症的更具代表性的模型。线体外。最近，在小鼠中开发了正位植入患者衍生的肿瘤异种移植（PDX）模型，以更好地复制患者肿瘤的特征。肝正交异种小鼠模型有望成为一个有用的癌症研究平台，为肿瘤生物学和药物治疗提供见解。然而，肝正交肿瘤的植入一般是复杂的。在这里，我们描述了我们治疗患者衍生的肝转移性黑色素瘤肿瘤的正射体植入方案。我们培养人类肝脏转移性黑色素瘤细胞系到免疫缺陷小鼠。这些方案可以使用具有患者衍生的Uveal黑色素瘤块的外科正交植入技术或具有培养人类细胞系的针注射技术，从而持续获得高技术成功率。我们还描述了CT扫描检测内部肝肿瘤和再植入技术使用冷冻肿瘤实现再移植的协议。这些方案共同为肝转移性黑色素瘤的肝正位肿瘤小鼠模型在转化研究中提供了更好的平台。
Introduction
乌韦黑色素瘤是西方成人最常见的眼内恶性肿瘤。在过去50年中，美国1、2个州的乌韦黑色素瘤发病率（百万分之5.1）保持稳定。乌韦黑素瘤产生于虹膜、纤毛体或胆囊中的黑色素细胞，当它发生转移时，它是一种极其致命的疾病。乌韦黑色素瘤转移患者1岁时死亡率为80%，转移初步诊断后2年死亡率为92%。从转移诊断到死亡的时间通常很短，不到6个月，没有治疗3，4。癌症通过血液扩散，并倾向于主要转移到肝脏（89-93%）4，5。为进一步研究肝转移性黑色素瘤，迫切需要一种有效的小鼠模型。对于转化研究，有一个明确的需求，以生成肝脏局部转移性黑色素瘤小鼠模型。
患者衍生的肿瘤异种移植（PDX）小鼠模型有望提供个性化的医学策略。这些模型可能是临床结果的预测，可用于临床前药物评估，并用于肿瘤生物学研究6。代表性的PDX模型是异位植入肿瘤的异种移植小鼠，它们在皮下部位有肿瘤。大多数研究人员可以做手术的皮下植入没有特殊的做法7，8。它们也可以很容易地监测皮下肿瘤。虽然皮下PDX模型在研究阶段开始流行，但在实际应用方面还是有一些障碍。皮下植入迫使患者衍生的肿瘤在肿瘤来源不同的微环境中移植，从而导致移植失败和减缓肿瘤生长9，10，11， 12，13，14.对于PDX模型来说，正交可能更理想和合理，因为它使用与原始肿瘤15、16相同的器官。
最近，我们开发了患者衍生肝转移性黑色素瘤肿瘤的外科正交植入技术，以及使用NOD培养的人类肝转移性黑色素瘤细胞系的针注射技术。Cg-PrkdcscidIl2rgtm1Wjl/SzJ （NSG） 小鼠17，18.该协议使技术成功率始终居高不下。我们还建立了CT扫描技术，用于检测内部肝肿瘤，并在PDX平台中开发了冷冻肿瘤的再植入。研究发现，乌韦黑素瘤肿瘤异种移植模型保持原患者肝肿瘤的特征，包括其组织病理学和分子特征。这些技术共同为肝正位肿瘤模型在转化研究中提供了更好的平台。
Protocol
根据机构审查委员会批准的协议，参与研究的患者应提供书面同意，允许将废弃的手术样本用于研究和基因研究。该协议严格按照国家卫生研究院《照料和使用实验室动物指南》中的建议执行，并经机构动物护理和使用委员会（IACUC）批准。 1. 新鲜患者衍生肿瘤组织的集合 从手术或医院手术室的针头活检中获得患者衍生的肿瘤组织。 将肿瘤组织放…
Representative Results
手术正交植入采用肝囊法，可在六个月内将人肝转移性黑色素瘤肿瘤移植到小鼠肝脏，成功率高达80%。异种移植肿瘤在肝脏中作为无女儿结节的单独肿瘤移植（图1和图3A）。手术正交注射技术进入肝脏使用微针成功地移植培养人类肝脏转移性黑色素瘤细胞在肝脏在所有情况下（图2和图3B）?…
Discussion
目前的正交异种异种移植模型是劳动密集型的，耗时的，而且创建成本很高。正交肿瘤异种移植小鼠模型建立于20多年前的19，20，21。然而，这种技术是复杂的，需要使用特殊设备，如微针支架和6-0至8-0在显微镜下细缝合。肿瘤和正常的肝脏组织必须仔细缝合，以便缝合不会损坏脆弱的肝脏组织。传统技术导致并发症，如血肿?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢Ohara、K.斋藤和太太郎审阅了手稿。作者承认福克斯大通癌症中心的Sato博士对这份手稿的编辑和英文协助进行了批判性评论。本文所述工作得到了邦尼·克罗尔研究基金、马克·文齐尔研究基金、托马斯·杰斐逊大学眼黑色素瘤研究基金、大阪社区基金会和大阪市JSPS KAKENHI赠款号JP 18K15596的支持。大学。A. Aplin博士实验室的研究得到了NIH资助R01 GM067893的支持。该项目还由院长的变革科学奖，托马斯杰斐逊大学方案倡议奖资助。
Materials
| Materials, tissues and animals | |||
| Buprenorphine | |||
| CO2 tank | |||
| Cryomedium | |||
| Exitron nano 12000 (Alkaline earth metal-based nanoparticle contrast agent) | Miltenyl Biotec | 130-095-700 | |
| HBSS 1X, with calcium & magnesium | Corning | 21-020-CM | |
| Human liver metastatic uveal melanoma cell line | |||
| Human uveal melanoma tissue in the liver | All tissue handling should be done in a Biosafety Level 2 hood. Be careful when working with human tissue; always use gloves and avoid direct skin contact. Assume patients may have been infected with HIV or other highly transmissible organisms. Do not process samples known to carry infections. | ||
| Iodine | |||
| Isoflurane | Purdue Products | 67618-150-17 | |
| Isopropanol | Fisher scientific | A416-1 | Avoid direct contact to skin and eye and inhalation of anesthetic agent. |
| Liquid nitrogen | |||
| Matrigel HC | BD | 354248 | |
| NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice | Jackson Lab | 5557 | 4 to 8 weeks old |
| PBS 1X, without calcium and magnesium | Corning | 21-031-CM | |
| RPMI 1640 | Corning | 10-013-CV | |
| Sterile alcohol prep pad (70% isopropyl alcohol) | Nice-Pak products | B603 | |
| 4% paraformaldehyde phosphate buffer solution | Wako | 163-20145 | |
| 70% Ethyl alcohol solution | Fisher Scientific | 04-355-122 | |
| Name | Company | Catalog Number | Comments |
| Equipments | |||
| Absorbable hemostat | Johnson and Johnson | 63713-0019-61 | |
| Autoclave | |||
| Body weight measure | |||
| Cautery | Bovie Medical | MC-23009 | |
| Cell counter | |||
| Centrifuzer | |||
| Cotton swab | |||
| Cryo freezing container | NALGENE | 5100-0001 | |
| Cryotube | SARSTEDT | 72.379 | |
| Curved scissors | World Precision Instruments | 503247 | |
| Curved ultrafine forceps | World Precision Instruments | 501302 | |
| Fabric sheet | |||
| Freezer | |||
| F/AIR Filter Canister | Harvard Apparatus | 600979 | |
| Heating pad | |||
| Isoflurane vaporizer | Artisan Scientific | 66317-1 | |
| Liquid nitrogen | |||
| Liquid nitrogen jar | Thermo Fisher Scientific | 2123 | |
| Micro-CT scan | Siemens | ||
| Needle holder | World Precision Instruments | 501246 | |
| Petri dishes | Fisher Scientific | FB0875713 | |
| Pipette | |||
| Spray bottle | |||
| Sterile hood | Biosafety level 2 cabinet | ||
| Sterile No.11 scalpel | AD Surgical | A300-11-0 | |
| Straight forceps | World Precision Instruments | 14226 | |
| Surgical drape | |||
| Tail vein restrainer | Braintree Scientific | TV-150-STD | |
| Water bath | |||
| 1 ml TB syringe with 27-gauge needle | BD | 309623 | |
| 1.7 ml tube | Bioexpress | C-3260-1 | |
| 5-0 PDO Suture | AD Surgical | S-D518R13 | |
| 15 mL conical tubes | AZER SCIENTIFIC | ES-9152N | |
| 27-gauge needle | BD | 780301 | |
| 27-gauge needle | Hamilton | 7803-01 | |
| 50 mL conical tubes | AZER SCIENTIFIC | ES-9502N | |
| 50 µl micro syringe | BD | 80630 | |
| 50 µl micro syringe | Hamilton | 7655-01 | |
| 100 mL container | Fisher Scientific | 12594997 | |
| 200μl tip |
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Citazione di questo articolo
Kageyama, K., Ozaki, S., Sato, T. Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice. J. Vis. Exp. (153), e59941, doi:10.3791/59941 (2019).