Nuclear Dynamics and Cancer Program, Fox Chase Cancer Center;
Nuclear Dynamics and Cancer Program, Fox Chase Cancer Center.
J Vis Exp. 2024 Jul 12(209). doi: 10.3791/66164.
In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.
体内肿瘤行为研究是癌症研究的重要组成部分；然而，使用小鼠在成本和时间上存在重大挑战。在这里，我们提出了幼鱼斑马鱼作为一种移植模型，它具有许多优于小鼠模型的优势，包括易于处理、成本低和实验周期短。此外，幼体阶段缺乏适应性免疫系统，无需生成和使用免疫缺陷品系。虽然已经存在斑马鱼胚胎异种移植的既定方案，但我们在此提出了一种改进的方法，涉及胚胎分期，以实现更快的转移、生存分析以及使用流式细胞术评估疾病负担。胚胎分期有助于快速将细胞注射到幼鱼的卵黄中，并进行细胞标记，以监测注射细胞团的一致性。注射后，评估胚胎的生存情况，直至注射后 7 天（dpi）。最后，还可以通过用荧光蛋白标记转移细胞并通过流式细胞术进行分析来评估疾病负担。流式细胞术通过一种标准化的方法从斑马鱼胚胎中制备细胞悬浮液来实现，该方法也可用于建立斑马鱼细胞的原代培养。总之，这里描述的程序允许更快速地评估肿瘤细胞在体内的行为，每个研究臂中使用更多的动物，并且成本效益更高。
