Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Summary

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Abstract

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Introduction

Xenografting of primary patient cancers or human cancer cell lines into model organisms is a widely used technique to study tumor progression and behavior in vivo, tumor response to drug treatment, and cancer cell interaction with the microenvironment, among others. Traditionally, cells are xenografted into immune-compromised mice, and this remains the standard in the field. However, this model system has several limitations, such as high cost, low replicate numbers, difficulties in accurately quantifying tumor burden in vivo, and the extended time that it takes for tumors to engraft and drug testing to be completed. In recent years, zebrafish have emerged as an alter....

Protocol

All procedures described in this protocol have been approved by the University of Kentucky’s Institutional Animal Care and Use Committee (protocol 2015-2225). Patient samples were collected under University of Kentucky’s Institutional Review Board (protocol 44672). All animal experiments performed following this protocol must be approved by the user’s Institutional Animal Care and Use Committee.

1. Thawing Primary Patient Acute Lymphoblastic Leukemia Cells

1. Thaw pr.......

Discussion

In this study, we demonstrated a standardized method for thawing and injection of primary patient leukemia cells into zebrafish as a xenograft model. We also established a protocol for high-throughput drug screening of xenografted zebrafish using a fluorescence microscope equipped imaging unit and automated sampler unit. Previously, xenografts have been reported with human cell lines, and quantification of xenografted tumors in a high-throughput manner has been a challenge in the field. This method serves as a basis for .......

Materials

Name	Company	Catalog Number	Comments
10x TBE Liquid Concentrate	VWR	0658-5L
96-well plate, flat bottom	CELLTREAT	229195	VAST is compatible with a variety of standard or deep well 24, 48, or 96 well plates
Agarose	Fisher Scientific	BP160-500
Borosilicate Glass Capillary without Filament	Sutter Instrument Company	B100-50-10
Dexamethasone	Enzo Life Sciences	BML-EI126-0001
DMSO	Sigma-Aldrich	D2438-5X10ML
E3 media		N/A	5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4
Femtotips Microloader Tips	Eppendorf	930001007
Fetal Bovine Serum (Premium Heat Inactivated)	Atlanta Biologicals	S11150H
ImageJ	FIJI	N/A	https://imagej.net/Fiji
Iscove's Modified Dulbecco's Medium	STEMCELL Technologies	36150
Large Particle (LP) Sampler	Union Biometrica	N/A	automated sampler unit http://www.unionbio.com/copas/features.aspx?id=8
Methotrexate	Sigma-Aldrich	A6770-10MG
Mineral Oil	Fisher Scientific	BP26291
Phosphate Buffered Saline (1x)	Caisson labs	PBL06-6X500ML
Stage Micrometer (400-Stage)	Hausser Scientific	400-S
Tricaine-S	Pentair Aquatic	TRS1
Trypan Blue	Thermo Fisher	T10282
VAST Bioimager	Union Biometrica	N/A	fluorescent equipped microscope imaging unit https://www.unionbio.com/vast/
Vincristine Sulfate	Enzo Life Sciences	BML-T117-0005
Vybrant DiI Stain	Thermo Fisher	V22885