The Role of Dermal Architecture in Wound Healing and Tumour Differentiation

Abstract

The microenvironment provides cues that can guide differentiation and suppress or support neoplasm and invasion. Defective or absent type VII collagen, caused by mutations in the COL7A1 gene, is responsible for the inherited Hallopeau-Siemens form of recessive dystrophic epidermolysis bullosa (RDEB) where trauma-induced blisters cause morbidity leading to skin cancer (SCC). Previous work done in the laboratory has identified that the dermal matrix composition drives tumour development in patients with RDEB (Ng, Pourreyron et al. 2012), showing that normal cells can inhibit the proliferation and expansion of tumour cells and that the proteins type 5 collagen alpha 1 (COL5A1) and thrombospondin 1 (TSP1) were upregulated in RDEBF and UVSCCF fibroblasts, and further upregulated in RDEBSCCF fibroblasts (Ng, Pourreyron et al. 2012).

Following these observations, the work done in this thesis attempts to further elucidate the contributions of these proteins in the development of SCC malignancies through the generation and study of shRNA knockdowns of COL7A1 and TSP1 in primary normal human fibroblasts. The knockdown models of COL7A1 and TSP1 showed altered mRNA expression profiles and displayed organoid disruption in co-cultures with SCCRDEB cells. These fibroblasts also appeared to accelerate wound closure and affect the migration of SCCRDEB cells in co-culture scratch wound assays. Attempts were also made to generate overexpression models of COL5A1 and TSP1 but this proved to be unexpectedly challenging and has yet to be successful.

Experiments using spheroid organoids to analyse the interaction between SCC keratinocytes and dermal fibroblasts showed morphologically distinct organoid formation amongst different SCC cell lines. These results also indicated a link between type VII collagen and spheroid organisation, demonstrating the usefulness of the spheroid model as a platform to analyse SCC-fibroblast interactions and the influence of type VII collagen. To more accurately model in vivo skin behaviour and architecture, a novel ‘sandwich’ method for culturing skin organotypics was also developed in the course of this thesis. While still in the early stages, a working prototype has been created as proof of concept to demonstrate the potential of the ‘Sandwich’ method for live-cell imaging and skin-mimic or multi-layered organotypics. This work highlights the importance of using genetic modulation to study the contributions of genes downstream of type VII collagen and the importance of using new models to gain new perspectives in the study of cSCC. Understanding the processes which lead to cSCC in RDEB will help in the development of new molecular targets for future therapeutic purposes.

Date of Award	2018
Original language	English
Awarding Institution	University of Dundee
Sponsors	Agency for Science, Technology and Research, Singapore
Supervisor	Ellen Lane (Supervisor) & Andrew South (Supervisor)