Cancer Modeling in Zebrafish: Tools for Oncology Drug Development

It’s been a long time since zebrafish was first used in cancer research back in 1965. Over the past six decades, the field has evolved dramatically, from inducing tumors with carcinogens to now being able to predict how a cancer patient might respond to chemotherapy using zebrafish cancer models. Zebrafish offers a unique combination of biological relevance, imaging accessibility, and speed in oncology drug development.

Why Zebrafish Are an Effective Model for Oncology Drug Development

Several features make zebrafish particularly effective for oncology drug development. First, its genetic homology to humans is remarkable, with around 80% of human disease-related genes having at least one zebrafish ortholog, with the conservation of even the epigenetic marks. Moreover, its small size, optical transparency during embryonic stages, and rapid development allow for high-resolution, real-time imaging of tumor growth and vascular interactions, which are critical for understanding the mechanisms of cancer invasion and metastasis.

One of the biggest advantages of using zebrafish for cancer research is the lack of an adaptive immune system in the first 30 days post-fertilization (dpf). This allows the implantation of living human tumor cells into a zebrafish embryo without rejection, a technique known as xenograft transplantation. Human cancer cells engraft, grow, disseminate, and respond to drugs in a living organism. Although its preferred temperature is 28°C, zebrafish larvae can survive up to 36°C, which is very close to human cell culture conditions. Compared to mammals, the number of cancer cells required for a xenotransplant is much lower, which represents an advantage when working with patient cells.

Figure 1. Summary of the advantages of Danio rerio as an animal model in research. Adapted from: Fontana, C.M., Van Doan, H. Zebrafish xenograft as a tool for the study of colorectal cancer: a review. (2024). Cell Death Dis. 15:23.

Another important advantage of using zebrafish as cancer models is the simplicity of generating transgenic and mutant models. This sums up to its high fecundity and low maintenance cost, further supporting its use in high-throughput drug screening platforms. Zebrafish is compatible with Petri dishes or 96-well plates, which only require microliter volumes of media, reducing the required amount of tested chemicals. These advantages were exemplified in the work we published a few years back, describing the development and validation of an improved xenograft method: ZeOncoTest (Cornet et al., 2020).

Cancer Modeling in Zebrafish: In Vivo Applications and Limitations

Zebrafish are used in two main types of cancer modeling: genetically engineered models and xenograft models.

Genetically engineered models

In genetic models, transgenic techniques are used to overexpress oncogenes or silence tumor suppressors, which can drive spontaneous tumor formation. These models are handy for studying tumor initiation, genetic interactions, and long-term disease progression. Among the available tools, Tol2-mediated integration and transgene electroporation in adult zebrafish (TEAZ) have been used for spatial and temporal control of oncogene expression. Morpholino antisense oligonucleotides, synthetic transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 can also be used to generate inheritable frameshift knockout mutations.  CRISPR/Cas9 has become the method of choice for genetic manipulation due to its precision and efficiency. It allows for the targeted editing of cancer-related genes to replicate specific mutations found in human tumors, enabling stable knockouts, knock-ins, and even fine-tuned base editing.

Figure 2. Pipeline for the generation of stable F1 and F2 KO using the CRISPR/Cas9 technique.

→ It may interest you: CRISPR/Cas9 vs. Tol2 Techniques for Zebrafish Genomic Manipulation

Zebrafish xenograft models 

Zebrafish xenografts, particularly patient-derived xenografts (PDXs) or zebrafish avatars, offer a more translational approach. In these models, fluorescently labeled human tumor cells are injected into zebrafish. It enables the monitoring of tumor behavior and the evaluation of treatment responses within a few days. Patient-derived xenografts (PDX) can be established using either 2-4 dpf larvae or juvenile zebrafish. Using juveniles offers the advantage of fully developed tissues, which better mimic the human physiological context. However, this approach requires immunosuppression and involves more complex imaging techniques due to the loss of tissue transparency at this stage.

Zebrafish models are not without limitations. Tested compounds are commonly administered via water immersion, which poses challenges for poorly water-soluble molecules. This method results in systemic exposure, differing significantly from the targeted routes of administration used in mammalian systems. Furthermore, the actual internal dose absorbed by the organism remains difficult to quantify with precision under these conditions.

Figure 3. Gastric cancer cells survived and induced angiogenesis in larval zebrafish (fli-eGFP). Typical images of subintestinal vessels of an uninjected embryo at 3 dpf. AGS cells (b) and SGC-7901 cells (c) were injected into the zebrafish embryos, and induced angiogenesis at 1 dpi. The white boxes in the lower right corner showed the higher magnification of the upper left white boxes. The arrow indicated the tumor cell-induced angiogenesis. Hpf: hours post fertilization; dpi: days post injection. Adapted from: Wu, JQ et al. Patient-derived xenograft in zebrafish embryos: a new platform for translational research in gastric cancer. (2017). J Exp Clin Cancer Res. 36:160. 

In Vivo Monitoring of Tumor Growth and Metastasis in Zebrafish

Zebrafish embryos provide an ideal platform for real-time, non-invasive imaging of tumor dynamics. Upon xenotransplantation, cancer cells can be monitored at single-cell resolution using confocal microscopy or light-sheet imaging. Angiographic zebrafish models with GFP-labeled blood vessels, such as Tg(fli:eGFP) zebrafish line, allow for the in vivo study of the interaction between endothelial and cancer cells. 

We can observe processes like angiogenesis, tumor invasion, and micrometastasis to distant organs such as the gills, eyes, or caudal hematopoietic tissue within just a few days post-injection.

Moreover, these models are highly informative for studying the metastatic potential of cancer subtypes. For instance, triple-negative breast cancer (TNBC) cell lines such as Hs578T and MDA-MB-468 have been shown to produce micrometastases in zebrafish embryos, mimicking the clinical aggressiveness of these subtypes (Mendes et al., 2025). The models also allow for the visualization of tumor-stroma interactions and the tumor microenvironment, particularly through the use of transgenic zebrafish lines expressing fluorescent markers in endothelial cells, macrophages, or fibroblasts.

Drug Screening in Zebrafish Models for Anticancer Therapy Development

Zebrafish models are instrumental in in vivo drug screening for anticancer therapies and personalized medicine. Their capacity for high-throughput screening enables the rapid testing of numerous compounds with minimal material requirements. In the case of PDXs, anticancer drug efficacy can be assessed by measuring cell apoptosis (e.g., activated caspase-3) after treatment. This approach has demonstrated the ability to distinguish between patient-specific responses to different chemotherapy regimens, including distinctions between drugs of the same family, such as doxorubicin versus epirubicin or paclitaxel versus docetaxel.

In a recent study, researchers microinjected patient-derived breast cancer tumor cells into zebrafish embryos and treated them with the same chemotherapy regimens the patients were receiving. Remarkably, apoptosis measurements in the zebrafish avatars matched the patient’s response to therapy in all 18 cases, highlighting the potential of this platform as a predictive tool in clinical oncology (Mendes et al., 2025). This not only accelerates therapeutic decision-making but also reduces the exposure of patients to ineffective treatments. 

This work, together with colorectal and ovarian cancer studies with similar results, has led to a unique clinical trial. In January 2025, a 5-year randomized clinical study kicked off to elucidate whether zebrafish could help predict treatment outcome. In the study, half of the patients will receive medications suggested by the zebrafish results, and the other half will receive the treatment picked by their doctors without any fish input.

Conclusion

It’s been 60 years since the first experiment using zebrafish to study cancer development, an early step that has since evolved into a powerful platform for cancer research. The growing use of zebrafish in oncology research reflects a deeper shift in how we understand and approach cancer: not just as a static disease of tissues, but as a dynamic process unfolding in time and space, influenced by genetic, cellular, and environmental factors. Zebrafish offers a window into that complexity, allowing us to see cancer as a whole in a living organism. As technological advances enhance the zebrafish toolbox and translational validation grows, their role in shaping the future of cancer therapeutics is only set to expand.

References

Astell KR, Sieger D. Zebrafish In Vivo Models of Cancer and Metastasis. (2020). Cold Spring Harb Perspect Med. 10(8):a037077. doi: 10.1101/cshperspect.a037077.

Cornet C, Dyballa S, Terriente J, Di Giacomo V. ZeOncoTest: Refining and Automating the Zebrafish Xenograft Model for Drug Discovery in Cancer. Pharmaceuticals (Basel). 2019 Dec 24;13(1):1. doi: 10.3390/ph13010001.

Fontana, C.M., Van Doan, H. Zebrafish xenograft as a tool for the study of colorectal cancer: a review. (2024). Cell Death Dis 15:23. doi.org/10.1038/s41419-023-06291-0

Leslie M. Fish could personalize cancer treatments. (2025). Science. 387(6730):122-123. doi: 10.1126/science.adv7960.

Mendes, R.V., Ribeiro, J.M., Gouveia, H. et al. Zebrafish Avatar testing preclinical study predicts chemotherapy response in breast cancer. (2025). npj Precis. Onc. 9:94. doi.org/10.1038/s41698-025-00882-0

Wu, JQ., Zhai, J., Li, CY. et al. Patient-derived xenograft in zebrafish embryos: a new platform for translational research in gastric cancer. (2017). J Exp Clin Cancer Res. 36:160 . doi:10.1186/s13046-017-0631-0

By Miriam Martínez

Miriam is a Human Biologist with a strong background in neuropharmacology and a passion for bridging science and innovation. After earning a master’s degree in the Pharmaceutical and Biotech Industry, she completed her PhD in Biomedicine at Pompeu Fabra University (Barcelona), where her research focused on the behavioral analysis of animal models for neurophenotypical characterization. Following her doctoral studies, Miriam transitioned into the healthcare marketing and communication sector, where she played a key role in developing impactful marketing strategies and educational campaigns for leading pharmaceutical brands. She now leverages her scientific expertise, strategic thinking, and creative communication skills in her current role at ZeClinics.