Complementary induction of immunogenic cell death by oncolytic parvovirus H-1PV and gemcitabine in pancreatic cancer.

UNLABELLED: 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-1ß (IL-1ß) 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-1ß points to the possible involvement of a danger-sensing inflammasome platform. Both provide a basis for further virus-oriented studies.

Improvement of gemcitabine-based therapy of pancreatic carcinoma by means of oncolytic parvovirus H-1PV.

UNLABELLED: 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.

Oncolytic rat parvovirus H-1PV, a candidate for the treatment of human lymphoma: In vitro and in vivo studies.

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.

Arming parvoviruses with CpG motifs to improve their oncosuppressive capacity.

Oncolytic viruses represent novel tools for cancer treatment. Besides specifically killing cancer cells (oncolysis), these agents also provide danger signals, prompting the immune system to eliminate virus-infected tumours. As a consequence of oncolytic events, the innate and adaptive immune systems gain access to tumour antigens, which result in cross-priming and vaccination effects. Here the aim was to see whether we could enhance this adjuvant capacity by incorporating immunostimulatory CpG motifs into the single-stranded genome of an oncolytic parvovirus (H-1PV). We engineered 2 CpG-enriched H-1PV variants (JabCG1 and JabCG2), preserving both the replication competence and the oncolytic features of the parental virus. In keeping with their increased CpG content, the JabCG1 and JabCG2 genomes proved in vitro to be more potent triggers of TLR-9-mediated signalling than wild-type H-1PV DNA. Antitumour activity was evaluated in a rat model of MH3924A hepatoma lung metastases, where an infection with parental or modified viruses served as an ex vivo adjuvant to a subcutaneously administered autologous cell vaccine. In this setup, which excludes direct oncolytic effects on metastases, the JabCG2 vector displayed enhanced immunogenicity, inducing markers of cellular immunity (IFN gamma) and dendritic cell activation (CD80, CD86) in mediastinal (tumour-draining) lymph nodes. This led to a significantly reduced metastatic rate (50%) as compared to other vaccination schedules (H-1PV-, JabCG1-, JabGC- or mock-treated cells). The data provide proof of principle that increasing the number of immunostimulatory CpG motifs within oncolytic viruses makes it possible to improve their overall anticancer effect by inducing antitumour vaccination.

Combined oncolytic and vaccination activities of parvovirus H-1 in a metastatic tumor model.

Oncolytic viruses have emerged as a novel class of potent anticancer agents offering an improvement on chemo- and radiotherapy in terms of tumor targeting and reduction of side-effects. Among these agents, autonomous parvoviruses have attracted the attention of researchers for their ability to preferentially replicate in and kill transformed cells, and to suppress tumors in the absence of adverse reactions in various animal models. We have previously shown that lethally irradiated autologous tumor cells can support parvovirus H-1PV production and serve as carriers to deliver progeny H-1PV into the vicinity of lung metastases in a rat tumor model, resulting in H-1PV infection of and multiplication in metastatic cells. It is known that irradiated autologous (neoplastic) cells can also act as a therapeutic vaccine against the original tumor. Yet the ability of these cells to suppress metastases in the above model was found to be much increased as a result of their H-1PV infection. This prompted us to determine whether H-1PV boosted the tumor-suppressing capacity of the autologous vaccine by increasing its immunogenic potential and/or by making it a factory of oncolytic viruses able to reach and destroy the metastases. Both effects could be dissociated in the presence of neutralising antibodies which either prevent the progeny viruses from spreading to metastatic cells, or deplete the CD8 effector cells from the immune system. This strategy revealed that the H-1PV infection of tumor cells enhanced their ability to trigger an immune response for which uninfected tumor cells could be the targets, thereby amplifying and taking over from the direct viral oncolytic activity. This dual oncolytic/vaccinal effect of H-1PV holds out promises of clinical applications to cancer therapy.

TLR-9 contributes to the antiviral innate immune sensing of rodent parvoviruses MVMp and H-1PV by normal human immune cells.

The oncotropism of Minute Virus of Mice (MVMp) is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs) in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR) involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs) as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN) of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9(+/+)), our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal sensing of the parvovirus genomes by TLR-9.

Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against cervical and pancreatic carcinomas.

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.

Parvoviruses-tools to fine-tune anticancer immune responses.

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).

Pancreatic cancer cell lines can induce prostaglandin e2 production from human blood mononuclear cells.

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, PGE(2), 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 PGE(2), some of them being even deficient in COX enzymes or other PGE(2) synthases. Here we describe that, beside pancreatic tumor cells or stromal fibroblasts, human peripheral blood mononuclear cells can also produce PGE(2) upon 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.

Interferon γ improves the vaccination potential of oncolytic parvovirus H-1PV for the treatment of peritoneal carcinomatosis in pancreatic cancer.

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.

Oncolytic parvoviruses as cancer therapeutics.

The experimental infectivity and excellent tolerance of some rodent autonomous parvoviruses in humans, together with their oncosuppressive effects in preclinical models, speak for the inclusion of these agents in the arsenal of oncolytic viruses under consideration for cancer therapy. In particular, wild-type parvovirus H-1PV can achieve a complete cure of various tumors in animal models and kill tumor cells that resist conventional anticancer treatments. There is growing evidence that H-1PV oncosuppression involves an immune component in addition to the direct viral oncolytic effect. This article summarizes the recent assessment of H-1PV antineoplastic activity in glioma, pancreatic ductal adenocarcinoma, and non-Hodgkin lymphoma models, laying the foundation for the present launch of a first phase I/IIa clinical trial on glioma patients.

Immune cells participate in the oncosuppressive activity of parvovirus H-1PV and are activated as a result of their abortive infection with this agent.

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 naïve 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.

B1 lymphocytes and myeloid dendritic cells in lymphoid organs are preferential extratumoral sites of parvovirus minute virus of mice prototype strain expression.

Due to their oncolytic properties and apathogenicity, autonomous parvoviruses have attracted significant interest as possible anticancer agents. Recent preclinical studies provided evidence of the therapeutic potential of minute virus of mice prototype strain (MVMp) and its recombinant derivatives. In a murine model of hemangiosarcoma, positive therapeutic outcome correlated with high intratumoral expression of MVMp-encoded genes in tumors and lymphoid organs, especially in tumor-draining lymph nodes. The source and relevance of this extratumoral expression, which came as a surprise because of the known fibrotropism of MVMp, remained unclear. In the present study, we investigated (i) whether the observed expression pattern occurs in different tumor models, (ii) which cell population is targeted by the virus, and (iii) the immunological consequences of this infection. Significant MVMp gene expression was detected in lymphoid tissues from infected tumor-free as well as melanoma-, lymphoma-, and hemangiosarcoma-bearing mice. This expression was especially marked in lymph nodes draining virus-injected tumors. Fluorescent in situ hybridization analysis, multicolor fluorescence-activated cell sorting, and quantitative reverse transcription-PCR revealed that MVMp was expressed in rare subpopulations of CD11b (Mac1)-positive cells displaying CD11c+ (myeloid dendritic cells [MDC]) or CD45B (B220+ [B1 lymphocytes]) markers. Apart from the late deletion of cytotoxic memory cells (CD8+ CD44+ CD62L-), this infection did not lead to significant alteration of the immunological profile of cells populating lymphoid organs. However, subtle changes were detected in the production of specific proinflammatory cytokines in lymph nodes from virus-treated animals. Considering the role of B1 lymphocytes and MDC in cancer and immunological surveillance, the specific ability of these cell types to sustain parvovirus-driven gene expression may be exploited in gene therapy protocols.

Carrier cell-mediated delivery of oncolytic parvoviruses for targeting metastases.

Over the last few years, naturally occurring or genetically manipulated oncolytic viruses gained increasing attention as novel therapeutics for cancer treatment. The present work provides proof of principle that an organotropic cell-based carrier system is suitable to deliver oncolytic parvoviruses to a tissue known to be a target for the formation of metastases. Carrier cells were inactivated by gamma-irradiation after infection, which was found not to affect the production and release of parvoviruses that were capable of lysing cocultured target neoplastic cells. Although systemically administered parvovirus H-1 showed a pronounced therapeutic effect against the development of established Morris hepatoma (MH3924A) lung metastases, the carrier cell strategy offered a number of advantages. Infected carriers were able to sustain H-1 virus expression for 6 days in the lungs of rats affected by metastatic disease and to reduce the spreading of the virus to peripheral organs. Compared to direct virus injection, the carrier cell protocol led to an improved therapeutic effect (metastases suppression) and a lesser generation of virus-neutralizing antibodies. These data support the use of carrier cells to deliver oncolytic viruses and/or viral vectors locally in tumors and, more particularly, metastases.