Development of the chick embryo as a replacement for rodent models of tumour metastasis.

The majority of cancer deaths are caused by aggressive cancer cells, which break away from the original (primary) tumour and spread to other parts of the body. This process is called metastasis. While the treatment of primary tumours has advanced greatly in the last decades, there is to date no effective cure for metastasis. Therefore, new therapies are urgently needed to increase patient survival. To understand how, when and why cancer cells spread, mouse or rat models of cancer are used in research. To study tumour formation and metastasis, adult mice or rats often have to undergo invasive procedures. These can cause pain, suffering and distress to the animal, which could be eliminated by using an immature model. Here, I propose to replace mouse and rat experiments with the chick embryo model. This model is cheaper, easier, quicker and enables the non-invasive study of metastasis, which is not possible with mouse or rat models. Cancer cells can be grown on a transparent membrane that is located directly beneath the eggshell, which is rich in blood vessels and located outside of the embryo and tumour formation occurs within days. If the cancer cells are aggressive, as in metastatic cancer, they will break away from the original tumour, travel through the vascular system and form a new tumour in organs of the chick embryo. I aim to study this process by implementing advanced imaging methods, some of which are used clinically, such as magnetic resonance imaging, and some that are only used for small animals. These include special techniques called bioluminescence, photoacoustic and intravital imaging. Because these imaging methods are predominantly used for mammals, I will adapt them for the imaging of tumours and metastasis in the chick embryo. Then, I will use them to study fundamental questions in cancer research, thus demonstrating that the chick embryo model is suitable to study tumour development, metastasis and treatment in a variety of tumour types. I have successfully developed this model during the past 6 years to elucidate the mechanisms of metastasis in the paediatric cancer neuroblastoma. It is now crucial that the wider cancer research community becomes aware of this model as it would accelerate their research and enable the replacement of a large number of animal experiments. I will therefore apply it to other tumour types during my fellowship, where I will test the role of different proteins involved in the metastasis of breast and pancreatic cancers and establish standard protocols and videos to share good practices. I will also explore how this model can be used by pharmaceutical companies to generate pharmacokinetics data; a requirement for clinical testing. Pharmaceutical companies are continuously developing a large number of new drugs against cancer and the most promising ones have to be tested in animals to evaluate their safety and efficacy. By testing novel compounds in the chick-embryo, these companies can quickly screen and eliminate ineffective drugs, testing only the most promising options in rats or mice. I am going to work with a pharmaceutical company, learn about the industrial needs and use the chick embryo model to test newly developed drugs targeting tumour formation and metastasis. If successful, the chick embryo model could not only be used by this company but also by the wider pharmaceutical industry, ultimately leading to a significant reduction in the use of mice and rats in research.

Metastasis accounts for the majority of cancer deaths, yet it is one of the most poorly understood aspects of tumour progression. In order to reduce metastasis associated mortality it is crucial to understand how, when and where metastasis occurs. Metastatic dissemination is a complex process that involves several steps which can currently not be recreated in in vitro culture systems. Thus, in vivo models are used, of which murine models of cancer are the most common. Here, I aim to develop the chick embryo as a replacement for murine models of cancer. The chick embryo model is an excellent tool to study tumourigenesis and metastasis non-invasively as tumour cells can be easily engrafted onto its chorioallantoic membrane (CAM), an extraembryonic and highly vascularised membrane that is located directly beneath the eggshell and thus easily accessible. Within days, tumourigenesis and invasion occur and in the case of aggressive tumour samples, metastasising cells can disseminate to chick embryo's organs through haematogenous metastasis. Further advantages over murine models include its cost effectiveness, its simplicity and immunodeficiency, which allows the engraftment of any xenogeneic material. As the experiments will be terminated at E14, the proposed model is classified as non-protected under the Animals Scientific Procedures Act 1986 (amended 2012) and hence it is a valid animal replacement technique. Moreover, the chick embryo has the potential to be readily imaged in vivo using preclinical imaging and longitudinal high-throughput screening. Here I propose to establish its broader use as an exemplary in vivo model to investigate and intervene in tumour metastasis. This will be achieved by developing standard operating procedures (SOPs) for generating and detecting primary tumours as well as metastasis, and for the evaluation of therapeutics, thus encouraging a wide replacement of murine models for this purpose.

The 3Rs are fundamental to this application and as such, promoting the chick embryo as 3R compliant alternative to murine models of cancer is the main goal of this project. In order to achieve wide adoption, the chick embryo will be used for the replacement of rodents in two cancer types: breast and pancreatic cancer. After initial optimisation and validation in breast cancer, it will be used to replace the currently used rat model to study the influence of S100 variant on metastatic progression, saving 240 rats that would have undergone a procedure classed as moderate (objective A1.2). Additionally, it will be used to test the efficacy of inhibitors of S100-induced metastasis. For each compound 3 different doses and administration routes will be tested. In the current model, each experimental point requires 20 rats, which would result in the replacement of 180 rats per compound. Here, I propose to test the efficacy of 4 newly synthesised and 2 known inhibitors of metastasis which will lead to the replacement of 1120 rats (6 x 180 rats plus 2 x 20 controls (untreated and untransfected cells)) that would have undergone a procedure classed as moderate to severe (objective A1.3). Together, the successful completion of objective A1.2 and A1.3 alone will lead to a replacement of 1360 rats. In order to generalise the approach to other tumour types the chick embryo will be used to replace a mouse model of pancreatic cancer, where the efficacy of new compounds targeting metastasis will be tested. For each compound 3 different doses and 2 administration routes will be tested, which would have required 10 mice per group, hence leading to the replacement of 60 mice per compound. Here, I plan to test 3 novel compounds, which will result in the replacement of 200 mice (3 x 60 plus 2 x 10 controls).

The goals outlined in objective A1 and 2 will be immediately beneficial to my collaborators as they will generate data that would otherwise have required the use of 1360 rats and 200 mice. Additionally, the successful completion of these objectives will lead to the generation of SOPs for the study of tumourigenesis, metastasis, intravital and preclinical imaging, data management as well as the establishment of fluorescent and luminescent cell lines that will be of direct benefit to the breast and pancreatic cancer research community. The publication of methods, protocols and results aims to advocate the chick embryo model to all cancer areas and could lead to a significant replacement of murine models by the chick embryo.

Through my collaboration with a pharmaceutical company (Redx oncology) I aim to further widen the impact of the project. The chick embryo will be implemented as a model for preclinical small molecule screening in industry via two steps. First, existing pharmacokinetic (PK) screening protocols for rodents will be adapted to the chick embryo. Then, in a second stage a panel of novel small molecule-based candidates will be tested for their PK profile and efficacy to inhibit tumour formation and metastasis. This will be directly beneficial to Redx oncology but also other pharmaceutical companies that aim to test newly developed compounds as to date no studies of PK screening in the chick embryo exist. In a typical PK screening 2 different doses and 8 time points are tested on an average of 3 animals per group. That results in 48 animals per compound without control. A medium sized company such as Redx oncology might test a number of 30 novel compounds per year in vivo, resulting in a reduction of 1440 animals per year for PK screening only. If a larger company would implement this method, an even bigger reduction in animal experiments could be achieved. It is expected that the economic advantage of the chick embryo (one fertilised egg costs £0.25 compared to £16 for a mouse before husbandry costs) will be a strong incentive for a wide implementation of the chick embryo model in industry.