Introducing the CAM Model as a 3Rs-compliant alternative for laboratory animal experiments
At the UMC Utrecht (department of Radiology and Nuclear Medicine) theranostic compounds for the treatment of cancer are being developed in the research group of Dr Alex Poot. These radioactive compounds are tested preclinically on tumor cells and organoids, on patient-derived tumor tissues and finally in an animal model of cancer.
Our student assistant Sol猫ne Feyzi has been working on an innovative drug testing method, the chicken chorioallantoic membrane (CAM) model. This model enables tumor growth and high-throughput biodistribution testing of drugs (like radiopharmaceuticals) and may provide a good alternative to the use of living animals.
What is the CAM model?
The CAM is an extra-embryonic membrane that is present in fertilized chicken eggs during embryonic development. It features a highly vascularized environment to which tumor cells can be added to grow small, well-vascularized tumors. Moreover, it lacks a mature immune system, making it an ideal environment for studying tumor growth, metastasis, and angiogenesis. As it takes only 14 days from the start of the fertilized egg incubation to the end of the experiment, the model can be used in a high-throughput setting. Since a fertilized egg costs about 1% of an immunodeficient mouse, it is also a cost effective model.
What did the internship result in?
The goal of Sol猫ne鈥檚 project was to develop a protocol for the CAM model and to assess its potential as a cost- and time-efficient method for drug evaluation between in vitro studies and preclinical testing in mice. Specifically, radioligands designed for diagnosing neuroblastoma, a form of childhood cancer, were tested in the model.
Key highlights of her work include successful 1) breeding of fertilized eggs up to day 14, 2) opening and closing the eggshell for administration of tumor cells, 2) growing viable tumors on top of the CAM with embryo survival rates >90%, 3) developing a microscopic injection system with in-house pulled and sharpened glass needles, 4) Injecting radioactive compounds and analyzing subsequent biodistribution.
As a result of her work, the CAM xenograft model was demonstrated to hold significant promise as a high-throughput, rapid alternative to mouse models, with tumors ready for analysis in just 14 days. During the project, the biodistribution patterns of various radioligands were successfully retrieved and analyzed across key organs, including the kidney, liver, and tumor, highlighting the model's utility in evaluating new radiopharmaceuticals.
How does this relate to the 3Rs?
Without access to the CAM model, every radioligand giving the desired in vitro results would be evaluated in a relevant cancer mouse model. The CAM model bridges the gap between in vitro and in vivo experiments.
Compounds that do not meet expected results in the CAM model will no longer be evaluated in mice. Although the CAM model makes use of a developing animal, the chicken embryo is not considered a laboratory animal yet in this early phase of development. It therefore contributes to the Replacement and Reduction of laboratory animal use.
The CAM model involves only a minor disturbance of the embryo during the 14 days in the egg breeding machine. At day 3, a little window in the eggshell is made and at day 9 tumor cells are added on top of the membrane. Then, at day 14, a radioactive compound is injected in the membrane vasculature. After a certain circulation and distribution time, embryos are euthanized and dissected. Tumors and radioactive compounds are added outside of the animal, which is a pain-free procedure. This in strong contrast to laboratory mice, where injections of tumor cells and radioactive compounds cause pain and stress.
In conclusion
The CAM model was successfully developed as a time- and cost-efficient screening method and will now be used in go/no-go decision making in advancing compounds into laboratory mouse models. If future results indicate that results in this model overlap well with mouse tumor models, experiments in tumor-bearing mice could be even further reduced.
