Abstracts
2000 Digestive Disease Week

# 2289 Gene Therapy for Pancreatic Cancer Using Soluble VEGF Receptor.
Tohru Hoshida, Makoto Sunamura, Dan G. Duda, Fuyuhiko Motoi, Masaru Onuma, Ryuzaburou Shineha, Susumu Satomi, Seiki Matsuno, Sendai, Japan

This study focuses on the anti-angiogenesis effect of intratumoral delivery of adenovirus encoding the soluble form of the extracellular domain of human flt-1 VEGF receptor. We measured the levels of VEGF protein in the conditioned media of ten human pancreatic cancer cell lines by ELISA and compared them to that of human umbilical vein endothelial cells. The expression of VEGF mRNA was assessed by RT-PCR and Northern blot analysis. Next, we transfected Panc-1 cells with the soluble flt-1 receptor and as control with the lacZ gene using the adenovirus vectors Adsflt and AdlacZ, respectively. Using the MTT assay we quantified the cytopathic effect of the adenoviral transfer at a multiplicity of infection of 0, 10, 50, 100 and, 150. The in vivo subcutaneous growth of Panc-1 cells was measured in NKdepleted SCID mice. Adsflt or AdlacZ (3.5X107 PFU) or 100 ml of PBS were injected directly into tumors daily from day 8 through day 12 after the implantation. Furthermore, we implanted in dorsal skinfold chamber the wild-type Panc-1 and its transfectants and monitored the tumor angiogenesis by our in vivo microscopy system and off-line analysis. Finally, we performed the biotinylated lectin staining of vessels in tumor sections. The VEGF levels in conditioned media were higher in all cell lines (mean, 1969 pg/ml; Panc-1, 980 pg/ml), compared to the level in media of HUVEC (8 pg/ml). The expression of VEGF mRNA was also detected by RT-PCR and Northern blot analysis in all cell lines and it was confirmed by DNA sequencing. In vitro, proliferation of Panc-1 cells infected with Adsflt was not suppressed as compared to those of AdlacZ group or wild-type group. In vivo, tumor growth rate of Adsflt-infected cells was lower than those of AdlacZ group and no treatment group, and the difference was statistically significant. Wild type Panc-1 and the LacZ transfectant implantation prompted strong tumor angiogenesis as observed in skinfold chamber, whereas soluble flt-1 transfectants failed to exert such an effect. The lectin staining showed a decrease in number and diameter of vessels in Adsflt treated tumors as compared to the AdlacZ treated or no treatment ones. Soluble flt-1 VEGF receptor intratumoral delivery by adenoviral vectors inhibited tumor growth in vivo, although it didn’t inhibit cell proliferation after tumor cell transfection in vitro. These results suggest that antiangiogenic gene therapy might be an effective approach for pancreatic cancer treatment.