Objectives: Tenascin-C (TNC) is a large extracellular matrix glycoprotein expressed during embryogenesis, wound healing, chronic inflammation and in various cancers. Prior pathology studies in oral head and neck squamous cell carcinoma (SCCa) have found the expression of TNC to be increased in metastatic oral SCCa. TNC is produced by both epithelial tumor cells as well as cancer-associated fibroblasts (CAFs) and deposited into the extracellular matrix (ECM) where it contributes to the remodeling of the tumor microenviroment (TME) and the epithelial-to-mesenchymal transition of tumor cells. The exact mechanism of action of TNC is unknown, however, TNC does increase expression of matrix metalloproteinases, which remodel the TME/ECM to facilitate cell migration and metastasis. The goal of this study was to determine if TNC was produced by head and neck SCCa cell lines isolated directly from patient tumor samples, and if expression of TNC was necessary for cancer cell invasion.
Materials/Methods: Expression of TNC was confirmed in various human head and neck SCCa tissue samples and low-passage cancer cell lines via RT-PCR, TNC specific immunohistochemistry (IHC), and western blot analysis. Location of primary tumor samples included tonsil, base of tongue, hypopharynx, oral tongue, and floor of mouth. Treatment with TNC-specific siRNA and shRNA was utilized to reduce the expression of TNC in cancer cell lines (CUHN013C, CUHN036C), which was confirmed by RT-PCR and western blot analysis. Matrigel invasion assays were performed comparing wild-type CUHN013C and TNC siRNA-treated cells. For the invasion assays, cells were plated in serum-free media on matrigel inserts placed in serum-containing media and allowed to incubate for 24 hours. Inserts were then stained and cell invasion was quantified. Scratch assays were also performed comparing wild-type CUHN013C and TNC siRNA-treated cells.
Results: TNC is present in the tumor microenvironment, especially at the tumor-stroma interface, in head and neck SCCa primary tumors as well as lymph node metastases. Immortalized cancer cell lines derived from patient tumor samples also express TNC, which can be significantly (p<0.05) decreased by TNC-specific siRNA or shRNA treatment. Decreased expression of TNC results in significantly (p<0.05) reduced invasion of CUHN013C cancer cells in matrigel invasion assays. Interestingly, decreased TNC expression did not affect migration of CUHN013C cancer cells in scratch assays.
Conclusion: TNC is expressed by head and neck SCCa cells from a variety of primary sites. TNC expression is necessary for invasion of head and neck SCCa cells in an in vitro invasion assay but does not affect migration. Therapies specifically targeting TNC may reduce the invasiveness and metastatic potential of head and neck SCCa.