一种简单、快速、有效的透明斑马鱼胚胎肿瘤异种移植分析方法
Summary
我们描述了一种将异种移植到透明斑马鱼胚胎卵黄中的方案，该方案通过一种简单、快速的分期方法进行了优化。注射后分析包括存活率和通过流式细胞术评估异种移植细胞的疾病负荷。
Abstract
肿瘤行为的体内研究是癌症研究的主要内容;然而，小鼠的使用在成本和时间上提出了重大挑战。在这里，我们将斑马鱼幼虫作为一种移植模型，与鼠类模型相比，该模型具有许多优点，包括易于处理、成本低和实验时间短。此外，在幼虫阶段缺乏适应性免疫系统，因此无需产生和使用免疫缺陷菌株。虽然存在斑马鱼胚胎异种移植的既定方案，但我们在这里提出了一种改进的方法，包括胚胎分期以加快移植、存活分析以及使用流式细胞术评估疾病负担。对胚胎进行分期以促进细胞快速注射到幼虫的卵黄中，并进行细胞标记以监测注射细胞团的一致性。注射后，在注射后 7 天 （dpi） 评估胚胎存活分析。最后，还通过用荧光蛋白标记转移的细胞并通过流式细胞术进行分析来评估疾病负担。流式细胞术是通过从斑马鱼胚胎制备细胞悬液的标准化方法实现的，该方法也可用于建立斑马鱼细胞的原代培养物。总之，此处描述的程序允许更快速地评估肿瘤细胞在体内的行为，每个研究组的动物数量更多，并且以更具成本效益的方式。
Introduction
分析肿瘤对体内基因改变或药物治疗的反应是癌症研究的基本要素1,2,3,4。此类研究最常涉及使用免疫功能低下的小鼠（Mus musculus）模型5;然而，小鼠异种移植研究在许多方面都存在局限性，包括容量有限、持续时间延长、费用高昂以及需要复杂的成像设备来监测内部肿瘤的进展 6,7。相比之下，斑马鱼模型（Danio rerio）可实现更大的容量、更短的持续时间、更低的费用，并且由于其透明度，可以简单地监测疾病进展8,9。
斑马鱼是一种发达的脊椎动物模型系统，具有子宫外发育和高繁殖力，单个雌性产生 100 多个胚胎10.此外，斑马鱼胚胎是透明的，可以使用荧光相关技术（如报告基因）轻松可视化发育过程。最后，关键发育过程的保守性使它们成为许多类型研究的理想模型，包括移植恶性细胞的行为11,12。野生型斑马鱼胚胎发育出黑素细胞，使它们在 2 周大时光学不透明，但这已被 casper 胚胎的产生所克服（roya9;MITFAW2），它们在整个生命周期内保持透明13.由于其光学特性，卡斯珀斑马鱼是移植肿瘤细胞的理想受体14,15,16。将肿瘤细胞异种移植到斑马鱼中在过去 2 年中变得越来越重要 17,18,19,20,21。斑马鱼胚胎具有先天免疫力;然而，它们在幼虫阶段缺乏适应性免疫，使它们在功能性免疫功能上受损，这使它们能够作为移植肿瘤异种移植物的有效宿主22。
已经制定了用于斑马鱼胚胎和成虫胚胎中肿瘤植入的方案，这些方案考虑了许多不同的变量 23,24,25,26,27。这些研究探索了斑马鱼肿瘤沉积的许多位点，包括在卵黄、卵黄周间隙和心脏以及不同发育阶段的注射16,28。斑马鱼异种移植物水产养殖的环境温度也很重要，因为斑马鱼的饲养通常在28°C下进行，而哺乳动物细胞在37°C下生长。 因此，必须采用鱼能耐受但又支持肿瘤生长的折衷温度，而 34 °C 似乎可以实现这两个目标29.异种移植后肿瘤行为和进展的分析是另一个重点领域，这涉及使用各种成像模式以及生存分析30。斑马鱼模型的主要优点之一是可以获得大量研究动物，为肿瘤行为的体内研究提供巨大的统计能力;然而，由于注射需要繁琐的安装程序，以前的方法严重限制了这种潜力。
在这里，我们通过开发一种简单、快速的方法来解决这一限制，该方法可用于分期胚胎，从而实现高通量并使用透明 的casper 斑马鱼系监测注射质量。这需要在受精后 2 天 （dpf） 将异种移植物注射到 casper 斑马鱼胚胎的卵黄囊中。作为肿瘤行为分析的一部分，我们观察异种移植后胚胎的存活情况。我们通过制作单细胞悬浮液和流式细胞术分析进一步展示了异种移植后疾病负担的评估（图1）。
Protocol
斑马鱼的维持、摄食和饲养是在28.5°C的标准水产养殖条件下进行的，如所述31。所有与斑马鱼相关的实验都是在这个温度下进行的;然而，在异种移植之后，根据机构动物护理和使用委员会（IACUC）批准的程序，在实验期间将动物在34°C下培养。 1.养殖（注射前3天） 在繁殖前一周为鱼对提供干饲料（额外饲料;每条鱼5-6个颗粒），以最大限?…
Representative Results
异种移植图1描绘了整个实验和分析的全面视图，从胚胎生产到通过流式细胞术分析生存和疾病负担分析来评估疾病进展。这种方法带来了一些改进，提高了异种移植的可重复性和可扩展性，并增加了一种评估疾病负担的新方法。这些实验的成功在很大程度上取决于移植细胞的健康状况，因为不健康且处于对数期的细胞在移植时无法繁殖。注射会话的持续时间…
Discussion
斑马鱼异种移植已成为小鼠研究的一种快速、稳健且具有成本效益的替代方案12.尽管已经报道了几种斑马鱼异种移植方法，但我们的适应已经带来了显着的改进。除了标准化程序的参数外，这些改进还特别侧重于加快肿瘤注射的速度，从而增加每个研究组的动物数量，并使用肿瘤标记来监测注射质量和注射后行为。
虽然这里描述的对这种方法的改进具有很?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院 R37AI110985 和 P30CA006927 拨款的支持，这是宾夕法尼亚联邦、白血病和淋巴瘤协会以及主教基金的拨款。这项研究还得到了 Fox Chase 核心设施的支持，包括细胞培养、流式细胞术和实验动物设施。我们感谢 Jennifer Rhodes 博士维护 FCCC 的斑马鱼和显微注射设施。
Materials
| 1-phenyl 2-thiourea (PTU) | Sigma | P7629 | |
| 70 micron cell strainer | Corning | CLS431751-50EA | |
| 90 mm Petri dish | Thermo Fisher Scientific | S43565 | |
| Agarose | Apex bioresearch | 20-102GP | |
| APC APC anti-mouse CD45.2 Antibody | Biolegend | 109814 | |
| BD FACSymphony A5 Cell Analyzer | BD Biosciences | BD FACSymphony A5 | |
| calibration capillaries | Sigma | P1424-1PAK | |
| Cell tracker CM-dil dye | Invitrogen | C7001 | |
| Collageanse IV | Gibco | 17104019 | |
| Dumont forceps number 55 | Fine science tools | 11255-20 | |
| FBS | Corning | 35-015-CV | |
| Fluorescence microscope | Nikon | model SMZ1500 | |
| Glass capillaries (Borosilicate) | World precision instruments | 1B100-4 | |
| HBSS | Corning | 21-023-CV | |
| Helix NP Blue | Biolegend | 425305 | |
| Instant Ocean Sea Salt | Instant ocean | SS15-10 | |
| Light microscope | Nikon | model SMZ1000 | |
| Methylene blue | Sigma | M9140-100G | |
| Microloader (long tips for laoding cells) | eppendorf | 930001007 | |
| P1000 micropipette puller | Sutter instruments | model P-97 | |
| PM 1000 cell microinjector | MicroData Instruments, Inc. (MDI) | PM1000 | |
| Tricaine methanesulphate (Ethyl 3- aminobenzoate methanesulphate) | Sigma | E10521-10G | |
| Trypsin-EDTA (0.5%), no phenol red | Gibco | 15400054 | |
| Zebrafish adult irradiated diet (dry feed) | Zeigler | 388763 |
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Cite This Article
Verma, M., Rhodes, M., Shinton, S., Wiest, D. L. A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos. J. Vis. Exp. (209), e66164, doi:10.3791/66164 (2024).