Intravenous Injection of Cancer Cells into Chicken Chorioallantoic Membrane Vasculature: A Procedure to Establish Chorioallantoic Membrane Cancer Model to Study Cancer Invasion and Metastasis
Published: April 30, 2023
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…
Divulgaciones
The authors have nothing to disclose.
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 |
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Intravenous Injection of Cancer Cells into Chicken Chorioallantoic Membrane Vasculature: A Procedure to Establish Chorioallantoic Membrane Cancer Model to Study Cancer Invasion and Metastasis. J. Vis. Exp. (Pending Publication), e20469, doi: (2023).