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

Abstract

The CYP2B1/cyclophosphamide (CPA) suicide gene therapy approach has been shown to be highly promising in clinical trials for the treatment of pancreatic cancer. However, delivering the therapeutic gene to a sufficient number of tumor cells able to trigger a complete response remains a challenge. Target-specific delivery of adenovirus to fibroblast growth factor receptors (FGFRs) has been obtained in a variety of tumor models and has been shown to highly increase transduction efficiency. In the present paper we have tested the therapeutic outcome of retargeting the adenoviral vector, Ad-CYP2B1, to FGFRs, using an FGF2-Fab conjugate, in pancreatic cancer models. First, we show a heterogeneous subcellular distribution of overexpressed FGFR-1 in pancreatic cancer cells. Higher transduction efficiency was observed in five of the six cell lines studied after FGF2-AdGFPLuc infection. Interestingly, an association between FGFR-1 membrane cell expression and viral entry was found. Moreover, tumors injected with FGF2-AdGFPLuc showed enhanced and persistent transgene expression. Importantly, we demonstrate the relevant enhanced cytotoxic effect of the FGF2-Ad-CYP2B1/CPA system in four of the six cell lines studied. Moreover, retargeting Ad-CYP2B1/CPA to FGFRs resulted in a potent antitumoral effect and in an increased survival rate, in two human pancreatic xenograft models. Thus, our results indicate that redirecting adenoviruses to FGFRs highly increases the potency of the suicide system CYP2B1/CPA. Consequently, it may constitute a promising approach to the treatment of patients with pancreatic tumors, in which a high proportion of FGF receptors precisely localize to the plasma membrane. OVERVIEW SUMMARYBioactivation of the CYP2B1 gene by ifosfamide after local injection of microencapsulated CYP2B1-producing cells has shown promising results in the treatment of inoperable pancreatic carcinoma, suggesting that this system is a potent inducer of cell death. In the present work we aimed to study CYB2B1/cyclophosphamide (CPA) antitumoral activity when delivered by a highly efficient gene transfer system based on retargeting adenoviruses to fibroblast growth factor receptors (FGFRs). We showed that although there was some degree of heterogeneity, pancreatic tumor cells highly expressed FGFRs at the plasma membrane. Moreover, retargeting adenovirus to FGFRs enhanced adenoviral transduction and increased CYP2B1/CPA cytotoxicity. Importantly, intratumoral delivery of the Ad-CYP2B1 through FGFRs in combination with cyclophosphamide resulted in a potent antitumoral effect and a significant increase in the survival rate when assessed in two independent xenograft models, indicating the efficient response of pancreatic tumors to this type of therapy.

Gene-Directed Enzyme Prodrug Cancer Therapy

“…Consequently, modification to redirect adenoviral tropism to receptors highly expressed in cancer cells might improve tumor transduction and lead to an increased antitumoral effect. Huch et al showed that in a pancreatic cancer model retargeting Ad-CYP2B1/CPA to fibroblast growth factor receptors resulted in a potent antitumoral effect and an increased survival rate, and it increased the potency of the CYP/CPA system [118].…”

Connexin-26 Is a Key Factor Mediating Gemcitabine Bystander Effect

“…Surgical specimens were obtained as formalin-fixed paraffin-embedded tissues as previously indicated (16). Sections (5-mm thick) were submitted to standard deparaffin processing and treated with 10 mmol/L citrate buffer (pH 6.0) for antigen retrieval.…”

Keratin 7 promoter selectively targets transgene expression to normal and neoplastic pancreatic ductal cellsin vitroandin vivo

“…Tumor cell lines (see Supplemental Information) were maintained as described elsewhere (10,14). Human pancreatic duct epithelial (HPDE) cells were maintained as described elsewhere (15).…”

“…Xenografts were generated as described elsewhere (14). Mice were anesthetized, and D-Firefly-Luciferin (Xenogen, Alameda, CA, USA) was administered intraperitoneally (i.p., 16 mg/kg).…”

“…Mice were anesthetized, and D-Firefly-Luciferin (Xenogen, Alameda, CA, USA) was administered intraperitoneally (i.p., 16 mg/kg). Luciferase activity was visualized and quantified using an in vivo bioluminescent system (IVIS, Xenogen) and the Living Image 2.20.1 software (Xenogen) overlay on Igor Pro4.06A software (Wavematrics, Seattle, WA, USA) as described elsewhere (14). Regions of interest (ROIs) were drawn over tumor or liver areas, and total photons captured were calculated.…”