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Single-cell imaging of human cancer xenografts using adult immunodeficient zebrafish

Nature Protocols volume 15, pages 3105–3128 (2020)Cite this article

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Abstract

Zebrafish are an ideal cell transplantation model. They are highly fecund, optically clear and an excellent platform for preclinical drug discovery studies. Traditionally, xenotransplantation has been carried out using larval zebrafish that have not yet developed adaptive immunity. Larval engraftment is a powerful short-term transplant platform amenable to high-throughput drug screening studies, yet animals eventually reject tumors and cannot be raised at 37 °C. To address these limitations, we have recently developed adult casper-strain prkdc−/−, il2rgα−/− immunocompromised zebrafish that robustly engraft human cancer cells for in excess of 28 d. Because the adult zebrafish can be administered drugs by oral gavage or i.p. injection, our model is suitable for achieving accurate, preclinical drug dosing. Our platform also allows facile visualization of drug effects in vivo at single-cell resolution over days. Here, we describe the procedures for xenograft cell transplantation into the prkdc−/−, il2rgα−/− model, including refined husbandry protocols for optimal growth and rearing of immunosuppressed zebrafish at 37 °C; optimized intraperitoneal and periocular muscle cell transplantation; and epifluorescence and confocal imaging approaches to visualize the effects of administering clinically relevant drug dosing at single-cell resolution in vivo. After identification of adult homozygous animals, this procedure takes 35 d to complete. 7 days are required to acclimate adult fish to 37 °C, and 28 d are required for engraftment studies. Our protocol provides a comprehensive guide for using immunocompromised zebrafish for xenograft cell transplantation and credentials the model as a new preclinical drug discovery platform.

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Fig. 1: Schematic of a xenotransplantation experiment using prkdc−/−, il2rgα−/− adult zebrafish.
Fig. 2: Genotyping for prkdc and il2rgα mutations.
Fig. 3: Acclimating prkdc−/−, il2rgα−/− zebrafish for long-term housing at 37 °C.
Fig. 4: Transplantation of EGFP-expressing human RD cells into prkdc−/−, il2rgα−/− zebrafish by either i.p. or periocular transplantation.
Fig. 5: Clinically relevant dosing of drugs in prkdc−/−, il2rgα−/− zebrafish.
Fig. 6: Engraftment of EGFP-expressing RD cells into prkdc−/−, il2rgα−/− zebrafish.

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Data availability

All data generated in this study are included in this published article. The original data files are available on request from the corresponding author.

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Acknowledgements

We thank the MGH histopathology core, as well as the MGH Cancer Center Molecular Pathology Confocal Core for their technical help. We thank Drs. Qin Tang, John Moore and Jessica McCann for their advice and intellectual support for the initial development of the protocol. This work is supported by NIH grants R24OD016761 (D.M.L.), R01CA154923 (D.M.L.), R01CA215118 (D.M.L.), R01CA211734 (D.M.L.) and R01CA226926 (D.M.L.); the Liddy Shriver Sarcoma Initiative (D.M.L.); the MGH Research Scholars Program (D.M.L.); the Tosteson & Fund for Medical Discovery Fellowship from MGH (C.Y.); and the Alex’s Lemonade Stand Foundation Young Investigator Award (C.Y.).

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Author notes

  1. These authors contributed equally: Chuan Yan, Daniel Do, Qiqi Yang.

Authors and Affiliations

  1. Molecular Pathology Unit, Mass General Research Institute, Charlestown, MA, USA

    Chuan Yan, Daniel Do, Qiqi Yang, Dalton C. Brunson & David M. Langenau

  2. Mass General Cancer Center, Harvard Medical School, Charlestown, MA, USA

    Chuan Yan, Daniel Do, Qiqi Yang, Dalton C. Brunson & David M. Langenau

  3. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA

    Chuan Yan, Daniel Do, Qiqi Yang, Dalton C. Brunson & David M. Langenau

  4. Harvard Stem Cell Institute, Cambridge, MA, USA

    Chuan Yan, Daniel Do, Qiqi Yang, Dalton C. Brunson & David M. Langenau

  5. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA

    John F. Rawls

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Contributions

C.Y., Q.Y., D.D. and D.M.L. conceived, designed and conducted the study; analyzed data; and prepared the manuscript. C.Y., Q.Y., D.D. and D.C.B performed most of the experiments. J.F.R. provided important intellectual contributions and designed experiments.

Corresponding author

Correspondence to David M. Langenau.

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Competing interests

The General Hospital Corporation has a patent pending on the creation and use of immune-compromised zebrafish for engraftment of human cancers. D.M.L. is also a co-inventor on patent application number USSN 14/903,940.

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Yan, C. et al. Cell 177, 1903–1914.e14 (2019): https://www.sciencedirect.com/science/article/pii/S0092867419303903

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Yan, C., Do, D., Yang, Q. et al. Single-cell imaging of human cancer xenografts using adult immunodeficient zebrafish. Nat Protoc 15, 3105–3128 (2020). https://doi.org/10.1038/s41596-020-0372-y

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