Gene Therapy of Established Medullary Thyroid Carcinoma with Herpes Simplex Viral Thymidine Kinase in a Rat Tumor Model: Relationship of Bystander Effect and Antitumor Efficacy

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Abstract
Bystander effect (BSE) refers to killing of cells adjacent to a cell engineered to express a killing gene segment. BSE is considered an important aspect of suicide gene therapy with thymidine kinase. We evaluated the BSE of adenovirus expressing herpes simplex thymidine kinase (AdCMVtk) in rat medullary thyroid carcinomas (rMTC) and three rat thyroid epithelial cancer cell lines using an in vitro BSE assay. In the assay, different proportions of infected and uninfected cells are mixed. Only the proportion of directly infected cells was inhibited in the proliferation assay using rMTC cells. This indicates that there is little BSE in this cell line. One rat thyroid epithelial cancer cell line (RTC-R2) has a high BSE, with BSE index (BSEi) of 7. In the proliferation assay a greater proportion of cells was inhibited than those directly infected. BSE was also evaluated during in vivo tumor growth by subcutaneous injection of mixtures of AdCMVtk infected and uninfected cells. Ganciclovir (GCV) treatment of tumors developing from a 1:1 mixture of infected to uninfected rMTC cells failed to inhibit their growth. In contrast, GCV treatment of a 2:8 mixture of infected to uninfected RTC-R2 cells completely inhibit tumor development, indicating a high BSE. BSE is related to in vivo antitumor efficacy when replicationdefective adenovirus AdCMVtk is directly injected into rMTC tumors. After treatment with 100 mg/kg per day of GCV, a growth-retardation effect was observed in small tumors (>100 mm3), but there was little antitumor activity in large tumors (3). Our results indicate that there is a good correlation between this in vitro BSE assay and in vivo treatment efficacy. Not all kinds of tumors are suitable for thymidine kinase (TK)/GCV gene therapy because some lack BSE. Methods to improve BSE and/or transduction efficiency are needed in order to obtain an effective therapeutic result. It will be appropriate to test the BSE in human tumor cells before performing clinical trials with current adenoviral vectors expressing TK.