Intravenous Injection of Cancer Cells into Chicken Chorioallantoic Membrane Vasculature: A Procedure to Establish Chorioallantoic Membrane Cancer Model to Study Cancer Invasion and Metastasis

Abstract

Source: Stoletov, K. et al. Discovery of Metastatic Regulators Using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model. J. Vis. Exp. (2021)

This video demonstrates the procedure of intravenously injecting cancer cells into the chicken chorioallantoic membrane vasculature to generate a CAM cancer metastasis model. The injected cells eventually form scattered single-cancer cell colonies, allowing for the study of cancer invasion and metastasis.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of the Cancer Cells for Injection NOTE: Metastatic colony selection is based on a phenotype established by fluorescent cancer cells, derived from an expression library or vector tagged with fluorescent protein such as green or red fluorescent protein (see the whole screen flow …

Representative Results

Figure 1: Outline of cancer cell injection and metastatic colony isolation. (A) Flowchart outlining the chicken embryo screening platform steps. (B) Cancer cell injection set up on the stereo fluorescent microscope stage. (C) Injection of the cancer cells into the CAM vasculature. (D) Image showing successful can…

Materials

1 mL disposable syringes	BD	BD309659
15 mL conical centrifuge tubes	Corning	CLS430791-500EA
18 gauge x 1 1/2  BD precision needle	BD	BD305196	We use 1.2mm x 40mm, it is possible to use shorter needles if preferred
2.5% Trypsin solution	many sources are available
4T1 mouse breast cancer	ATCC	CRL-2539
B16F10 mouse melanoma cell line	ATCC	CRL-6475
Benchtop centrifuge	Many sources are available		Any TC compatible centrifuge that can be used to spin down the cells is suitable
Circular coverslips, 22 mm	Fisher Scientific	12-545-101
Collagenase	Sigma	C0130-100MG
Confocal microscope			We use Nikon A1r
Cotton swabs	Many sources are available		Must be sterilized before use
Culture media appropriate for the cell lines used	Many sources are available		We grow HT1080, HEp3 and b16 cell lines in DMEM, 10% FBS media
HT1080 human fibrosarcoma cell line	ATCC	CCL-121
Egg incubator	Many sources are available		An exact model that is necessary depends on the scale of the screen. Available sources are MGF Company Inc., Savannah, GA, or  Lyon Electric Company Inc., Chula Vista, CA
Eppendor tubes , 1.5ml	Sigma	T4816-250EA
Fertilized White Leghorn eggs	Any local supplier
Plastic weighting dishes	Simport	CA11006-614	Dimensions are 78x78x25mm; many other sources are available
Square petri dishes (used as lids for the weighting dishes)	VWR	CA25378-115	Dimensions are 100x100x15mm; many other sources are available
Stereo fluorescent microscope			We use Zeiss Lumar v12
Sodium borosilicate glass capillary tubes, outer diameter 1.0 mm, inner diameter 0.58 mm, 10 cm length	Sutter Instrument	BF100-58-10
Tygon R-3603 laboratory tubing	Cole-Parmer	AAC00001	1/32 in inner diameter, 3/32 in. outer diameter, 1/32 in. wall thickness
Fine forceps	Many sources are available		Must be sterilized before use
Hemocytometer	Millipore-Sigma	MDH-4N1-50PK