Source: Stoletov, K. et al. Discovery of Metastatic Regulators Using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model. J. Vis. Exp. (2021)
In this video, we describe the isolation of metastatic cancer cell colonies from the chicken chorioallantoic membrane (CAM) using shell-less egg culture. This method helps screen for factors that may be responsible for cancer metastasis.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Isolation of Metastatic Colonies
Figure 1. Representative results for different steps of the screening protocol. (A) Metastatic colonies formed by heterogenous (library transduced cancer cell line HEp3 cells), 5 days post injection. Red arrow shows compact colony (potential positive hit) that should be excised. (B) Metastatic colonies formed by one of the isolated screen hits (KIF3B) after reinjection, 5 days post injection. Insets show digitally cut out metastatic colony images from the dashed squares. This is acceptable image quality for C.I quantification. Average C.I. value for hit reinjection (KIF3B) is shown. (C) Isolation of the metastatic colony of interest from the CAM tissue. Representative optical sections of (D) a control colony, and (E) a KIF3B shRNA overexpressing colony, both showing cancer cell-blood vessel contact measurements. CAM vasculature is labeled with fluorescent lectin-649. Scale bars = 1 cm (A–C) or 50 µm (D, E)
Figure 2: 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 cancer cell injection (acceptable cancer cell density), taken immediately after injection. (E) Image showing poor cancer cell injection, seen as an over-injected embryo. Note cancer cell build up in the blood capillaries (white arrows). Scale bars = 1 cm.
The authors have nothing to disclose.
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 |
Benchtop centrifuge. | many sources are available | Any TC compatible centrifuge that can be used to spin down the cells is suitable |
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Collagenase | Sigma | C0130-100MG | |
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 |
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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 |
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eppendor tubes , 1.5ml | Sigma | T4816-250EA | |
Fertilized White Leghorn eggs | any local supplyer | ||
fine forceps | many sources are available | must be sterilized begfore use | |
Image analysis software | We use Nikon Elements | ||
PBS (1x) | many sources are available | ||
Plastic weighting dishes | Simport | CA11006-614 | dimensions are 78x78x25mm; many other sources are available |
small surgical scissors | many sources are available | must be sterilized before use | |
Stereo fluorescent microscope | We use Zeiss Lumar v12 |