In ovo xenograft model to study tumor dormancy, tumor cell dissemination and metastasis

1. Introduction

Metastasis is a multistep process in where tumor cells undergo invasion, intravasation, extravasation, seeding and metastatic growth after a period of dormancy[1,2]. The study of tumor dissemination and metastasis requires the use of in vivo systems to recapitulate the complex composition of the tumor microenvironment including the vasculature and the extracellular matrix. While mice have been widely used as a model organism to answer questions related to dissemination and metastasis, other in vivo models such as the chicken embryo chorioallantoic membrane (CAM) has multiple advantages that make it a suitable in vivo system to study different aspects of the metastatic cascade.

The CAM assay can be used for different metastasis-related experiments such as study of extravasation [3], metastatic growth[4,5] and to explore the mechanisms driving tumor cell dormancy and the dormancy- awakening transitions[6–8]. Their main advantages rely on it is a highly reproducible, cost-effective, versatile and rapid in vivo xenograft method, in where results are obtained within a week period.

The CAM is a vascular extra-embryonic membrane present in chicken eggs, and formed by the fusion of two embryonic membranes, the chorion and the allantois. It also presents an immature immune system. The CAM plays several important roles during chicken embryonic development, including the exchange of gases and nutrients, as well as the formation of blood vessels[9,10]. It is attached to the surface of the eggshell removing calcium and obtaining oxygen through the porous shell. This attachment to the shell inner membrane occurs around day 4–5 of embryonic development, shortly after fertilization. Initially, it is avascular, and develops into a rich vascular plexus that contains arteries and veins[10]. The growth of the CAM begins on embryonic development day three and continues until day ten when the CAM reaches its full size. The CAM is fully differentiated by day thirteen, when it acquires all it’s physiological functions[10]. All these characteristics have facilitated the use of the CAM for xenograft tumor models and to interrogate different aspects of the metastatic process such as tumor cell growth, invasion, angiogenesis or hypoxia in various tumor types [11–16].

Here, we describe a detailed protocol for the use of the CAM model in metastasis assays. We outline general methods that can be applied to the study of tumor growth and tumor dormancy (by using tumor engraftment methods), metastasis formation and tumor cell dissemination (by performing ex-vivo imaging of metastatic organs in the chicken embryo) and cancer cell extravasation (by performing intravenously injection of tumor cells) (Fig. 1). We have used two different cellular systems representing two different dormancy tumor models: a) T-HEp3, a tumorogenic (T) head and neck squamous cell carcinoma cell line and it’s dormancy (D) counterpart D-HEp3[17,18]; b) D2A1, triple negative breast cancer cell line and it’s dormancy counterpart D2.0R[19,20].

Fig. 1.

Model of the different metastasis assays used in the CAM model.

2. Materials

2.1. General material

1. Egg incubator with rotating function (Ref.1502 from GQF manufacturing). Fig. 2a

2. Tabletop egg incubator (Ref. USAG47C from Brinsea). Fig. 2b

3. Egg rack. Fig. 2c

4. Egg candler. Fig. 2d

5. Sterile angle serrated forceps.

6. Sterile push pin. Fig. 2e

7. Suction device. Fig. 2e

8. Sterile rotatory saw tool with a 15/16-inch cut-off wheel attached. Fig. 2f

9. Clear tape.

Fig. 2.

Photographs of the material required for egg incubation and preparation. a) Egg incubator with rotating function; b) tabletop egg incubator; c) egg rack; d) egg candler e) sterile push pin (bottom) and suction device (up); and f) sterile rotatory saw tool with a 15/16-inch cut-off wheel attached.

2.2. Tumor xenograft and harvesting

1. Sterile phosphate buffered saline containing calcium and magnesium (PBS +/+).

2. P100 and p200 pipettes and filtered tips.

3. 60mm petri dishes.

4. Tray or bucket with ice

5. Sterile 1.5ml Eppendorf tubes.

6. Sterile syringe and 30G needle.

7. 10% Formalin.

8. Dissection scissors.

9. Angled serrated forceps.

10. Media: For T-HEp3 and D-HEp3: DMEM, high glucose, GlutaMAX^™ Supplement, pyruvate (Ref. 10569044 from Thermo Fisher scientific) and for D2A1 and D2.0R: DMEM high glucose, GlutaMAX^™ Supplement media (Ref. 10566024 from Thermo Fisher scientific).

11. Trypsin: Trypsin-EDTA (0.25%), phenol red, Gibco^™

12. Trypan blue: Trypan blue stain 0.4%, Invitrogen

13. BSA: Bovine Serum Albumin, Heat Shock Treated, Fisher scientific

14. Collagenase: Collagenase from Clostridium histolyticum Type IA, 0.5–5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, Sigma Aldrich

2.3. Imaging

1. For analysis of extravasation at the lower CAM we recommended to use a stereoscope system. The one used in this study is an Olympus Macroscope MVX10.

2. For Imaging dissemination and metastasis at different organs in the chicken embryo we recommend using a two-photon system to perform deep tissue ex-vivo imaging of dissected organs. The one used in our study is an Olympus FluoView FVMPE-RS multi-photon laser scanning microscope. Two-photon microscopy allows to perform deep tissue microscopy and image disseminated tumor cells and metastasis.

Material needed to prepare imaging samples include:

1. Sterile phosphate buffered saline.

2. Dissection scissors.

3. Angled serrated forceps.

4. 38 × 10 mm plates.

5. Incubator chamber to place tissue. Silicone gaskets that can be stack and form hydrophobic reagent barriers around tissues (from Electron microscopy Sciences Ref. SKU: 70325–52) (see also Fig. 19).

Fig. 19.

Representative images of the extravasated cells in the lower CAM. Tumor cells are T-HEp3-GFP. Arrows point to single extravasated cells (left) or growing mass (right).

3. Methods

3.1. Egg incubation

1. Obtain 8 days fertilized chicken (white leghorn chicken) eggs pathogen free (Specific pathogen-free Premium Incubated Eggs, Charles River)

2. Place the eggs in a rotator tray inside an egg incubator for 48h at 37°C and 50% humidity.

3.2. Xenograft engraftment:

3.2.1. Dropping the CAM and opening the eggs

1. At day 10 post fertilization take the eggs out the incubator by placing them in an egg rack and bring them to a laminar flow hood.

2. Turn off the lights and hold the stamped end of the egg close to the egg candler. Search for the air sac and the CAM vasculature. The embryo will be visible inside the egg (Fig. 3, see Note 1).

3. Make a pencil mark between two blood vessels at the top, and another one in the side region where the air sac is located. Draw with a pencil a small rectangular region around the vessel area at the top in where the opening will be made (Fig. 4, See Note 2).

4. Use a sterile push pin to perform one hole at the air sac, where the pencil mark was made, and one hole at the blood vessels area in the top (Fig. 4, see Note 4).

5. Press the egg firmly against the sucking device and apply suction into the air sac area to pull out the air facilitating the air enter through the hole at the top and allowing the CAM to be dropped (Fig. 5). Make sure the CAM is still attached at the edges (arrows in Fig. 6) and creating a physical space in where tumor cells will be inoculated (See Note 4).

6. Remove the eggshell by cutting with the electric drill following the rectangular drawing made (Fig. 7). (See Note 5).

7. Slip the sterile forceps under the shell piece and while holding it, remove the entire piece cleanly and firmly (Fig. 8).

8. Place clear tape over the window opening and fold the tape at one end to facilitate removal later.

9. Place eggs in the tabletop egg incubator until time of inoculation of cancer cells (See Note 6).

Fig. 3.

Fertilized egg illuminated by the egg candler showing: the embryo (E), the CAM vasculature (V) and the air sac (AS).

Fig. 4.

Image of an egg in which the hole is being made in the shell (in the air sac mark) using the sterile pin. Note the whole already done in the top part where the square region is drawn.

Fig. 5.

Image of an egg in which suction is applied with the suction device in the orifice of the natural air sac.

Fig. 6.

Image of a successfully dropped the CAM. Note that the CAM is attached at the edges and air fill the space.

Fig. 7.

Egg shell cutting procedure. The small rectangular pencil mark around the blood vessel is shown.

Fig. 8.

Opened window in the egg. CAM is observed under the generated air bubble space.

3.2.2. Preparation of tumor cells and inoculation in the eggs

1. Aspirate the cell media and rinse the dish with 5mL of sterile PBS.

2. Add Trypsin-EDTA (0.05%) (2mL per 100mm dish) and incubate the dishes in the tissue culture incubator at 37°C for 5 min (incubation can vary depending on the type of cells used).

3. Resuspend the detached cells in complete media and count them using trypan blue. Use between 150.000 to 2.000.000 of cells per egg. These concentrations will vary depending on the cell type (See Note 7).

4. Measure the adequate volume with the required number of cells and centrifuge at 1355g for 5min.

5. Discard the supernatant and resuspend the pellet of cells in 50μL of PBS +/+ per engrafted egg and keep them on ice (See Note 8).

6. Place the egg in the laminar flow hood and peel off the clear tape of the opened window.

7. With the use of p200 pipettes and filtered tips, gently drop 50μL of cell solution directly into the center of the CAM (Fig. 9, see Note 9).

8. Re-tape the opened window and place again the eggs in the tabletop egg incubator (at 37°C, humidity level 50%) (See Note 10).

9. Allow tumors to grow from 6–7 days.

Fig. 9.

Dropping of the cell suspension solution onto the CAM using a pipette through the open window.

3.2.3. Tumor harvesting

1. Place the chicken embryo eggs at 4°C for 30 min prior to harvesting tumors to induce hypothermia sedation.

2. Prepare 38 × 10 mm plates with 4ml of PBS +/+. If the tumor tissues will be needed for histology also prepare a container with 10% formalin solution.

3. Remove the clear tape over the opened window and using sterile dissection scissors cut around the entire egg longitudinally (Fig. 10, see Note 11).

4. Employing the dissection scissors and angled serrated forceps, dissect the engrafted xenograft tumor away from the CAM and place it in the 38 × 10 mm plates with 4ml of PBS +/+.

5. Measure the tumor diameter by using a millimeter gridded sheet to calculate volume, and weight tumors by using a precision scale (Fig. 11).

6. For histology and immunohistochemistry experiments, fix tumors in formalin 10% (See Note 12).

Fig. 10.

Image of the opened egg showing the shell prior to dissecting out the tumor from the CAM.

Fig. 11.

Representative images of engrafted tumors in CAMs: dormant D20.R and proliferative D2A1 breast tumor cell lines; and proliferative T-HEp3 and dormant D-HEp3 head and neck tumor cells.

3.2.4. Tumor cell dissociation and counting

1. For tumor cell dissociation, mince the dissected tumors in the 38 × 10 mm plates.

2. Transfer the minced tumor into a 15ml centrifuge tube and add between 2–5 ml (depending on tumor size) of 2.5% BSA and 1.5mg/ml Collagenase A1 in PBS +/+.

3. Using a 5 ml pipette mix the solution multiple times to allow a proper dissociation of the tissue.

4. Incubate the tube at 37 °C for at least 30min (time of incubation can vary and depends on the tissue type).

5. Add 5ml of complete media per tube and resuspending completely by pipetting up and down over 20–40 times (See Note 13).

6. Allow the remain debris to settle (5–10 minutes), transfer the supernatant to a fresh tube and centrifuge at 1355g for 5min.

7. Discard the supernatant and leave the cell pellet (See Note 14).

8. Resuspend the cells in complete medium and count using trypan blue in order to obtain the total number of live cells (See Note 15, Fig. 12). Fig. 13 show representative results of this analysis by using tumor engraftment of head and neck cancer cell lines to assess tumor growth and dormancy (by using T-HEp3 proliferative and D-HEp3 dormant head and neck cancer cell lines). Note the highest proliferation of T-HEp3 compared with the dormant counterpart D-HEp3.

Fig. 12.

Representative image of cells extracted from CAM tumors in the Neubauer chamber. Note the oval shape of the chicken embryo cells compared with the human T-HEp3 tumor cells.

Fig. 13.

Representative graph of T-HEp3 and D-HEp3 tumor cell counts after 6 days growing in CAMs.

3.3. Tumor cell intravenous injection:

3.3.1. Preparations of the cells for injection

1. Aspirate the media from the cells and rinse the dish with 5mL of sterile PBS.

2. Add Trypsin-EDTA (0.05%) (2mL per 100mm dish) and incubate the dishes in tissue culture incubator for 5 min.

3. Resuspend the detached cells in media and count using trypan blue to count the number of live cells. Use between 100.000–200.000 cells, depending on the cell line, per egg (See Note 7).

4. Measure the adequate volume with the required number of cells and centrifuge at 1355g for 5min.

5. Discard the supernatant and resuspend the pellet of cells in 50μL of PBS +/+ per engrafted egg and keep them on ice (See Note 8).

6. Place the egg in the laminar flow hood. Turn off the lights and hold the end of the egg to the egg candler. Search for the air sac, the vasculature of the CAM and the embryo inside the egg (as in Fig. 3, see Note 1).

7. Make a small rectangular pencil mark around the larger vessel (Fig. 14) (See Note 2).

8. Turn on the lights and by holding the egg in one hand, make two transversal cuts in the shell over the small rectangular pencil mark with an electric drill (See Note 16). Next, make an extra cut on the longitudinal lines of the small rectangular pencil mark by lightly touching the eggshell with the drill. (Fig. 15).

9. Slip the sterile forceps under the shell piece and hold it with the forceps parallel to the shell. Remove it entirely and cleanly, with special care to do not disrupt the CAM and the white membrane located underneath the eggshell. This membrane needs to remain intact to not break the CAM.

10. Add a few drops of PBS +/+ on the exposed white membrane, to clean it and check that it is still intact.

11. Turn off the lights and hold the stamped end of the egg to the egg candler to identify the larger blood vessels of the CAM located below the opened window (Fig. 15).

12. Using a sterile syringe and 30G needle, inject the 50μL of cell suspension into the blood vessel (Fig. 16, See Note 17).

13. Place clear tape over the open window and fold the tape at one end to facilitate removal later.

14. Place eggs in the tabletop egg incubator (See Note 6).

15. Allow cells to extravasate and to metastasize (depends on cell type, between 2 and 3 days).

Fig. 14.

Image of the egg with the cuts made on the shell, showing the intact CAM attached to the shell.

Fig. 15.

Image of the egg with the entire piece cleanly removed, illuminated with the egg candler. The major blood vessels of the CAM can be seen located below the opened window and the intact CAM attached to the shell.

Fig. 16.

Image of the injection of the cell suspension into one of the two main blood vessels of the CAM located below the open window.

3.4. Imaging tumor metastasis and extravasation

For imaging extravasation at the lower CAM:

1. Place the eggs on ice and incubate for 15 minutes.

2. Prepare 38 × 10 mm plates with 4ml of PBS +/+.

3. Remove the clear tape over the opened window and using sterile dissection scissors cut around the entire egg longitudinally (See Note 11).

4. Employing the dissection scissors and angled serrated forceps, carefully dissect the lower CAM (See Note 18).

5. Transfer the collected lower CAM in 38 × 10 mm plates with 4ml of PBS +/+ to be carefully cleaned from blood residues.

6. Carefully spread the collected lower CAM on the bottom of a 38 × 10 mm plates (Fig. 17).

7. Draw a 1 cm by 1 cm square on the base of the lid with a permanent marker pen and place the lid on top of the bottom part of the dish with the lower CAM previously assembled. (Fig. 17,18 and See Note 19).

8. Place the assembled sample on the Olympus Macroscope MVX10 to image the extravasated tumor cells at the lower CAM (Fig. 19). Blood vessels can be seen as dark shadows and cells in this assay can be visualized when expressing fluorescent proteins such as GFP. Fluorescence intensity per field or number of extravasated cells per field can be measured to quantify extravasation.

Fig. 17.

Image of the lid of a 38 × 10 mm dish with the 1 cm by 1 cm square drawn.

Fig. 18.

Image of the CAM extended on the base of a 38 × 10 mm dish with the lid containing the 1 cm by 1 cm square mounted on top.

For imaging dissemination at different metastatic organs in the chicken embryo:

1. Place the eggs on ice and incubate for 15 minutes.

2. Prepare 38 × 10 mm plates with 4ml of PBS +/+

3. Remove the clear tape over the opened window and using sterile dissection scissors cut around the entire egg longitudinally (See Note 11). (Fig. 20).

4. Employing the dissection scissors and angled serrated forceps to dissect the chicken embryo and collect the brain, liver and lungs.

5. Transfer the collected organs in 38 × 10 mm plates with 4ml of PBS +/+ to be cleaned from blood residues.

6. Place the collected organs into the microscope chamber (Fig. 21).

7. Place the chamber on the microscope stage to image the disseminated cells in different organs from the chicken embryo (Fig. 22). We recommend using a two-photon system to facilitate penetrance into the tissue (Fig. 23)

Fig. 20.

Images of the collected organs of interest placed in the microscope chamber. a) brain; b) lung; c) liver.

Fig. 21.

Representative image of the egg opened with the chicken embryo inside.

Fig. 22.

Representative image of the chamber with the brain from the chicken embryo place on the microscope stage.

Fig. 23.

Representative images of disseminated T-HEp3-GFP cells in the lungs, brain and liver. a) growing micrometastasis; b) single disseminated tumor cells.

4. Notes

Note 1: The egg candler is a source of light used to visualize the chicken embryo and its associated air sac, CAM and vasculature.

Note 2: The size of the rectangle pencil mark should be around 1 cm in length and 0.7 cm in width.

Note 3: Do not insert the push pin too deep, a hole about 3 mm in depth is usually sufficient, in order to avoid damaging the CAM.

Note 4: If the air sac has not been displaced and the dropping of the CAM is not immediately achieved, use the push pin to make both holes slightly deeper and re-apply suction with the suction device. This procedure needs to be done very carefully to avoid the CAM breakage: if too much suction is applied, the CAM will break or not drop properly, or bubbles of air can be introduced into the egg resulting in a not viable egg embryo. If you will see an air bubble moving between the CAM and the embryo you should discard the egg.

Note 5: Make the two cuts just deep enough to cut through the shell but not too deep to cut all the way down the lowered CAM.

Note 6: Once the egg has been opened, it should be incubated in the tabletop egg incubator, not in the egg incubator with rotating function. Eggs should not be rotating at this point. No antibiotics are added to the CAM.

Note 7: Optimize the appropriate number of cells for each tumor type by doing a growth curve with increasing concentrations of cells.

Note 8: At this point, the cells can be treated (i.e inhibitors) as needed depending on the experiment to be perform. Tumor cells can be resuspended in a solution containing the treatment. Additionally, treatments can be added onto the cells after engraftment through the egg window.

Note 9: At this point, the cell suspension should be done very gently and dropwise.

Note 10: At this point, use caution when moving the manipulated eggs, as the cell suspension can easily be displaced.

Note 11: The CAM with the engrafted tumor should stick to the top half of the shell with the window.

Note 12: Alternatively, tumors can be flash frozen after removing the CAM for future biological assays.

Note 13: Thorough pipetting is essential for complete cell dissociation.

Note 14: At this point, the obtained pellets of cells can be used for several experiments including flow cytometry, Western blotting, RNA isolation and cell culture.

Note 15: A smaller portion of oval-shaped cells are chicken cells from the CAM and should not be counted as tumor cells. There should be a minimal number of non-tumor cells if the tumor is carefully dissected from the CAM (Fig. 22)

Note 16: Ensure that the wheel does not cut too deep into the egg to avoid damaging the CAM and that the CAM remains attached to the shell.

Note 17. The needle should be bent at a 30° angle. Make the first injection at one end of the visible blood vessel to be able to observe the blood vessel clearly in case you have to repeat the injection. A correct injection into the blood vessels is corroborated by the color change of the blood vessels due to the passage of the cell suspension in the vessel that replace the blood at the moment of the injection. Blood will immediately flow back into the vessel soon after the injection.

Note 18. Ensure that the CAM remains intact when collecting the specimen and remove as many chicken cells as possible.

Note 19. The size of the square mark should be around 1 cm by 1 cm and will allow to analyze the same area in different samples. For this assay, fluorescent cells should be used to allow imaging with the microscope by using the adequate filters.