The Role Of Adipocytes In The Oral Squamous Cell Carcinoma Microenvironment

Introduction: Oral squamous cell carcinoma (OSCC) is a significant cause of death and morbidity in patients with head and neck cancer. Despite technical improvements in treatments, the outcome for such patients has remained poor over recent years. This poor prognosis relates to the late diagnosis, invasion of critical local structures, complicated anatomy of the head and neck region and the high frequency of recurrence. The presence of lymph node metastasis is common although the precise mechanism of this spread is poorly understood. In particular the role of the interaction between malignant cells and adipocytes in the stroma surrounding the tumour is unclear. This has been shown to have an important role in the progression of other tumour types. Adipocytes can act as a source of energy for tumour cells and also represent a reservoir of chemical signals such as adipocytokines. Therefore, the hypothesis of this study is that adipocytes may represent an important candidate in tumour-stromal crosstalk in OSCC. Aim: The present study aims to investigate the role of adipocytes and adipokines in OSCC cell behaviour. Materials and Methods: The effect of adipocyte conditioned media (ACM) from differentiated mouse 3T3-L1 adipocytes, and the most abundant adipocyte-secreted adipokines (adiponectin (APN) and leptin (LEP)), on oral cancer cell lines (H357 and SCC-9) proliferation and migration was assessed using MTS proliferation assays and Transwell migration assays. The presence of adipocyte ligands and their receptors on H357 and SCC-9 cell lines and OSCC tissue sections was investigated using flow cytometry (FCM) and immunohistochemistry (IHC). The impact of ACM on activating oral fibroblasts, and inducing epithelial-mesenchymal transition (EMT) in H357 and SCC-9 cells was examined with immunofluorescence (IF), Western blot (WB) and reverse transcriptase polymerase chain reaction (RT-PCR). Adipokine arrays were used to screen and examine the difference between culture supernates of adipocytes cultured alone and adipocytes co-cultured with H357 or SCC-9 cells using an interactive co-culture assay. Results: The results showed that ACM, APN and LEP stimulate migration of oral cancer cells with no significant influence on cell proliferation. FCM results revealed that APN and LEP receptors are widely expressed on the surface of the H357 and SCC-9 cells. IHC results showed a variable expression of LEP receptor (OBR), APN and its receptor AdipoR2 in different grades of OSCC tissue sections. WB findings demonstrated that ACM induced an increase in the expression of EMT transcription factors and mesenchymal markers in H357 and SCC-9 cells, and a decrease in the epithelial marker, E-cadherin, in both cell lines. However, the changes were not statistically significant. Whereas, RT-PCR results showed a significant decrease in the E-cadherin in both cells (P=0.0085 and P=0.0026, respectively), and no significant changes in the other EMT markers. The adipokine array results revealed that H357 and SCC-9 cells increase the expression of a wide variety of adipocytokines in co-cultured adipocytes as compared to those cultured separately. Conclusion: Adipocytes may influence oral cancer spread by secreting adipokines, which the results have shown can enhance oral cancer cell motility in vitro. APN and LEP receptors are widely expressed on OSCC cell lines, which explains the response of these cells to their ligands. The IF results indicated that adipocytes may be capable of activating normal fibroblasts, giving them the characteristics of myofibroblasts which have been previously shown to be an important marker of poor prognosis in OSCC. ACM also stimulated partial EMT changes in OSCC cell lines suggesting a potential role of adipocytes within the OSCC microenvironment that favours the spread of cancer. Furthermore, oral tumour cells stimulate adipocytes to produce a higher amount of different adipocytokines suggesting a potential alteration in adipocyte phenotype that may further promote cancer progression and metastasis. Future research can now build on the foundation established by this study to examine in depth the role of adipocytes in OSCC progression and metastasis.