Dr David Hill was awarded funding to develop a 3D in vitro human melanoma skin equivalent and a zebrafish xenograft model to investigate melanoma invasion, migration and metastasis.
Principal Investigator: Dr David Hill
Organisation: Newcastle University
Award type: David Sainsbury fellowship
Start date: 2014
Duration: 3 years
Malignant melanoma is the most lethal form of skin cancer and the fifth most common cancer in the UK. It 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, late stage metastatic melanoma is highly invasive and currently incurable.
Melanoma research, like that for most other cancers, has a heavy reliance on mouse xenograft models for investigating tumour development and progression, and responses to therapeutics. Studies of melanoma initiation and invasion into the skin typically involve the engraftment of human skin, or skin reconstructs containing human melanoma cell lines or patientderived melanoma cells, onto the backs of immunodeficient mice. In some cases, transgenic mice that develop spontaneous tumours are used. For the study of metastatic disease, melanoma cells are implanted subcutaneously and allowed to spread to distant and multiple sites. The primary engrafted tumour may be surgically removed to prevent humane endpoints based on tumour size being breached (and thus requiring animals to be killed) while the study of the metastasis is ongoing. Alternatively, melanoma cells may be injected directly into the circulation via the tail vein or the metastatic site of interest (e.g. intracranially). Depending on the cell line or patient source, metastasis to the lungs, liver, kidney or brain are common and careful monitoring of the mice is essential as the potential for pain and suffering is high.
3Rs benefits (actual and potential)
During his Fellowship, David focused on two approaches that have replaced the use of mice in his melanoma research, saving around 200 animals a year. David has optimised and validated a 3D in vitro model of human skin that recapitulates the early melanoma microenvironment. The model contains both a dermal and epidermal equivalent into which cancer cells can be introduced. These form groups or nests at the dermal/epidermal interface before breaching the basement membrane and invading the dermis. This mirrors early tumour invasion with, for example, the characteristic breakdown of type IV and VII collagens as is observed in human cutaneous tumours in vivo. Details of the model were published in Molecular Cancer Therapeutics in 2015.
David has also established a xenograft model for investigating cancer cell invasion and metastasis in zebrafish embryos which can be completed prior to five days post-fertilisation (the point when they become regulated under the Animals (Scientific Procedures) Act 1986). Fluorescently-labelled human melanoma cells injected into the yolk sac can be tracked in real time by time-lapse imaging. Using transgenic fish, for example with GFP-labelled endothelial blood vessels, David has been able to track local nonvascular spread, intravasation of metastatic cells and subsequent haematological and lymphatic metastatic dissemination. The latter requires interaction with the endothelium during entry and exit, and the metastatic melanoma cells show characteristic sticking and rolling behaviours on the surface of vascular endothelium indicative of specific interactions between human melanoma and zebrafish endothelial cells. Details of the zebrafish model were published on the NC3Rs gateway in 2018 – the paper has been downloaded 100 times to date.
Scientific and technological benefits
In patients with malignant melanoma, a subset of tumour cells often become resistant to mitogen-activated protein kinase kinase enzymes (MEK) inhibiting drugs, such as trametinib. This drug resistance correlates with an increase in autophagy and leads to re-growth of the tumour. Using the zebrafish embryo model, David and his colleagues have demonstrated that combined treatment with drugs that specifically block both MEK and autophagy reduced the invasion of trametinibresistant melanoma cells – this is the first time that inhibition of autophagy has been shown to overcome resistance to targeted MEK inhibitors. The work was published in the British Journal of Dermatology in 2019 and could have clinical relevance. David is now using the in vitro human skin model to determine the effect of autophagy modulation on the invasion of melanoma cells as a potential preventative therapy for metastasis in high risk patients.
The 3D skin model is now routinely used by Alcyomics, a Newcastle-based company which conducts preclinical skin tests on chemicals and pharmaceutical compounds to assess immune hypersensitive reactions. This has replaced the use of up to 200 rodents a year in skin contact hypersensitivity experiments.
David has received funding for two PhD studentships from the BBSRC and EPSRC to further develop the in vitro skin model. The first, in collaboration with Professor Stefan Przyborski at Durham University, is to apply the model to study skin ageing to replace the use of mice, and the second in collaboration with Alcyomics focuses on bioprinting to increase the throughput of the assay. In 2015, David was awarded the British Society of Investigative Dermatology Young Investigator of the Year award.
This case study was published in our 2019 Research Review.