在斑马鱼肿瘤异种移植物中宿主血管反应的Vivo成像和定量
Summary
这种方法的目的是通过异种移植哺乳动物肿瘤细胞进入带有荧光标记血管的斑马鱼胚胎,生成肿瘤血管生成体内模型。通过成像异种移植物和相关血管,可以得到血管原反应的定量测量。
Abstract
肿瘤血管生成是抗癌治疗的主要目标,该方法的研制为研究体内肿瘤治疗过程提供了新的模型。斑马鱼异种移植是通过将哺乳动物肿瘤细胞植入受精后两天的斑马鱼胚胎的围观空间,然后测量在实验端点观察到的血管生成反应的程度,长达两天植入后。这种方法的主要优点是能够准确量化斑马鱼宿主血管生成反应的移植物。这使得详细的检查分子机制以及宿主与肿瘤对血管生成反应的贡献。异种移植的胚胎可以进行各种治疗,如用潜在的抗血管生成药物孵育,以研究抑制肿瘤血管生成的策略。血管生成反应也可以进行实时成像,以检查更动态的细胞过程。相对不高的实验技术、廉价的斑马鱼维护成本以及较短的实验时间,使得该模型对于开发控制肿瘤血管生成的策略特别有用。
Introduction
血管生成是癌症的经典特征之一,是抗癌治疗的目标1,2。为了研究这一过程,通过将哺乳动物肿瘤细胞植入小鼠3等动物体内,建立了癌症异种移植模型。还开发了斑马鱼异种移植模型,包括将肿瘤细胞植入受精后2天(dpi)斑马鱼体内,导致斑马鱼血管快速生长到异种移植4中。
该协议描述了一个体内斑马鱼胚胎肿瘤异种移植模型,其中血管生成反应可以在整个异种移植中精确定量。这种方法允许研究者在体内检查支撑肿瘤血管生成反应的分子机制。斑马鱼的遗传可分性允许对宿主的贡献进行询问,而选择不同的肿瘤细胞系允许肿瘤对血管生成的贡献也检查5,6,7。此外,由于斑马鱼幼虫可渗透到小分子中,可以使用特定的通路抑制剂,也可以筛选药物库,以识别肿瘤血管生成8、9、10的新抑制剂。 11.
斑马鱼胚胎异种移植模型与其他哺乳动物异种移植模型相比具有独特的优势。斑马鱼异种移植更便宜,更容易执行,大量的动物可以检查和活细胞成像允许详细检查细胞行为4。不像其他体内模型,需要几个星期来观察显着的血管生长,斑马鱼异种移植的血管生成可以在植入后24小时内观察到植入3,4。然而,胚胎斑马鱼缺乏适应性免疫系统,虽然有利于维持异种移植,但意味着不能检查适应性免疫反应及其对肿瘤血管生成的贡献。此外,缺乏肿瘤基质细胞、无法正射植入肿瘤以及斑马鱼和哺乳动物细胞维持温度的差异是这种方法的潜在弱点。尽管如此,该模型对活成像的可幸存性以及准确定量血管生成反应的能力,使得它对于研究调节体内肿瘤血管生成细胞过程具有独特的益处。
Protocol
1. 显微注射针的制备 打开微移液拉拔器并设置以下参数(为材料表中列出的微移液器拉拔器型号校准):热,680;拉, 75;速度, 40;时间, 55;压力: 530. 将硼硅酸盐玻璃毛细管固定到微移液器拉拔器中,然后拉毛细管,制成两根针。根据需要重复多针。 2. 用于植入的细胞培养 注:当使用该协议时,任何哺乳动物的癌细胞系都可用…
Representative Results
通过在 6、24 和 48 hpi 处成像单个异种移植物,可以计算出不同时间点的血管生成反应,如图1A-C所示。最大的血管生成反应是在植入后24-48小时之间观察到的,移植血管的最大水平在2 dpi左右(图1A-C)。在补充电影1中可以看到一个典型的血管生成反应B16-F1异种移植从20.75 hpi到46 hpi的延时电影。这部…
Discussion
该协议的第一个关键步骤是肿瘤细胞的植入。至关重要的是,细胞被注射到一个位置,将允许异种移植成功植入胚胎,而不使胚胎水肿。过于前导的注射可以让细胞向心脏移动,阻塞血液并导致水肿,而太后注射将导致植入不良的异种移植。最好避免前注射,将针头插入蛋黄囊,使其在注射时指向远离心脏。最好通过在蛋黄囊圆部分的前半部分注射细胞来避免后注射。此外,细胞必须注射到蛋黄囊的腹腔,而?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Alhad Mahagaonkar先生管理奥克兰大学斑马鱼设施和奥克兰大学医学院生物医学成像研究股,在延时共聚焦显微镜方面提供协助。这项工作得到了新西兰卫生研究理事会项目赠款(14/105)、新西兰皇家学会马斯登基金项目赠款(UOA1602)和奥克兰医学研究基金会项目赠款(1116012)给予J.W.A.的支持。
Materials
| Air cylinder | BOC | 011G | Xenotransplantation |
| B16-F1 cells | ATCC | Cell culture | |
| BD Matrigel LDEV-free (extracellular matrix mixture) | Corning | 356235 | Xenotransplantation |
| Borosillicate glass capillaries | Warner Instruments | G100T-4 | OD=1.00 mm, ID=0.78 mm, Length =10 cm Cell injection |
| Cell culture dish -35 mm diameter | Thermofisher NZ | NUN153066 | Fish husbandry |
| Cell culture dish -100 mm diameter | Sigma-Aldrich | CLS430167-500EA | Fish husbandry |
| Cell culture flask 75 cm2 | In Vitro Technologies | COR430641 | Cell culture |
| CellTracker Green | Invitrogen | C2925 | Cell labelling, Stock concentration (10 mM in DMSO), working concentration (0.2 μM in serum-free media) |
| Dimethyl sulfoxide | Sigma-Aldrich | D8418 | Drug treatment, Cell labelling |
| E3 Media (60x in 2 L of water) 34.8 g NaCl 1.6 g KCl 5.8 g CaCl2·2H2O 9.78 g MgCl2·6H2O adjust to pH 7.2 with NaOH |
In house [1] | Fish husbandry | |
| Ethyl-3-aminobenzoate methanesulfonate (Tricaine) | Sigma-Aldrich | E10521 | Xenotransplantation, Imaging |
| Filter tip 1000 μL | VWR | 732-1491 | Used during multiple steps |
| Filter tip 200 μL | VWR | 732-1489 | Used during multiple steps |
| Filter tip 10 μL | VWR | 732-1487 | Used during multiple steps |
| Fluorescence microscope | Leica | MZ16FA | Preparation of embryos |
| FBS (NZ origin) | Thermofisher Scientific | 10091148 | Cell culture |
| Gloves | Any commercial brand | Used during multiple steps | |
| Haemocytometer cell counting chamber Improved Neubauer | HawksleyVet | AC1000 | Xenotransplantation |
| Heraeus Multifuge X3R Centrifuge | Thermofisher Scientific | 75004500 | Cell culture, Cell labelling |
| Hoechst 33342 | Thermofisher Scientific | 62249 | Cell labelling, Stock concentration (1 mg/ml in DMSO), working concentration (6 μg/ml in serum-free media) |
| Low Melting Point, UltraPure Agarose | Thermofisher Scientific | 16520050 | Imaging |
| Methycellulose | Sigma-Aldrich | 9004 67 5 | Xenotransplantation |
| Methylene blue | sigma-Aldrich | M9140 | Fish husbandry |
| Microloader 0.5-20 μL pipette tip for loading microcapillaries | Eppendorf | 5242956003 | Xenotransplantation |
| Micropipettes | Any commercial brand | Used during multiple steps | |
| Micropipette puller P 87 | Sutter Instruments | Xenotransplantation | |
| Microscope cage incubator | Okolab | Time-lapse imaging | |
| Microwave | Any commercial brand | Imaging | |
| Mineral oil | Sigma-Aldrich | M3516 | Xenotransplantation |
| Minimal Essential Media (MEM) – alpha | Thermofisher Scientfic | 12561056 | Cell Culture |
| MPPI-2 Pressure Injector | Applied Scientific Instrumentation | Xenotransplantation | |
| Narishige micromanipulator | Narishige Group | Xenotransplantation | |
| Nikon D Eclipse C1 Confocal Microscope | Nikon | Imaging | |
| N-Phenylthiourea (PTU) | Sigma-Aldrich | P7629 | Fish husbandry |
| PBS | Gibco | 10010023 | Cell culture |
| Penicillin Streptomycin | Life Technologies | 15140122 | Cell culture |
| S1 pipet filler | Thermoscientific | 9501 | Cell culture |
| Serological stripette 10 mL | Corning | 4488 | Cell culture |
| Serological stripette 25 mL | Corning | 4489 | Cell culture |
| Serological stripette 5 mL | Corning | 4485 | Cell culture |
| Serological stripette 2 mL | Corning | 4486 | Cell culture |
| Terumo Needle 22 gauge | Amtech | SH 182 | Fish husbandry |
| Tissue culture incubator | Thermofisher Scientfic | HeraCell 150i | Cell culture |
| Tivozanib (AV951) | AVEO Pharmaceuticals | Drug treatment | |
| Transfer pipette 3 mL | Mediray | RL200C | Fish husbandry |
| Trypsin/EDTA (0.25% ) | Life Technologies | T4049 | Cell culture |
| Tweezers | Fine Science Tools | 11295-10 | Fish husbandry |
| Volocity Software (v6.3) | Improvision/Perkin Elmer | Image analysis |
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Cite This Article
Britto, D. D., Hall, C. J., Astin, J. W. In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts. J. Vis. Exp. (150), e59849, doi:10.3791/59849 (2019).