Name: Rapid in vivo Drug Response Prediction Using Leukemia Cell Grafts in Zebrafish Embryos (Video) | JoVE Journal
Uploaded: 2025-05-23T12:40:00.000+00:00
Duration: 10 min 46 s
Description: Learn a rapid seven-day workflow for predicting drug responses in human acute lymphoblastic leukemia using zebrafish embryo xenografts. This method enables precise preclinical assessment of pediatric cancer treatments.

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5 minutes

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the Collaborative Research Center CRC1588, project number 493872418 and the Dr. Kleist Stiftung, Berlin, as well as by the Deutsche Jos&#233; Carreras Leuk&#228;mie Stiftung (R03/2016), the Berliner Krebsgesellschaft (HEFF201633KK) and the German Cancer Consortium (DKTK, Joint Funding Call 2016). We thank Julia K&#246;ppke and Mareike Wolff for their critical reading of the manuscript.

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</ol>

All authors declare no conflicts of interest.

Zebrafish embryos have become an increasingly popular xenograft model for drug screening and cancer research due to their high throughput capacity and cost-effectiveness. These xenografts hold promise as a critical pillar of translational medicine, aiding preclinical research and decision-making9,21. However, zebrafish xenograft models for human leukemia cell expansion and treatment remain underrepresented compared to the extensive body of work on solid tumor gra...

Zebrafish xenotransplantation has become a crucial in vivo model for understanding cancer pathogenesis and predicting drug responses1,2,3,4,5. Animal models remain critical for preclinical drug testing, and the zebrafish model offers significant advantages over other in vivo systems, including high throughput and cost-efficiency6,7,8. &#160;This model could also aid personalized treatment response predictions, including molecular targeted therapies and CAR-T cell therapy9,10,11,12.
BCP-ALL can particularly benefit from zebrafish xenografting, as expanding primary patient cells in culture remains challenging13. There is an undeniable need for novel treatment approaches in ALL. Despite a high remission rate of 80%-85% in children with BCP-ALL, the long-term survival rates for patients with relapsed or refractory disease range only between approximately 30%-60 %14,15,16. In such cases, drug testing using the proposed pipeline could be integrated into the clinical setting to identify the optimal patient-specific therapy14,15. This personalized approach can be crucial when dealing with multiple drug resistances, significantly reducing the treatment burden for patients by avoiding ineffective or suboptimal drugs with severe side effects.
Several features make zebrafish embryo xenografting a suitable model. The genetic similarities between humans and zebrafish - 70% genetic homology and 84% shared disease-linked genes - support gene-drug interaction studies17. Using a transgenic host embryo can thus reveal genetic predispositions affecting drug susceptibility18. Alternatively, cells with specific genetic modifications can be transplanted to evaluate whether the drug sensitivity or resistance aligns with in vitro findings. Zebrafish embryo xenografts also provide insights into the potential systemic effects of drugs. Although organ development in 2-3 days old embryos is not fully mature, the organs are correctly localized and partly share the cellular composition with their adult counterparts19.
Further advantages of this model include that only a few cancer cells are needed for engraftment, maintaining host embryos is simple, as no feeding is required within the first 5 days of life, and injection success can be rapidly assessed due to the transparency and size of the embryos. A unique feature is that only innate immunity is active at this developmental stage, facilitating efficient engraftment20. In the ZefiX protocol described here (see summary in Figure 1), immunodeficiency is further enhanced by suppressing the innate immune system during the first 4 days of life using stable Morpholino antisense oligonucleotides targeting spi1 and csf3r, which block macrophage and neutrophil differentiation21,22,23.
This protocol also differs from previous zebrafish xenotransplantation protocols, which were primarily developed for solid tumor grafts and typically use whole-mount imaging-based drug response assessment methods. ZefiX is optimized for liquid cancer cells, such as BCP-ALL cells, and has been successfully used to expand fresh or fresh-frozen patient material21. ZefiX can also be adapted for adherent cancer cells by selecting appropriate enzymes for tissue dissociation.
Another major advantage is the downstream analysis using flow cytometry, which offers several benefits: (i) a large number of graft cells can be processed quickly, allowing for robust statistical analysis at the single-cell level, (ii) proliferation rate and viability can be assessed simultaneously in individual cells, and (iii) flow cytometers are commonly available in clinical research settings, enabling drug response evaluation of graft cells on a single cell level within a few hours. To ensure reproducibility, this protocol provides a standardized pipeline from preparation through transplantation to flow cytometry analysis, allowing for drug response prediction in ALL cells within a week.

<table><tbody><tr><td>Petri dish&amp;nbsp; (10 cm)</td><td>Greiner</td><td>P7237</td><td></td></tr><tr><td>7-AAD viability staining solution&amp;nbsp;</td><td>Invitrogen</td><td>00-6993-50</td><td></td></tr><tr><td>Agarose (LE, analytic grade)</td><td>Biozym&amp;nbsp;</td><td>840004</td><td></td></tr><tr><td>Air pressure injector</td><td>Narishige</td><td>IM400</td><td>&amp;nbsp;with external gas supply</td></tr><tr><td>Alexa Fluor&amp;nbsp;488 anti-human CD19 antibody&amp;nbsp;</td><td>Biolegend</td><td>302219</td><td></td></tr><tr><td>Annexin binding buffer</td><td>Biolegend</td><td>422201</td><td>Or see solutions for preparation</td></tr><tr><td>APC annexin V&amp;nbsp;</td><td>Biolegend</td><td>640941</td><td></td></tr><tr><td>Capillaries (10 cm, OD 1.0 mm, with filaments)</td><td>WPIINC</td><td>TW100F-4</td><td>1.0 OD; 0.75 ID</td></tr><tr><td>Cell culture flask (T-175)</td><td>Sarstedt</td><td>83,39,12,002</td><td></td></tr><tr><td>CellTrace Violet&amp;nbsp;</td><td>Invitrogen</td><td>C34557</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Roth</td><td>A994.1</td><td></td></tr><tr><td>Dispase II&amp;nbsp;</td><td>Sigma Aldrich</td><td>D4693-1g</td><td></td></tr><tr><td>DNase I</td><td>AppliChem GmbH</td><td>A3778</td><td></td></tr><tr><td>Eppendorf tubes (1.5 ml)</td><td>Eppendorf</td><td>30120086</td><td></td></tr><tr><td>FACS tube (Polystyrene round botton Tube with Cell strainer Cap, 5 ml)</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>Falcon tubes (50 ml)</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Sigma Aldrich</td><td>C8056</td><td></td></tr><tr><td>Fine mesh filter&amp;nbsp;(10 &amp;micro;m)</td><td>PluriStrainer</td><td>435001050</td><td></td></tr><tr><td>Fine mesh filter&amp;nbsp;(20 &amp;micro;m)</td><td>PluriStrainer</td><td>431002040</td><td></td></tr><tr><td>Flow cytometer&amp;nbsp;</td><td>Becton Dickinson</td><td>BD LSRFortessa X-20</td><td></td></tr><tr><td>Fluorescent stereomicroscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Fluorescent stereomicroscope with camera</td><td>Leica</td><td>M165 FC</td><td>Camera: DFC7000 T</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS, Calcium and Magnesium free )</td><td>Sigma Aldrich</td><td>88284</td><td></td></tr><tr><td>Injection mold (Zebrafish MI/Transplant KIT)</td><td>World Precision Instruments&amp;nbsp;</td><td>Z-MOLDS</td><td></td></tr><tr><td>Injection needles (without filament)</td><td>Biomedical instruments</td><td>VZIPbl-20-10-55</td><td>Zebrafish injection pipette, blunt, OD: 20&amp;mu;m &amp;plusmn; 1, TL:~10mm, PL: 55mm, Glass: BM100T-10P</td></tr><tr><td>Macro-centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Micro-centrifuge</td><td></td><td></td><td></td></tr><tr><td>Morpholino (csf3r)</td><td>Gene Tools LLC</td><td>csf3r (GAAGCACAAGCGA&lt;br/&gt; GACGGATGCCA)</td><td></td></tr><tr><td>Morpholino (spi1)</td><td>Gene Tools LLC</td><td>spi1(GATATACTGATAC&lt;br/&gt; TCCATTGGTGGT)</td><td></td></tr><tr><td>Papain</td><td>Sigma Aldrich</td><td>P3125</td><td></td></tr><tr><td>Penicillin-Streptomycin (Penstrep; 10.000&amp;nbsp;U/ml)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Plates (4-well)</td><td>Greiner Bio one&amp;nbsp;</td><td>657160</td><td></td></tr><tr><td>Plates (96-well)</td><td>Greiner Bio one&amp;nbsp;</td><td>657180</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640 Medium</td><td>Gibco</td><td>21875-034</td><td></td></tr><tr><td>Tricaine&amp;nbsp; (MS-222)</td><td>Sigma Aldrich</td><td>E10521-50G</td><td>Ethy-3 aminobenzoate methanesulfenate</td></tr></tbody></table>

rapid in vivo drug response prediction using leukemia cell grafts in zebrafish embryos

Rapid in vivo 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&#160;is completed within 7&#160;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.

For a detailed scientific evaluation of the ZefiX protocol, including the xenograft and drug treatment of fresh-frozen, primary BCP-ALL cell samples, please refer to the previously published manuscript21. Approval for the use of patient samples in research for preclinical drug testing was granted as part of add-on studies to the ALL-REZ BFM 2002 trial (NCT00114348) and the ALL-REZ BFM registry and biobank (EA2/055/12) by the local medical research ethics committees, as well as to the IntReALL SR 2...

This protocol provides step-by-step instructions for generating and troubleshooting human acute lymphoblastic leukemia (ALL) xenografts from cell lines and fresh patient material in transiently immunosuppressed zebrafish embryos, along with guidelines for drug response assessment using flow cytometry. The experimental pipeline can also be adapted for solid tumors.

Zebrafish xenotransplantation is a pivotal technique for investigating human cancer pathogenesis and predicting individual drug responses. This document ...

rapid-vivo-drug-response-prediction-using-leukemia-cell-grafts

Research

JoVE Journal

Cancer Research

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A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

We used cell proliferation staining to identify quiescent cells in the zebrafish T-acute lymphoblastic leukemia model. The stain is retained in non-dividing cells and reduced during cell proliferation, enabling the selection of dormant cells for further interrogation. This protocol provides a functional tool to study self-renewal in the context of cellular quiescence.

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Learn a rapid seven-day workflow for predicting drug responses in human acute lymphoblastic leukemia using zebrafish embryo xenografts. This method enables precise preclinical assessment of pediatric cancer treatments.

Rapid in vivo Drug Response Prediction Using Leukemia Cell Grafts in Zebrafish Embryos

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