Rapid <em>in vivo</em> Drug Response Prediction Using Leukemia Cell Grafts in Zebrafish Embryos

Zebrafish xenotransplantation is a pivotal technique for investigating human cancer pathogenesis and predicting individual drug responses. This document introduces a streamlined protocol (ZefiX) for expanding primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patient samples or immortalized cell lines in transiently immunosuppressed zebrafish embryos, utilizing flow cytometry for high-resolution single-cell analysis of treatment responses. Compared to solid tumor engraftments, leukemia cells profit significantly from a morpholino antisense oligonucleotide-based suppression of macrophage and neutrophil differentiating factors during the assay. Flow cytometry analysis of dissociated graft cells enables precise evaluation of cell count, proliferation rate, and vitality after treatment on a per-cell basis. This approach has been validated using targeted therapeutics such as venetoclax and dasatinib, with treatment outcomes compared to clinical records of related patient samples and traditional 2D culture controls. Notably, the protocol is completed within 7 days, aligning with clinical decision-making timelines. The methodology is adaptable for testing selected drugs in various cancer types, including solid tumors, thereby supporting personalized therapeutic strategies. However, limitations on the number of drugs that can be assessed, likely due to pharmacokinetic constraints in zebrafish embryos, should be considered.