Eicher S A, Clayman G L, Liu T J, Shillitoe E J, Storthz K A, Roth J A, Lotan R
Departments of Head and Neck Surgery, Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Clin Cancer Res. 1996 Oct;2(10):1659-64.
The organotypic (raft) culture system has been shown to be a useful model 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 as a model for topical gene therapy, we cultured the oral head and neck squamous cell carcinoma cell line TR146 on fibroblast-containing collagen gels at the air-medium interface and assessed the efficiency of transduction of a topically applied adenoviral vector containing beta-galactosidase cDNA. Diffuse expression of -galactosidase activity in multiple cell layers demonstrated effective penetration of the vector. 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 organotypic cultures transduced by topical application. Twenty-four, 48, and 72 h after transduction, the cultures were harvested, and residual cell number and FLAG peptide expression were determined. The number of cells 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 the 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 squamous carcinomas and premalignancies.
器官型（筏式）培养系统已被证明是一种有用的模型，可用于在模拟起源组织体内环境的体外条件下，研究生化操作对各种上皮细胞类型的影响。为了研究这种方法作为局部基因治疗的模型，我们将口腔头颈部鳞状细胞癌细胞系TR146培养在含成纤维细胞的胶原凝胶上，置于气-液界面，并评估局部应用含β-半乳糖苷酶cDNA的腺病毒载体的转导效率。多个细胞层中β-半乳糖苷酶活性的弥漫性表达证明了载体的有效渗透。然后在通过局部应用转导的TR146器官型培养物中评估含与FLAG标记相连的野生型p53 cDNA的复制缺陷型重组腺病毒（AdCMV-p53-FLAG）的转导效率和治疗活性。转导后24、48和72小时，收获培养物，测定残留细胞数和FLAG肽表达。在所有三个时间点，p53转导培养物中的细胞数与对照相比均显著减少（P < 0.001），这是由原位DNA末端标记确定的细胞凋亡诱导所致。此外，FLAG肽在残留细胞中弥漫性表达，进一步证实了外源基因产物在多个细胞层中的有效转导和表达。我们得出结论，器官型培养是评估局部应用基因治疗对头颈部鳞状细胞癌和癌前病变疗效的一种有效的体外模型。
