Abstract PR12: Dynamic single-cell imaging of human cancer growth and therapy responses following engraftment into immunodeficient zebrafish
Authors: Yan Chuan , Yang Qiqi , Do Daniel , Brunson Dalton , Iafrate John , Rawls John , Langenau David M. 
Published: June 2020

Cancer xenograft engraftment studies using immune-deficient mice are indispensable for preclinical drug discovery and are required for IND filings that lead to clinical trials. While immune-deficient mice robustly engraft a wide variety of human cancers, high-resolution intravital imaging of transplanted cells is tedious and its high husbandry costs often limit the scale of experiments. In contrast, zebrafish are an ideal cell transplantation model. They are highly fecund; optically clear, permitting dynamic single-cell imaging of fluorescent cancer cells; and are an excellent platform for high-throughput, large-scale studies. We have recently generated two optically clear prkdc-/-, il2rga-/- and rag2-/-, il2rga-/- immune-compromised zebrafish models that lack T-, B-, and NK-cells. These immune-deficient animals can be grown at 37°C and robustly engraft a variety of human cancers including patient-derived xenografts for &gt;30 days. Importantly, tumors grown in immune-deficient zebrafish and mice are indistinguishable in terms of morphology, proliferation rates, apoptosis, and response to therapy. Engraftment of human cells into the superficial periocular muscle also allowed high-resolution, single-cell imaging using conventional confocal microscopy. Using this approach, we performed photoconversion cell lineage tracing of human rhabdomyosarcoma (RMS), a pediatric cancer of the muscle, and identified mutually exclusive migratory and proliferative cell states. We also demonstrated the preclinical efficacy of combination therapy involving olaparib (PARP-inhibitor) and temozolomide (DNA-damaging agent), including the dynamic visualization of therapeutic responses at single-cell resolution through use of the four-color FUCCI cell cycle fluorescent reporter and serial confocal imaging over days. Importantly, this same drug combination also exhibited remarkable efficacy in studies performed in NSG mice and is now moving forward for clinical evaluation in RMS patients. Finally, recent work has focused on assessing the efficacy of immunotherapies in curbing tumor growth, including assessing CAR-T cell and bispecific T-cell engager antibodies (BITES) in vivo. In both of these platforms, dynamic live cell imaging and automated 3D modeling allowed quantification of migratory potential of T cells into the tumor, dynamic remodeling of T-cell morphology changes following interaction with tumor cells, and real-time visualization of T cell-mediated cancer cell killing using fluorescent caspase reporters. In total, our studies have credentialed the immune-deficient zebrafish as a new platform for preclinical drug studies and provides novel technologies that utilize real-time, single-cell imaging for early endpoint analysis. These models are likely to be particularly useful for discovery of immunomodulatory antibodies and immunomedicines in the future.

Citation Format: Chuan Yan, Qiqi Yang, Daniel Do, Dalton Brunson, John Iafrate, John Rawls, David M. Langenau. Dynamic single-cell imaging of human cancer growth and therapy responses following engraftment into immunodeficient zebrafish [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR12.