Imaging Normal and Malignant Cell Engraftment at Single Cell Resolution Using Optically Clear Immune Compromised Zebrafish

Abstract

Cell transplantation and engraftment into immunodeficient mice has revolutionized our understanding of regenerative biology and cancer. Research conducted with these models has identified stem and progenitor cells in multiple tissue types and organs, and facilitated key discoveries in cancer biology. Despite the great utility of mice for cell transplantation studies, the high cost of these animals limits their applicability in high-throughput in vivo studies. Moreover, direct imaging of cellular processes at high resolution is challenging with furred rodents, requiring surgical construction of imaging windows to gain optical access to internal organs and use of complex intravital microscopy. In the past decade, the zebrafish has emerged as a promising model for transplantation studies. Their high evolution conservation with human, high fecundity and whole-body transparency are ideal attributes for large-scale transplantation studies. In a quest to develop immune compromised zebrafish models for optimized cell transplantation, we used multiple genome editing methods to create mutations in genes that regulate T, B and Natural Killer cell functions, including rag2 (recombination activating gene 2), prkdc (protein kinase, DNA-activated, catalytic polypeptide), and jak3 (Janus kinase 3), leading to variable defects in innate and adaptive immunities which largely recapitulates human pathology. Capitalizing on the optical transparency of these mutant lines created in the casper background, we also developed methods to perform allograft transplantation of zebrafish tumor cells into immunodeficient recipients, and directly visualized dynamic restructuring of tumor-associated neovascularization, emergence of tumor cell heterogeneity, micro-metastatic melanoma dissemination, and competition between genetically-defined leukemia clones. From these studies, we discovered that clonal dominance could emerge stochastically in T-cell acute lymphoblastic leukemia as a result of dynamic competition between clones, lending new cell biological insights into how human leukemia acquire elevated growth potential and dominate at relapse. In summary, my work has developed immune deficient zebrafish as a powerful new tool to dissect the behavior of engrafted tumor cells at single cell resolution in live animals. Our long-term goal is to develop zebrafish models that allow robust engraftment of xenograft transplantation of human cells to facilitate large-scale drug screening with patient-derived xenografts (PDX) at affordable costs.