Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Previous studies have shown that therapeutic resistance arises from the interactions between tumor cells and the tumor microenvironment (TME). In particular, cancer-associated fibroblasts (CAFs) have been shown to induce therapeutic resistance and worsen prognosis through various mechanisms, including the release of growth factors and cytokines, as well as promoting epithelial-mesenchymal transition (EMT) in tumor cells. However, our understanding of CAFs in HPV+ HNSCC compared to HPV- HNSCC in the TME remains limited.
Methods: CAFs were generated by dissociation of fresh tissue collected from surgery from four patients, two of whom has HPV+ HNSCC. After dissociation, cells were cultured in a collagen-coated plate for 3 to 4 weeks. To confirm the identity of CAFs, immunofluorescence and Western blotting were performed using previously identified CAF markers, including FAP and α-SMA. To co-culture CAFs with tumor cells, UM-SCC-47, an HPV-16 positive cell line, was stably transfected with EGFP to enable distinction from CAFs after co-culture. Cells were co-cultured at 1:1 ratio and treated with various concentrations of cisplatin and WST-1 viability assay was performed. Furthermore, the co-cultured cells were enriched through FACS then further analyzed for epithelial markers, mesenchymal markers, cytokines, and membrane receptors using qPCR.?
Results: In this study, CAFs were successfully derived from four HNSCC patients. These CAFs exhibited an elongated spindle shape with multiple branched projection from the cytoplasm. The patient-derived CAFs showed the expression of previously identified CAF markers, FAP and α-SMA via Western blotting and vimentin via immunofluorescence. A greater increase in vimentin was observed when SCC47 was co-cultured with HPV+ CAF (FC 658.58; p < 0.001) compared with SCC47 cells that were not co-cultured with CAFs. Similar trends were observed with other mesenchymal markers, indicating that SCC47 cells were experiencing EMT when co-cultured with CAFs. Lastly, we observed increased cisplatin resistance when SCC47 and CAFs were co-cultured (IC50 7.9μM) compared to non-co-cultured SCC47 cells (13.18μM). We are currently investigating the differences between HPV+ and HPV- CAFs and their differential influence on SCC47.
Conclusion: HPV+ CAFs provide an environment that promotes epithelial-mesenchymal transition and therapeutic resistance in SCC47 cell line. This effect is amplified in co-culture compared to conditioned media, supporting the unique direct cell contact and secreted factors in enhancing invasive properties. Future studies will elucidate the mechanisms underlying the divergent properties of HPV+ CAFs from HPV- CAFs.