Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Summary

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

Abstract

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Introduction

Metastatic disease is a major cause of cancer mortality and many mechanisms that enable cancer cell dissemination remain to be discovered¹. In order for a cancer cell to successfully metastasize, it must first invade through the stroma that surrounds a primary tumor, enter (intravasate) into the circulatory system, survive in transit, exit (extravasate) from the circulation, and lastly establish a viable colony at the distant organ site². Intravasation and extravasation are thus crucial steps in the metastatic cascade, yet every cancer cell is not inherently adept at disrupting and migrating through endothelial junctions³. In fact, the....

Protocol

Ethics Statement: Zebrafish embryos were generated according to an approved IACUC protocol. These experiments were carried out in compliance with recommendations by the Georgetown University Animal Care and Use Committee.

1. Organize Embryos for Injection and Create Stock Solutions

1. Generate requisite zebrafish larvae to assess cancer cell vascular invasion.
   1. Set up pair-wise or group in-cross mating with Tg(kdrl:grcfp)zn1;mitfa^b692;ednrb1^b140 fish.
      NOTE: We generated Tg(kdrl:grcfp)zn1;mitfa^b692;ednrb1^b140 zebrafis....

Results

Here we tested the vascular invasive ability of commonly used breast cancer cell lines in a zebrafish embryo model (Figure 1). Rigorous criteria were employed in scoring extravasation for these different cell lines, where positive events were only counted if the cancer cells had clearly extravasated, this being done chiefly to limit any false-positives that could arise from scoring cellular debris.

Our analysis .......

Discussion

This technique utilizes the zebrafish model to efficiently test the vascular invasive ability of cancer cells (see Figure 1). Here we applied the technique to a panel of breast cancer cell lines in order to provide a baseline onto which other investigators can then build their own studies (see Table 1; Figures 2 - 3). The observation that MDA-MB-231 cells readily invaded into the caudal region of zebrafish embryos would make this cell line ideal for testing agents that m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	Life Technologies	25300-054
100 mm Dishes	Corning Incorporated	3160-100
5 3/4" Disposable Pastur Pipets, borosilicate Glass	Fisher Brand	13-678-20B
60 mm Dish	Corning Incorporated	3160-60
Agarose, Low Melting	Fisher	BP165-25
Agarose, Molecular Grade	Bioline	BIO-41026
Capillary Glass, Standard, 1.2 mm x 0.68 mm, 4"	A-M Systems, Inc	627000
David Kopf 700C Vertical Pipette Puller	Hofstra Group	3600
DMEM	Life Technologies	11995-065
Electrode Storage Jar, 1.0 mm	World Precision Instruments, Inc	E210
Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine, MS-222)	Fluka	A5040
Eyelash Brush	Ted Pella, Inc	113
Fetal Bovine Serum, Heat Inactivated	Omega Scientific	FB-12
Fisherbrand Transfer Pipettes	ThermoFisher Scientific	13-711-7M
Gel Loading Pipet Tips	Fisher Brand	02-707-181
Glass Bottom Dishes (12.0 mm)	ThermoFisher Scientific	150680
Glass Depression Slide	VWR	470005-634
Instant Ocean Salt, Sea Salt	Pentair	IS50
Latex Rubber Bulbs, 2 ml, Pack of 72	Heathrow Scientific	HS20622B
SP8 Confocal Microscope	Leica
Micromanipulator	Narishige
Eclipse E600	Nikon
PBS	Life Technologies	10010-023
Penicillin-G Potassium	Fisher Biotech	BP914-100
Petri Plates, 100 mm x 15 mm	Fisher Brand	FB0875713
Picospritzer II	General Valve Corporation
RPMI 1640 Medium	Life Technologies	11875-093
Streptomycin Sulfate	Fisher Biotech	BP910-50
Vybrant DiI	ThermoFisher Scientific	V22885
Vybrant DiO	ThermoFisher Scientific	V22886
Zebrafish	Georgetown Zebrafish Shared Resources
Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and HCC18-6 cells, which were mantained in RPMI + 10% FBS.