Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Summary

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

Abstract

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Introduction

Overt cancer metastasis in the clinic comprises a series of complex and multi-step events known as the "metastatic cascade". The cascade has been extensively reviewed and can be dissected into successive steps: local invasion, intravasation, dissemination, arrest, extravasation, and colonization^1,2. A better understanding of the pathogenesis of cancer metastasis and the development of potential treatment strategies in vivo require robust host models of cancer cell spread. Rodent models are well established and are widely used to evaluate metastasis³, but these approache....

Protocol

All research using the transgenic fluorescent zebrafish Tg (fli:EGFP) strain, which has enhanced green fluorescent protein (EGFP)-labeled vasculature²⁰, including housing and experiments, was carried out according to the international guidelines and was approved by the local Institutional Committee for Animal Welfare (Dier Ethische Commissie (DEC) of the Leiden University Medical Center.

NOTE: As summarized in Figure 1, the protocol is roughly broken down into four steps: embryo collection (Figure 1A), microinjection (Figure 1B), screening (Figure 1C), and analysis (Figure 1....

Results

In the embryonic xenograft zebrafish model with a perivitelline space injection, the hematogenous dissemination of labeled cancer cells in the fish body is considered as active migration. This process can be detected and quantified under a fluorescent microscope, as described in the methods above. To illustrate this xenograft model, we followed the dissemination process of different breast cancer cell lines with known (or without) invasion/metastasis potential according to in vitro

Discussion

Here, we described two methods to investigate the invasive behavior of breast cancer cells in Tg (fli1:EGFP) zebrafish embryos, with perivitelline space and Doc injections. By injecting cancer cells labeled with chemical dye or fluorescent protein into transgenic zebrafish embryos, the dynamic and spatial characteristics of invasion and metastasis can be clearly tracked in real-time at the single-cell or cluster level under a fluorescence microscope. In most cases, the rapid progression of metastasis in zebrafis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	MP Biomedicals	AGAF0500
Borosilicate glass capillary	Harvard Apparatus	300038
Cholera enterotoxin	Calbiochem	227035
Confocal microscope	Leica	SP5 STED
DMEM-high glucose media containing L-glutamine	ThermoFisher Scientific	11965092
DMEM/F-12 media containing L-glutamine	ThermoFisher Scientific	21041025
Dumont #5 forceps	Fine Science Tools Inc	11252-20
Epidermal growth factor	Merck Millipore	01-107
Fetal bovine serum	ThermoFisher Scientific	16140071
Fluorescent stereo microscope	Leica	M165 FC
HEK293T cell line	American Type Culture Collection	CRL-1573
Hydrocortisone	SigmaAldrich	227035
Horse serum	ThermoFisher Scientific	26050088
Insulin	SigmaAldrich	I-6634
MCF10A (M1) cell line			Kindly provided by Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA)
MCF10Aras (M2) cell line			Kindly provided by Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA)
MDA-MB-231 cell line	American Type Culture Collection	CRM-HTB-26
Manual micromanipulator	World Precision Instruments	M3301R
Micropipette puller	Sutter Instruments	P-97
Wide-tip Pasteur pipette (0.5-20 µL)	Eppendorf	F276456I
pCMV-VSVG plasmid			Kindly provided by Prof. Dr. Rob Hoeben (Leiden University Medical Center, Leiden, The Netherlands)
Penicillin-Streptomycin (10,000 U/mL)	ThermoFisher Scientific	15140122
PLV-mCherry plasmid	Addgene	36084
pMDLg-RRE (gag/pol) plasmid			Kindly provided by Prof. Dr. Rob Houben (Leiden University Medical Center, Leiden, The Netherlands)
Pneumatic picoPump	World Precision Instruments	SYS-PV820
Polybrene	SigmaAldrich	107689
Prism 4 software	GraphPad Software
pRSV-REV plasmid			Kindly provided by Prof. Dr. Rob Hoeben (Leiden University Medical Center, Leiden, The Netherlands)
Stereo microscope	Leica	MZ16FA
Tg (fli:EGFP) zebrafish strain			Kindly provided by Dr. Ewa Snaar-Jagalska (Institute of Biology, Leiden University, Leiden, The Netherlands)
Tris-base	SigmaAldrich	11814273001
Tricaine (3-aminobenzoic acid)	SigmaAldrich	A-5040
Trypsin-EDTA (0.5%)	ThermoFisher Scientific	15400054
Petri dishes, polystyrene (60 × 15 mm)	SigmaAldrich	P5481-500EA
Polystyrene dish with glass bottom	WillCo	GWST-5040