Contribution of autophagy to the maintenance, invasion and metastasis of melanoma stem-like sub populations.

Mice are often used as a tool to investigate cancer, including malignant melanoma, the most deadly form of skin cancer, and the the 5th most common cancer in the UK, resulting in more than 2000 deaths annually. Although melanoma localised to the skin can be cured by surgical excision, when the tumour spreads to other parts of the body (metastasis) it is incurable, emphasising the acute need for better ways to identify and treat potentially aggressive tumours. While the use of animals in research is vital to improve our understanding of cancer biology, and to the development of new drugs to treat cancer, researchers nevertheless aim to minimise the use of animals in cancer research and wherever possible develop alternative ways through which to generate meaningful results. The principal goal of the current research proposal is therefore to develop models that replace the use of mice for the investigation of melanoma, specifically, by creating a 3D human skin equivalent model, that recreates the precise environmental conditions of the skin, to model the early stages of melanoma development and progression, as well as a zebrafish model that mimics metastasis of melanoma in the human body during later disease stages. When fully developed, these models will be used to answer important biological questions and generate crucially required novel treatment strategies for melanoma. Preliminary research has identified a particular subpopulation of tumour cells (melanoma stem-like cells) that are able to self-renew and drive tumour progression. Understanding how specific cell survival mechanisms (autophagy) contribute to the growth of these subpopulations, will enable the development of novel therapeutic strategies with improved clinical outcome.

Mouse models of cancer are a valuable tool for the investigation of tumour growth, progression and response to therapy. However, as these models likely cause pain and distress to the mouse, researchers have an obligation to minimise the use of mice, and if possible replace them with non-animal alternatives. The current proposal aims to develop and optimise two novel models, a 3D in vitro human melanoma skin equivalent (MSE) and an in vivo zebrafish xenograft of human melanoma, to replace the use of mice in research. Malignant melanoma is a growing world health concern, with an incidence that has risen more than any other malignancy in the last 40 years. Although curable by surgical resection at early disease stages, metastatic disease is highly invasive, and evolves with an extensive repertoire of molecular defences against immunological and cytotoxic attack, rendering this type of tumour, as yet, incurable. Targeting melanoma stem-like subpopulations, including those expressing stem cell markers CD271 and ABCB5, which constitute a self-renewing, tumour-maintaining subpopulation, that sustain melanoma growth and drive tumour progression, may represent a novel therapeutic strategy for melanoma. Since pilot data also suggests CD271/ABCB5 expressing melanoma subpopulations use autophagy, a critical recycling mechanism for the maintainence of cellular homeostasis, modulating autophagy may offer a novel therapeutic approach to target these cells. This project will thus use the developed MSE and zebrafish xenograft models of human melanoma to test the hypothesis that autophagy contributes to the maintenance, invasiveness and metastatic potential of CD271/ABCB5 expressing stem-like melanoma subpopulations.

The primary objective of the proposed research will be to develop and optimise two novel models; a 3D in vitro human melanoma skin equivalent (MSE) and an in vivo zebrafish xenograft of human melanoma, to Replace the use of mice for the investigation of human melanoma development, invasion and metastasis. Secondary objectives will be to use a phylogenetically lower order animal and anaesthesia regimes to Refine the melanoma xenograft model, and also adopt time-lapse imaging techniques to Reduce the use of protected zebrafish for cancer research.

1) Replacement: Development and optimisation of a 3D full-thickness MSE model and a zebrafish xenograft model of human melanoma has the potential to replace over 25,000 mice currently used to investigate the early stages of melanoma development, as well as subsequent invasion and metastasis. Further development will promote the potential of these models to replace the use of mice for immunology and toxicology assays, not only for melanoma studies but also for the study of other cancer types, further increasing the numbers of mice which these models may replace.
2) Refinement: This project aims to refine the melanoma xenograft model, by exchanging mice for less sentient, more immature zebrafish, and by employing a non-recovery anaesthesia regime. These modifications will additionally benefit animal welfare by lowering the Home Office severity limit for this protocol
3) Reduction: This project also aims to reduce the use of zebrafish greater than 5 days post-fertilisation by employing time-lapse imaging techniques such that individual animals may be analysed over multiple time-points, thereby reducing animal usage by as much as 92%.

Impact metrics to assess the progress of the research project objectives include number of publications, collaborations and up-take of these models by other research groups, both within the institute and globally.