Zebrafish tumor xenograft models for drug-screening: a systematic review of methods for treatment assessment

Zebrafish xenotransplantation models are increasingly employed as drug-screening platforms in precision oncology, offering the advantages of high replicate numbers and rapid data acquisition. Multiple strategies have been developed to evaluate anticancer therapies in zebrafish xenografts, varying in experimental design, methodology, and analytical approaches. However, a persistent limitation is the complexity of quantifying and automating drug-response measurements, which limits scalability and increases experimental workload. We conducted a systematic review to summarize methodologies for evaluating drug efficacy in human cells xenografted into zebrafish larvae. A comprehensive search of PubMed, Scopus, and Web of Science yielded 113 eligible studies after a two-step screening process. The review focused on two major aspects: xenotransplantation procedures and methods of drug-effect quantification. The analysis describes the injection of approximately 200 tumor cells into the embryos’ yolk sac, followed by a 72-hour treatment, representing the parameters most commonly employed. Drug efficacy was most frequently assessed by measuring tumor fluorescence intensity with a fluorescence stereomicroscope. Notably, 86.75% of studies tested five or fewer conditions per experiment. In conclusion, there is a clear need to develop standardized protocols that allow testing of a larger number of treatment conditions, thereby supporting the advancement of high-throughput drug screening platforms for personalized medicine.