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Pancreatic carcinoma is a gastrointestinal malignancy with poor prognosis. Treatment with gemcitabine, the most potent chemotherapeutic against this cancer up to date, is not curative, and resistance may appear. Complementary treatment with an oncolytic virus, such as the rat parvovirus H-1PV, which is infectious but nonpathogenic in humans, emerges as an innovative option.Purpose: To prove that combining gemcitabine and H-1PV in a model of pancreatic carcinoma may reduce the dosage of the toxic drug and/or improve the overall anticancer effect.Experimental Design: Pancreatic tumors were implanted orthotopically in Lewis rats or subcutaneously in nude mice and treated with gemcitabine, H-1PV, or both according to different regimens. Tumor size was monitored by micro-computed tomography, whereas bone marrow, liver, and kidney functions were monitored by measuring clinically relevant markers. Human pancreatic cell lines and gemcitabine-resistant derivatives were tested in vitro for sensitivity to H-1PV infection with or without gemcitabine.Results:In vitro studies proved that combining gemcitabine with H-1PV resulted in synergistic cytotoxic effects and achieved an up to 15-fold reduction in the 50% effective concentration of the drug, with drug-resistant cells remaining sensitive to virus killing. Toxicologic screening showed that H-1PV had an excellent safety profile when applied alone or in combination with gemcitabine. The benefits of applying H-1PV as a second-line treatment after gemcitabine included reduction of tumor growth, prolonged survival of the animals, and absence of metastases on CT-scans.Conclusion: In addition to their potential use as monotherapy for pancreatic cancer, parvoviruses can be best combined with gemcitabine in a two-step protocol.

Supplementary Data from Improvement of Gemcitabine-Based Therapy of Pancreatic Carcinoma by Means of Oncolytic Parvovirus H-1PV

Hypoxia and dysfunctional tumor vessels represent a prominent feature of pancreatic cancer, being, at least in part, responsible for chemotherapy resistance and immune suppression in these tumors. We tested whether the increase of oxygen delivery induced in vivo by myo-inositol trispyrophosphate (ITPP) can reverse hypoxia, control tumor growth and improve chemotherapy response. Tumor size, metastatic development (microcomputed tomography scan follow-up) and the survival of rats and nude or NOD.SCID mice, (bearing syngenic rat and MiaPaCa2- or patient-derived pancreatic tumors), were determined on ITPP and/or gemcitabine treatment. Partial oxygen pressure, expression of angiogenic factors and tumor histology were evaluated. Infiltration and oxidative status of immune cells, as well as chemotherapy penetration in tumors, were determined by fluorescence-activated cell sorting, fluorometry, nitric oxide release assays, Western blot and confocal microscopy. Weekly intravenous ITPP application resulted in the inhibition of metastasis development and restricted primary tumor growth, showing a superior effect on the rats' survival compared with gemcitabine. ITPP treatment restored tumor normoxia and caused a reduction in hypoxia inducible factor-1α levels, with subsequent VEGF and Lox downregulation, resulting in improved vessel structure and decreased desmoplasia. The latter effects translated into elevated immune cells influx and improved susceptibility to gemcitabine treatment. Growth of human pancreatic tumor xenografts was strongly inhibited by administration of ITPP. ITPP exploits a two-stage mechanism causing rapid, early and sustainable late stage normoxia. This is due to the angiogenic factor modulation and vascular normalization, leading to enhanced chemotherapy delivery and synergistic life prolongation, on combination with low doses of gemcitabine.

The rat parvovirus H-1PV has oncolytic and tumour-suppressive properties potentially exploitable in cancer therapy. This possibility is being explored and results are encouraging, but it is necessary to improve the oncotoxicity of the virus. Here we show that this can be achieved by co-treating cancer cells with H-1PV and histone deacetylase inhibitors (HDACIs) such as valproic acid (VPA). We demonstrate that these agents act synergistically to kill a range of human cervical carcinoma and pancreatic carcinoma cell lines by inducing oxidative stress, DNA damage and apoptosis. Strikingly, in rat and mouse xenograft models, H-1PV/VPA co-treatment strongly inhibits tumour growth promoting complete tumour remission in all co-treated animals. At the molecular level, we found acetylation of the parvovirus nonstructural protein NS1 at residues K85 and K257 to modulate NS1-mediated transcription and cytotoxicity, both of which are enhanced by VPA treatment. These results warrant clinical evaluation of H-1PV/VPA co-treatment against cervical and pancreatic ductal carcinomas.

Our aim was to establish and characterize a novel pancreatic ductal adenocarcinoma cell line from a patient in whom the origin of the invasive carcinoma could be traced back to the intraductal papillary mucinous neoplasm (IPMN) precursor lesion.The primary patient-derived tumor was propagated in immunocompromised mice for 2 generations and used to establish a continuous in vitro culture termed ASAN-PaCa. Transplantation to fertilized chicken eggs confirmed the tumorigenic potential in vivo. Molecular analyses included karyotyping, next-generation genomic sequencing, expression analysis of marker proteins, and mucin-profiling.The analysis of marker proteins confirmed the epithelial nature of the established cell line, and revealed that the expression of the mucin MUC1 was higher than that of MUC2 and MUC5AC. ASAN-PaCa cells showed rapid in vitro and in vivo growth and multiple chromosomal aberrations. They harbored mutations in KRAS (Q61H), TP53 (Y220C), and RNF43 (I47V and L418M) but lacked either IPMN-specific GNAS or presumed pancreatic ductal adenocarcinoma-driving mutations in KRAS (codons 12/13), SMAD, and CDKN2A genes.ASAN-PaCa cell line represents a novel preclinical model of pancreatic adenocarcinoma arising in the background of IPMN, and offers an opportunity to study how further introduction of known driver mutations might contribute to pancreatic carcinogenesis.

Oncolytic viruses with their capacity to specifically replicate in and kill tumor cells emerged as a novel class of cancer therapeutics. Rat oncolytic parvovirus (H-1PV) was used to treat different types of cancer in preclinical settings and was lately successfully combined with standard gemcitabine chemotherapy in treating pancreatic ductal adenocarcinoma (PDAC) in rats. Our previous work showed that the immune system and particularly the release of interferon-gamma (IFNγ) seem to mediate the anticancer effect of H-1PV in that model. Therefore, we reasoned that the therapeutic properties of H-1PV can be boosted with IFNγ for the treatment of late incurable stages of PDAC like peritoneal carcinomatosis. Rats bearing established orthotopic pancreatic carcinomas with peritoneal metastases were treated with a single intratumoral (i.t.) or intraperitoneal (i.p.) injection of 5 x 10⁸ plaque forming units of H-1PV with or without concomitant IFNγ application. Intratumoral injection proved to be more effective than the intraperitoneal route in controlling the growth of both the primary pancreatic tumors and peritoneal carcinomatosis, accompanied by migration of virus from primary to metastatic deposits. Concomitant i.p. treatment of H-1PV with recIFNγ resulted in improved therapeutic effect yielding an extended animal survival, compared with i.p. treatment with H-1PV alone. IFNγ application enhanced the H-1PV-induced peritoneal macrophage and splenocyte responses against tumor cells while causing a significant reduction in the titers of H1-PV-neutralising antibodies in ascitic fluid. Thus, IFNγ co-application together with H-1PV might be considered as a novel therapeutic option to improve the survival of PDAC patients with peritoneal carcinomatosis.

Pancreatic carcinoma is a gastrointestinal malignancy with poor prognosis. Treatment with gemcitabine, the most potent chemotherapeutic against this cancer up to date, is not curative, and resistance may appear. Complementary treatment with an oncolytic virus, such as the rat parvovirus H-1PV, which is infectious but nonpathogenic in humans, emerges as an innovative option. Purpose: To prove that combining gemcitabine and H-1PV in a model of pancreatic carcinoma may reduce the dosage of the toxic drug and/or improve the overall anticancer effect. Experimental Design: Pancreatic tumors were implanted orthotopically in Lewis rats or subcutaneously in nude mice and treated with gemcitabine, H-1PV, or both according to different regimens. Tumor size was monitored by micro-computed tomography, whereas bone marrow, liver, and kidney functions were monitored by measuring clinically relevant markers. Human pancreatic cell lines and gemcitabine-resistant derivatives were tested in vitro for sensitivity to H-1PV infection with or without gemcitabine. Results: In vitro studies proved that combining gemcitabine with H-1PV resulted in synergistic cytotoxic effects and achieved an up to 15-fold reduction in the 50% effective concentration of the drug, with drug-resistant cells remaining sensitive to virus killing. Toxicologic screening showed that H-1PV had an excellent safety profile when applied alone or in combination with gemcitabine. The benefits of applying H-1PV as a second-line treatment after gemcitabine included reduction of tumor growth, prolonged survival of the animals, and absence of metastases on CT-scans. Conclusion: In addition to their potential use as monotherapy for pancreatic cancer, parvoviruses can be best combined with gemcitabine in a two-step protocol.

Novel therapies employing oncolytic viruses have emerged as promising anticancer modalities. The cure of particularly aggressive malignancies requires induction of immunogenic cell death (ICD), coupling oncolysis with immune responses via calreticulin, ATP, and high-mobility group box protein B1 (HMGB1) release from dying tumor cells. The present study shows that in human pancreatic cancer cells (pancreatic ductal adenocarcinoma [PDAC] cells; n = 4), oncolytic parvovirus H-1 (H-1PV) activated multiple interconnected death pathways but failed to induce calreticulin exposure or ATP release. In contrast, H-1PV elevated extracellular HMGB1 levels by 4.0 ± 0.5 times (58% ± 9% of total content; up to 100 ng/ml) in all infected cultures, whether nondying, necrotic, or apoptotic. An alternative secretory route allowed H-1PV to overcome the failure of gemcitabine to trigger HMGB1 release, without impeding cytotoxicity or other ICD activities of the standard PDAC medication. Such broad resistance of H-1PV-induced HMGB1 release to apoptotic blockage coincided with but was uncoupled from an autocrine interleukin-1β (IL-1β) loop. That and the pattern of viral determinants maintained in gemcitabine-treated cells suggested the activation of an inflammasome/caspase 1 (CASP1) platform alongside DNA detachment and/or nuclear exclusion of HMGB1 during early stages of the viral life cycle. We concluded that H-1PV infection of PDAC cells is signaled through secretion of the alarmin HMGB1 and, besides its own oncolytic effect, might convert drug-induced apoptosis into an ICD process. A transient arrest of cells in the cyclin A1-rich S phase would suffice to support compatibility of proliferation-dependent H-1PV with cytotoxic regimens. These properties warrant incorporation of the oncolytic virus H-1PV, which is not pathogenic in humans, into multimodal anticancer treatments. IMPORTANCE The current therapeutic concepts targeting aggressive malignancies require an induction of immunogenic cell death characterized by exposure of calreticulin (CRT) as well as release of ATP and HMGB1 from dying cells. In pancreatic tumor cells (PDAC cells) infected with the oncolytic parvovirus H-1PV, only HMGB1 was released by all infected cells, whether nondying, necrotic, or succumbing to one of the programmed death pathways, including contraproductive apoptosis. Our data suggest that active secretion of HMGB1 from PDAC cells is a sentinel reaction emerging during early stages of the viral life cycle, irrespective of cell death, that is compatible with and complements cytotoxic regimens. Consistent induction of HMGB1 secretion raised the possibility that this reaction might be a general “alarming” phenomenon characteristic of H-1PV's interaction with the host cell; release of IL-1β points to the possible involvement of a danger-sensing inflammasome platform. Both provide a basis for further virus-oriented studies.

Objective: For quite a long time oncolytic viruses (OVs) have been regarded merely as specific tumor cell killers, while disregarding the fact that all oncolytic activities take place in the context of a functional immune system. Oncolytic parvoviruses (PV) represent non-pathogenic, naturally oncolytic (non-modified), animal (rodent) viruses with a tropism that extends to a number of transformed human cells. Our recent work using various animal models substantiates the contention that H-1PV acts as both an oncolytic agent and an adjuvant, by direct cytoreduction in the tumor and bystander antitumor immunity. ImmunostimulatoryCpG motifs were incorporated into the singlestranded DNA genome of H-1PV and our current objective was to test whether the CpG-armed virus was in possession of an enhanced adjuvant capacity. Methods: The immunogenic potential of the CpG-enriched parvoviral derivative (JabCG) was tested in in vitro infection of human PBMCs or coculture of DCs and T-cells. In vivo tumor xenografts were raised in NOD.SCID mice that were later reconstituted with an autologous DCs and T-cells mix primed with an infected or chemovirotherapy (gemcitabine and H-1PV)-treated pancreatic cancer line vaccine. The therapeutic activity of the native and modified viruses was evaluated upon systemic application in pancreatic cancer-bearing immunocompetent Lewis rats. Results: Compared with wt H-1PV, JabCG displayed enhanced immunotherapeutic capacity to activate human immune cells ex vivo (PBMCs or DCs and T-cells isolated from pancreatic cancer patients) with a striking increase in the capacity of the latter cells for suppressing autologous tumorxenografts in NOD.SCID mice. Furthermore, intravenous application of JabCG in immunocompetent rats caused early NK and T-cell infiltration into tumors, elevated IFNγ levels in serum and spleens, and notably prolonged survival, as compared to control-treated animals. Conclusion: Taken together, data indicate that CpG-enrichment of OVs represents a potent strategy to enhance their immunotherapeutic properties.

Hypoxia and dysfunctional tumor vessels represent a prominent feature of pancreatic cancer, being, at least in part, responsible for chemotherapy resistance and immune suppression in these tumors. We tested whether the increase of oxygen delivery induced in vivo by myo-inositol trispyrophosphate (ITPP) can reverse hypoxia, control tumor growth and improve chemotherapy response. Tumor size, metastatic development (microcomputed tomography scan follow-up) and the survival of rats and nude or NOD.SCID mice, (bearing syngenic rat and MiaPaCa2- or patient-derived pancreatic tumors), were determined on ITPP and/or gemcitabine treatment. Partial oxygen pressure, expression of angiogenic factors and tumor histology were evaluated. Infiltration and oxidative status of immune cells, as well as chemotherapy penetration in tumors, were determined by fluorescence-activated cell sorting, fluorometry, nitric oxide release assays, Western blot and confocal microscopy. Weekly intravenous ITPP application resulted in the inhibition of metastasis development and restricted primary tumor growth, showing a superior effect on the rats' survival compared with gemcitabine. ITPP treatment restored tumor normoxia and caused a reduction in hypoxia inducible factor-1α levels, with subsequent VEGF and Lox downregulation, resulting in improved vessel structure and decreased desmoplasia. The latter effects translated into elevated immune cells influx and improved susceptibility to gemcitabine treatment. Growth of human pancreatic tumor xenografts was strongly inhibited by administration of ITPP. ITPP exploits a two-stage mechanism causing rapid, early and sustainable late stage normoxia. This is due to the angiogenic factor modulation and vascular normalization, leading to enhanced chemotherapy delivery and synergistic life prolongation, on combination with low doses of gemcitabine.

Oncolytic viruses represent versatile tools that through natural mechanisms or upon genetic manipulation can specifically target and kill tumor cells. In the last ten years it became clear that one of the major modes of action of these agents is their effect as an in situ (intratumoral) anticancer vaccine.

Oncolytic virotherapy represents a recent approach to anticancer therapy. Rodent autonomous parvoviruses (PVs) represent naturally oncolytic viruses that are non-pathogenic for humans but possess and extended tropism, being capable of infecting transformed cells of both rodent and human origin. Recent work from our group demonstrate that PVs can act as direct lytic agents and adjuvants, stimulating antitumor immune responses against glioma and pancreatic ductal adenocarcinoma (PDAC).

Rodent autonomous parvoviruses (PVs) are endowed with oncotropic properties and represent virotherapeutics with inherent oncolytic features. This work aimed to evaluate the capacity of Minute Virus of Mice (MVMp) to act as an adjuvant stimulating a mouse glioblastoma-specific immune response. MVMp was shown to induce cell death through apoptosis in glioma GL261 cells. Antigen-presenting cells (APCs) provide the initial cue for innate and adaptive immune responses, and thus MVMp-infected GL261 cells were tested for their ability to activate dendritic cells (DCs) and microglia (MG), two distinct cell types that are able to act as APCs. MG and discrete DC subsets were activated after co-culture with MVMp-infected glioma GL261 cells, as evidenced by upregulation of specific activation markers (CD80, CD86) and release of proinflammatory cytokines (tumor necrosis factor-a and interleukin-6). The in vivo analysis of immunodeficient and immunocompetent mice revealed a clear difference in their susceptibility to MVMp-mediated tumor suppression. Immunocompetent mice were fully protected from tumor outgrowth of GL261 cells infected ex vivo with MVMp. In contrast, immunodeficient animals were less competent for MVMp-dependent tumor inhibition, with only 20% of the recipients being protected, arguing for an additional immune component to allow full tumor suppression. In keeping with this conclusion, immunocompetent mice engrafted with MVMp-infected glioma cells developed a level of anti-tumor immunity with isolated splenocytes producing elevated levels of interferon-gamma. In rechallenge experiments using uninfected GL261 cells, we could show complete protection against the tumor, arguing for the induction of a T-cell-mediated, tumor-specific, long-term memory response. These findings indicate that the anticancer effect of PVs can be traced back not only for their direct oncolytic effect, but also to their ability to break tumor tolerance.

Accumulating evidence suggests an important role for cyclooxygenase-2 (COX-2) in the pathogenesis of a wide range of malignancies. The protumorigenic properties of COX-2 are generally thought to be mediated by its product, PGE2, which is shown to promote tumor spread and growth by multiple mechanisms but most importantly through modulation of the local immune response in the tumor. Pancreatic tumor cells produce various amounts of PGE2, some of them being even deficient in COX enzymes or other PGE2synthases. Here we describe that, beside pancreatic tumor cells or stromal fibroblasts, human peripheral blood mononuclear cells can also produce PGE2upon coculture with pancreatic cancer cells. Stimulating of cellular cPLA2 within PBMCs by secreted factors, presumably sPLA2, from tumor cells appeared crucial, while the direct contact between PBMCs and PDACs seemed to be dispensable for this effect. Our data is emphasizing the complex interactions participating in the formation of the tolerogenic immune milieu within pancreatic tumors.

Treatment of cancers by means of viruses, that specifically replicate in (oncotropism) and kill (oncolysis) neoplastic cells, is increasingly gaining acceptance in the clinic. Among these agents, parvoviruses have been shown to possess not only direct oncolytic but also immunomodulating properties, serving as an adjuvant to prime the immune system to react against infected tumors. Here, we aimed to establish whether immunomodulating mechanisms participate in the recently reported therapeutic potential of parvoviruses against pancreatic carcinoma. Using adoptive transfer experiments we discovered that the transfer of splenocytes of donor rats harboring H-1PV-treated orthotopic PDAC tumors could significantly prolong the survival of naive tumor-bearing recipients, compared to those receiving cells from mock-treated donors. Closer investigation of immunological parameters in infected donor rats revealed that virus-induced interferon gamma production and cellular immune response played an important role in this effect. These data have also preclinical relevance since abortive H-1PV infection of human peripheral blood mononuclear cells or cocultivation of these cells with H-1PV-preinfected pancreatic cancer cells, resulted in enhancement of innate and adaptive immune reactivity. Taken together our data reveal that oncolytic H-1PV modulates the immune system into an anticancer state, and further support the concept of using parvoviruses in the fight against pancreatic cancer.

Parvovirus minute virus of mice (MVMp) is endowed with oncotropic properties so far ascribed only to the dependency of the virus life cycle on cellular factors expressed during S phase and/or modulated by malignant transformation. For other viruses oncotropism relies on their inability to circumvent type I interferon (IFN)-induced innate antiviral mechanisms, the first line of defense triggered by normal cells against viral infections. These agents propagate, therefore, preferentially in transformed/tumor cells, which often lack functional antiviral mechanisms. The present study aimed at investigating whether antiviral processes also contribute to MVMp oncotropism. Our results demonstrate that in contrast to MVMp-permissive transformed mouse A9 fibroblasts, freshly isolated normal counterparts (mouse embryonic fibroblasts [MEFs]) mount, through production and release of type I IFNs upon their infection, an antiviral response against MVMp lytic multiplication. Pretreatment of MEFs with a type I IFN-β-neutralizing antibody, prior to MVMp infection, inhibits the virus-triggered antiviral response and improves the fulfillment of the MVMp life cycle. Our results also show that part of the A9 permissiveness to MVMp relies on the inability to produce type I IFNs upon parvovirus infection, a feature related either to an A9 intrinsic deficiency of this process or to an MVMp-triggered inhibitory mechanism, since stimulation of these cells by exogenous IFN-β strongly inhibits the parvovirus life cycle. Taken together, our results demonstrate for the first time that parvovirus infection triggers an innate antiviral response in normal cells and suggest that the MVMp oncotropism depends at least in part on the failure of infected transformed cells to mount such a response.

International audience; The incidence of lymphomas developing in both immunocompetent and immunosuppressed patients continues to steadily increase worldwide. Current chemotherapy and immunotherapy approaches have several limitations, such as severe side toxicity and selection of resistant cell variants. Autonomous parvoviruses (PVs), in particular the rat parvovirus H-1PV, have emerged as promising anticancer agents. Although it is apathogenic in humans, H-1PV has been shown to infect and suppress various rat and human tumors in animal models. In this study, we demonstrate the capacity of H-1PV for efficiently killing, through necrosis, cell cultures originating from Burkitt's lymphoma (BL), while sparing normal B lymphocytes. The cytotoxic effect was generally accompanied by a productive H-1PV infection. Remarkably, parvovirus-based monotherapy efficiently suppressed established BL at an advanced stage in a severe combined immunodeficient (SCID) mouse model of the disease. The data show for the first time that an oncolytic parvovirus deserves further consideration as a potential tool for the treatment of some non-Hodgkin B-cell lymphomas, including those resistant to apoptosis induction by rituximab.