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.