Targeting of suicide gene delivery in pancreatic cancer cells via FGF receptors

Abstract

Pancreatic ductal adenocarcinomas ( PDACs ) overexpress various cell -surface tyrosine kinase receptors, including the type I highaffinity fibroblast growth factor receptor ( FGFR -1 ). The purpose of this study was to determine whether FGFR -targeted gene therapy is feasible in this disorder. Accordingly, the effects of a conjugate consisting of fibroblast growth factor ( FGF ) -2 linked to a Fab 0 fragment against the adenovirus knob region were evaluated in human pancreatic cancer cell lines treated with an adenoviral vector containing the herpes simplex virus thymidine kinase ( AdTK ) gene. An adenoviral vector containing the firefly luciferase reporter gene ( AdLuc ) served to assess infection efficiency, and was initially tested in L6 rat myoblasts. In parental L6 cells that express exceedingly low levels of high -affinity FGFRs, transduction with AdLuc was enhanced 7 -to 10 -fold with the FGF2 -Fab 0 conjugate, whereas in L6 cells transfected to express FGFR -1, it was enhanced 39 -to 52 -fold. The pancreatic cancer cell lines expressed variable levels of the four high -affinity FGF receptors, and exhibited 2 -to 34 -fold increases in gene transduction in the presence of the FGF2 -Fab 0 conjugate. In the absence of FGF2 -Fab 0 there was no correlation between surface binding of FGF2 and AdLuc transduction efficiency, whereas in the presence of FGF2 -Fab 0 , enhanced AdLuc transduction efficiency correlated with greater surface binding of FGF2. In the absence of AdTK, all the cell lines were insensitive to ganciclovir, whereas after AdTK transduction, only ASPC -1 and PANC -1 cells were resistant to ganciclovir even in the presence of FGF2 -Fab 0 . Ganciclovir -mediated inhibition was dependent on the conjugate in CAPAN -1 and COLO -357 cells, but was independent of the conjugate in T3M4 and MIA -PaCa -2 cells. Real -time quantitative PCR of laser -captured cancer cells revealed high levels of various FGFR mRNA species in six of seven PDAC tumor samples. These findings indicate that transduction efficiency with FGF2 -Fab 0 in pancreatic cancer cells is independent of native adenoviral transduction efficiency and is greatest in cells that exhibit concomitant expression of various highaffinity FGFRs. In view of the overexpression of high -affinity FGFRs in the cancer cells in PDAC, our findings also suggest that the combined use of AdTK, ganciclovir, and FGF2 -Fab 0 may ultimately be a promising therapeutic approach in a subgroup of patients with PDAC.

Aberrant Expression of Neuropilin-1 and -2 in Human Pancreatic Cancer Cells

“…Frozen pancreatic cancer tissues sections (5 m) were mounted on uncoated glass slides, and immediately fixed sequentially in 70%, 95%, and 100% ethanol with a final 5 min dehydration step in xylene, followed by air-drying. Morphologically identified pancreatic cancer cells, inflammatory cells, and connective tissues were laser captured separately, using the PixCell I LCM system from Arcturus Engineering (Mountain View, CA), as reported previously (18). Total RNA from each population of laser captured cells, from normal pancreatic tissues, or from pancreatic cancer cell lines was extracted as reported previously (18).…”

“…Morphologically identified pancreatic cancer cells, inflammatory cells, and connective tissues were laser captured separately, using the PixCell I LCM system from Arcturus Engineering (Mountain View, CA), as reported previously (18). Total RNA from each population of laser captured cells, from normal pancreatic tissues, or from pancreatic cancer cell lines was extracted as reported previously (18). DNA was removed by incubating with DNase (10 units) for 2 h at 37°C in the presence of RNase inhibitor (10 units).…”

“…Sequences for target genes were obtained from the National Center for Biotechnology Information GenBank databases (Table 1). Q-PCR analysis was performed using an ABI Prism 7700 Sequence Detection System, as reported previously (18). RNA expression was calculated based on a relative standard curve representing 5-fold dilutions of human cDNA.…”

Targeting the CYP2B1/Cyclophosphamide Suicide System to Fibroblast Growth Factor Receptors Results in a Potent Antitumoral Response in Pancreatic Cancer Models

“…This targeting system has previously been shown to enhance adenoviral transduction of cells that express high-affinity FGF receptors (Goldman et al, 1997;Rancourt et al, 1998;Wang et al, 2005). Several studies have also shown that pancreatic tumor cells and pancreatic adenocarcinoma overexpress FGFRs (Kobrin et al, 1993;Leung et al, 1994;Kleeff et al, 2002;Tyminski et al, FIG. 5.…”

“…Retargeting adenovirus to FGFRs through basic fibroblast growth factor-2 (FGF2) has been shown to enhance cell transduction, both in vivo and in vitro, in various tumor models (Sosnowski et al, 1996;Goldman et al, 1997;Rancourt et al, 1998;Gu et al, 1999;Wang et al, 2005). In pancreatic cancer, it has been shown that targeting AdTK/GCV (adenoviral vector carrying the herpes simplex virus thymidine kinase gene/ganciclovir) enhances the cytotoxic effect of ganciclovir in vitro, although in only a limited number of pancreatic cell lines (Kleeff et al, 2002).…”