The Complex Role of Infectious Agents in Human Cutaneous T-Cell Lymphoma Pathogenesis: From Candidate Etiological Factors to Potential Therapeutics.

Cutaneous T-cell lymphoma (CTCL) is a devastating, potentially fatal T-lymphocyte malignancy affecting the skin. Despite all efforts, the etiology of this disease remains unknown. Infectious agents have long been suspected as factors or co-factors in CTCL pathogenesis. This review deals with the panel of bacterial and viral pathogens that have been investigated so far in an attempt to establish a potential link between infection/carriage and CTCL development. A special focus is given to a recently discovered human protoparvovirus, namely the cutavirus (CutaV), which has emerged as a plausible CTCL etiological agent. Available evidence in support of this hypothesis as well as alternative interpretations and uncertainties raised by some conflicting data are discussed. The complexity and multifacetedness of the Parvoviridae family of viruses are illustrated by presenting another protoparvovirus, the rat H-1 parvovirus (H-1PV). H-1PV belongs to the same genus as the CutaV but carries considerable potential for therapeutic applications in cutaneous lymphoma.

Oncolytic Rodent Protoparvoviruses Evade a TLR- and RLR-Independent Antiviral Response in Transformed Cells.

The oncolytic rodent protoparvoviruses (PVs) minute virus of mice (MVMp) and H-1 parvovirus (H-1PV) are promising cancer viro-immunotherapy candidates capable of both exhibiting direct oncolytic activities and inducing anticancer immune responses (AIRs). Type-I interferon (IFN) production is instrumental for the activation of an efficient AIR. The present study aims at characterizing the molecular mechanisms underlying PV modulation of IFN induction in host cells. MVMp and H-1PV triggered IFN production in semi-permissive normal mouse embryonic fibroblasts (MEFs) and human peripheral blood mononuclear cells (PBMCs), but not in permissive transformed/tumor cells. IFN production triggered by MVMp in primary MEFs required PV replication and was independent of the pattern recognition receptors (PRRs) Toll-like (TLR) and RIG-like (RLR) receptors. PV infection of (semi-)permissive cells, whether transformed or not, led to nuclear translocation of the transcription factors NFĸB and IRF3, hallmarks of PRR signaling activation. Further evidence showed that PV replication in (semi-)permissive cells resulted in nuclear accumulation of dsRNAs capable of activating mitochondrial antiviral signaling (MAVS)-dependent cytosolic RLR signaling upon transfection into naïve cells. This PRR signaling was aborted in PV-infected neoplastic cells, in which no IFN production was detected. Furthermore, MEF immortalization was sufficient to strongly reduce PV-induced IFN production. Pre-infection of transformed/tumor but not of normal cells with MVMp or H-1PV prevented IFN production by classical RLR ligands. Altogether, our data indicate that natural rodent PVs regulate the antiviral innate immune machinery in infected host cells through a complex mechanism. In particular, while rodent PV replication in (semi-)permissive cells engages a TLR-/RLR-independent PRR pathway, in transformed/tumor cells this process is arrested prior to IFN production. This virus-triggered evasion mechanism involves a viral factor(s), which exert(s) an inhibitory action on IFN production, particularly in transformed/tumor cells. These findings pave the way for the development of second-generation PVs that are defective in this evasion mechanism and therefore endowed with increased immunostimulatory potential through their ability to induce IFN production in infected tumor cells.

Generation and Validation of Monoclonal Antibodies Suitable for Detecting and Monitoring Parvovirus Infections.

For many applications it is necessary to detect target proteins in living cells. This is particularly the case when monitoring viral infections, in which the presence (or absence) of distinct target polypeptides potentially provides vital information about the pathology caused by the agent. To obtain suitable tools with which to monitor parvoviral infections, we thus generated monoclonal antibodies (mAbs) in order to detect the major non-structural protein NS1 in the intracellular environment and tested them for sensitivity and specificity, as well as for cross-reactivity towards related species. Using different immunogens and screening approaches based on indirect immunofluorescence, we describe here a panel of mAbs suitable for monitoring active infections with various parvovirus species by targeting the major non-structural protein NS1. In addition to mAbs detecting the NS1 of parvovirus H-1 (H-1PV) (belonging to the Rodent protoparvovirus 1 species, which is currently under validation as an anti-cancer agent), we generated tools with which to monitor infections by human cutavirus (CuV) and B19 virus (B19V) (belonging to the Primate protoparvovirus 3 and the Primate erythroparvovirus 1 species, respectively, which were both found to persistently infect human tissues). As well as mAbs able to detect NS1 from a broad range of parvoviruses, we obtained entities specific for either (distinct) members of the Rodent protoparvovirus 1 species, human CuV, or human B19V.

Virotherapy in Germany-Recent Activities in Virus Engineering, Preclinical Development, and Clinical Studies.

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review-as part of the special edition on "State-of-the-Art Viral Vector Gene Therapy in Germany"-the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.

Phase 2 Trial of Oncolytic H-1 Parvovirus Therapy Shows Safety and Signs of Immune System Activation in Patients With Metastatic Pancreatic Ductal Adenocarcinoma.

PURPOSE: To investigate the safety, clinical efficacy, virus pharmacokinetics, shedding, and immune response after administration of an oncolytic parvovirus (H-1PV, ParvOryx) to patients with metastatic pancreatic ductal adenocarcinoma (PDAC) refractory to first-line therapy. PATIENTS AND METHODS: This is a noncontrolled, single-arm, open-label, dose-escalating, single-center clinical trial. Seven patients with PDAC and at least one liver metastasis were included. ParvOryx was administered intravenously on 4 consecutive days and as an intralesional injection, 6 to 13 days thereafter. Altogether, three escalating dose levels were investigated. In addition, gemcitabine treatment was initiated on day 28. RESULTS: ParvOryx showed excellent tolerability with no dose-limiting toxicities. One patient had a confirmed partial response and one patient revealed an unconfirmed partial response according to RECIST criteria. Both patients showed remarkably long surivial of 326 and 555 days, respectively. Investigation of pharmacokinetics and virus shedding revealed dose dependency with no excretion of active virus particles in saliva or urine and very limited excretion in feces. H-1PV nucleic acids were detected in tumor samples of four patients. All patients showed T-cell responses to viral proteins. An interesting immunologic pattern developed in tumor tissues and in blood of both patients with partial response suggesting immune activation after administration of ParvOryx. CONCLUSIONS: The trial met all primary objectives, revealed no environmental risks, and indicated favorable immune modulation after administration of ParvOryx. It can be considered a good basis for further systematic clinical development alone or in combination with immunomodulatory compounds.

Parvovirus-Based Combinatorial Immunotherapy: A Reinforced Therapeutic Strategy against Poor-Prognosis Solid Cancers.

Resistance to anticancer treatments poses continuing challenges to oncology researchers and clinicians. The underlying mechanisms are complex and multifactorial. However, the immunologically "cold" tumor microenvironment (TME) has recently emerged as one of the critical players in cancer progression and therapeutic resistance. Therefore, TME modulation through induction of an immunological switch towards inflammation ("warming up") is among the leading approaches in modern oncology. Oncolytic viruses (OVs) are seen today not merely as tumor cell-killing (oncolytic) agents, but also as cancer therapeutics with multimodal antitumor action. Due to their intrinsic or engineered capacity for overcoming immune escape mechanisms, warming up the TME and promoting antitumor immune responses, OVs hold the potential for creating a proinflammatory background, which may in turn facilitate the action of other (immunomodulating) drugs. The latter provides the basis for the development of OV-based immunostimulatory anticancer combinations. This review deals with the smallest among all OVs, the H-1 parvovirus (H-1PV), and focuses on H-1PV-based combinatorial approaches, whose efficiency has been proven in preclinical and/or clinical settings. Special focus is given to cancer types with the most devastating impact on life expectancy that urgently call for novel therapies.

Fluorescence In Situ Hybridization (FISH) Detection of Viral Nucleic Acids in Oncolytic H-1 Parvovirus-Treated Human Brain Tumors.

Fluorescence in situ hybridization (FISH) is a specific, sensitive, accurate, and reliable technique widely applied in both research and clinic. Here we describe the detailed protocol of a FISH method established by us to serve the scientific purposes of the first oncolytic parvovirus clinical trial (ParvOryx01). This trial was launched in Germany in 2011. After trial completion in 2015, results were published in Molecular Therapy in 2017. The primary purpose of the trial was to evaluate the safety of an oncolytic parvovirus, H-1PV (ParvOryx), in recurrent glioblastoma patients. In addition, the efficiency of H-1PV tumor targeting after intratumoral or systemic virus administration was assessed by FISH detection of viral nucleic acids (genomic single-stranded DNA, mRNA and parvovirus double-stranded replicative forms) in formalin-fixed paraffin-embedded glioblastoma tissues resected at day 10 after ParvOryx treatment. The FISH method allowed the detection-for the first time in humans-of H-1PV replication markers in brain tumors of parvovirus-treated patients. A protocol combining mRNA FISH with simultaneous immunofluorescent staining for tumor and tumor microenvironment markers was also developed and is described here, in order to better characterize H-1PV cellular targets and H-1PV treatment-associated tumor microenvironment changes.

Immune Conversion of Tumor Microenvironment by Oncolytic Viruses: The Protoparvovirus H-1PV Case Study.

Cancer cells utilize multiple mechanisms to evade and suppress anticancer immune responses creating a "cold" immunosuppressive tumor microenvironment. Oncolytic virotherapy is emerging as a promising approach to revert tumor immunosuppression and enhance the efficacy of other forms of immunotherapy. Growing evidence indicates that oncolytic viruses (OVs) act in a multimodal fashion, inducing immunogenic cell death and thereby eliciting robust anticancer immune responses. In this review, we summarize information about OV-mediated immune conversion of the tumor microenvironment. As a case study we focus on the rodent protoparvovirus H-1PV and its dual role as an oncolytic and immune modulatory agent. Potential strategies to improve H-1PV anticancer efficacy are also discussed.

Immune System Stimulation by Oncolytic Rodent Protoparvoviruses.

Rodent protoparvoviruses (PVs), parvovirus H-1 (H-1PV) in particular, are naturally endowed with oncolytic properties. While being historically described as agents that selectively replicate in and kill cancer cells, recent yet growing evidence demonstrates that these viruses are able to reverse tumor-driven immune suppression through induction of immunogenic tumor cell death, and the establishment of antitumorigenic, proinflammatory milieu within the tumor microenvironment. This review summarizes the most important preclinical proofs of the interplay and the cooperation between PVs and the host immune system. The molecular mechanisms of PV-induced immunostimulation are also discussed. Furthermore, initial encouraging in-human observations from clinical trials and compassionate virus uses are presented, and speak in favor of further H-1PV clinical development as partner drug in combined immunotherapeutic protocols.

Tailoring acyclovir prodrugs with enhanced antiviral activity: rational design, synthesis, human plasma stability and in vitro evaluation.

Bile acid prodrugs have served as a viable strategy for refining the pharmaceutical profile of parent drugs through utilizing bile acid transporters. A series of three ester prodrugs of the antiherpetic drug acyclovir (ACV) with the bile acids cholic, chenodeoxycholic and deoxycholic were synthesized and evaluated along with valacyclovir for their in vitro antiviral activity against herpes simplex viruses type 1 and type 2 (HSV-1, HSV-2). The in vitro antiviral activity of the three bile acid prodrugs was also evaluated against Epstein-Barr virus (EBV). Plasma stability assays, utilizing ultra-high performance liquid chromatography coupled with tandem mass spectrometry, in vitro cytotoxicity and inhibitory experiments were conducted in order to establish the biological profile of ACV prodrugs. The antiviral assays demonstrated that ACV-cholate had slightly better antiviral activity than ACV against HSV-1, while it presented an eight-fold higher activity with respect to ACV against HSV-2. ACV-chenodeoxycholate presented a six-fold higher antiviral activity against HSV-2 with respect to ACV. Concerning EBV, the highest antiviral effect was demonstrated by ACV-chenodeoxycholate. Human plasma stability assays revealed that ACV-deoxycholate was more stable than the other two prodrugs. These results suggest that decorating the core structure of ACV with bile acids could deliver prodrugs with amplified antiviral activity.

Immunotherapeutic Potential of Oncolytic H-1 Parvovirus: Hints of Glioblastoma Microenvironment Conversion towards Immunogenicity.

Glioblastoma, one of the most aggressive primary brain tumors, is characterized by highly immunosuppressive microenvironment. This contributes to glioblastoma resistance to standard treatment modalities and allows tumor growth and recurrence. Several immune-targeted approaches have been recently developed and are currently under preclinical and clinical investigation. Oncolytic viruses, including the autonomous protoparvovirus H-1 (H-1PV), show great promise as novel immunotherapeutic tools. In a first phase I/IIa clinical trial (ParvOryx01), H-1PV was safe and well tolerated when locally or systemically administered to recurrent glioblastoma patients. The virus was able to cross the blood-brain (tumor) barrier after intravenous infusion. Importantly, H-1PV treatment of glioblastoma patients was associated with immunogenic changes in the tumor microenvironment. Tumor infiltration with activated cytotoxic T cells, induction of cathepsin B and inducible nitric oxide (NO) synthase (iNOS) expression in tumor-associated microglia/macrophages (TAM), and accumulation of activated TAM in cluster of differentiation (CD) 40 ligand (CD40L)-positive glioblastoma regions was detected. These are the first-in-human observations of H-1PV capacity to switch the immunosuppressed tumor microenvironment towards immunogenicity. Based on this pilot study, we present a tentative model of H-1PV-mediated modulation of glioblastoma microenvironment and propose a combinatorial therapeutic approach taking advantage of H-1PV-induced microglia/macrophage activation for further (pre)clinical testing.

Oncolytic H-1 Parvovirus Shows Safety and Signs of Immunogenic Activity in a First Phase I/IIa Glioblastoma Trial.

Oncolytic virotherapy may be a means of improving the dismal prognosis of malignant brain tumors. The rat H-1 parvovirus (H-1PV) suppresses tumors in preclinical glioma models, through both direct oncolysis and stimulation of anticancer immune responses. This was the basis of ParvOryx01, the first phase I/IIa clinical trial of an oncolytic parvovirus in recurrent glioblastoma patients. H-1PV (escalating dose) was administered via intratumoral or intravenous injection. Tumors were resected 9 days after treatment, and virus was re-administered around the resection cavity. Primary endpoints were safety and tolerability, virus distribution, and maximum tolerated dose (MTD). Progression-free and overall survival and levels of viral and immunological markers in the tumor and peripheral blood were also investigated. H-1PV treatment was safe and well tolerated, and no MTD was reached. The virus could cross the blood-brain/tumor barrier and spread widely through the tumor. It showed favorable pharmacokinetics, induced antibody formation in a dose-dependent manner, and triggered specific T cell responses. Markers of virus replication, microglia/macrophage activation, and cytotoxic T cell infiltration were detected in infected tumors, suggesting that H-1PV may trigger an immunogenic stimulus. Median survival was extended in comparison with recent meta-analyses. Altogether, ParvOryx01 results provide an impetus for further H-1PV clinical development.

A non-controlled, single arm, open label, phase II study of intravenous and intratumoral administration of ParvOryx in patients with metastatic, inoperable pancreatic cancer: ParvOryx02 protocol.

BACKGROUND: Metastatic pancreatic cancer has a dismal prognosis, with a mean six-month progression-free survival of approximately 50% and a median survival of about 11 months. Despite intensive research, only slight improvements of clinical outcome could be achieved over the last decades. Hence, new and innovative therapeutic strategies are urgently required. ParvOryx is a drug product containing native parvovirus H-1 (H-1PV). Since H-1PV was shown to exert pronounced anti-neoplastic effects in pre-clinical models of pancreatic cancer, the drug appears to be a promising candidate for treatment of this malignancy. METHODS: ParvOryx02 is a non-controlled, single arm, open label, dose-escalating, single center trial. In total seven patients with pancreatic cancer showing at least one hepatic metastasis are to be treated with escalating doses of ParvOryx according to the following schedule: i) 40% of the total dose infused intravenously in equal fractions on four consecutive days, ii) 60% of the total dose injected on a single occasion directly into the hepatic metastasis at varying intervals after intravenous infusions. The main eligibility criteria are: age ≥ 18 years, disease progression despite first-line chemotherapy, and at least one hepatic metastasis. Since it is the second trial within the drug development program, the study primarily explores safety and tolerability after further dose escalation of ParvOryx. The secondary objectives are related to the evaluation of certain aspects of anti-tumor activity and clinical efficacy of the drug. DISCUSSION: This trial strongly contributes to the clinical development program of ParvOryx. The individual hazards for patients included in the current study and the environmental risks are addressed and counteracted adequately. Besides information on safety and tolerability of the treatment after further dose escalation, thorough evaluations of pharmacokinetics and intratumoral spread as well as proof-of-concept (PoC) in pancreatic cancer will be gained in the course of the trial. TRIAL REGISTRATION: ClinicalTrials.gov-ID: NCT02653313 , Registration date: Dec. 4th, 2015.

The Oncolytic Virotherapy Era in Cancer Management: Prospects of Applying H-1 Parvovirus to Treat Blood and Solid Cancers.

Non-Hodgkin lymphoma (NHL) and leukemia are among the most common cancers worldwide. While the treatment of NHL/leukemia of B-cell origin has much progressed with the introduction of targeted therapies, few treatment standards have been established for T-NHL/leukemia. As presentation in both B- and T-NHL/leukemia patients is often aggressive and as prognosis for relapsed disease is especially dismal, this cancer entity poses major challenges and requires innovative therapeutic approaches. In clinical trials, oncolytic viruses (OVs) have been used against refractory multiple myeloma (MM). In preclinical settings, a number of OVs have demonstrated a remarkable ability to suppress various types of hematological cancers. Most studies dealing with this approach have used MM or B- or myeloid-cell-derived malignancies as models. Only a few describe susceptibility of T-cell lymphoma/leukemia to OV infection and killing. The rat H-1 parvovirus (H-1PV) is an OV with considerable promise as a novel therapeutic agent against both solid tumors (pancreatic cancer and glioblastoma) and hematological malignancies. The present perspective article builds on previous reports of H-1PV-driven regression of Burkitt's lymphoma xenografts and on unpublished observations demonstrating effective killing by H-1PV of cells from CHOP-resistant diffuse large B-cell lymphoma, cutaneous T-cell lymphoma, and T-cell acute lymphoblastic leukemia. On the basis of these studies, H-1PV is proposed for use as an adjuvant to (chemo)therapeutic regimens. Furthermore, in the light of a recently completed first parvovirus clinical trial in glioblastoma patients, the advantages of H-1PV for systemic application are discussed.

Establishment and Characterization of a Novel Cell Line, ASAN-PaCa, Derived From Human Adenocarcinoma Arising in Intraductal Papillary Mucinous Neoplasm of the Pancreas.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The Promise of Gene Therapy for Pancreatic Cancer.

Unlike for other digestive cancer entities, chemotherapy, radiotherapy, and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for patients with PDAC. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using nonviral therapeutic gene delivery, targeted transgene expression, or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC but may also significantly influence the field of gene-based molecular treatment of cancer.

Tumor Selectivity of Oncolytic Parvoviruses: From in vitro and Animal Models to Cancer Patients.

Oncolytic virotherapy of cancer is among the innovative modalities being under development and especially promising for targeting tumors, which are resistant to conventional treatments. Presently, at least a dozen of viruses, belonging to nine different virus families, are being tested within the frames of various clinical studies in cancer patients. Continuously growing preclinical evidence showing that the autonomous rat parvovirus H-1 (H-1PV) is able to kill tumor cells that resist conventional treatments and to achieve a complete cure of various human tumors in animal models argues for its inclusion in the arsenal of oncolytic viruses with an especially promising bench to bedside translation potential. Oncolytic parvovirus safe administration to humans relies on the intrinsic preference of these agents for quickly proliferating, metabolically, and biochemically disturbed tumor versus normal cells (tumor selectivity or oncotropism). The present review summarizes and discusses (i) preclinical evidence of H-1PV innocuousness for normal cells and healthy tissues in vitro and in animals, respectively, (ii) toxicological assessments of H-1PV mono- or combined therapy in tumor-bearing virus-permissive animal models, as well as (iii) historical results of experimental infection of human cancer patients with H-1PV. Altogether, these data argue against a risk of H-1PV inducing significant toxic effects in human patients. This highly favorable safety profile allowed the translation of H-1PV preclinical research into a Phase I/IIa clinical trial being currently in progress.

Double-faceted mechanism of parvoviral oncosuppression.

The H-1 parvovirus (H-1PV) exerts oncosuppressive action that has two components: oncotoxicity and immunostimulation. While many human tumor cells, including conventional drug-resistant ones, can be killed by H-1PV, some fail to support progeny virus production, necessary for infection propagation in neoplastic tissues. This limitation can be overcome through forced selection of H-1PV variants capable of enhanced multiplication and spreading in human tumor cells. In the context of further developing H-1PV for use in cancer therapy, arming it with immunostimulatory CpG motifs under conditions preserving replication and oncolysis enhances its action as an anticancer vaccine adjuvant. A first clinical study of H-1PV treatment in glioma patients has yielded evidence of intratumoral synthesis of the viral oncotoxic protein NS1 and immune cell infiltration.

Oncolytic parvoviruses: from basic virology to clinical applications.

Accumulated evidence gathered over recent decades demonstrated that some members of the Parvoviridae family, in particular the rodent protoparvoviruses H-1PV, the minute virus of mice and LuIII have natural anticancer activity while being nonpathogenic to humans. These studies have laid the foundations for the launch of a first phase I/IIa clinical trial, in which the rat H-1 parvovirus is presently undergoing evaluation for its safety and first signs of efficacy in patients with glioblastoma multiforme. After a brief overview of the biology of parvoviruses, this review focuses on the studies which unraveled the antineoplastic properties of these agents and supported their clinical use as anticancer therapeutics. Furthermore, the development of novel parvovirus-based anticancer strategies with enhanced specificity and efficacy is discussed, in particular the development of second and third generation vectors and the combination of parvoviruses with other anticancer agents. Lastly, we address the key challenges that remain towards a more rational and efficient use of oncolytic parvoviruses in clinical settings, and discuss how a better understanding of the virus life-cycle and of the cellular factors involved in virus infection, replication and cytotoxicity may promote the further development of parvovirus-based anticancer therapies, open new prospects for treatment and hopefully improve clinical outcome.

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.