Gaétan Bour

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

ABSTRACT 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-1PVs 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.

Myo-InositolTrisPyroPhosphate treatment leads to HIF-1α suppression and eradication of early hepatoma tumors in rats,

Myo‐inositol trispyrophosphate (ITPP), a synthetic allosteric effector of hemoglobin, increases the regulated oxygen‐releasing capacity of red blood cells (RBCs), leading to suppression of hypoxia‐inducible factor 1α (HIF‐1α) and to down‐regulation of hypoxia‐inducible genes such as vascular endothelial growth factor (VEGF). As a consequence, tumor growth is markedly affected. The effect of weekly intravenous injection of ITPP on an orthotopic, syngenic rat hepatocellular carcinoma (HCC) model was compared to that for untreated animals and animals subjected to conventional Doxorubicin chemotherapy. The longitudinal examination of HCC was performed by microCT imaging, and the cellular and molecular changes were evaluated by histology and Western blotting analysis of HIF‐1α, VEGF, and caspase‐3 gene expression in the tumor and in the surrounding liver. Hematologic impact was evaluated by blood cell‐count measurement and determination of P50 (oxygen partial pressure for a 50 % oxygen saturation of hemoglobin). The HCC evaluation by microCT revealed a high potency of ITPP for tumor growth inhibition, thus allowing long‐term survival and even cure of almost all the treated animals. The P50 value of hemoglobin in RBCs underwent a shift of 30 % following ITPP injection. Under these conditions, HIF‐1α activity was strongly decreased, VEGF expression was down‐regulated, and apoptosis was induced in HCC and surrounding liver cells, as indicated by Caspase‐3 expression. ITPP did not affect hematologic parameters during treatment. The observations of in vivo tumor eradication suggest a significant clinical potential for ITPP in cancer therapy.

In Vivo Proof of Concept of Adoptive Immunotherapy for Hepatocellular Carcinoma Using Allogeneic Suicide Gene-modified Killer Cells

Cell therapy based on alloreactivity has completed clinical proof of concept against hematological malignancies. However, the efficacy of alloreactivity as a therapeutic approach to treat solid tumors is unknown. Using cell culture and animal models, we aimed to investigate the efficacy and safety of allogeneic suicide gene-modified killer cells as a cell-based therapy for hepatocellular carcinoma (HCC), for which treatment options are limited. Allogeneic killer cells from healthy donors were isolated, expanded, and phenotypically characterized. Antitumor cytotoxic activity and safety were studied using a panel of human or murine HCC cell lines engrafted in immunodeficient or immunocompetent mouse models. Human allogeneic suicide gene-modified killer cells (aSGMKCs) exhibit a high, rapid, interleukin-2-dependent, and non-major histocompatibility complex class I-restricted in vitro cytotoxicity toward human hepatoma cells, mainly mediated by natural killer (NK) and NK-like T cells. In vivo evaluation of this cell therapy product demonstrates a marked, rapid, and sustained regression of HCC. Preferential liver homing of effector cells contributed to its marked efficacy. Calcineurin inhibitors allowed preventing rejection of allogeneic lymphocytes by the host immune system without impairing their antitumor activity. Our results demonstrate proof of concept for aSGMKCs as immunotherapy for HCC and open perspectives for the clinical development of this approach.

Design and In Vivo Evaluation of a Robotized Needle Insertion System for Small Animals

The development of imaging devices adapted to small animals has opened the way to image-guided procedures in biomedical research. In this paper, we focus on automated procedures to study the effects of the recurrent administration of substances to the same animal over time. A dedicated system and the associated workflow have been designed to percutaneously position a needle into the abdominal organs of mice. Every step of the procedure has been automated: the camera calibration, the needle access planning, the robotized needle positioning, and the respiratory-gated needle insertion. Specific devices have been developed for the registration, the animal binding under anesthesia, and the skin puncture. Among the presented results, the system accuracy is particularly emphasized, both in vitro using gelose phantoms and in vivo by injecting substances into various abdominal organs. The study shows that robotic assistance could be routinely used in biomedical research laboratories to improve existing procedures, allowing automated accurate treatments and limited animal sacrifices.

Design and Development of a Robotized System Coupled to µCT Imaging for Intratumoral Drug Evaluation in a HCC Mouse Model

Hepatocellular carcinoma (HCC) is one of the most common cancer related deaths worldwide. One of the main challenges in cancer treatment is drug delivery to target cancer cells specifically. Preclinical evaluation of intratumoral drugs in orthotopic liver cancer mouse models is difficult, as percutaneous injection hardly can be precisely performed manually. In the present study we have characterized a hepatoma model developing a single tumor nodule by implantation of Hep55.1C cells in the liver of syngeneic C57BL/6J mice. Tumor evolution was followed up by µCT imaging, and at the histological and molecular levels. This orthotopic, poorly differentiated mouse HCC model expressing fibrosis, inflammation and cancer markers was used to assess the efficacy of drugs. We took advantage of the high precision of a previously developed robotized system for automated, image-guided intratumoral needle insertion, to administer every week in the tumor of the Hep55.1C mouse model. A significant tumor growth inhibition was observed using our robotized system, whereas manual intraperitoneal administration had no effect, by comparison to untreated control mice.

Genomic CpG enrichment of oncolytic parvoviruses as a potent anticancer vaccination strategy for the treatment of pancreatic adenocarcinoma

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.

Apport de l’imagerie structurale par micro scanner X dans l’évaluation pré-clinique de chimiothérapies du carcinome hépatocellulaire sur greffe orthotopique chez le rat ACI

Animal experimentation is a prerequisite for preclinical evaluation of treatments such as chemotherapy. Its strictly regulated with the purpose of reducing the number of experimental animal as well as their pain. Small animal imaging should provide a painless longitudinal follow up of tumor progression on a single animal. The aim of the study is to validate small animal imaging by microscanner (μscan) in longitudinal follow up of a hepatocellular carcinoma (HCC) and to demonstrate its interest for in vivo evaluation of tumor response to different therapeutics. An HCC model achieved by orthotopic graft of the MH3924A cell line in ACI rats was followed using a Imtek/Siemens microscanner (μscan) with contrast agents (Fenestra(®) LC/VC). The procedures giving the optimal enhancement of the liver as well as a reliable determination of tumor volumes by μscan were validated. Three protocols for therapeutic assessment through μscan longitudinal follow up were performed. Each consisted in three groups testing a chemotherapy (gemcitabine, gemcitabine-oxaliplatine or sorafenib) versus two control groups (placebo and doxorubicine). Comparison was done on tumor volumes, median and actual survivals. There was a significant correlation between tumor volumes measured by μscan and autopsy. Treatment by sorafenib, at the contrary of gemcitabine alone or with oxaliplatine, resulted in a significant reduction in tumor volumes and prolongation of actuarial survival. These results are consistent with available clinical data for these diverse therapeutics. In conclusion, small animal imaging with μscan is a non-invasive, reliable, and reproducible method for preclinical evaluation of antitumor agents.

Apport de l’imagerie structurale par micro scanner X dans l’évaluation pré-clinique de chimiothérapies du carcinome hépatocellulaire sur greffe orthotopique chez le rat ACIContribution of microCT structural imaging to preclinical evaluation of hepatocellular carcinoma chemotherapeutics on orthotopic graft in ACI rats

Animal experimentation is a prerequisite for preclinical evaluation of treatments such as chemotherapy. Its strictly regulated with the purpose of reducing the number of experimental animal as well as their pain. Small animal imaging should provide a painless longitudinal follow up of tumor progression on a single animal. The aim of the study is to validate small animal imaging by microscanner (μscan) in longitudinal follow up of a hepatocellular carcinoma (HCC) and to demonstrate its interest for in vivo evaluation of tumor response to different therapeutics. An HCC model achieved by orthotopic graft of the MH3924A cell line in ACI rats was followed using a Imtek/Siemens microscanner (μscan) with contrast agents (Fenestra(®) LC/VC). The procedures giving the optimal enhancement of the liver as well as a reliable determination of tumor volumes by μscan were validated. Three protocols for therapeutic assessment through μscan longitudinal follow up were performed. Each consisted in three groups testing a chemotherapy (gemcitabine, gemcitabine-oxaliplatine or sorafenib) versus two control groups (placebo and doxorubicine). Comparison was done on tumor volumes, median and actual survivals. There was a significant correlation between tumor volumes measured by μscan and autopsy. Treatment by sorafenib, at the contrary of gemcitabine alone or with oxaliplatine, resulted in a significant reduction in tumor volumes and prolongation of actuarial survival. These results are consistent with available clinical data for these diverse therapeutics. In conclusion, small animal imaging with μscan is a non-invasive, reliable, and reproducible method for preclinical evaluation of antitumor agents.