Developing preclinical xenograft models in zebrafish.

项目摘要 异种移植细胞移植改变了我们对人类疾病的理解， 广泛评估再生，干细胞自我更新和癌症。然而，小鼠异种移植研究 昂贵且不易于以单细胞分辨率对植入进行成像。相比之下，斑马鱼 便宜，可以大量饲养，并且能够对荧光标记的细胞进行实时成像 单细胞分辨率。我们小组最近率先使用成年免疫缺陷斑马鱼， 在37 ℃下饲养时，人类癌症和血细胞的异种移植。尽管取得了这些成功， 需要做更多的工作来开发下一代免疫受损的斑马鱼， 异种移植细胞移植研究。我们工作的长期目标是开发一种通用的斑马鱼 移植模型移植广泛的人类再生和癌细胞类型。整体 目的是i）开发新的免疫缺陷斑马鱼模型，用于优化人的异种移植物植入， 癌症、胚胎和诱导多能干细胞（ES和iPSC）和血细胞，以及ii）提供许多 需要的工具、方法和细胞生物学读数，以直接评估 放射、药物和体内细胞生物免疫疗法。我们研究的基本原理是， 血液发育是高度保守的，开发斑马鱼移植模型将提供新的 在体内和单细胞分辨率下快速评估临床前治疗的工具。目标1将开发化合物 用于优化异种移植细胞移植的突变体和转基因斑马鱼。我们将开发新的模型， 缺乏T、B、NK和巨噬细胞功能以及转基因表达人细胞因子以支持 人类血液的生长。我们还将产生敲入的“无基因型rag 2/，il 2 rga −/−斑马鱼”， 在鉴定双纯合突变动物中的通量。Aim 2将测试这些模型， 移植人癌症、ES、iPSC和血细胞。这项工作很重要，因为它将提供 新的模型和实验方案来移植广泛的再生细胞类型。目标3将 动态可视化异种移植物单细胞对放射、联合药物治疗的反应， 临床前建模研究中的免疫疗法。这项工作将提供急需的细胞生物学读数， 直接评估多种疗法中单细胞分辨率的药效学反应。这些 相同的成像工具和方法可以用于许多异种移植模型-包括患者来源的 异种移植物（PDX）、ES/IPSC和血液。我们的工作意义重大，因为它将培养下一代 低成本，高通量的细胞移植模型，可以直接观察移植的细胞 临床前治疗中的行为。这项工作将产生积极的翻译影响， 开发临床前动物模型，有效地移植广泛的人体组织。如此广泛 获得免疫受损斑马鱼的应用跨越了许多NIH研究所的使命。

PROJECT SUMMARY Xenograft cell transplantation has transformed our understanding of human disease and has been used extensively to assess regeneration, stem cell self-renewal, and cancer. Yet, mouse xenograft studies are expensive and not easily amenable to imaging engraftment at single cell resolution. By contrast, zebrafish are inexpensive, can be reared in large numbers, and are capable of real-time imaging of fluorescent-labeled cells at single cell resolution. Our group has recently pioneered the use of adult immune-deficient zebrafish for xenograft transplantation of human cancers and blood cells when reared at 37OC. Despite these successes, more needs to be done to develop the next generation of immune compromised zebrafish for long-term xenograft cell transplantation studies. The long-term goal of our work is to develop a universal zebrafish transplantation model to engraft of a wide array of human regenerative and cancer cell types. The overall objective is i) to develop new immune deficient zebrafish models for optimized xenograft engraftment of human cancer, embryonic and induced-pluripotent stem cells (ES and iPSCs), and blood cells and ii) provide much needed tools, methods, and cell biological readouts to directly assess pharmacodynamic responses to radiation, drugs, and cell biological immunotherapies in vivo. The rationale for our research is that zebrafish blood development is highly conserved and that developing zebrafish transplantation models will provide new tools to rapidly assess preclinical therapies in vivo and at single cell resolution. Aim 1 will develop compound mutant and transgenic zebrafish for optimized xenograft cell transplantation. We will develop new models that lack T, B, NK, and macrophage cell function and that transgenically express human cytokines to support the growth of human blood. We will also generate knock-in “genotype-less rag2∆/∆, il2rga−/− zebrafish” to increase throughput in identifying double homozygous mutant animals. Aim 2 will test these models for enhanced engraftment of human cancers, ES, iPSCs, and blood cells. This work is important, because it will provide novel models and experimental protocols to engraft a wide array of regenerative cell types. Aim 3 will dynamically visualize xenograft single cell responses to radiation, combination drug therapies, and immunotherapy in preclinical modeling studies. This work will provide much needed cell biological readouts to directly assess pharmacodynamic responses at single cell resolution across a wide array of therapies. These same imaging tools and approaches can be used in many xenograft models – including patient-derived xenografts (PDXs), ES/IPSCs, and blood. Our work is significant because it will develop the next generation of low-cost, high throughput cell transplantation models that allow direct visualization of engrafted cell behaviors in the context of preclinical therapies. This work will have a positive translational impact by developing preclinical animal models that efficiently engraft a wide array of human tissues. Such broad reaching applications for immune compromised zebrafish spans the mission of many NIH institutes.