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IGF-I modulates liver tissue homeostasis. It is produced by hepatocytes and signals within the liver through IGF-I receptor expressed on hepatic stellate cells (HSCs). Liver cirrhosis is characterized by marked IGF-I deficiency. Here we compared the effect of two different gene therapy vectors encoding IGF-I as a potential treatment for cirrhotic patients. Rats with carbon tetrachloride-induced liver cirrhosis were treated with controls or with adeno-associated virus 1 (AAV) or simian virus 40 (SV40) vectors expressing IGF-I (AAVIGF-I or SVIGF-I) and molecular and histological studies were performed at 4 days, 8 weeks and 16 weeks. Increased levels of IGF-I were observed in the liver as soon as 4 days after vector administration. Control cirrhotic rats showed increased hepatic expression of pro-inflammatory and pro-fibrogenic factors including transforming growth factor beta (TGFβ), tumor necrosis factor-alpha (TNFα), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF) together with upregulation of α-smooth muscle actin (αSMA), a marker of HSC activation. In IGF-I-treated rats the levels of all these molecules were similar to those of healthy controls by week 8 post-therapy. Of note, the decline of TGFβ, CTGF, VEGF and αSMA expression was more rapid in AAVIGF-I treated animals reaching statistical significance by day 4 post-therapy. IGF-I-treated rats showed similar improvement of liver function tests in parallel with upregulation of hepatocyte nuclear factor 4α (HNF4α), a factor that promotes hepatocellular differentiation. A significant decrease of liver fibrosis, accompanied by upregulation of the hepatoprotective and anti-fibrogenic hepatocyte growth factor (HGF), occurred in all IGF-I-treated rats but complete reversal of liver cirrhosis took place only in AAVIGF-I group. Therefore, AAVIGF-I reverts liver cirrhosis in rats, a capability which deserves clinical testing.

We investigated whether gene transfer of insulin-like growth factor I (IGF-I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver cirrhosis induced by carbon tetrachloride (CCl4) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF-I (SVIGF-I), or luciferase (SVLuc). Animals were sacrificed 8 weeks after vector injection. In cirrhotic rats we observed that, whereas IGF-I was synthesized by hepatocytes, IGF-I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF-I showed increased hepatic levels of IGF-I, improved liver function tests, and reduced fibrosis in association with diminished α-smooth muscle actin expression, up-regulation of matrix metalloproteases (MMPs) and decreased expression of the tissue inhibitors of MMPs TIM-1 and TIM-2. SVIGF-I therapy induced down-regulation of the profibrogenic molecules transforming growth factor beta (TGFβ), amphiregulin, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endothelium growth factor (VEGF) and induction of the antifibrogenic and cytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF-I-treated animals showed decreased expression of Wilms tumor-1 (WT-1; a nuclear factor involved in hepatocyte dedifferentiation) and up-regulation of hepatocyte nuclear factor 4 alpha (HNF4α) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF-I was also tested in rats with thioacetamide-induced liver cirrhosis. Also in this model, SVIGF-I improved liver function and reduced liver fibrosis in association with up-regulation of HGF and MMPs and down-regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1). Conclusion: IGF-I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF-I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation. (HEPATOLOGY 2010;51:912–921.)

// Anabel Jose 1,2 , Luciano Sobrevals 1,2 , Juan Miguel Camacho-Sanchez 3 , Meritxell Huch 2 , Nuria Andreu 2 , Eduard Ayuso 4 , Pilar Navarro 5 , Ramon Alemany 3 , Cristina Fillat 1,2 1 Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Barcelona. 2 Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Barcelona. 3 Laboratori de Recerca Traslacional IDIBELL-Institut Catala d’Oncologia, L’Hospitalet de Llobregat. 4 Centre de Biotecnologia Animal i Terapia Genica and Centro de Investigacion Biomedica en Red de Diabetes y Enfermedades Metabolicas Asociadas CIBERDEM, Barcelona. 5 Institut de Recerca Hospital del Mar-IMIM, Barcelona, Spain. Correspondence: Cristina Fillat, email: // Keywords : Pancreatic cancer, adenovirus, orthotopic xenografts, transgenic mice, thymidine kinase. Received : December 21, 2012, Accepted : January 10, 2013, Published : January 12, 2013 Abstract Gene-based anticancer therapies delivered by adenoviruses are limited by the poor viral distribution into the tumor. In the current work we have explored the feasibility of targeting pancreatic tumors through a loco-regional route. We have taken advantage of the ductal network in the pancreas to retrogradelly inject adenoviruses through the common bile duct in two different mouse models of pancreatic carcinogenesis: The transgenic Ela-myc mice that develop mixed neoplasms displaying both acinar-like and duct-like neoplastic cells affecting the whole pancreas; and mice bearing PANC-1 and BxPC-3 orthotopic xenografts that constitute a model of localized human neoplastic tumors. We studied tumor targeting and the anticancer effects of newly thymidine kinase-engineered adenoviruses both in vitro and in vivo, and conducted comparative studies between intraductal or intravenous administration. Our data indicate that the intraductal delivery of adenovirus efficiently targets pancreatic tumors in the two mouse models. The in vivo application of AduPARTK T plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TK T oncolytic adenoviruses significantly improved mean survival of mice bearing PANC-1 and BxPC-3 pancreatic xenografts from 30 to 52 days and from 20 to 68 days respectively (p

Pancreatic Ductal Adenocarcinoma (PDAC) therapies show limited success./nIrreversible electroporation (IRE) is an innovative loco-regional therapy in which highvoltage pulses are applied to induce plasma membrane defects leading to cellular death. In the present study we evaluated the feasibility of IRE against PDAC. IRE/ntreatment exhibited significant antitumor effects and prolonged survival in mice with orthotopic xenografts. Extensive tumor necrosis, reduced tumor cell proliferation and disruption of microvessels were observed at different days post-IRE. Animals had transient increases in transaminases, amylase and lipase enzymes that normalized at/n24h post-IRE. These results suggest that IRE could be an effective treatment for locally advanced pancreatic tumors. This work was supported by the Spanish/nMinistry of Science and Innovation (MICINN), BIO2008-04692-C03-02/01 and received/npartial support from the Generalitat de Catalunya SGR091527. Anabel José was a/nrecipient of a FPU fellowship from the MICINN. Fillat’s group is also partially funded by/nCIBERER and by IIS10/00014 from Instituto de Salud Carlos III. Ivorra’s research is/ncurrently supported by a Ramón y Cajal fellowship from the Spanish Ministry for/nScience and Innovation and a Marie Curie IRG grant from the European Commission.

Background Combined treatment of oncolytic adenoviruses with chemotherapeutic agents is foreseen as a therapeutic option for cancer. Here we have investigated the potential to use gemcitabine in combination with the oncolytic adenovirus AduPARE1A to treat pancreatic cancer and evaluate the underlying mechanism. Methods We treated pancreatic cancer cell lines BxPC-3 and PANC-1 with AduPARE1A and gemcitabine individually or in combination and analyzed cell viability, combination index, apoptosis and viral production. We also investigated the effects of the combination on tumor growth and mice survival in two xenograft models. Furthermore, we analyzed uPAR promoter activity from different uPAR-controlled adenovirus and studied NF-κB mediated effects. Results Synergistic cell killing from the combination AduPARE1A/Gemcitabine was observed in BxPC-3 and PANC-1 cells. Moreover, the combination treatment produced therapeutic benefits over either individual modality in two mouse models bearing orthotopic tumors, showing reduced tumor progression and significant prolonged mouse survival. Mechanistic studies showed that the synergistic cell death was not due to an increase in viral replication but occurred through an enhancement of apoptotic cell death. Gemcitabine stimulation increased the transcription of uPAR-controlled transgenes through the induction of NF-κB acting on the uPAR promoter. Interestingly, NF-κB gemcitabine-mediated induction of AduPAR adenoviruses interfered with the activation of NF-κB regulated genes, probably as a result of an intracellular competition for NF-κB DNA binding. Consequently, AduPARE1A infection sensitized cells to gemcitabine-induced apoptosis in the combined treatment. Conclusions These data highlights the potential of the combination as a treatment modality for pancreatic cancer patients. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0413-2) contains supplementary material, which is available to authorized users.