Development of a Tissue-Engineered Oral Mucosa Model to Investigate the Role of Tumour-Associated Macrophages in Oral Cancer Progression

Tumour-associated macrophages (TAM) represent a prominent component of the leukocytic infiltrate of human tumours and their accumulation in oral squamous cell carcinoma (OSCC) has been shown to be a predictor of poor prognosis. Evidence suggests that the tumour microenvironment drives TAM into a pro-tumour phenotype which exacerbates tumour growth. Three-dimensional (3D) models are gaining in popularity as they can replicate complex in vivo environments without the regulatory or financial barriers presented by in vivo experiments, whilst improving clinical relevance. The research described in this thesis addresses a lack of oral mucosa models containing human macrophages or tools with which to analyse cells cultured within 3D models by developing (i) a 3D model of the oral mucosa containing primary human macrophages, and (ii) an optimised multi-colour flow cytometry panel to quantitatively measure the expression of CD14, CD36, CD45, CD80, CD86, CD163 and CD206 on macrophages cultured within the tissue-engineered oral mucosa. An oral mucosa model containing FNB6 epithelial cells and monocyte-derived macrophages (MDM) was developed and characterised using immunohistochemistry; E-cadherin, AE1/3 and Ki67 were visualised in the epithelium and CD68 staining was visualised in the connective tissue. The expression of vimentin and collagen IV was also analysed. In addition, flow cytometry confirmed the viability of MDM cultured within this model, and ELISA detected a significant increase in IL-6 release following LPS challenge. The use of 8-colour flow cytometry and rheometry provided preliminary results to demonstrate that models containing the OSCC cell line, H357, contained an increased number of CD163+ MDM and a stiffer extracellular matrix compared to those models cultured with normal oral keratinocytes or immortalised keratinocyte cell line, FNB6. These results demonstrate that the macrophage-containing oral mucosa model and molecular tools presented in this thesis will be useful in improving understanding of the role macrophages play in oral cancer progression.