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

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 in Figure 1A). It is not recommended to use cells transiently transfected with fluorescent proteins or labeled with cell permeable fluorescent dyes due to rapid fading of signal caused by cancer cell proliferation and uneven staining.

1. Culture cancer cells to 60–70% confluency at the time of injection. Using cancer cells at higher levels of confluency will decrease their viability, resulting in low efficiency of metastatic colony formation. Ensure that the cancer cells used are mycoplasma-free since mycoplasma contamination may lead to decreased chicken embryo viability.
2. Rinse cells twice with 1x PBS, pH 7.4. Remove the remaining PBS, then add 0.5% Trypsin-EDTA and incubate at 37 °C for 2–5 min until all cells are lifted from the culture dish surface.
3. Transfer the cell suspension into a 15 mL conical tube and centrifuge cells at room temperature at 200 x g for 5 min.
4. Resuspend cells in 10 mL of PBS to and centrifuge the cells again as in step 1.3, to remove trypsin and other culture media components such as antibiotics.
   NOTE: The presence of trypsin or cell culture components such as antibiotics in the cancer cell suspension may result in decreased chicken embryo viability.
5. Carefully aspirate the supernatant and resuspend cells with 1 mL of ice-cold PBS.
6. Count the number of cells using a hemocytometer or any other cell counting equipment available.
   NOTE: This screening protocol requires that each metastatic colony be initiated by a single cell. When counting the cells make sure that the cells are fully trypsinized and exist as a single cell suspension (no cell clumps are present).
7. For intravenous (IV) injection, concentrate cells to 0.5 x 10⁶ – 1.0 x 10⁶ cells/mL. Use ice-cold 1x PBS to dilute and/or resuspend cell concentrates. Expect that approximately 1 mL of cell suspension will be needed for every ten embryos.
   NOTE: Necessary cell suspension concentration in suspension is mainly determined by the level of experimenter experience. Please see the next section for more details.

2. Intravenous Injection of Cancer Cells for Metastatic Colony Formation

NOTE: Following this protocol as many as hundred embryos can be injected within one day. It generally takes longer time to isolate the metastatic colonies than to inject the cancer cells and, therefore, it is recommended to perform an initial experiment to estimate the time needed to complete all the steps. Using differential cancer cell fluorescent labels helps to reduce animal numbers and the time required for each experiment. For example, control cells can be labeled with RFP (red fluorescent protein) while mutant tumor cells can be alternatively labeled with GFP (green fluorescent protein). In this case, each experiment has a built-in control that corrects for inter-embryo variability.

1. Assemble the injection apparatus as shown in Figure 1B. First, mount a needle onto the syringe and then extend the syringe needle with a 3–5 cm long piece of tubing.
2. Break the tip of the borosilicate needle off using the fine forceps (~20–60 µL wide).
3. Load the syringe with the cancer cell suspension (50–200 µL) and insert the borosilicate needle into the tubing (Figure 1B).
4. Inspect the borosilicate needle for cell clogging and air bubbles. It is critical to inject cells as a single cell suspension to ensure that each metastatic colony is initiated by a single cell.
   NOTE: Cancer cells tend to aggregate in PBS within 1–2 h post trypsinization and, therefore, should be prepared immediately before injection. If necessary, cells can be resuspended periodically using the 1 mL syringe used for injections (without the borosilicate needle).
5. If cell clogging is observed within the capillary, remove the capillary, resuspend the cells, and replace it with a new capillary. Use the plunger to push out any bubbles. If no cell clogging or bubbles are observed, proceed to the next step.
6. Remove the cover lid and transfer the embryo under the stereoscope. Identify the appropriate vein to be injected on the CAM surface. Veins and arteries form the intricate network within the CAM tissue (Figure 1C). Veins are distinguished by the brighter red color because they carry oxygen-rich blood to the embryo.
   NOTE: For general embryo manipulation, cancer cell injection and metastatic colony excision, use a fluorescent stereomicroscope equipped with 0.8x and 1.5x objectives and 10x eyepieces for visualization. Less advanced microscopes can also be successfully used for these procedures, depending on the users' experience level. Readers can contact the authors for more detailed recommendations.
7. Find the ideal vein to inject cells that is normally only slightly wider (10–20%) than the diameter of borosilicate needle tip and located midway between the embryo and the weighing dish wall. It is generally easier to inject at the point immediately adjacent to the vein bifurcation.
   NOTE: Injection into a larger vein may appear easier but will lead to excessive bleeding and decreased embryo survival.
8. Press the tip of the needle against the blood vessel wall and apply gentle pressure in the same direction as the blood flow. If necessary, use a cotton swab (held in other hand) to help anchor or stabilize the vessel that is being injected.
9. Gently depress the syringe plunger. One can visualize a successful injection by observing a "clearing" of the blood vessel when the cancer cell suspension enters the bloodstream.
10. Continue depressing the syringe plunger for 2–10 s until the desired suspension volume is injected into the bloodstream of the vein. All together the injection of a single embryo may require 1–10 min depending on the experience level of the user. If excessive bleeding or clear liquid accumulation appears at the injection site, discard the embryo.
    NOTE: It is easy to "over-inject" an embryo. The general consideration that should be kept in mind is that metastatic colonies should not touch each other after a 4–5 days growth period. Ideally, ~5–10% of terminal CAM vein capillaries should have cells immobilized in them after a successful injection (Figure 1D, E). As mentioned in the section 1, a range of cell concentration can be used (0.5 x 10⁶ – 1.0 x 10⁶ cells/mL). Lower concentrations will require longer injection time but will decrease the needle clogging. Higher concentrations will require shorter injection times but will increase the needle clogging. It is recommended to try several concentrations to find the one that is most comfortable for an operator. Generally, a higher concentration (1.0 x 10⁶ cells/mL) is preferred to decrease the injection time.
11. Remove the needle from the CAM and gently dab the injection site with a cotton swab to remove any blood or excess cancer cells.
12. Cover the embryo in the weighing dish with a lid and return the injected chicken embryo into the incubator.
13. Repeat the procedure with the next embryo until all embryos are injected.

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 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.