IADR Abstract Archives
Novel tissue-engineered constructs to examine the role of fibroblast-derived extracellular matrix in oral cancer progression
Objectives: Cancer progression is determined by the interaction between tumour cells, stromal cells (fibroblasts and leukocytes) and other components of the microenvironment such as the extracellular matrix (ECM). The ECM is predominantly deposited by cancer-associated fibroblasts (CAF) and plays a key role in cancer progression. Although evidence exists demonstrating a role for CAF in oral squamous cell carcinoma (OSCC), little is known about their influence on ECM: tumour interactions. This project aims to generate novel tissue-engineered 3D constructs using normal oral fibroblast- (NOF) and CAF-derived ECM, as a native scaffold to accurately model ECM: tumour interactions in OSCC progression.
Methods: NOF isolated from normal oral mucosa, CAF isolated from OSCC and NOF stimulated with TGFβ-1 (to induce transdifferentiation into an αSMA-positive ‘CAF-like’ phenotype) were cultured as monolayers and their phenotype analysed using qPCR and immunoblotting both short (24-72 hours) and longer-term (14 days). Different culture conditions were investigated to stimulate NOF- and CAF-derived ECM deposition and full-thickness epithelium models produced using normal and OSCC cell lines. Models were characterised by immunoblotting and immunohistochemistry for markers of proliferation (Ki67), epithelial differentiation (AE1/3) and ECM deposition (collagen I).
Results: TGFβ-1 treatment of NOF stimulated transdifferentiation into a CAF-like phenotype with cells expressing αSMA-rich stress fibres and fibronectin extra domain A (FN1-EDA). Tumour-derived CAF expressed αSMA, FN1-EDA, collagen I and versican. NOF stimulated to produce ECM over a four-week culture period, generated an organised matrix with an average thickness of ~200 µm compared to TGFβ-1-treated NOF and CAF that produced thicker (350 µm), irregular ECM. Immunoblotting and immunohistochemical analysis of the models revealed a significantly different protein deposition between the NOF and CAF phenotypes.
Conclusions: Using tissue-engineering techniques it is possible to model fibroblast-mediated ECM deposition providing a novel, pathophysiologically relevant in vitro tool for the study of OSCC progression.
Methods: NOF isolated from normal oral mucosa, CAF isolated from OSCC and NOF stimulated with TGFβ-1 (to induce transdifferentiation into an αSMA-positive ‘CAF-like’ phenotype) were cultured as monolayers and their phenotype analysed using qPCR and immunoblotting both short (24-72 hours) and longer-term (14 days). Different culture conditions were investigated to stimulate NOF- and CAF-derived ECM deposition and full-thickness epithelium models produced using normal and OSCC cell lines. Models were characterised by immunoblotting and immunohistochemistry for markers of proliferation (Ki67), epithelial differentiation (AE1/3) and ECM deposition (collagen I).
Results: TGFβ-1 treatment of NOF stimulated transdifferentiation into a CAF-like phenotype with cells expressing αSMA-rich stress fibres and fibronectin extra domain A (FN1-EDA). Tumour-derived CAF expressed αSMA, FN1-EDA, collagen I and versican. NOF stimulated to produce ECM over a four-week culture period, generated an organised matrix with an average thickness of ~200 µm compared to TGFβ-1-treated NOF and CAF that produced thicker (350 µm), irregular ECM. Immunoblotting and immunohistochemical analysis of the models revealed a significantly different protein deposition between the NOF and CAF phenotypes.
Conclusions: Using tissue-engineering techniques it is possible to model fibroblast-mediated ECM deposition providing a novel, pathophysiologically relevant in vitro tool for the study of OSCC progression.