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Cancer Research

矫形子宫内膜癌模型，具有从 Vivo 传播和培养的 VX2 细胞中制成的再生淋巴病

Published: September 12, 2019
doi:
10.3791/59340
, , , , , , , , ,
1Institute of Medical Science,University of Toronto, 2Department of Obstetrics and Gynecology,University of Toronto, 3Guided Therapeutics Laboratory, TECHNA Institute,University Health Network, 4Department of Pathology, Princess Margaret Cancer Center,University Health Network, 5Department of Medical Biophysics,University of Toronto, 6Princess Margaret Cancer Center,University Health Network, 7Techna Institute,University Health Network, 8Department of Surgery,University of Toronto, 9Department of Otolaryngology, Head and Neck Surgery,University of Toronto, 10Division of Gynecologic Oncology, University of Toronto /Princess Margaret Cancer Center,University Health Network, 11Princess Margaret Cancer Center,University Health Network

Summary

该协议提出了一种标准化的方法，用于培养VX2细胞，并创建子宫内膜癌的正交VX2模型，在兔子中具有逆转淋巴结转移。正位子宫内膜癌模型对于对诊断淋巴结转移的新型成像模式进行临床前研究具有重要意义。

Abstract

子宫内膜癌是北美最常见的妇科恶性肿瘤，其发病率正在全球上升。治疗包括手术与或不辅助治疗取决于淋巴结参与由淋巴腺切除术确定。淋巴腺切除术是一种病态手术，在许多患者中尚未证明具有治疗作用，因此需要一种诊断淋巴结转移的新方法。为了测试新的成像剂，需要一个可靠的子宫内膜癌模型与逆转淋巴结转移。VX2子宫内膜癌模型在文献中经常被描述;然而，在模型建立方法方面存在着很大的差异。此外，没有研究报告使用培养的VX2细胞来创建这个模型，因为以前只使用在体内繁殖的细胞。本文介绍了建立VX2子宫内膜癌模型的标准化手术方法和术后监测方法，并报告首次使用培养的VX2细胞创建该模型。

Introduction

子宫内膜癌，或子宫内膜癌，是全球第二大最常见的妇科恶性肿瘤，也是发达国家最常见的恶性肿瘤1。子宫内膜癌的发病率稳步上升，在2005-2013年间每年上升2.3%，死亡率为1、2、3，相应上升2.2%。淋巴结转移的诊断是最重要的，因为正淋巴结的存在是生存4，5，6，7的强烈负预测，并可以指导管理辅助疗法8，9，10，11，12，13。淋巴结转移目前通过手术切除骨盆和腹部主要血管的淋巴组织进行诊断。这个程序，被称为淋巴腺切除术，是有争议的，因为冲突的生存数据从两个大型试验14，15，16，17，18和已知的术中15、19、20和术后发病风险21、22、23。由于目前的非侵入性成像模式没有所需的灵敏度和特异性来取代淋巴结解剖24，因此一直在推动开发新的诊断成像技术。为了在临床前环境中测试这些新技术，需要一个可靠的子宫内膜癌模型与逆转淋巴结转移。

兔子VX2肿瘤模型是一个成熟的模型，已被广泛用于研究多个人体固体器官肿瘤25，包括肺26，头颈部27，28，肝脏29，肾脏30，骨31，32，脑33，胰腺34和子宫35，36，37。VX2模型最初由基德和鲁斯38于1940年开发，成功移植了1933年Shope发现的棉尾兔辣椒瘤病毒。自那时以来，VX2模型一直维持在体内，需要连续通过白新西兰兔子40的四头肌。然而，最近，多个组已经成功地在体外40，41，42中生长VX2细胞，并证明了培养细胞系31的保留性。42，43.VX2肿瘤被组织学定义为肿瘤鳞状细胞癌44，并含有类似于腺癌26的腺体特征。肿瘤的特点是易于植入，快速生长和超血管44，45和可靠的转移，最常见的区域和遥远的淋巴结45。子宫血管和淋巴解剖46以及正交生长部位的相似性确保了兔子VX2癌的转移模式与人类子宫内膜癌的转移模式相仿，使VX2模型成为研究人类的可靠模型转移性疾病。此外，组织学特征，如异常微血管增殖47，以及免疫学48和基因相似性49，50人与兔子之间的表明肿瘤微环境可能反映人类子宫内膜癌。

多个小组已经报告使用VX2创建子宫内膜癌模型与逆转性转移与高报告成功率36，51，52;然而，在模型创建方法方面，当前文献中存在着显著的变化。细胞悬浮剂量低至4 x 105细胞/子宫角51和高达5 x 109细胞/子宫角37，53已报告没有标准共识所需的VX2细胞剂量。此外，还报告了多种接种方法，包括将肿瘤微手术植入子宫肌膜36，注射VX2细胞悬浮液37，44，52,53，在某些情况下，在接种前添加子宫角缝合52。最后，没有组报告使用培养的 VX2 单元格来创建此模型。因此，本研究的目的是展示VX2模型创建的成功标准化方法，并报告首次使用培养的VX2细胞创建子宫内膜癌模型与在兔子的逆转转移。

Protocol

深腹壁闭合：识别腹膜切口的顶点，用组织钳子抓住腹膜、整管肌肉和筋膜。所有动物研究均在大学卫生网络动物资源中心（ARC）批准的设施中进行，并符合经批准的动物使用和护理协议（AUP #3994/#4299）。VX2细胞系是从大学健康网络的Aken博士实验室获得的。 1. 创建体外VX2细胞系 从兔子四头肌和小鼠侧翼生长中收获VX2肿瘤。 解冻冷冻VX2肿瘤块（1厘米x1厘米），?…

Representative Results

28只兔子被用于建立子宫内膜癌模型。在实验时，兔子的平均体重为2.83公斤（2.71-3.58公斤）。子宫肿瘤在21只兔子中成功生长，总体模型成功率为75%。在将子宫缝合纳入协议之前，成功率为57%，而添加子宫缝合后为81%。第7只兔子之后，由于最初的低模型成功率，在协议中加入了子宫缝合。五个模型是从培养的VX2细胞（在8只兔子中尝试，成功率为63%）和16个兔子模?…

Discussion

在此，我们报告了建立VX2子宫内膜癌模型的标准化手术方法，并报告了首次使用培养的VX2细胞创建此模型。肿瘤的切除率75%低于文献35、37、53、56所报告的100%;肿瘤的接受率是75%的。”37、53、56″的肿瘤取肿瘤率低于75%的发病率。然而，90%的病理确认淋巴结转移率与之前对该模型35、53<su…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究由特里·福克斯研究所（PPG#1075）、加拿大健康研究所（基金会赠款#154326）、加拿大癌症学会研究所（704718）、加拿大自然科学和工程研究理事会资助，[阿纳达创新基金会和玛格丽特公主癌症基金会。

我要感谢玛格丽特·阿肯斯博士为建立初始VX2模型和冷冻VX2肿瘤块提供了初始VX2细胞。我要感谢马可·迪格拉帕帮助进行初始VX2细胞培养实验，并感谢丁丽丽帮助VX2细胞培养。

Materials

11-blade scalpel, Sterile, Disposible Aspen Surgical (VWR) 80094-086
22-gague ear vein catheter CDMV 14332
3-0 absorbable poly-filament suture (Polysorb) Covidien 356718
3-0 braided absorbable suture (Polysorb) Covidien 356718
70uM cell strainer, Individually wrapped, Nylon Falcon 352350
Acepromazine (Atravet) CDMV 1047
Betadine soap (Poviodone iodine 7.5%) CDMV 4363
Betadine solution (Poviodone iodine 10%) UHN Stores 457955
Buprenorphine McGill University
Cefazolin UHN in-patient pharmacy No Cat # Needed
Chlorhexidine solution CDMV 119872
Corning BioCoatCellware, Collagen Type I, 100mm dishes Corning 354450 brand not important
Corning BioCoatCellware, Collagen Type I, 24-well plates Corning 354408 brand not important
Corning BioCoatCellware, Collagen Type I, 6-well plates Corning 354400 brand not important
Corning Matrigel Basement Membrane Matrix, *LDEV-free, 10 mL Corning 354234
DMEM/HAM F12 1:1 Life Technologies 11320 brand not important
DMSO Caledon Lab Chem 1/10/4100
Enrofloxacin (Baytril injectable) CDMV 11242
Falcon Tube Corning Centri-Star 430828
Fetal Bovine Serum, Qualified, Canadian Origin, 500ml Life Technologies 12483020 brand/source not important
Isoflurane UHN in-patient pharmacy No Cat # Needed
Isohexol contrast GE Healthcare 407141210
Meloxicam (Metacam 0.5%) CDMV 104674
Normal Saline House Brand (UofT Medstore) 1011
PBS Multicell or Sigma 331-010-CL or D8537-500mL
Penicillin/Streptomycin (100mL; 10000U Penicillin, 10000ug Streptomycin) Corning-Cellgro CA45000-652
Sterile Hanks Balanced Salt Solution (-Ca++, -Mg++, -Phenol Red) T.C.M.F (Dr Bristow) 28-Jan-11
Surgical Glue (Tissue Adhesive) 3M Vetbond 14695B
Trypsin (0.25%), Proteomics Grade Sigma T-6567-5X20UG
Trypsin-EDTA, 0.05%, 100ml Wisent Inc 325-542-EL brand not important
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Tags

Keywords: Orthotopic Endometrial Cancer ModelVX2 CellsRetroperitoneal Lymph Node MetastasisImage-guided SurgeryIn Vitro PropagationIn Vivo PropagationCell CultureCell SuspensionCell CountingCell Injection
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Philp, L., Chan, H., Rouzbahman, M., Rostami, A., Ding, L., Bratman, S. V., Akens, M. K., Irish, J. C., Bernardini, M. Q., Zheng, G. An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells. J. Vis. Exp. (151), e59340, doi:10.3791/59340 (2019).
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