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

Summary

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.

Abstract

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.

Introduction

Zebrafish xenotransplantation has become a crucial in vivo model for understanding cancer pathogenesis and predicting drug responses^1,2,3,4,5. 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-efficiency^6,7,8.  This model could also aid personalized treatment response....

Protocol

All zebrafish experiments comply with the Charité-Universitätsmedizin Berlin Research Institutes for Experimental Medicine guidelines and official authorities. All studies involved zebrafish embryos < 6 days post fertilization (dpf), exempting them from the Protection of Animals Act. Zebrafish (Danio rerio) were raised and maintained at the animal facility of Charité-Universitätsmedizin Berlin, Berlin, Germany, according to standard protocols. They were housed at 28 °C with a 14 h light and 10 h dark cycle. Wild-type fish of AB or TüLF strains were used for all experiments.

NOTE: Establishing optim....

Results

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.......

Discussion

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-making^9,21. However, zebrafish xenograft models for human leukemia cell expansion and treatment remain underrepresented compared to the extensive body of work on solid tumor gra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Petri dish  (10 cm)	Greiner	P7237
7-AAD viability staining solution	Invitrogen	00-6993-50
Agarose (LE, analytic grade)	Biozym	840004
Air pressure injector	Narishige	IM400	with external gas supply
Alexa Fluor 488 anti-human CD19 antibody	Biolegend	302219
Annexin binding buffer	Biolegend	422201	Or see solutions for preparation
APC annexin V	Biolegend	640941
Capillaries (10 cm, OD 1.0 mm, with filaments)	WPIINC	TW100F-4	1.0 OD; 0.75 ID
Cell culture flask (T-175)	Sarstedt	83,39,12,002
CellTrace Violet	Invitrogen	C34557
Dimethyl sulphoxide (DMSO)	Roth	A994.1
Dispase II	Sigma Aldrich	D4693-1g
DNase I	AppliChem GmbH	A3778
Eppendorf tubes (1.5 ml)	Eppendorf	30120086
FACS tube (Polystyrene round botton Tube with Cell strainer Cap, 5 ml)	Falcon	352235
Falcon tubes (50 ml)	Falcon	352070
Fetal calf serum (FCS)	Sigma Aldrich	C8056
Fine mesh filter (10 µm)	PluriStrainer	435001050
Fine mesh filter (20 µm)	PluriStrainer	431002040
Flow cytometer	Becton Dickinson	BD LSRFortessa X-20
Fluorescent stereomicroscope	Leica
Fluorescent stereomicroscope with camera	Leica	M165 FC	Camera: DFC7000 T
Hank’s Balanced Salt Solution (HBSS, Calcium and Magnesium free )	Sigma Aldrich	88284
Injection mold (Zebrafish MI/Transplant KIT)	World Precision Instruments	Z-MOLDS
Injection needles (without filament)	Biomedical instruments	VZIPbl-20-10-55	Zebrafish injection pipette, blunt, OD: 20μm ± 1, TL:~10mm, PL: 55mm, Glass: BM100T-10P
Macro-centrifuge	Eppendorf
Micro-centrifuge
Morpholino (csf3r)	Gene Tools LLC	csf3r (GAAGCACAAGCGA GACGGATGCCA)
Morpholino (spi1)	Gene Tools LLC	spi1(GATATACTGATAC TCCATTGGTGGT)
Papain	Sigma Aldrich	P3125
Penicillin-Streptomycin (Penstrep; 10.000 U/ml)	Gibco	15140122
Plates (4-well)	Greiner Bio one	657160
Plates (96-well)	Greiner Bio one	657180
Roswell Park Memorial Institute (RPMI) 1640 Medium	Gibco	21875-034
Tricaine  (MS-222)	Sigma Aldrich	E10521-50G	Ethy-3 aminobenzoate methanesulfenate