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

Summary

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

Abstract

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

Introduction

Analysis of the behavior of tumors in response to genetic alteration or drug treatment in vivo is an essential element of cancer research^1,2,3,4. Such studies most often involve the use of immunocompromised mouse (Mus musculus) models⁵; however, xenotransplantation studies in mice are limited in many respects, including limited capacity, extended duration, significant expense, and the requirement for sophisticated imaging equipment to monitor the progression of internal tumors⁶

Protocol

Zebrafish maintenance, feeding, and husbandry occurred under standard aquaculture conditions at 28.5 °C, as described³¹. All zebrafish-related experiments were done at this temperature; however, following xenotransplantation, the animals were cultured at 34 °C for the duration of the experiment, in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC).

1. Breeding (3 days before injection)

1. P.......

Discussion

Zebrafish xenotransplantation has emerged as a rapid, robust, and cost-effective alternative to mouse studies¹². Though several approaches to zebrafish xenotransplantation have been reported, our adaptation has resulted in significant improvements. In addition to standardizing parameters around the procedure, these improvements specifically focus on accelerating the rate at which tumor injections can be performed, thus enabling an increase in the number of animals per study arm and using tumor lab.......

Materials

Name	Company	Catalog Number	Comments
1-phenyl 2-thiourea (PTU)	Sigma	P7629
70 micron cell strainer	Corning	CLS431751-50EA
90 mm Petri dish	Thermo Fisher Scientific	S43565
Agarose	Apex bioresearch	20-102GP
APC APC anti-mouse CD45.2 Antibody	Biolegend	109814
BD FACSymphony A5 Cell Analyzer	BD Biosciences	BD FACSymphony A5
calibration capillaries	Sigma	P1424-1PAK
Cell tracker CM-dil dye	Invitrogen	C7001
Collageanse IV	Gibco	17104019
Dumont forceps number 55	Fine science tools	11255-20
FBS	Corning	35-015-CV
Fluorescence microscope	Nikon	model SMZ1500
Glass capillaries (Borosilicate)	World precision instruments	1B100-4
HBSS	Corning	21-023-CV
Helix NP Blue	Biolegend	425305
Instant Ocean Sea Salt	Instant ocean	SS15-10
Light microscope	Nikon	model SMZ1000
Methylene blue	Sigma	M9140-100G
Microloader (long tips for laoding cells)	eppendorf	930001007
P1000 micropipette puller	Sutter instruments	model P-97
PM 1000 cell microinjector	MicroData Instruments, Inc. (MDI)	PM1000
Tricaine methanesulphate (Ethyl 3- aminobenzoate methanesulphate)	Sigma	E10521-10G
Trypsin-EDTA (0.5%), no phenol red	Gibco	15400054
Zebrafish adult irradiated diet (dry feed)	Zeigler	388763