Improving zebrafish embryo xenotransplantation conditions by increasing incubation temperature and establishing a proliferation index with ZFtool
Improving zebrafish embryo xenotransplantation conditions by increasing incubation temperature and establishing a proliferation index with ZFtool
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DOI：
10.1186/s12885-017-3919-8
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发表时间：
2018-01-02
期刊：
影响因子：
3.8
通讯作者：
Sanchez, Laura
中科院分区：
文献类型：
--
作者：
Cabezas-Sainz, Pablo;Guerra-Varela, Jorge;Sanchez, Laura
背景：斑马鱼（Danio rerio）是一种模式生物，已成为癌症研究的工具，癌症是发达国家人类仅次于心血管疾病的第二大常见死亡原因。斑马鱼是人类癌细胞异种移植和不同化疗药物在体内毒性研究的有用模型。与小鼠模型相比，斑马鱼模型速度更快，可以使用高通量方法进行筛选，并且维护成本较低，使创建个性化治疗成为可能并且负担得起。虽然基于图像采集和定量的细胞增殖测定方法已经发展起来，但仍然存在一些缺陷。在异种移植技术中，体内细胞增殖的量化对于模型未来临床前应用的标准化过程至关重要。方法：本研究改进了异种移植技术的条件，通过ZFtool软件进行图像处理，优化温度，定量测定细胞在体内的增殖情况，以规范过程，为将来的临床前应用奠定基础。开发ZFtool是为了建立消除胚胎自身荧光的基本阈值，并测量标记细胞（GFP）的面积和这些细胞的强度，以确定增殖指数。结果：肿瘤细胞在不同温度（34℃和36℃）下的增殖分析与体外细胞增殖的比较提供了更好的增殖率，在36℃下达到预期的增殖率，这是迄今为止尚未证明的维持温度。胚胎的死亡率保持在5%到15%之间。5-氟尿嘧啶测试2天，溶解于培养培养基中，以量化注射肿瘤块的减少。在36℃和5-氟尿嘧啶孵育的几乎所有胚胎中，与对照组相比，肿瘤细胞明显减少。结论：我们的研究结果表明，注射细胞在36℃时增殖更好，这个温度最适合测试5-氟尿嘧啶等化疗药物。
Background: Zebrafish (Danio rerio) is a model organism that has emerged as a tool for cancer research, cancer being the second most common cause of death after cardiovascular disease for humans in the developed world. Zebrafish is a useful model for xenotransplantation of human cancer cells and toxicity studies of different chemotherapeutic compounds in vivo. Compared to the murine model, the zebrafish model is faster, can be screened using high-throughput methods and has a lower maintenance cost, making it possible and affordable to create personalized therapies. While several methods for cell proliferation determination based on image acquisition and quantification have been developed, some drawbacks still remain. In the xenotransplantation technique, quantification of cellular proliferation in vivo is critical to standardize the process for future preclinical applications of the model.Methods: This study improved the conditions of the xenotransplantation technique - quantification of cellular proliferation in vivo was performed through image processing with our ZFtool software and optimization of temperature in order to standardize the process for a future preclinical applications. ZFtool was developed to establish a base threshold that eliminates embryo auto-fluorescence and measures the area of marked cells (GFP) and the intensity of those cells to define a proliferation index'.Results: The analysis of tumor cell proliferation at different temperatures (34 degrees C and 36 degrees C) in comparison to in vitro cell proliferation provides of a better proliferation rate, achieved as expected at 36 degrees, a maintenance temperature not demonstrated up to now. The mortality of the embryos remained between 5% and 15%. 5-Fluorouracil was tested for 2 days, dissolved in the incubation medium, in order to quantify the reduction of the tumor mass injected. In almost all of the embryos incubated at 36 degrees C and incubated with 5-Fluorouracil, there was a significant tumor cell reduction compared with the control group. This was not the case at 34 degrees C.Conclusions: Our results demonstrate that the proliferation of the injected cells is better at 36 degrees C and that this temperature is the most suitable for testing chemotherapeutic drugs like the 5-Fluorouracil.