Cost-Effective and Functional Alternatives to Thermanox Cover Rings for Tumor Xenotransplantation on Fertilized Chicken Eggs

Tumor xenotransplantation to the chorioallantois membrane (CAM) of fertilized chicken eggs is excellently suited to short-time analysis of xenograft growth and can reduce mouse experiments. However, the limiting factor with egg xenograft experiments is the high price and limited availability of Thermanox plastic cell culture coverslips, which are often sold out and unavailable for weeks. According to the regular protocol, rings are punched from Thermanox plastic cell culture coverslips 13 mm ø and sticked to the CAM. The xenografts are then sown into these rings in order to prevent that they slide around on the CAM. Thermanox rings are well tolerated and do not interfere with the development of chicken embryos. Here we present a method for preparation of comparable, cheaper and always available alternative rings. We evaluated self-made rings from agar and PVC tube and compared their suitability to Thermanox rings. We found that rings of agar and PVC tube achieved the same xenograft sizes and had no side effects on chick embryonal growth, and therefore seem to be as as good as Thermanox rings.


Introduction
Usually, human tumor xenografts are transplanted to immunode cient mice, which is the most common animal model for tumor xenotransplantation, although it is associated with high costs and administrative and ethical barriers. An avian alternative is the xenotransplantation of tumor cells to the chorioallantoic membrane (CAM) of fertilized chicken eggs. The egg model is natural immunode cient, because immunocompetence in birds develops only after hatching. 1 The CAM is an extra embryonic membrane, in which blood vessels start to grow on day 3 of development. Around day 8 of development the blood vessel network is dense enough to support the growth of a tumor xenograft, 2-4 just like in immunecompromised mice. The CAM is noninnervated and allows painless tumor inoculation, growth and injections. Other advantages compared to mice are the faster tumor growth and a tumor microenvironment, which resembles those of primary patient tissue and the derived mouse xenografts. [5][6][7][8] Experiments with fertilized chicken eggs can be easily performed in any laboratory, as an animal application is not required until day 18 of embryonic development, when the xenografts have to be resected because the chick hatches on day 21. Thus, the chicken egg xenotransplantation model is well suited for short-term in vivo xenograft growth for up to 10 days.
For xenotransplantation of tumor cells to fertilized chicken eggs, we and others have until recently used small rings, which we punched from ThermanoxÔ plastic cell culture coverslips 13 mm ø. [9][10][11][12] These rings stuck onto the CAM and avoid the sliding of inserted tumor cells around and enable the growth of threedimensional tumor xenografts. The latter are supplied by blood vessels of the CAM and chick broblasts contribute to the formation of a tumor stroma. 10 Unfortunately, we have often had the problem that the ThermanoxÔ coverslips were not available for months. This was annoying because it prevented us from conducting egg xenograft experiments. In addition, the ThermanoxÔ coverslips rings are with 0.75 €/ring relatively expensive. Therefore we looked for alternatives and evaluated the suitability of self-made rings from agar and PVC tube. We found that such rings were as good as the before used ThermanoxÔ rings and describe here the procedure for preparation and handling. Thermanox rings were punched from coverslips and the ring hole was enlarged with scissors to a diameter of approximately 9 mm. The rings were washed with 70% EtOH and stored in a sterile petri dish. Agar rings were prepared by boiling 3% agarose in 1× PBS, followed by the pouring of a gel of about 2 mm high. After solidi cation of the gel, platelets were carefully cut out using the open end of 15 ml Falcon tubes and rings were prepared by cutting out the inlet with FACS tubes. The agar rings were stored in sterile PBS at 4°C. PVC tube rings were cutted out from PVC tubes by the use of sterile scissors, followed by washing with 70% ethanol and storage in a sterile petri dish. The basic material used for preparation of each ring type is shown in Fig. 1A, the preparation of rings in Fig. 1B and the nal rings stored in Petri dishes in Fig. 1C.

EGG PREPARATION
This step was performed as described in our recent ProtocolExchange manuscript. 11 TUMOR XENOGRAFT TRANSPLANTATION AsanPaCa cells were transplanted to fertilized chicken eggs at day 9 of development as described in our recent Nature Protocol Exchange manuscript. 11 In detail, each ring type was placed on 10 chicken eggs each, followed by seeding of 1×10 6 AsanPaCa cells in 25 µl Matrigel™ into each ring, resulting in xenografts growing in Thermanox rings, agar rings, or PVC rings ( Fig. 2A).

RESECTION OF XENOGRAFT TUMORS
The chick embryos were euthanized at day 18 of development followed by tumor xenograft resection, as described in our recent ProtocolExchange manuscript. 11 Images of the resected tumor xenografts and their individual volumes are shown (Fig. 2B) and there was no signi cant different size of the tumor xenografts detectable. Likewise, H&E staining and microscopic evaluation of tumor xenograft sections revealed no obvious difference, as exempli ed by representative images at 400× magni cation (Fig. 2C).

EVALUATION OF SIDE EFFECTS
To determine if there were differences in the tolerance of the different ring types on the CAM, we examined the weight of the embryos and any abnormalities. No abnormalities were detectable, because there was no obvious malformation (Fig. 3A), nor was a difference in the mean embryonal weight detectable (Fig. 3B).

Troubleshooting
Fertilized chicken eggs should not be delivered at temperatures colder than 8°C -or should not be stored colder than 8°C. Also, storage should be limited to only a few days, because otherwise the percentage of dead embryos would increase. The primary human pancreatic ductal adenocarcinoma cancer cell line AsanPaCa was obtained from the European Pancreatic Center Heidelberg, Germany and was cultured as recently described. 14 Time Taken

Anticipated Results
Compared to the normally used Thermanox rings, both tested alternative self-made rings are well suited for tumor xenograft growth on the CAM. Regarding Thermanox rings, the punching of the coverslips the preparation of a hole with scissors takes a lot of time. It is important to ensure that the correct side of the coverslights is placed on the CAM, as only one side carries a coating for the cell culture. However, to stick this ring type to the CAM is easy. Regarding agar rings, they can be produced uncomplicated and quickly. However, a minor problem is, that agar rings dry out a little on the CAM, but that has not hindered tumor growth. Regarding PVC tube rings, they are easy to make and handle and also this ring type was well tolerated from the CAM and chick embryo. In conclusion, both, agar and PVC tube rings offer a cheaper and simple alternative to rings prepared from Thermanox plastic cell culture coverslips 13 mm ø.