Head and Neck Squamous Cell Carcinoma (HNSCC) Effects on Dendritic Cell Phenotype in vitro

Innate immune system dendritic cells (DC) are critical to the development of an immune response to infections and cancer. HNSCC produce factors that can affect immune system cell behavior. Specific HNSCC effects on DC have not been examined. Objective: Develop in vitro model to study HNSCC effects on DC precursor migration and DC phenotype and function. Methods: Cells: 1) Human HNSCC cell lines Cal27 and FaDu (ATCC). 2) Immature DC were generated in vitro from normal peripheral blood monocytes in the presence of GM-CSF and IL-4. Immature DC phenotype (CD1a+CD83-CD14+/-) was confirmed by flow cytometry (BD FACS Vantage). Migration-function assays: Immature DC-enriched populations were plated into 3mm porous inserts, above HNSCC cells pre-equilibrated overnight in bottom wells in Gelfoam sponges, with or without DC maturation-inducing factor lipopolysaccharide (LPS). After 3-4 day incubation, cells remaining in inserts were analyzed by flow cytometry for CD1a, CD83 and CCR6 expression. Supernatants were analyzed by ELISA for chemokine MIP-3a and TNFa. Sponges were fixed with 1% paraformaldehyde, sectioned and stained by immunohistochemistry for CD1a and CD83. Results: Presence of HNSCC in bottom chambers significantly increased CD1a expression in non-migrating DC, suggesting that HNSCC may secrete factors that support DC phenotype. Few if any DC migrated to empty sponges, but numerous CD1a+ DC were detected in HNSCC sponges, correlating with HNSCC production of DC chemoattractant MIP-3a. CD83+ mature DC were extremely rare or absent from all sponges, which correlated with low CD83 expression on non-migrating cells, with or without LPS. Lack of predicted LPS effect on CD83 up-regulation may be due to serum-free conditions and lack of CD14 expression. Conclusions: Our in vitro model with removable gelatin matrix is useful for in vitro analysis of HNSCC-DC interactions. HNSCC factors influence DC phenotype. Supported by NIDCR Dental Student Research Fellowship and American Cancer Society Grant #IN-122V.