EVALUATION OF TOPICAL GENE-THERAPY FOR HEAD AND NECK SQUAMOUS-CELL CARCINOMA IN AN ORGANOTYPIC MODEL

The organotypic (raft) culture system has been shown to be a useful mo del for examining the effects of biochemical manipulations on various epithelial cell types, using in vitro conditions that simulate the in vivo environment of the tissue of origin, To investigate this method a s a model for topical gene therapy, we cultured the oral head and neck squamous cell carcinoma cell line TR146 on fibroblast-containing coll agen gels at the air-medium interface and assessed the efficiency of t ransduction of a topically applied adenoviral vector containing beta-g alactosidase cDNA, Diffuse expression of beta(-)galactosidase activity in multiple cell layers demonstrated effective penetration of the vec tor, Transduction efficiency and therapeutic activity of a replication -defective recombinant adenovirus containing wild-type p53 cDNA linked to a FLAG marker (AdCMV-p53-FLAG) were then assessed in TR146 organot ypic cultures transduced by topical application, Twenty-four, 48, and 72 h after transduction, the cultures were harvested, and residual cel l number and FLAG peptide expression were determined, The number of ce lls in p53 transduced cultures was significantly reduced in comparison to controls at all three time points (P < 0.001), which resulted from the induction of apoptosis as determined by in situ DNA end labeling, In addition, the FLAG peptide was expressed diffusely in the residual cells, further confirming effective transduction and expression of th e exogenous gene products throughout multiple layers, We conclude that the organotypic culture is an effective in vitro model for assessing the efficacy of topically applied gene therapy on head and neck squamo us carcinomas and premalignancies.