体内模型对缺氧对肿瘤转移的影响测试
Summary
This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.
Abstract
Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.
Introduction
尤文肉瘤（ES）是影响儿童和青少年积极恶性肿瘤。 1肿瘤的软组织和骨骼发育，常见于四肢。虽然转移的存在是ES患者最强大的不良预后因素，其发展背后的机制仍不清楚。 2肿瘤缺氧是在ES进展牵连的几个因素之一。在ES患者，非灌注区域的肿瘤组织中存在与预后不良相关联。 3 在体外 ，缺氧增加ES细胞的侵袭，并触发亲转移性基因的表达。 4-6然而，尽管这些证据，对缺氧诱导的ES进展和蔓延没有直接的证据存在。此外，由缺氧产生如此效果，目前的机制尚不清楚。因此，我们已经创建了一个体内模型，以填补现有体外数据和临床安装前后之间的间隙vations。此模型系统使得在它们的天然环境中存在的肿瘤的缺氧的影响直接检测，利用磁共振成像（MRI），以按照与体外病理学和分子分析（ 图1）组合的体内肿瘤进展和转移。
自的ES没有建立转基因模型是目前可用的，这些肿瘤的转移特性的体内研究依赖于人的细胞注射到免疫缺陷的小鼠。而采用免疫受损的动物可能会低估对疾病进展的免疫系统的影响，用人类细胞的能力提高了这些研究的译。在不同的异种移植模型，全身注射到尾静脉是最容易执行，但他们忽略肿瘤细胞血管内的最初步骤，并从经济增长的主站点逃脱。 7-12另一方面，orthoto PIC异种移植，其中涉及肿瘤细胞注射到骨头（股骨，肋骨）或肌肉，更多的是技术上具有挑战性，也更生物学相关的人类癌症。 13-16转移发展之前然而，在过去，与原发肿瘤的快速生长相关的高发病率常常必要动物安乐死。在这项研究中，我们采用的细胞注射的先前建立模型到腓肠肌接着将得到的原发肿瘤与通过MRI纵向监测转移进展的组合的切除。 17,18这种注射成靠近胫骨腓肠肌允许在两个天然ES环境肿瘤生长-肌肉和骨骼-并导致远处转移到通常受在人类中的位置。 18因此，这种模式概括准确疾病进展过程中ES患者发生转移的进程。
帐篷“>原发性肿瘤中的下后肢的定位也有助于血液供应肿瘤组织的精确控制。股动脉结扎（FAL）是血管生成的研究用来阻止血液流向的远侧区域中的良好建立的技术腿和调查组织血管响应于缺血19,20重要的是，在血流量的初始下降之后是侧枝血管开口和组织灌注FAL后约3天观察到20因此，在荷瘤肢执行时，该模型再现自然出现缺氧/再灌注事件在迅速生长的肿瘤，使由于灌注到经由新打开的侧支血管的下后肢的恢复转移性肿瘤细胞的逃逸。21重要的是，当肿瘤大小是必须执行此程序足够小，以防止在对照肿瘤过度缺氧（通常在荷瘤小牛体积150 UME – 250 立方毫米），确保控制和FAL治疗组之间的肿瘤缺氧的水平显著的差异。除了缺氧对ES延迟的影响和转移的频率纵向监测，这种模式也允许组织的收集和从原发肿瘤和转移两个新的细胞系的开发。重要的是，以前的工作确定，转移来源的细胞系后，再引入显示出增强转移潜力动物，这表明肿瘤传播与在肿瘤细胞中的表型永久改变，并由此确认使用这些细胞系的破译转移性进程相关联。 18总的来说，这些模型现在可以用于识别缺氧诱导转移性途径所需的遗传和分子分析。
由于缺氧是一个亲转移因子增强各种T的恶性肿瘤umors，我们的模型可以作为一个平台来调查缺氧在其他类型的肿瘤，在四肢自然发展，如骨肉瘤和横纹肌肉瘤的作用。 21-23此外，类似的方法可以适用于恶性肿瘤在其它解剖学位置具有定义良好的血液供应的路线发展。最终，模型可以被修改，并且它的效用进一步延长，这取决于个体的研究需要。
Protocol
Representative Results
Discussion
我们的模型涉及转移的两个实验组的比较-对照组，其中，肿瘤被允许在后肢在达到250 立方毫米小牛体积随后截肢开发和缺氧暴露组，其中，肿瘤轴承后肢经受FAL在相同体积，随后截肢3天后。即使在这些实验中FAL治疗的肿瘤都具有稍微延迟的截肢，相比于对照肿瘤，其大小不期间结扎和截肢之间的3天期间增加。因此，这种方法能够相媲美的大小但显着不同程度的缺氧肿瘤的转移潜能的比?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Institutes of Health (NIH) grants: UL1TR000101 (previously UL1RR031975) through the Clinical and Translational Science Awards Program, 1RO1CA123211, 1R03CA178809, R01CA197964 and 1R21CA198698 to JK. MRI was performed in the Georgetown-Lombardi Comprehensive Cancer Center’s Preclinical Imaging Research Laboratory (PIRL) and tissue processing in the Georgetown-Lombardi Comprehensive Cancer Center’s Histopathology & Tissue Shared Resource, both supported by NIH/NCI grant P30-CA051008. The authors thank Dan Chalothorn and James E. Faber, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, for their assistance with postmortem x-ray angiography, and providing insight and expertise on collaterogenesis.
Materials
| SK-ES1 Human Ewing sarcoma (ES) cells | ATCC | ||
| TC71 Human ES cells | Kindly provided from Dr. Toretsky | ||
| McCoy's 5A (modified) Medium | Gibco by Life Technologies | 12330-031 | |
| RPMI-1640 | ATCC | 30-2001 | |
| PBS | Corning Cellgro | 21-040-CV | |
| FBS | Sigma-Aldrich | F2442-500mL | |
| 0.25% Trypsin-EDTA (1X) | Gibco by Life Technologies | 25200-056 | |
| Penicillin-Streptomycin | Gibco by Life Technologies | 15140-122 | |
| Fungizone® Antimycotic | Gibco by Life Technologies | 15290-018 | |
| MycoZap™ Prophylactic | Lonza | VZA-2032 | |
| Collagen Type I Rat tail high concetration | BD Biosciences | 354249 | |
| SCID/beige mice | Harlan or Charles River | 250 (Charles River) or 18602F (Harlan) | |
| 1 mL Insulin syringes with permanently attached 28G½ needle | BD | 329424 | |
| Saline (0.9% Sodium Chloride Injection, USP) | Hospira, INC | NDC 0409-7984-37 | |
| Digital calipers | World Precision Instruments, Inc | 501601 | |
| Surgical Tools | Fine Science Tools | ||
| Rimadyl (Carprofen) Injectable | Zoetis | ||
| Hypoxyprobe-1 (Pimonidazole Hydrochloride solid) | HPI, Inc | HP-100mg | |
| hypoxyprobe-2 (CCI-103F-250mg) | HPI, Inc | CCI-103F-250mg | |
| Povidone-iodine Swabstick | PDI | S41350 | |
| Sterile alcohol prep pad | Fisher HealthCare | 22-363-750 | |
| LubriFresh P.M. (eye lubricant ointment) | Major Pharaceuticals | NDC 0904-5168-38 | |
| VWR Absorbent Underpads with Waterproof Moisture Barrier | VWR | 56617-014 | |
| Oster Golden A5 Single Speed Vet Clipper with size 50 blade | Oster | 078005-050-002 (clipper), 078919-006-005 (blade) | |
| Nair Lotion with baby oil | Church & Dwight Co., Inc. | ||
| Silk 6-0 | Surgical Specialties Corp | 752B | |
| Prolene (polypropylene) suture 6-0 | Ethicon | 8680G | |
| Vicryl (Polyglactin 910) suture 4-0 | Ethicon | J386H | |
| Fisherbrand Applicators (Purified cotton) | Fisher Scientific | 23-400-115 | |
| GelFoam Absorbable Dental Sponges – Size 4 | Pfizer Pharmaceutical | 9039605 | |
| Autoclip Wound Clip Applier | BD | 427630 | |
| Stereo Microscope | Olympus | SZ61 | |
| Autoclip remover | BD | 427637 | |
| Aound clip | BD | 427631 | |
| MRI 7 Tesla | Bruker Corporation | ||
| Paravision 5.0 software | Bruker Corporation | ||
| CO2 Euthanasia system | VetEquip | ||
| 25G 5/8 Needle (for heart-puncture) | BD | 305122 | |
| 0.1 mL syringe (for heart-puncture) | Terumo | SS-01T | |
| K3 EDTA Micro tube 1.3ml | Sarstedt | 41.1395.105 | |
| 10% Neutral Buttered Formalin | Fisher Scientific | SF100-4 |
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