患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型
Summary
该协议描述了患者衍生的正交异种移植模型,通过内在灌输高级尿细胞癌细胞或直肠内注射结肠直肠癌细胞到非肥胖糖尿病/严重组合免疫缺陷(NOD/SCID)小鼠原发性肿瘤生长和自发转移在淋巴结基质细胞的影响下,模拟人类转移性疾病的进展。
Abstract
当膀胱和结肠直肠癌 (CRC) 的高级别泌尿细胞癌 (HG-UCC) 中存在淋巴结 (LN) 参与时,癌症患者的预后较差。超过50%的肌肉侵入性UCC患者,尽管对临床局部疾病进行治疗,但会发展转移并在5年内死亡,而转移性CRC是美国癌症相关死亡的主要原因。需要持续模仿UCC和CRC转移的患者异种移植模型。本研究旨在生成UCC和CRC的由患者衍生的正射异种移植(PDOX)模型,用于在LN基质细胞的影响下原发性肿瘤生长和自发转移,模拟药物筛选中的人类转移性疾病的进展。新鲜UCC和CRC肿瘤分别从接受HG-UCC和结肠直肠腺癌切除的同意患者身上获得。与LN基质细胞(LNSC)模拟HK细胞共接种,荧光素酶标记UCC细胞被注射到女性非肥胖糖尿病/严重合并免疫缺陷(NOD/SCID)小鼠中,CRC细胞被注射到内直肠(IR)雄性NOD/SCID小鼠。利用生物发光成像(BLI)每周监测肿瘤生长和转移。牺牲后,对血氧林和欧辛和免疫组织化学染色进行收获、称重和正式固定原发性肿瘤和小鼠器官。在我们独特的PDOX模型中,异种移植肿瘤类似于患者植入前肿瘤。在港细胞中,两种模型均具有高肿瘤植入率,以BLI和肿瘤重量测量,UCC为83.3%,CRC为96.9%,高远器官转移率(UCC检测到肝或肺转移33.3%,CRC检测为53.1%)。此外,两种型号的死亡率均为零。我们为人类HG-UCC和CRC建立了独特的、可重复的PDOX模型,允许肿瘤形成、生长和转移研究。有了这些模型,新型治疗药物的测试可以有效地进行,临床模拟的方式。
Introduction
研究表明,淋巴结(LN)转移是许多固体器官恶性肿瘤的预后指标差,包括膀胱的高档泌尿细胞癌(UCC)和结肠直肠癌(CRC)1、2。超过一半的肌肉侵入性UCC(MIUCC)患者,尽管对临床局部疾病进行治疗,但会发展转移并在5年内死亡。转移性CRC是美国癌症相关死亡的主要原因。
据估计,2018年美国将有81,190名新患者和17,240名癌症特定死亡,原因是膀胱3、4的UCC。而患者将主要 (70%)目前与非肌肉侵入性疾病,30%将有MIUCC5。尽管临床局部性疾病的用于治疗(根囊切除术[RC],无论是否进行全身化疗),膀胱MIUCC患者的一半仍然会发展转移,并在5年内死亡3。淋巴结参与发现在大约20%-25%的患者经历了RC6,7,8。即使经过 RC,LN 阳性患者的五年生存率也低于 35%,这表明 LN 参与是 UCC 患者预后的关键负预测因素。
结肠直肠癌是美国男性和女性诊断的第三大常见癌症。患者结果主要取决于肿瘤特征和肿瘤微环境,如入侵深度、LN参与和远器官转移。虽然过去十年,由于筛检及有效手术,CRC的死亡率有所下降,但估计近50%的CRC患者会发展为转移或复发性疾病9。
小型动物模型提供了一个快速、可重复和可修改的平台,用于研究肿瘤进展和不同的转移模式。目前没有描述的异种移植模型,持续模仿CRC和UCC转移在患者看到。癌症远距离转移的主要途径是通过淋巴扩散。新的研究表明,LAN为肿瘤提供了独特的微环境,它不仅是癌细胞瞬时通过的固定靶点,而且通过在转移过程中与癌细胞相互作用而起着不可或缺的作用。事实上,我们的研究发现,除了教育和促进肿瘤进展和转移,LN基质微环境也负责在CRC10,11的耐药性。我们的实验室最近使用患者衍生的正交异种移植(PDOX)小鼠模型12、13,确认了LN基质细胞(LNSCs)对CRC和UC的肿瘤效应。
开发PDOX模型为转化癌症研究提供了一个重要的平台。PDOX模型通过保持其供体肿瘤的主要组织学和遗传特征,在各通道之间保持稳定,为转化性癌症研究提供了良好的平台。PDOX 模型正用于临床前药物评估、生物标志物鉴定和个性化药物策略的临床前评估,从而预测临床结果。目前,没有描述异种移植模型考虑LN参与的重要性,并能够持续复制原发肿瘤和遥远的器官转移在CRC和UCC。在这项研究中,我们描述了在NOD/SCID小鼠中,在LNSC参与下,在转移性CRC和UCC疾病的繁殖下,PDOX模型的发展。
Protocol
Representative Results
Discussion
转移性疾病是造成大多数癌症患者死亡的原因。在临床前治疗测试中,建立与自发性远器官转移最密切地模拟人类肿瘤生长的小鼠模型至关重要。使用小鼠模型与植入的患者肿瘤衍生癌细胞(异种移植物)允许更好地了解肿瘤生物学和预测生物标志物,以及测试和预测抗肿瘤效果的新疗法18。许多模型已经用于显示UCC和CRC转移在鼠实验中,如静脉尾静脉注射显示有能力产生…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢布赖恩·鲁特、丹妮尔·贝尔托尼、彼得·米勒和香农·麦切斯尼,他们帮助启动了这些研究,感谢他们出色的技术支持。作者还感谢希瑟·格林·马特拉纳、玛格丽特·瓦里亚诺、苏尼尔·塔尔瓦尔和玛丽亚·拉茨在同意患者和提供肿瘤标本方面给予的帮助。
Materials
| Avidin-biotin-peroxidase | Vector Labs Inc | PK-6100 | |
| Biotinylated secondary antibody | Vector Labs Inc | BA-1000 | |
| Collagenase IV (1.5 mg/mL) | Worthington Biochemical Corporation | LS004189 | |
| Deoxyribonuclease I (0.1 mg/mL) | Sigma | D4263 | |
| D-Luciferin (150 mg/kg) | Perkin Elmer | 122796 | |
| Formalin (10% neutral buffered) | Leica | 46129 | |
| glutamine (2 nM) | Fisher Scientific | 35050061 | |
| Hair Removal Cream | Church & Dwight Co., Inc | 1 (800) 248-8820 | |
| Hanks Balanced Salt Solution (HBSS) | Fisher Scientific | SH30016.02 | |
| Hyaluronidase (20 mg/mL) | Sigma | H3884 | |
| Isoflurane | Henry Schein Animal Health | 108333 | |
| Luc/RFP-lentivirus | From our collaborators. See reference 13: Gills, J. et al. A patient-derived orthotopic xenograft model enabling human high-grade urothelial cell carcinoma of the bladder tumor implantation, growth, angiogenesis, and metastasis. Oncotarget. 9, 32718-32729, doi:10.18632/oncotarget.26024 (2018). | ||
| McCoy’s medium | Life Technologies | 110862 | |
| penicillin/streptomycin 100 mL (100 U/mL) | Fisher Scientific | 15140-122 | |
| RPMI-1640 Medium | American Type Culture Collection | 110636 | |
| Trypan Blue | Sigma | T6146 | |
| Trypsin/EDTA | Life Technologies | 15400-054 | |
| Name | Company | Catalog Number | Comments |
| Gas | |||
| 100% Oxygen | Airgas Inc | OX USP200 | |
| 100% CO2 | Airgas Inc | CD USPE | |
| Name | Company | Catalog Number | Comments |
| Mice | |||
| 6-8 week old NOD/SCID Mice (male) | Jackson Lab | 001303 | |
| 6-8 week old NOD/SCID Mice (female) | Jackson Lab | 001303 | |
| Name | Company | Catalog Number | Comments |
| Immunohistochemistry | |||
| Hematoxylin | Sigma | GHS232 | |
| Ki-67 Rabbit Monoclonal Antibody | Thermo Scientific | RM-9106-S | |
| Name | Company | Catalog Number | Comments |
| Tools | |||
| 40 µm cell strainer | Fisher Scientific | 08-771-1 | |
| 100 µm cell strainer | Fisher Scientific | 08-771-19 | |
| 15 mL Conical Tube | Sarstedt | 11799 | |
| 50 mL Conical tube | Sarstedt | 15762 | |
| 150 mm Tissue Culture Dish | USA Scientific Inc | CC7682-3614 | |
| 96 Well plate | USA Scientific Inc | CC7682-7596 | |
| Forceps | Symmetry Surgical Inc | 06-0011 | |
| Surgical scissors | Symmetry Surgical Inc | 02-2011 | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| 5% CO2 humidified incubator | Thermo Scientific | 3110 | |
| Bioluminescent (BLI) Imaging Machine | Perkin Elmer | CLS136334 | |
| BLI Imaging Machine Software | Perkin Elmer | CLS136334 | |
| Centrifuge | Beckman | 366830 | |
| Deconvoluting Microscope | Intelligent Imaging Innovations | Marianas | |
| Deconvoluting Microscope Imaging Software | Intelligent Imaging Innovations | +1 (303) 607-9429 x1 | |
| Digital caliper | Fowler Tools and Instruments | 54-115-330 | |
| Dissecting microscope | Precision Instruments LLC | (504) 228-0076 | |
| Electrosurgical generator | ValleyLab | FORCE1C20 | |
| Isoflurane Induction Chamber | Perkin Elmer | 119038 | |
| Microtome | American Optical Corporation | 829 | |
| Pipet Aid | Fisher Healthcare | 13-681-15E | |
| Serological pipet (10 mL) | Sarstedt | 86.1254.001 |
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