(0616) Cancer-Associated Fibroblasts Demonstrate Aberrant Cytokine Secretion and Promote Cell Proliferation of Head and Neck Squamous Cell Carcinoma

Introduction: In head and neck squamous cell carcinoma (HNSCC), high densities of cancer-associated fibroblasts (CAFs) are associated with advanced stage disease, vascular and perineural invasion, and other poor prognostic factors. The molecular crosstalk between cancer and CAFs is complex and not fully elucidated. In our study, we compared the secretomes of normal dermal fibroblasts (HDFs) and CAFs from patient-derived HNSCCs, and test how CAFs affect HNSCC cell proliferation and spread in vitro.

Methods: Fresh HNSCC tumor was harvested in the operating room, enzymatically digested, and plated. Fibroblast and epithelial cell expansion media were used to culture primary CAF and primary HNSCC cells, respectively. HDF and two oral cavity SCC (SCC9, UCSF-OT-1109) cell lines were cultured in parallel. Immunocytochemistry (ICC) was performed to confirm expression of S100A4 (fibroblast marker) in fibroblasts and EpCAM (epithelial carcinoma marker) in HNSCC cells. Conditioned media was collected at 48 hours for secretome analysis. HNSCC spheroids were cultured alone or with CAFs and live-cell imaging was performed to assess fibroblast effect on cancer proliferation in vitro.

Results: Patient-derived CAFs and HNSCC cells were successfully isolated and cultured. On ICC, fibroblast and cancer cell lines expressed high levels of S100A4 and EpCAM, respectively. Compared to normal fibroblasts, CAFs secreted significantly higher levels of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and epidermal growth factor (EGF). Additionally, HNSCC cultures with CAFs facilitated more cancer cell proliferation and spread than HNSCC cultures without fibroblasts.

Conclusions: We describe a technique for developing primary CAF and HNSCC cell lines from HNSCC tumor samples. Compared to normal fibroblasts, CAFs secreted higher levels of multiple cytokines that have been linked to tumorigenesis and metastasis. CAFs also promoted cell proliferation and spread of HNSCC in vitro. Further investigations into the role of CAFs within the TME may lead to the development of novel therapeutic targets for HNSCC.