The effect of thymidine kinase transduction and ganciclovir therapy on tumor vasculature and growth of 9L gliomas in rats

Abstract

✓ Eradication of malignant brain tumors by in situ intratumoral, retrovirally mediated transfer of the herpes simplex virus thymidine kinase (HSVtk) gene, which sensitizes the tumor cells to ganciclovir, has recently been demonstrated in animal models. The observation that tumors studied in vitro and in animals can be completely eliminated despite only partial transduction of the tumor suggests a bystander mechanism that affects nontransduced tumor cells. Such a bystander effect is not completely understood and may represent a combination of several factors that lead to tumor eradication. Endothelial cells of the tumor blood vessels were shown to occasionally integrate the retroviral vector and thus become sensitized to ganciclovir. In the presence of vector-producer cells, which continuously release infectious viral particles, diffuse multifocal hemorrhages occurred during ganciclovir administration. When the tumor was composed of cells that had been transduced with the thymidine kinase gene before inoculation, no infectious viral particles were present within the tumor, no transduction of endothelial cells occurred, and no hemorrhages were observed during ganciclovir therapy. These observations suggest that tumor regression may be due, in part, to destruction of in vivo HSVtk-transduced endothelial cells after exposure to ganciclovir, resulting in tumor ischemia as one possible bystander mechanism. The authors investigated this hypothesis using the subcutaneous 9L gliosarcoma tumor model in Fischer rats. The tumors were evaluated with Doppler color-flow and ultrasound imaging during the various phases of the study. Twenty rats received intratumoral injections of HSVtk retroviral vector-producer cells (6 × 107 cells/ml) 21 days after bilateral flank tumor inoculation. Ten rats were subsequently treated with intraperitoneal ganciclovir (15 mg/kg/ml twice a day) for 14 days starting on Day 7 after producer cell injection; 10 control rats received intraperitoneal saline injections (1 ml twice a day) instead of ganciclovir. Ultrasound and flow images were obtained before cell injection, before and during ganciclovir or saline administration, and after cessation of treatment. The number, location, and ultrasonographic appearance of tumor vessels and the tumor volumes were recorded. The number of blood vessels in the tumors increased over time in both groups before treatment. Intratumoral cell injection without ganciclovir administration did not influence tumor growth or intratumoral vasculature. However, tumor vasculature decreased after initiation of ganciclovir therapy in the HSVtk-transduced tumors (p < 0.05). Early patchy or diffuse necrotic changes associated with ultrasonographic evidence of scattered intratumoral hemorrhage occurred in tumors treated with ganciclovir. Reduction of the tumor blood supply may be an important feature of HSVtk transduction-mediated tumor regression and may, at least partially, account for the degree of tumor destruction that occurs despite the lack of transduction of all tumor cells.