uPAR-controlled oncolytic adenoviruses eliminate cancer stem cells in human pancreatic tumors

Abstract Pancreatic tumors contain cancer stem cells highly resistant to chemotherapy. The identification of therapies that can eliminate this population of cells might provide with more effective treatments. In the current work we evaluated the potential of oncolytic adenoviruses to act against pancreatic cancer stem cells (PCSC). PCSC from two patient-derived xenograft models were isolated from orthotopic pancreatic tumors treated with saline, or with the chemotherapeutic agent gemcitabine. An enrichment in the number of PCSC expressing the cell surface marker CD133 and a marked enhancement on tumorsphere formation was observed in gemcitabine treated tumors. No significant increase in the CD44, CD24, and epithelial-specific antigen (ESA) positive cells was observed. Neoplastic sphere-forming cells were susceptible to adenoviral infection and exposure to oncolytic adenoviruses resulted in elevated cytotoxicity with both Adwt and the tumor specific AduPARE1A adenovirus. In vivo, intravenous administration of a single dose of AduPARE1A in human-derived pancreatic xenografts led to a remarkable anti-tumor effect. In contrast to gemcitabine AduPARE1A treatment did not result in PCSC enrichment. No enrichment on tumorspheres neither on the CD133+ population was detected. Therefore our data provide evidences of the relevance of uPAR-controlled oncolytic adenoviruses for the elimination of pancreatic cancer stem cells

Intraductal Delivery Of Adenoviruses Targets Pancreatic Tumors In Transgenic Ela-myc Mice And Orthotopic Xenografts

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-TKT 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<0.0001) when combined with GCV. Of notice, both AduPARTK(T) and ICOVIR15-TKT antitumoral responses were stronger by ductal viral application than intravenously, in line with the 38-fold increase in pancreas transduction observed upon ductal administration. In summary our data show that cytotoxic adenoviruses retrogradelly injected to the pancreas can be a feasible approach to treat localized pancreatic tumors

uPAR-controlled oncolytic adenoviruses eliminate cancer stem cells in human pancreatic tumors

Pancreatic tumors contain cancer stem cells highly resistant to chemotherapy. The identification of therapies that can eliminate this population of cells might provide with more effective treatments. In the current work we evaluated the potential of oncolytic adenoviruses to act against pancreatic cancer stem cells (PCSC). PCSC from two patient-derived xenograft models were isolated from orthotopic pancreatic tumors treated with saline, or with the chemotherapeutic agent gemcitabine. An enrichment in the number of PCSC expressing the cell surface marker CD133 and a marked enhancement on tumorsphere formation was observed in gemcitabine treated tumors. No significant increase in the CD44, CD24, and epithelial-specific antigen (ESA) positive cells was observed. Neoplastic sphere-forming cells were susceptible to adenoviral infection and exposure to oncolytic adenoviruses resulted in elevated cytotoxicity with both Adwt and the tumor specific AduPARE1A adenovirus. In vivo, intravenous administration of a single dose of AduPARE1A in human-derived pancreatic xenografts led to a remarkable anti-tumor effect. In contrast to gemcitabine AduPARE1A treatment did not result in PCSC enrichment. No enrichment on tumorspheres neither on the CD133+ population was detected. Therefore our data provide evidences of the relevance of uPAR-controlled oncolytic adenoviruses for the elimination of pancreatic cancer stem cells

Irreversible electroporation shows efficacy against pancreatic carcinoma without systemic toxicity in mouse models

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

Insulin-Like Growth Factor I (IGF-I) Expressed from an AAV1 Vector Leads to a Complete Reversion of Liver Cirrhosis in Rats

<div><p>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.</p></div

Irreversible electroporation shows efficacy against pancreatic carcinoma without systemic toxicity in mouse models

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

Analysis of MMPs and MMP inhibitors.

<p>Animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162955#pone.0162955.g001" target="_blank">Fig 1</a> and MMP-1, 2, 9 and 14 mRNAs (a) and MMP-2 and 9 proteins (b) were evaluated in liver extracts by qRT-PCR or ELISA respectively. Total MMP activity in liver extracts (c) and the levels of MMP inhibitor TIMP-2 mRNA (d) were also evaluated. Transcript levels shown are relative to GAPDH mRNA levels. Error bars denote standard deviations. Significant and non-significant (ns) differences are highlighted.</p

Functional IGF-I is expressed in the liver after administration of AAVIGF-I or SVIGF-I.

<p>(a) Schematic of the experimental protocol used. Healthy animals were untreated, or used to induce liver cirrhosis by intragastric administration of CCl₄ once a week for eight weeks (w). One week after the end of the cirrhosis induction protocol, cirrhotic animals were treated with saline, AAVLuc, AAVIGF-I or SVIGF-I by intra-arterial administration. Animals were sacrificed 4 days, 8 weeks and 16 weeks after vector inoculation. Healthy animals were sacrificed in parallel as controls. Blood was collected and liver samples were processed for histology, and purification of RNA and proteins for further analysis. (b) Analysis of liver IGF-I expression and activity. Liver samples were obtained from healthy, cirrhotic animals treated with saline (Ci), AAVLuc (Ci+AAVLuc), AAVIGF-I (Ci+AAVIGF-I) or SVIGF-I (Ci+SVIGF-I). Total IGF-I protein (a) and IGF-I, IGFBP3 and IGF-IR mRNAs were quantified by ELISA and qRT-PCR in liver extracts. Transcript levels shown are relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. Samples were obtained 4 days or 8 weeks after vector inoculation. Error bars denote standard deviations. Significant and non-significant (ns) differences are highlighted.</p

Analysis of liver damage, pro-inflammatory and pro-fibrogenic factors.

<p>Animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162955#pone.0162955.g001" target="_blank">Fig 1</a> and TGFβ (a), TNFα (b) and IL-6 (c) mRNA and protein levels were evaluated in liver extracts by qRT-PCR or ELISA. CTGF, VEGF and PDGF mRNA levels were also measured. Transcript levels shown are relative to GAPDH mRNA levels. (e) Transaminases AST, ALT and ALP were quantified in the serum of the animals 8 weeks after vector administration. Error bars denote standard deviations. Significant and non-significant (ns) differences are highlighted.</p