Short-Term Culture and In-Vivo Modeling of Primary HNSCC

Abstract

Problem

Head and neck squamous cell carcinoma (HNSCC) accounts for 4% of annual U.S. cancer deaths. In-vivo models exist using established HNSCC lines, but currently there is no such model that allows consistent growth of HNSCC from primary tumors.

Methods

Primary HNSCC tissue was obtained from 103 patients at biopsy/resection, disaggregated and seeded onto collagen-coated plates in keratinocyte growth media with 10% FBS, additives and antibiotics. After short-term growth in culture, cells were transferred to denuded rat tracheas and implanted subcutaneously in nude mice. Indirect immunofluorescent staining using antibodies specific to cytokeratin, vimentin and nuclear Ku was performed to determine cell lineage and origin.

Results

Cultured cells exhibited morphology consistent with epithelial or stromal derivation. 80% of cultures had viable cells present at 10 days and 24% were maintained 30 days or longer. 5 cultures (5%) proliferated after multiple passages and thrived on uncoated plates in the absence of mesenchymal cells. The xenograft model was able to successfully establish tumors in vivo from 59% of primary tumors. Immunostaining for nuclear Ku and cytokeratin confirmed human origin and epithelial cell lineage, respectively.

Conclusion

The high success rate indicates that selective growth and survival pressures for short-term culture of primary HNSCC may be considerably less than for establishment of cell lines. Additionally, the techniques permit tumor-derived epithelial and mesenchymal cells to be cultured simultaneously. Preliminary data for the in-vivo trachea xenograft model is promising. A luciferase lentiviral system has been created to allow monitoring of tumor growth in vivo with serial live animal imaging.

Significance

These short-term culture techniques may more accurately characterize both the biological diversity of HNSCC and tumor-stromal cell interactions. Once optimized, the trachea xenograft model can be used to determine the in-vivo response of a heterogeneous group of HNSCC to standard and novel therapies.

Support

Funds provided by an endowment for the Barry Baker Laboratory for Head and Neck Oncology, the Vanderbilt Ingram Cancer Center Endowed Professorship Fund, and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation.