Zebrafish patient avatars in cancer biology and precision cancer therapy

Abstract

In precision oncology, two major strategies are being pursued for predicting clinically relevant tumour behaviours, such as treatment response and emergence of drug resistance: inference based on genomic, transcriptomic, epigenomic and/or proteomic analysis of patient samples, and phenotypic assays in personalized cancer avatars. The latter approach has historically relied on in vivo mouse xenografts and in vitro organoids or 2D cell cultures. Recent progress in rapid combinatorial genetic modelling, the development of a genetically immunocompromised strain for xenotransplantation of human patient samples in adult zebrafish and the first clinical trial using xenotransplantation in zebrafish larvae for phenotypic testing of drug response bring this tiny vertebrate to the forefront of the precision medicine arena. In this Review, we discuss advances in transgenic and transplantation-based zebrafish cancer avatars, and how these models compare with and complement mouse xenografts and human organoids. We also outline the unique opportunities that these different models present for prediction studies and current challenges they face for future clinical deployment.

Fig. 1 |. Timeline of key developments in zebrafish cancer avatars.

Timeline highlighting key milestones in the development of zebrafish cancer models towards personalized patient avatars (beige boxes). Salient milestones in clinical translation of cancer therapy response prediction using mouse, organoid and zebrafish patient avatars (purple boxes). PDX, patient-derived xenograft; zPDX, zebrafish patient-derived xenograft.

Fig. 2 |. Generation of zebrafish avatars.

a | Mosaic transgenic zebrafish can be created using embryo microinjection or electroporation in adults. In embryos (top panel), Tol2 mRNA is co-injected with plasmid DNA vector(s) containing Tol2 sites, which are recognized by Tol2 protein and inserted in the host cell genome. For transgene electroporation in adult zebrafish (bottom panel), gene delivery is accomplished by co-injection of a DNA plasmid expressing Tol2 with transgenesis vector(s) before electroporation. Between Tol2 sites, transgenesis vectors typically include a (1) tissue-specific promoter driving a coding sequence, often a human oncogene^,,,, or Cas9 for CRISPR knockout of tumour suppressors^,, and (2) a transgenesis marker. The marker is often composed of a tissue-specific promoter and a fluorescent protein (for example, heart marker cmlc2:EGFP) or, in the case of melanoma, an mitfa minigene (promoter plus coding sequence to rescue the loss of mitfa), which allows melanocyte development itself to be used as a transgenesis marker in F0 zebrafish^,. b | Zebrafish patient-derived xenografts can be created by transplantation of cancer cells (which are usually labelled) in 48-hour post-fertilization larvae raised at 34 °C or genetically immunocompromised adult zebrafish (prkdc^−/−;il2rga^−/−) raised at 37 °C. Zebrafish embryos and adults are kept at 26–28.5 °C, while human cells grow at 37 °C. To support human cell engraftment, embryos are raised at 34 °C (ref.), which, while below the normal physiological temperature for human cells, is the upper limit tolerated by zebrafish embryos and larvae. Adult fish can tolerate living at 37 °C for months if the temperature is slowly raised from 28.5 to 37 °C by about 0.5–1 °C per day.

Fig. 3 |. Drug administration in zebrafish.

a | Submersion in a water and drug solution is used to treat larval zebrafish patient-derived xenografts (zPDXs) (10–20 24-hour post-fertilization embryos per well in a 24-well plate or one embryo per 96-well plate) or adult avatars (two adults in a 10-cm Petri dish). Example compounds shown were used by Fior et al. (5-fluorouracil treatment of larvae) and Ablain et al. (treatment of adults with the BRAF inhibitor dabrafenib). b | Oral gavage can also be used to treat adult avatars (mosaic transgenic zebrafish or zPDXs). Zebrafish are anaesthetized and immobilized using MS-222 alone or in combination with isoflurane. Oral gavage using a syringe with flexible tubing allows ~3 μl of drug solution to be dispensed (described by Dang et al.). c | Intraperitoneal injection is another alternative to treat adult avatars (mosaic transgenic zebrafish or zPDXs). Zebrafish are anaesthetized and immobilized using MS-222 or MS-222 and isoflurane. Free-hand injections are performed with 10-μl capacity syringes to deliver up to 5 μl of drug solution. Approximately 3–5 μl is the tolerable range reported^,.