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Oral squamous cell carcinomas (OSCC) contain a high number of immune cells with tumour associated macrophages (TAMs) being elevated compared to normal oral mucosa suggesting a role in tumour development (Xue Y et al 2022). A higher density of TAMs is observed at the more advanced stages of a number of tumour types and they also correlate with poor clinical outcomes in cancer including OSCC (Hu Y et al 2016).

The overall hypothesis of the proposed study is that there is a bi-directional relationship between macrophages and OSCC cells promoting tumourigenesis that can be modelled in vitro. We propose that the presence of M2 like macrophages in a reconstituted tumour microenvironment can influence the invasive behaviour of OSCC cell lines and invasive OSCC cells influence the phenotype of macrophages.

To commence our work, simple co-culture experiments will be performed using the monocytic cell line THP-1 which is commonly using to model macrophage phenotypes (Daigneault M et al 2010). These will be treated with phorbol myristate acetate (PMA) to generate M0 macrophages which then can be treated with interferon-ϒ and interleukin-4 to generate M1 and M2 like phenotypes respectively. OSCC cell lines can then be exposed to conditioned medium generated from the different macrophage phenotypes and their migration and invasion behaviour followed using ‘scratch’ and transwell migration and invasion assays. Expression of markers of epithelial to mesenchymal transition and matrix metalloproteinases will also be followed.

Oral mucosal models will be generated using collagen I gels with and without gingival fibroblasts as previously described (Jennings LR et al 2016). Macrophages of different phenotypes can also be included in this connective tissue equivalent and differentiation markers used to confirm their phenotype. Localisation of differentiated THP-1 cells can be followed using cytoplasmic loading of fluorescent CFSE. The matrices will be overlayed with OSCC cell lines and invasion of these cells into the connective tissue equivalent followed using histological processing and quantified using image analysis.