Protocol for xenografting of BxPC-3 pancreatic tumors using a chorioallantoic membrane model

Summary

The chorioallantoic membrane (CAM) model is an increasingly attractive model for the study of human tumors. However, concise techniques for the use of pancreatic ductal adenocarcinoma BxPC-3 xenografts in CAM assays are not yet available. Here, we present a protocol for the induction of BxPC-3 xenograft tumors with high grafting efficiency. We describe steps for embryo incubation, egg handling, and grafting, each of which has been optimized to prevent fungal contamination and minimize mortality.

Graphical abstract

Highlights

• •Steps for embryo incubation and preparation
• •Precautions to ensure optimal embryo viability and experimental success
• •Generation of single-cell grafting suspension for tumor induction
• •Instructions for CAM abrasion

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The chorioallantoic membrane (CAM) model is an increasingly attractive model for the study of human tumors. However, concise techniques for the use of pancreatic ductal adenocarcinoma BxPC-3 xenografts in CAM assays are not yet available. Here, we present a protocol for the induction of BxPC-3 xenograft tumors with high grafting efficiency. We describe steps for embryo incubation, egg handling, and grafting, each of which has been optimized to prevent fungal contamination and minimize mortality.

Before you begin

• 1.Calculate how many chicken embryos will be used in the upcoming experiment, depending on the desired experimental groups.

Note: A significant amount of growth-arrested embryos and embryo death caused by preparation prior to the actual experiment, is expected. Therefore, we suggest acquiring at least 12 fertilized eggs per experimental group. To achieve 100% grafting efficiency, 4 × 10⁶ cells will be grafted per egg. Therefore, it is adamant to have a sufficient number of dividing cells in culture at embryonic day of development (ED) 7.

Note: This protocol applies a horizontal positioning of the eggs after opening, causing an approximate reduction in available incubator space of 25%. I.e., if the egg incubator fits 100 eggs, only approximately 75 eggs will fit after the egg opening procedure.

• 2.
  Timing of the different steps.

  Note: The chicken embryo develops over a time span of 21 days. Generally, CAM experiments are started during the second week of development, starting as early as ED6 and ended prior to ED18. To ensure cells are harvested before reaching high confluency and guarantee enough cells for tumor induction among all embryos, it is essential to time the expansion of the cells required for the grafting procedure correctly.

  • a.
    Contact your local egg supplier and inform when they can provide ED0 fertilized chicken eggs and base the start of the cell line expansion on this date.
  • b.
    Ensure to have the necessary number of cells ready in culture 7 days after the start of incubation (ED7) of the chicken embryos in your egg incubator.
  • c.
    Ensure that enough staff is available to perform the procedures at ED3 (egg opening), ED7 (tumor grafting) and at termination of the experiment.

    Note: In a typical CAM experiment, two experienced researchers can perform these procedures for approximately 60–80 embryos during normal working hours.

• 3.
  Validate the accuracy of the incubator temperature set point.

  Note: Although chicken embryos develop optimally at an incubation temperature of 37.5°C–37.7°C, we recommend maintaining a constant embryo temperature of 37°C. This ensures that the human tumor cells are not exposed to supra-physiological temperature conditions which may affect their response to therapies. Additionally, we have not observed any adverse effects on the CAM development or embryo mortality by this lower incubation temperature. The accuracy of the temperature set point of commercially available egg incubators is subject to a large degree of variability. Therefore, we suggest performing the following steps:

  • a.
    Set the incubation temperature of the egg incubator to 37°C and the humidity to 50%–60% relative humidity (RH).

    Note: Higher RH values are not required and increase the chances of fungal contamination.

  • b.
    Drill a small hole into the convex side of a fertilized chicken egg.
  • c.
    Insert a temperature sensor into the egg.
  • d.
    Monitor the internal egg temperature over a period of at least 24 h and,

    • i.
      Ensure that this internal temperature averages around 37°C.
    • ii.
      If there is a significant offset, adjust the incubator set point and monitor for another period of at least 24 h. Repeat if necessary.

      Note: During the actual experiment, one or multiple fertilized eggs should be used as described above to steer the temperature set point for the entire duration of incubation.

  • e.
    Perform steps c and d for the centermost incubator position and at least two extreme positions (e.g., bottom and top).

Recommended:

Determine the toxicity of compounds to be used (if applicable). Many compounds and drugs have never been tested in the CAM model. Therefore, information on embryo-toxicity is often absent and drugs can exhibit higher levels of toxicity when doses are extrapolated from data in rodents or other non-avian model systems. For that reason, we suggest determining a dose-toxicity curve for each compound based on embryonic deaths and macroscopic evaluation, necropsy, and histological characterization of the developing embryo, even if literature is available describing its toxicity profile in other animal models.

Institutional permissions

Before starting any experiments, please confer with your local ethical committee on the local legislation of the use of chicken embryos as a model system and acquire the necessary approval when required. Additionally, ensure your lab has the correct biosafety permissions to carry out experiments with human cell lines.

Experiments were performed in accordance with the Directive 2010/63/EU of the European Parliament and the Belgian Royal Decree of 29 May 2013.

In vitro expansion of BxPC-3 cells

Timing: 2–4 weeks

• 4.
  Thaw at least 5 million BxPC-3 cells in large culture vessels (e.g., T175-flask).

  • a.
    Grow in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin until cells are 70–80% confluent and,
• 5.
  Split at a 1:4 ratio.

  • a.
    Rinse each flask well with calcium-free PBS prior to passaging.
  • b.
    Detach cells with a prewarmed 1:1 dilution of Trypsin-EDTA (0.25%) in calcium-free PBS.
  • c.
    Count cells and ensure high viability (e.g., via trypan blue exclusion assay).
• 6.Expect a yield of 15–20 million BxPC-3 cells per T175.
• 7.Perform the last passage on ED3-4 to ensure sufficient BxPC-3 cells at ED7.

Start of chicken embryo incubation

CRITICAL: Work as cleanly as possible, fungal contamination is the leading cause of failing experiments. If possible, perform all main steps from ED3 onwards in a laminar flow cabinet. Work as quickly as possible and provide heating blocks or infrared lamps to avoid an excessive drop in embryo temperature which can negatively impact their viability.

ED0 (7 days prior to grafting)

Timing: 1 h

• 8.
  Preparation of the egg incubator.

  • a.
    Clean the incubator and egg trays thoroughly with a germicide with anti-fungal properties (e.g., a 2.5% solution of UMONIUM 38 Master in water).

Note: Regardless of the method of humidifying, ensure a humidity level within the incubator of 50–60% RH. Higher levels of humidity will facilitate fungal contamination. Additives to inhibit microbial growth (e.g., Aqua-stabil at a final concentration of 0.2%) can be added to the water, especially when using evaporation-based humidifiers.

• 9.
  Incubation of fertilized chicken eggs.

  • a.
    Acquire high-quality fertilized chicken eggs from a reputable supplier.

    Note: Incubation of the eggs should start as soon as possible to ensure optimal viability. If the incubation needs to be postponed, the fertilized eggs can be stored for up to one week at a temperature of 13°C–18°C in a room with a high RH (80%).¹ Storage of fertilized eggs at even lower temperatures (e.g., 4°C) is not recommended as this might result in substantially decreased embryo viability and development.

  • b.
    Remove feathers, excrements, and any other dirt from the eggs by thoroughly washing with lukewarm water.
  • c.
    Allow eggs to dry and wipe the eggshells with germicide.
  • d.
    Place the eggs vertically in the incubator with the air pocket facing upwards (usually marked with an identifier stamp).
  • e.
    Activate the tilting function of the incubator (Figure 1).

    Note: When no stamp is present, the air pocket can be identified as a translucent area when candling the egg with a light source. If the incubator does not possess an automatic tilting functionality, tilt eggs approximately 90 degrees at least three times daily.

    CRITICAL: Tilting of the eggs during the first three days of incubation is crucial for optimal embryo development.²

• 10.Recommended: Weigh each egg and write down its mass (on the shell in pencil).
• 11.Recommended: Reposition the eggs frequently (at least once per day) to counteract the effect of temperature gradients present within the incubator.
• 12.Cut enough flexible polyurethane-based adhesive (e.g., Opsite Flexifix) into rectangular shapes of approximately 3 × 4 cm to be applied on the eggshell during the opening at ED3 to prevent debris falling into the egg.
• 13.Cut an excess of Parafilm to similar dimensions to make a resealable cover of the created opening in the eggshell to prevent embryo contamination and dehydration.

Figure 1.

Correct positioning of the eggs at start of incubation (ED0)

Left: eggs placed with stamped side upwards and tilted 45°. Right: eggs turned 90° to the opposite side.

Opening of the eggshell and dropping of the embryo

ED3 (4 days prior to grafting)

Timing: approximately 2 min per person per egg

During this step, a working opening in the eggshell is created, the embryo is subsequently dropped and detached from the inner eggshell membrane, ensuring its viability when cutting open the eggshell.

• 14.At least 2 h prior to the opening procedure, stop the automatic tilting function if present.
• 15.
  Position the eggs horizontally within the incubator.

  • a.
    Mark the uppermost side with a pencil line. The embryo will shift position and be located approximately centrally along the axis of this line (Figure 2).
  • b.
    Disinfect the stamped side (air pocket) and the surface along the pencil line with a germicide.
• 16.
  After at least 2 h, take out an egg and place horizontally in the egg tray in the flow cabinet.

  • a.
    Disinfect the stamped side (air pocket) and the surface along the pencil line with a germicide.
  • b.
    Puncture a small hole into the stamped side using an egg pricker.
• 17.Place the egg back horizontally in the egg tray.

Note: Thumbtacks for puncturing will also work but entail a higher risk of fracturing the eggshell.

Note: If no stamp is present, the air pocket is located at the spherical end of the egg. This can be verified by candling the egg with a light source and identification of a translucent area.

• 18.Insert a sterile 19G needle coupled to a sterile syringe (volume = 10 mL) through the created hole under an angle of at least 45° relative to the horizontal axis of the egg (Figure 3). This ensures there will be no puncturing of the yolk and safeguards the embryo.
• 19.
  Pierce the air cell completely.

  • a.
    Carefully aspirate and discard a predetermined volume of albumen.

Note: For small eggs (<50 g) remove 4 mL, for medium eggs (50–60 g) 4–5 mL and for large eggs (>60 g) 5–6 mL.

Note: Along with the horizontal positioning, removal of albumen allows the creation of a large working area compared to vertical incubation.

• 20.Apply an adhesive strip along the long axis of the pencil mark.
• 21.Place the egg back horizontally into the incubator.
• 22.Continue until all eggs have been punctured and the necessary amount of albumen has been extracted.
• 23.
  Starting from the egg that was handled first: pierce the eggshell through the adhesive from the top with sharp, fine-point scissors.

  • a.
    Cut a (round) hole of approximately 2 × 2 cm, starting from the egg that was handled first.
  • b.
    Remove the off-cut.
  • c.
    Confirm the presence of a viable embryo.

Note: Small, fine scissors are preferred over larger ones as they generate fewer eggshell debris. At this stage (ED3), blood vessels and a beating heart are present and are a sign of viability.

Note: At times, the embryo is not situated directly beneath the opening (Figure 4). Inspect carefully with the aid of an LED light source to confirm the presence (or absence) of a developing embryo.

Figure 2.

Left: schematic drawing of an egg, view from the top showing the approximate embryo positioning. Right: photograph of an opened egg at ED3, showing the actual embryo position indicated by the arrow

Figure 3.

Correct positioning and insertion of the needle into the egg avoids damaging the yolk or embryo

Figure 4.

Left: Top view showing an embryo that is not located at the expected position. Right: oblique view into the same egg, revealing the viable, developing embryo

• 24.
  Use a parafilm strip to cover the opening that has been created.

  • a.
    Avoid stretching the film too much as this could cause it to rupture during incubation.
• 25.Place the eggs back in the horizontal position in the incubator with the created window facing upwards.
• 26.Inspect daily until the grafting day.
• 27.Remove any non-developing embryos or when contamination is suspected.

Note: A foul-smelling egg can be indicative for bacterial contamination while fungal contamination can manifest itself as the presence of bluish or white spores on the eggshell, near the egg window or in the egg itself (Figure 5).

Figure 5.

Left. Bluish fungal spores (arrow) present on the outer eggshell during incubation, signifying contamination. Right. Fungal contamination on the CAM with visible mycelia

ED6 preparations (1 day prior to grafting)

• 28.
  Place a sufficient amount (total volume needed = 25 μL x number of expected grafts to be performed) of Basement Membrane Extract or Matrigel on ice.

  • a.
    Let thaw in a fridge at 4°C for at least 8 h.
• 29.Additionally, place sterile micropipette tips (100 μL and 1 mL) at 4°C.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium, high glucose, + L-glutamine, no pyruvate	Gibco	Cat#41965039
Gibco fetal bovine serum, qualified, heat inactivated (HI), E.U.-approved, South America	Gibco	Cat#10500064
Trypsin-EDTA (0.25%), phenol red	Gibco	Cat#25200072
Cell culture flask, 550 mL, 175 cm2	Greiner Bio-One	Cat#660175
Penicillin-Streptomycin (10,000 U/mL)	Gibco	Cat#15140122
Umonium Master	Laboratoire Huckert’s	PF 10118
Aqua Stabil	Julabo	Cat#8940006
Cultrex basement membrane extract, type 3, Pathclear	R&D Systems	Cat#3632-010-02
DPBS, no calcium, no magnesium	Gibco	Cat#14190169
Critical commercial assays
Trypan blue	Gibco	Cat#15250061
Experimental models: Cell lines
Human BxPC-3 pancreatic adenocarcinoma cells	ATCC	CRL-1687
Experimental models: Organisms/strains
Fertilized chicken eggs	Belgabroed NV	BE1752
Other
Counting chamber, Bürker-Türk	VWR	HECH40444702
Egg pricker	Amazon.com, Inc	B09VYQHCMV
Reusable, sterilizable egg tray or holders	Borotto	N/A
LED light source	Power-Lux	SL-PL
Stainless steel fine-tip dissection scissors	VWR	N/A
Stainless steel curved dissection scissors	VWR	N/A
Stainless steel tweezers	VWR	N/A
Seeker with bent-end (abrasion tool)	Fisherbrand	Cat#08-995
Egg incubator	Fiem	MG140/200 Rurale
Automatic humidifier Limpia 4	Olimpia Splendid	Cat#99424
Parafilm M sealing film	BRAND	Cat#701605
Opsite Flexifix 5 cm × 10 m	Smith & Nephew	Cat#66000040
19G sterile needles	Terumo	NN-1950R
10 mL sterile syringes with rubber plunger stoppers	Terumo	SS∗10LE1
Sterile micropipette tips 100 μL	Sarstedt	Cat#70.3050.255
Sterile micropipette tips 1,000 μL	Sarstedt	Cat#70.3050.355
Sterile 1.5 mL Eppendorf tubes	Eppendorf	Cat#0030121503
Polystyrene culture dish 100 mm	Corning	Cat#430167
Polystyrene box	N/A	N/A
Wet ice	N/A	N/A
Egg pricker	Amazon.com, Inc	B09VYQHCMV
Reusable, sterilizable egg tray or holders	Borotto	N/A
Infrared heating lamp	Philips	HP3616

Materials and equipment

Grafting suspension

Reagent	Final concentration	Amount
Cultrex Basement Membrane Extract (BME), Type 3, Pathclear (10–12 mg/mL)	5–6 mg/mL	50 μL per embryo
Complete medium (DMEM containing 10% of HI-FBS and 1% penicillin/streptomycin)	N/A	50 μL per embryo
BxPC-3 cells suspended in complete medium (cell suspension)	4 × 107/mL	4 × 106 per embryo
Final grafting suspension	N/A	100 μL per embryo

Note: Store the BxPC-3-containing cell suspension A in aliquots of 1 mL in a CO₂ incubator at 37°C until use. These aliquots can be stored for at least 2 h without discernable loss in protocol success. Prepare both suspensions as closely in time as possible to the actual procedure grafting itself.

Recommended: Abrasion tool

An abrasion tool can be made by modifying a blunt dissection probe.

With a metal file, make 2–3 passes against the tip at an angle of approximately 90°. This will result in the creation of sharp ridges (Figure 6). Test the abrasion potential on tissue paper or on bare skin. If the process was not successful, it can be repeated multiple times, until the desired sharpness is obtained.

Alternatives: Cultrex Basement Membrane Extract, Type 3, Pathclear can be substituted with Matrigel.

Figure 6.

Modified dissection probe with abrasive edges (arrows)

Magnified 20×.

Fiem egg incubator can be substituted with Brinsea Ova-Easy 100 Advance or 190 Advance. Umonium Master can be replaced by Fermacidal D2 (Labotect). The CAM could also be abraded with filter or tissue paper or with a sharp needle, albeit with varying consistency.

Step-by-step method details

Preparation of the BxPC-3 grafting cell suspension

Timing: cell handling 2–3 h, performed at ED7 (grafting day)

Generation of a single cell grafting suspension to be used as the basis for tumor induction.

• 1.
  Cell dissociation & counting.

  • a.
    Discard old medium.
  • b.
    Create a single cell suspension.

    • i.
      Add trypsin-EDTA 0.25% and place at 37°C until all cells have detached.
    • ii.
      Deactivate trypsin with complete medium.
    • iii.
      Transfer to Falcon tubes.
    • iv.
      Centrifuge cells at 250 g for 7 min.
    • v.
      Discard the supernatant.
    • vi.
      Resuspend and pool all cells in complete medium.
  • c.
    Count cells and assess viability (e.g., via trypan-blue assay).
  • d.
    Centrifuge the pooled cells at 250 g for 7 min.
  • e.
    Resuspend in complete medium to a concentration of 8 × 10⁷ cells/mL (50 μL will contain 4 × 10⁶ cells).
  • f.
    Aliquot the pooled cells into Eppendorf tubes at manageable volumes (e.g., 500 μL in a 1.5 mL Eppendorf tube).
  • g.
    Place into a humidified CO₂ incubator at 37°C until further use.

Note: Cells can be left pooled for at least 2 h without noticeable changes in grafting efficiency.

• 2.
  Grafting cell suspension.

  • a.
    Place the cooled micropipette tips and thawed BME on ice in a clean flow cabinet.
  • b.
    Take 1 aliquot of the cell suspension, containing the BxPC-3 cells, out of the 37°C incubator, and place on ice in the flow cabinet.

    Optional: prior to mixing, let the cell suspension cool for approximately 5 min.

  • c.
    Create the grafting suspension by adding an equal volume of ice-cold BME to the cell suspension-containing Eppendorf, using the pre-chilled tips.
  • d.
    Mix gently with a chilled 1 mL micropipette tip, ensuring the entire cell pellet is resuspended well.

    Note: Avoid the introduction of bubbles into the suspension through pipetting carefully, as BME/Matrigel can gel surrounding any introduced air bubbles, thereby resulting in non-representative tumor architecture (Figure 7).

    CRITICAL: Keep the grafting suspension on ice throughout the entire process to prevent gelation prior to grafting.

    Note: By aliquoting the grafting suspension into smaller aliquots (step 1.e), cold-time to which the BxPC-3 cells are exposed is reduced to a minimum.

Figure 7.

Hematoxylin-Eosin staining on a BxPC-3 xenograft tumor presenting large vacuolar structures (arrows) formed by BME gelation around air bubbles

The latter can be lined with tumor or chicken embryonal cells. Image acquired via the ZEISS Axioscan platform at 40× magnification.

Suspension grafting of BxPC-3 cells

Timing: 2 min per graft

Confirming embryo viability, determining the grafting location and induction of the tumor.

• 3.
  Embryo viability and CAM abrasion.

  • a.
    Place a windowed egg in the flow cabinet.
  • b.
    Open the parafilm window and confirm the embryo viability.

    Note: Deceased embryos are recognized by atrophic, degenerating blood vessels, pale embryo color, the absence of (spontaneous) movement and heartbeat (Figure 8).

  • c.
    Identify the exact location of the CAM and determine where the graft can be placed. Ideally, the grafting site should be situated in between a major vessel bifurcation (Figure 9A).

    CRITICAL: Vitelline blood vessels originating from the yolk sac can sometimes be mistaken for CAM-derived vessels which lie more superficially and are highly branching (Figure 9B). Grafting on the yolk sac membrane will not result in tumor induction.

    Note: when the CAM is consistently (too) small to allow easy grafting, the procedure can be delayed until ED8. This, however, shortens the experimental window by one day.

  • d.
    Wipe the modified dissection probe/other semi-sharp tool with germicide, allow to dry and lightly abrade as follows (Figure 10):

    • i.
      Place the abrasion tool onto the desired grafting location on the CAM.
    • ii.
      Exert a slight downward pressure onto the CAM with the tool and drag towards self over a distance of approximately 1 cm.
    • iii.
      Observe for a few seconds and look for minimal punctiform bleeding.
    • iv.
      If bleeding is absent, repeat but allow for sufficient time between abrasions for the bleeding to become overt.

      CRITICAL: Excessive abrasion of the CAM blood vessels can easily lead to embryo death. Therefore, one should aim to err on the side of caution when applying the abrasion.

      Note: The abrasion is carried out to facilitate attachment and subsequent growth of the cancer cells intro the stromal CAM layer. A flattened, semi-sharp tool surface will allow delamination of the superficial epithelial CAM layer, whereas sharp, pointed tools can lead to the complete penetration of the three embryonic layers. This is not always directly and clearly visible. Consequently, the grafting will be unsuccessful as the cell suspension will be deposited directly into the allantois.

• 4.
  Cell line grafting.

  • a.
    Using a chilled pipette tip, take 100 μL of the final grafting suspension.

    • i.
      Slowly dispense onto the abraded region while maintaining contact between the tip and the membrane.
    • ii.
      Hold in place for approximately 5–10 s and remove the tip.

      Note: Avoid holding the grafting suspension at the bottom of the Eppendorf tube since this will cause warming and potential gelation of the suspension.

  • b.
    Re-seal the egg window with parafilm.
  • c.
    Number the egg on the shell in pencil.
  • d.
    Gently place back into the horizontal incubation position in the incubator.
• 5.Repeat for the remaining eggs.

Figure 8.

Photograph of a deceased embryo (blue arrow) and atrophic blood vessels (black arrow) at ED7

Figure 9.

Overview of the CAM at ED7

(A) Possible suited grafting locations on the CAM (circles).

(B) Visualization of the distinction between CAM (C) and yolk sac (Y). The extreme edge of the CAM is indicated by the white arrows.

Figure 10.

Left: Example of abrasion-induced punctiform bleeding. Right: Schematic depiction of the abrasion procedure. A = abrasion tool

Viability assessment and successful grafting rate determination at ED10

Timing: 2–3 min per embryo

Counting of the number of viable embryos with developing tumors.

• 6.Take out eggs and determine embryo viability. Limited mortality (< 10% from ED3 through ED9) should be observed at this point.
• 7.Inspect each egg carefully for the presence of a developing tumor, as the movement of the embryo and expansion of the CAM may cause it to grow on a CAM portion attached to the upper eggshell.
• 8.
  Divide the successfully grafted embryos at random into the different treatment groups.

  • a.
    subsequently label each egg with the respective group identifier.
  • b.
    Assign random positions to each embryo to minimize gradient effects within the incubator.
• 9.Treatment can now be started.

Note: Treatments and investigations can be postponed until later EDs, but this will result in a shorter experimental follow up.

Sacrificing the embryo

Note: Timing of embryo sacrifice should be performed according to local legislation and regulations.

• 10.Cut out the tumor from the CAM with small and sharp scissors.
• 11.Crack open the egg and gently slide the embryo into a 10 cm dish.

Note: During the sliding, avoid rupturing the membranes on the eggshell edges. This will ensure the integrity of the vitelline blood vessels. Failure to do so will cause extensive bleeding and may cause unnecessary embryo stress.

• 12.Bluntly dissect the membranes surrounding the embryo head with scissors.
• 13.Sacrifice via decapitation before moving on to further processing of the isolated tumor.

Expected outcomes

Following the above-described protocol, we obtain a grafting efficiency of at least 95%. (In case of lower grafting efficiency, see section “troubleshooting”). At 2 days post grafting (ED9), superficially located amorphous growth can be observed (Figure 11 A) which over time will develop into rounded, invasive, and vascularized tumors (Figure 11B). Depending on the timing of embryo sacrifice, tumors with a mass of 15–100 mg can be obtained. The tumors are characterized by well-defined dense cellular nodules which are engulfed by collagenous capsules (Figures 11C and 11D).

Note: Quantitative assessment of growth by eye can be deceptive as the tumor cells can invade downwards into the CAM rather than grown upwards.

Figure 11.

Stereomicroscopic and histological evaluation of BxPC-3 tumors

(A) BxPC-3 xenograft tumor growing on the CAM at ED14.

(B) Fixed for 24 hours in 4% formalin & showing clear presence of vascularization.

(C and D) H&E, respectively trichrome staining of BxPC-3 xenograft tumor, excised and fixed at ED18. Images acquired via the ZEISS Axioscan platform at 20× magnification.

Limitations

Although our CAM assay protocol ensures both a high grafting efficiency and embryo survival, it has some minor limitations. Firstly, the horizontal positioning of the eggs can lead to slightly lowered viability and causes a reduction in available space within the incubator. The use of animal-derived products such as FBS and basement membrane extract are subject to biological and batch-to-batch variability which may impact the reproducibility of the obtained results.

This protocol has not yet been applied rigorously for other (PDAC) cell lines. Therefore, lower grafting efficiencies could be expected when using other cell lines than BxPC-3.

Troubleshooting

Problem 1

No albumen is being aspirated during the dropping of the CAM, or a large force needs to be exerted to aspirate.

Potential solution

The needle lumen is likely obstructed by collapsed eggshell membrane, chalaza or the air cell has not been completely penetrated.

• •Remove the needle from the egg, purge the needle and reinsert at a slightly different angle relative to the sagittal plane.
• •If excessive albumen leakage occurs after needle removal; wipe down and cover with an adhesive strip (related to “Dropping of the embryo”).

Problem 2

A substantial amount (>10%) of embryos are deceased in the days following the egg opening procedure.

Potential solution

• •Verify the absence of contamination.
• •Safeguard clean/sterile working conditions for all steps.
• •Verify incubator temperature and humidity levels.
• •Ensure correct needle positioning and verify that no yolk or embryo is aspirated (related to “Dropping of the embryo”).

Problem 3

A substantial amount (>10%) of embryos are deceased in the days following the grafting procedure.

Potential solution

The CAM abrasion was likely carried out too roughly, resulting in excessive bleeding and hemodynamic embryo stress.

• •Abrade the CAM in a gentler manner and ensure only punctiform bleeding can be observed.

Alternatively, high embryo mortality can be observed during contamination.

• •Ensure sterility of all steps and reagents (and cell line) in the protocol (related to “suspension grafting of BxPC-3 cells”).

Problem 4

Low grafting efficiency (<75%).

Potential solutions

• •Ensure that the grafting suspension is deposited correctly on the CAM, and not on the yolk sac membrane. (Related to “expected outcome” see Figure 9).
• •Dispense the grafting suspension in a slow and controlled manner onto the CAM. Quick and harsh dispensing can lead to dispersion of the suspension over the CAM, leading to superficial, non-invasive, tumor-like growth which can be mistaken for metastatic cells at later EDs.
• •Ensure that the grafting suspension is deposited onto the abraded area.
• •Rough handling directly following the grafting procedure may cause dispersion of the grafting suspension. Therefore, move the egg slowly during handling.
• •Do not allow the cells to become overly confluent during the in vitro expansion.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Robin Colenbier (robin.colenbier@uantwerpen.be).

Technical contact

Further information and requests on thecnical aspects of the protocol should be directed to Robin Colenbier (robin.colenbier@uantwerpen.be) and Lenny Coppens (lenny.coppens@uantwerpen.be).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate/analyze datasets or code.