Anti-angiogenesis is a novel treatment strategy for pancreatic cancer (PaCa). Activated endothelial cells of the tumor vasculature abundantly express receptors for vascular endothelial growth factor (VEGF), a key mediator of angiogenesis. The aim of the study was to specifically target and damage the vasculature of PaCa by fusing the VEGF isoform VEGF165 to diphtheria toxin (DT), which inhibits the protein synthesis of target cells.
Methods: VEGF165 fusion protein containing the enzymatic and translocation domains of DT was produced with GST in vector pGEX-KG and expressed in E. coli SG12036. In vitro: Two human PaCa cell lines (AsPC-1, poorly differentiated; HPAF-2, moderately differentiated) and human endothelial cells (HUVEC) were exposed to increasing concentrations (1 - 10000 ng/ml) of DT-VEGF. Cell proliferation was assessed after 3 days by cell count and MTT-assay. In vivo: 1 mm3 fragments of sc. PaCa donor tumors were implanted into the pancreas of nude mice which received either DT-VEGF (200 µg/kg, every other day) or vehicle (Con) ip. for 14 weeks. Volume of primary tumor (TU-Vol), metastatic spread (Met-Score), and animal weight were determined at autopsy. Microvessel density (MVD) was analyzed in CD31-stained tumor sections.
Results: In vitro: Proliferation of the PaCa cells was significantly inhibited at high concentrations of DT-VEGF ( 1000 ng/ml). In contrast, DT-VEGF effectively decreased the growth of HUVEC at 10 ng/ml. In vivo: Table (p<0.05 vs. Con).
Conclusions: DT-VEGF fusion protein reduced tumor growth and metastatic spread of PaCa in vivo, resulting in increased survival of the animals. In vitro data and reduced MVD indicate that this effect was due to the toxic effect of DT-VEGF on endothelial cells of the tumor vasculature rather than to direct inhibition of PaCa cell growth. DT-VEGF therapy was not associated with systemic side effects such as weight loss.
in-vivo AsPC/Con AsPC/DT-VEGF HPAF/Con HPAF/DT-VEGF
TU-Vol. (mm3) 1386±155 652±73* 3920±495 927±122*
Met-Score (pts) 16.5±1.3 8.3±1.1* 7.2±1.3 0.75±0.16*
Survival (n/n) 1 / 8 6 / 8* 4 / 8 7 / 8
Weight (gram) 28.2±0.8 27.3±0.9 30.0±0.7 29.6±1.3
MVD /0.74 mm2 69.8±5.1 26.6±3.8* 85.7±7.2 39.6±4.3*