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Rheumatoid arthritis is frequently associated with chronic pain that still remains difficult to treat. Targeting nerve growth factor (NGF) seems very effective to reduce pain in at least osteoarthritis and chronic low back pain but leads to some potential adverse events. Our aim was to better understand the involvement of the intracellular signalling pathways activated by NGF through its specific tyrosine kinase type A (TrkA) receptor in the pathophysiology of rheumatoid arthritis using the complete Freund adjuvant model in our knock-in TrkA/C mice. Our multimodal study demonstrated that knock-in TrkA/C mice exhibited a specific decrease of mechanical allodynia, weight-bearing deficit, peptidergic (CGRP+) and sympathetic (TH+) peripheral nerve sprouting in the joints, a reduction in osteoclast activity and bone resorption markers, and a decrease of CD68-positive cells in the joint with no apparent changes in joint inflammation compared with wild-type mice after arthritis. Finally, transcriptomic analysis shows several differences in dorsal root ganglion mRNA expression of putative mechanotransducers, such as acid-sensing ionic channel 3 and TWIK-related arachidonic acid activated K+ channel, as well as intracellular pathways, such as c-Jun, in the joint or dorsal root ganglia. These results suggest that TrkA-specific intracellular signalling pathways are specifically involved in mechanical hypersensitivity and bone alterations after arthritis using TrkA/C mice.

International audience; Over the last decade, upconversion nanoparticles (UCNP) have been widely investigated in nanomedicine due to their high potential as imaging agents in the near-infrared (NIR) optical window of biological tissues. Here, we successfully develop active targeted UCNP as potential probes for dual NIR-NIR fluorescence and radioactive-guided surgery of prostate-specific membrane antigen (PSMA)(+) prostate cancers. We designed a one-pot thermolysis synthesis method to obtain oleic acid-coated spherical NaYF4:Yb,Tm@NaYF4 core/shell UCNP with narrow particle size distribution (30.0 ± 0.1 nm, as estimated by SAXS analysis) and efficient upconversion luminescence. Polyethylene glycol (PEG) ligands bearing different anchoring groups (phosphate, bis- and tetra-phosphonate-based) were synthesized and used to hydrophilize the UCNP. DLS studies led to the selection of a tetra-phosphonate PEG(2000) ligand affording water-dispersible UCNP with sustained colloidal stability in several aqueous media. PSMA-targeting ligands (i.e., glutamate-urea-lysine derivatives called KuEs) and fluorescent or radiolabelled prosthetic group were grafted onto the UCNP surface by strain-promoted azide-alkyne cycloaddition (SPAAC). These UCNP, coated with 10 or 100% surface density of KuE ligands, did not induce cytotoxicity over 24 h incubation in LNCaP-Luc or PC3-Luc prostate cancer cell lines or in human fibroblasts for any of the concentrations evaluated. Competitive binding assays and flow cytometry demonstrated the excellent affinity of UCNP@KuE for PSMA-positive LNCaP-Luc cells compared with non-targeted UCNP@CO2H. Furthermore, the binding of UCNP@KuE to prostate tumour cells was positively correlated with the surface density of PSMA-targeting ligands and maintained after 125I-radiolabelling. Finally, a preliminary biodistribution study in LNCaP-Luc-bearing mice demonstrated the radiochemical stability of non-targeted [125I]UCNP paving the way for future in vivo assessments.

Myocardial insulin resistance (IR) could be a predictive factor of cardiovascular events. This study aimed to introduce a new method using 123I-6-deoxy-6-iodo-d-glucose (6DIG), a pure tracer of glucose transport, for the assessment of IR using cardiac dynamic nuclear imaging. The protocol evaluated first in rat-models consisted in two 6DIG injections and one of insulin associated with planar imaging and blood sampling. Compartmental modeling was used to analyze 6DIG kinetics in basal and insulin conditions and to obtain an index of IR. As a part of a translational approach, a clinical study was then performed in 5 healthy and 6 diabetic volunteers. In rodent models, the method revealed reproducible when performed twice at 7 days apart in the same animal. Rosiglitazone, an insulin-sensitizing drug, induced a significant increase of myocardial IR index in obese Zucker rats from 0.96 ± 0.18 to 2.26 ± 0.44 (P

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International audience; Colibactin-producing E. coli (CoPEC) are frequently detected in colorectal cancer (CRC) and exhibit procarcinogenic properties. Because increasing evidence show the role of immune environment and especially of antitumor T-cells in CRC development, we investigated the impact of CoPEC on these cells in human CRC and in the APC(Min/+) mice colon. T-cell density was evaluated by immunohistochemistry in human tumors known for their CoPEC status. APC(min/+) mice were chronically infected with a CoPEC strain (11G5). Immune cells (neutrophils and T-cell populations) were then quantified by immunofluorescent staining of the colon. The quantification of lymphoid populations was also performed in the mesenteric lymph nodes (MLNs). Here, we show that the colonization of CRC patients by CoPEC is associated with a decrease of tumor-infiltrating T lymphocytes (CD3(+) T-cells). Similarly, we demonstrated, in mice, that CoPEC chronic infection decreases CD3(+) and CD8(+) T-cells and increases colonic inflammation. In addition, we noticed a significant decrease in antitumor T-cells in the MLNs of CoPEC-infected mice compared to that of controls. Moreover, we show that CoPEC infection decreases the antimouse PD-1 immunotherapy efficacy in MC38 tumor model. Our findings suggest that CoPEC could promote a procarcinogenic immune environment through impairment of antitumor T-cell response, leading to tumoral resistance to immunotherapy. CoPEC could thus be a new biomarker predicting the anti-PD-1 response in CRC.

International audience; Rationale: Pretargeted radioimmunotherapy (PRIT) based upon bioorthogonal click chemistry has been investigated for the first time in the context of peritoneal carcinomatosis using a CEA-targeting 35A7 mAb bearing trans-cyclooctene (TCO) moieties and several 177 Lu-labeled tetrazine (Tz) radioligands. Starting from three Tz probes containing PEG linkers of varying lengths between the DOTA and Tz groups (i.e. PEGn = 3, 7, or 11, respectively, for Tz-1, Tz-2, and Tz-3), we selected [ 177 Lu]Lu-Tz-2 as the most appropriate for pretargeted SPECT imaging and demonstrated its efficacy in tumor growth control.Methods: An orthotopic model of peritoneal carcinomatosis (PC) was obtained following the intraperitoneal (i.p.) injection of A431-CEA-Luc cells in nude mice. Tumor growth was assessed using bioluminescence imaging. Anti-CEA 35A7 mAb was grafted with 2-3 TCO per immunoglobulin. Pretargeted SPECT imaging and biodistribution experiments were performed to quantify the activity concentrations of [ 177 Lu]Lu-Tz-1-3 in tumors and non-target organs to determine the optimal Tz probe for the PRIT of PC.Results: The pharmacokinetic profiles of [ 177 Lu]Lu-Tz-1-3 alone were determined using both SPECT imaging and biodistribution experiments. These data revealed that [ 177 Lu]Lu-Tz-1 was cleared via both the renal and hepatic systems, while [ 177 Lu]Lu-Tz-2 and [ 177 Lu]Lu-Tz-3 were predominantly excreted via the renal system. In addition, these results illuminated that the longer the PEG linker, the more rapidly the Tz radioligand was cleared from the peritoneal cavity. The absorbed radiation dose corresponding to pretargeting with 35A7-TCO followed 24 h later by [ 177 Lu]Lu-Tz-1-4 was higher for tumors following the administration of [ 177 Lu]Lu-Tz-2 (i.e. 0.59 Gy/MBq) compared to either [ 177 Lu]Lu-Tz-1 (i.e. 0.25 Gy/MBq) and [ 177 Lu]Lu-Tz-3 (i.e. 0.18 Gy/MBq). In a longitudinal PRIT study, we showed that the i.p. injection of 40 MBq of [ 177 Lu]Lu-Tz-2 24 hours after the systemic administration of 35A7-TCO significantly slowed tumor growth compared to control mice receiving only saline or 40 MBq of [ 177 Lu]Lu-Tz-2 alone. Ex vivo measurement of the peritoneal carcinomatosis index (PCI) confirmed that PRIT significantly reduced tumor growth (PCI = 15.5 ± 2.3 after PRIT vs 30.0 ± 2.3 and 30.8 ± 1.4 for the NaCl and [ 177 Lu]Lu-Tz-2 alone groups, respectively).Conclusion: Our results clearly demonstrate the impact of the length of PEG linkers upon the biodistribution profiles of 177 Lu-labeled Tz radioligands. Furthermore, we demonstrated for the first time the possibility of using bioorthogonal chemistry for both the pretargeted SPECT and PRIT of peritoneal carcinomatosis.

International audience; Triple negative breast cancers (TNBC) without BRCA1/2 gene mutation or BRCAness are nowadays the breast malignancies most difficult to treat. Improvement of their treatment, for all phases of the disease, is an important unmet medical need. We analyzed the effect of homoharringtonine (HHT), a natural protein synthesis inhibitor approved for treatment of chronic myeloid leukemia, on four cell lines representing aggressive, BRCA1/2 non-mutated, TNBC genomic categories. We show that HHT inhibits in vitro growth of all cell lines for more than 80%, after 48-72 h exposure to 20-100 ng/mL, the concentrations achievable in human plasma after subcutaneous administration of the drug. HHT, at 100 ng/mL, strongly reduced levels of a major TNBC survival factor, anti-apoptotic protein Mcl-1, after only 2 h of exposure, in all cell lines except MDA-MB-231. Other anti-apoptotic proteins, Bcl-2, survivin and XIAP, were also strongly downregulated. Moreover, in vivo growth of the least sensitive cell line to HHT in vitro, MDA-MB-231, was inhibited for 36.5% in mice, by 1 mg/kg of the drug, given subcutaneously, bi-daily, over 7 days. These results demonstrate marked antineoplastic activity of homoharringtonine in TNBC, making further development of the drug in this disease highly warranted.

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Bioorthogonal chemistry represents a challenging approach in pretargeted radioimmunotherapy (PRIT). We focus here on mAb modifications by grafting an increase amount of trans-cyclooctene (TCO) derivatives (0 to 30 equivalents with respect to mAb) bearing different polyethylene glycol (PEG) linkers between mAb and TCO (i.e. PEG0 (1), PEG4 (2) and PEG12 (3)) and assessing their functionality. We used colorectal xenograft (HT29/Ts29.2) and peritoneal carcinomatosis (A431-CEA-Luc/35A7) as tumor cells/mAbs models and fluorescent tetrazines (TZ). MALDI-TOF MS shows that grafting with 2,3 increases significantly the number of TCO per mAb compared with no PEG. In vitro immunofluorescence showed that Ts29.2 and 35A7 labeling intensity is correlated with the number of TCO when using 1,3 while signals reach a maximum at 10 equivalents when using 2. Under 10 equivalents conditions, the capacity of resulting mAbs-1–3 for antigen recognition is similar when reported per grafted TCO and comparable to mAbs without TCO. In vivo, on both models, pretargeting with mAbs-2,3 followed by TZ injection induced a fluorescent signal two times lower than with mAbs-1. These findings suggest that while PEG linkers allow a better accessibility for TCO grafting, it might decrease the number of reactive TCO. In conclusion, mAb-1 represents the best candidate for PRIT.

Uniform, hydrophilic 50 nm diameter Nd

Uniform, hydrophilic 50 nm diameter Nd3+-doped Ba0.3Lu0.7F2.7 nanospheres are synthesized at 120 °C using a singular one-pot method based on the use of ethylene glycol as solvent, in the absence of any additive. The composition and crystal structure of the undoped material are analyzed in detail using ICP and XRD, which reveals a BaF2 cubic crystal structure that is able to incorporate 70 mol% of Lu ions. This finding contrasts with the reported phase diagram of the system, where the maximum solubility is around 30 mol% Lu. XRD proves as well that the Ba0.3Lu0.7F2.7 structure is able to incorporate Nd3+ ions up to, at least 10 mol%, without altering the uniform particles morphology. The Nd-doped particles exhibit nearinfrared luminescence when excited at 810 nm. The maximum emission intensity with the minimum concentration quenching effect is obtained at 1.5% Nd doping level. X-ray computed tomography experiments are carried out on powder samples of the latter composition. The sample significantly absorbs X-ray photons, thus demonstrating that the Nd3+-doped Ba0.3Lu0.7F2.7 nanospheres are good candidates as contrast agents in computed tomography.

Tetraspanin 8 (TSPAN8) overexpression is correlated with poor prognosis in human colorectal cancer (CRC). A murine mAb Ts29.2 specific for human TSPAN8 provided significant efficiency for immunotherapy in CRC pre-clinical models. We therefore evaluate the feasability of targeting TSPAN8 in CRC with radiolabeled Ts29.2. Staining of tissue micro-arrays with Ts29.2 revealed that TSPAN8 espression was restricted to a few human healthy tissues. DOTA-Ts29.2 was radiolabeled with 111In or 177Lu with radiochemical purities >95%, specific activity ranging from 300 to 600 MBq/mg, and radioimmunoreactive fractions >80%. The biodistribution of [111In]DOTA-Ts29.2 in nude mice bearing HT29 or SW480 CRC xenografts showed a high specificity of tumor localization with high tumor/blood ratios (HT29: 4.3; SW480-TSPAN8: 3.9 at 72h and 120h post injection respectively). Tumor-specific absorbed dose calculations for [177Lu]DOTA-Ts29.2 was 1.89 Gy/MBq, establishing the feasibility of using radioimmunotherapy of CRC with this radiolabeled antibody. A significant inhibition of tumor growth in HT29 tumor-bearing mice treated with [177Lu]DOTA-Ts29.2 was observed compared to control groups. Ex vivo experiments revealed specific DNA double strand breaks associated with cell apoptosis in [177Lu]DOTA-Ts29.2 treated tumors compared to controls. Overall, we provide a proof-of-concept for the use of [111In/177Lu]DOTA-Ts29.2 that specifically target in vivo aggressive TSPAN8-positive cells in CRC.

Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.

Introduction: Triple negative breast cancer (TNBC) is a heterogeneous disease for which new therapeutic approaches are urgently needed. Homoharringtonine (HHT) is a protein synthesis inhibitor clinically approved in chronic myeloid leukemia. Its multi-factor mechanism of action includes downregulation of short-lived cellular proteins like Mcl-1. As increased protein synthesis lies behind several hallmarks of cancer present in TNBC, we investigated the effect of HHT on TNBC in vitro and in vivo. Methods: TNBC cell lines CAL-51, MDA-MB-231 and MDA-MB-468, representative of aggressive, BRCA1 non-mutated TNBC (Lehmann, J Clin Invest 2011) were used. HHT was provided by LeukePharma, Houston, TX, USA. Cell viability was assessed by sulphorhodamine B assay. Number of cells in different cell cycle phases or in apoptosis was determined by flow cytometry. Protein expression was evaluated by western blot. For in vivo experiments, xenografts of MDA-MB-231 were established in Swiss nu/nu mice, treated for 7 days by subcutaneous HHT (1 mg/kg, bi-daily), and analyzed on day 10. Evaluation of HHT on MDA-MB-468 and CAL-51 xenografts is ongoing. Results: Already after 24h of exposure to 100 ng/ml HHT (mean plasma HHT concentration after 5 mg/m2/day in humans (Sentenac S et al, Blood 2003)), viability of CAL-51 and MDA-MB-468 cells was strongly reduced (10.4±1.0% and 19.2±2.2% of control, respectively). This inhibition level was obtained in MDA-MB-231 cells only after 72h (18.7±4.8%). After 48h in 100 ng/ml HHT, MDA-MB-231 and -468 cells accumulated in the S phase of the cell cycle, whereas the CAL-51 accumulated in the G1/0 phase. This was associated with an increase of apoptosis in CAL-51 (42.0±2.4% vs 9.2±1.3%, untreated vs treated cells, respectively) and in MDA-MB-468 (24.2±2.8% vs 12.2±3.6%) but not in MDA-MB-231 (9.0±2.2% vs 3.6±1.7%). Concordantly, 100 ng/ml of HHT induced, after only 2h, a marked decrease of the level of anti-apoptotic proteins Mcl-1, Bcl-2, survivin and XIAP in CAL-51 and MDA-MB-468 cells, which preceded by 2h the reduction of caspase-3 level. No change in expression of those proteins was observed in MDA-MB-231 during all 48h of exposure to HHT. On the other side, the quantity of phosphorylated S6 increased in MDA-MB-231 but decreased in CAL-51 and MDA-MB-468 already after 24h of incubation with 100 ng/ml HHT. In vivo, HHT reduced growth of MDA-MB-231 xenografts for 36.5% (tumor volume on day 10: 230.4±27.7% mm3 vs 146.2±15.5% mm3, untreated vs treated, respectively). Conclusion: HHT exerts cytotoxic action on TNBC cell lines CAL-51 and MDA-MB-468 whereas its effect on MDA-MB-231 is dominantly cytostatic. Because of its capacity to rapidly reduce the levels of anti-apoptotic proteins, HHT is worth further studies as potential sensitizer of TNBC to chemo- and/or targeted therapy. Citation Format: Mohamad Yakhni, Arnaud Briat, Elisabeth Miot-Noirault, Florent Cachin, Frederique Penault-Llorca, Nina Radosevic-Robin. Homoharringtonine, a natural protein synthesis inhibitor, inhibits growth of triple negative breast cancer in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5803.

Summary Survival following pancreatic cancer remains poor despite incremental advances in surgical and adjuvant therapy, and new strategies for treatment are needed. Oncolytic virotherapy is an attractive approach for cancer treatment. In this study, we have evaluated the effectiveness of the Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapeutic approach for pancreatic cancer. The Lister vaccine strain of vaccinia virus was effective against all human pancreatic carcinoma cells tested in vitro, especially those insensitive to oncolytic adenovirus. The virus displayed inherently high selectivity for cancer cells, sparing normal cells both in vitro and in vivo, with effective infection of tumors after both intravenous (IV) and intratumoral (IT) administration. The expression of endostatin-angiostatin fusion protein was confirmed in a pancreatic cancer model both in vitro and in vivo, with evidence of inhibition of angiogenesis. This novel vaccinia virus demonstrated significant antitumor potency in vivo against the Suit-2 model by IT administration. The present study suggests that the novel Lister strain of vaccinia virus armed with the endostatin-angiostatin fusion gene is a potential therapeutic agent for pancreatic cancer.

Insulin resistance, implying depressed cellular sensitivity to insulin, is a risk factor for type 2 diabetes and cardiovascular disease. This study is the first step towards the development of a technique of insulin resistance measurement in humans with a new tracer of glucose transport, [(123)I]6-deoxy-6-iodo-D-glucose (6DIG).We investigated 6DIG kinetics in anaesthetised control rats and in three models of insulin-resistant rats: fructose fed, Zucker and ZDF. The study of myocardial 6DIG activity was performed under two conditions: first, 6DIG was injected under the baseline condition and then it was injected after a bolus injection of insulin. After each injection, radioactivity was measured over 45 min by external detection via NaI probes, in the heart and blood. A tri-compartment model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the heart.These coefficients were significantly increased with insulin in control rats and did not change significantly in insulin-resistant rats. The ratio of the coefficient obtained under insulin to that obtained under basal conditions gave an index of cardiac insulin resistance for each animal. The mean values of these ratios were significantly lower in insulin-resistant than in control rats: 1.16 +/- 0.06 vs 2.28 +/- 0.18 (p0.001) for the fructose-fed group, 0.92 +/- 0.05 vs 1.62 +/- 0.25 (p0.01) for the Zucker group and 1.34 +/- 0.06 vs 2.01 +/- 0.26 (p0.05) for the ZDF group.These results show that 6DIG could be a useful tracer to image cardiac insulin resistance.

Introduction RNA interference is efficient in in vitro studies, and appears as a therapeutic tool of major clinical interest. Nevertheless, the clinical utilisation of siRNAs is restrained by the poor availability of biodistribution data on this new class of pharmaceutics. This study aimed at defining the biodistribution and pharmacokinetics properties of an siRNA directed to the Casein Kinase-2 beta (CK2β) subunit, a potential target in cancer therapy. Methods Four CK2β siRNAs were chemically modified on each extremity of sense or anti-sense strand and radioiodinated. The biodistribution of each entity was analysed in glioblastoma-bearing mice using nuclear imaging and compared to a control GFP siRNA. Results The labelling process was associated with preservation of interference activity, except when applied to the 5 ' antisense terminus. Radioactivity was predominantly observed in organs of the excretory system after intravenous administration: liver, kidneys and bladder. Tumor/Contralateral muscle ratio showed significant differences depending on the labelling site. Activity associated with CK2β 5 ' s was quite constant over 2hours, while CK2β 3 ' as activity decreased by 40% in tumor. Finally, synchrotron X-ray analysis showed that CK2β 3 ' s is more abundant in tumor than in liver, brain or muscle, and uniformly distributed between intra- and extracellular compartments. Conclusions In this study, we highlighted the large influence of siRNAs radiolabelling position on their biodistribution and pharmacokinetic profiles, and proposed a systematic approach for the imaging of all siRNAs of clinical interest.

De nombreuses pathologies sont associees aux anomalies du metabolisme du glucose ou aux perturbations de son transport (diabete de type 2 ou insulino-resistance). L'etat pre-diabetique est caracterise par un etat d'insulino-resistance ou, en d'autres termes, par un defaut de transport de glucose vers les tissus insulino-sensibles. tels que les muscles et le tissu adipeux. La modelisation mathematique des donnees experimentales peut etre une excellente methode pour etudier les mecanismes impliques dans le phenomene biologique concerne. Ainsi, a partir d'une representation symbolique comme la modelisation compartimentale, il est possible de developper une base theorique pour l'observation et de considerer les experiences les mieux adaptees pour l'etude. Nous avons montre, avec des modeles compartimentaux, que le [ 123 I]-6-deoxy-6-iodo-D-glucose (6-DIG), valide comme traceur du transport du glucose in vitro, peut permettre la verification de cette anomalie de transport. Pour mesurer cette insulino-resistance, nous avons estime les coefficients de transfert fractionnel du 6-DIG du sang vers les differents organes de l'etude. Nous avons realise plusieurs etudes avant d'arriver a un modele satisfaisant et une attention toute particuliere a ete portee a la precision des parametres estimes, afin de choisir le meilleur modele. Les resultats ont montre qu'en associant les donnees experimentales obtenues avec les activites du 6-DIG et un modele mathematique adapte, des parametres discriminants (coefficients de transfert fractionnel) entre les deux groupes, rats temoins et insulinoresistants, peuvent etre degages.

Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has played a crucial role in the eradication of smallpox. It continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer. However, the mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. By studying the effect of hypoxia on VV infection, we found that vascular endothelial growth factor A (VEGF-A) augments oncolytic VV cytotoxicity. VEGF derived from tumor cells acts to increase VV internalization, resulting in increased replication and cytotoxicity in an AKT-dependent manner in both tumor cells and normal respiratory epithelial cells. Overexpression of VEGF also enhances VV infection within tumor tissue in vivo after systemic delivery. These results highlight the importance of VEGF expression in VV infection and have potential implications for the design of new strategies to prevent poxvirus infection and the development of future generations of oncolytic VV in combination with conventional or biological therapies.

Pancreatic adenocarcinoma (PDAC) is an aggressive malignancy which is resistant to conventional treatments. PDAC typically contains areas that are profoundly hypoxic which both drives early epithelial to mesenchymal transition, with subsequent metastasis, and is an important determinant of resistance to chemotherapy and radiotherapy. Oncolytic Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has demonstrated efficacy as an anti-tumor therapy in clinical trials. It is currently the only oncolytic viral therapy to show efficacy on systemic administration, which would be crucial for the treatment of metastatic PDAC (1). No unique host cell surface receptor has been identified for VV and the reasons for its tropism for PDAC and other cancers are unclear. We have investigated VV as a vector for targeting the hypoxic regions in PDAC, as other viral vectors have been found to replicate poorly in hypoxia. We found that cytotoxicity was enhanced in hypoxia in PDAC cells up to 10 fold (2). This increase in cytotoxicity was only seen in PDAC cell lines where there was hypoxic induction of vascular endothelial growth factor-A (VEGF). Stable MiaPaca2 cell lines expressing VEGF (MPVe-165) and a paired vector control (MPVC) were used to demonstrate that VEGF can augment viral transgene expression, cytotoxicity and replication of oncolytic VV. SUIT2 cells treated with VEGF specific siRNA or control were used to demonstrate that conversely, VEGF suppression reduces viral transgene expression, cytotoxicity and replication in vitro. MPVe-165 and MPVC xenografts were established in BALB/c nude mice before treatment with systemic oncolytic VV. Increased viral replication and gene expression in MPVe-165 xenografts demonstrates this novel finding is relevant in vivo. Using qPCR, confocal microscopy of a fluorescently tagged VV (VVL-488) and specific tyrosine kinase inhibition we found that VEGF acts in an autocrine fashion to increase VV internalization, via Akt phospohorylation in PDAC. These results show that VEGF is a novel factor involved in the tropism and pathogenesis of VV for PDAC, and other tumor types, and has relevance for potential combination therapy regimes. Given these findings we then constructed an oncolytic VV to target hypoxic PDAC cells using the HIF-1α oxygen degradation domain, encephalomyocarditis virus internal ribosomal entry site and the VEGF 3’ un-translated region to regulate gene expression in hypoxia. A conventional hypoxia response element promoter driven construct was not feasible given that the VV life cycle is located in the host cell cytoplasm and gene expression does not rely on eukaryotic transcription factors. We have shown a dose-, time- and oxygen-dependent effect using this construct in hypoxic cell culture, on exposure to hypoxia mimetics (DMOG/CoCl) and using a proteasomal inhibitor (MG132). We propose this concept may be adapted to regulate therapeutic genes, or produce a conditionally replicating VV, in hypoxic conditions, and enhance the specificity of this targeted therapy. References: 1. Breitbach CJ, Burke J, Jonker D, Stephenson J, Haas AR et al. Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature. 2011 Aug 31;477(7362):99-102. 2. Hiley CT, Yuan M, Lemoine NR, Wang Y. Lister strain vaccinia virus, a potential therapeutic vector targeting hypoxic tumours. Gene Ther. 2010 Feb;17(2):281-7. Citation Format: Crispin T. Hiley, Louisa Chard, James Tysome, Arnaud Briat, Nicholas R. Lemoine, Yaohe Wang. Increased efficacy of oncolytic vaccinia virus, an emerging therapy for pancreatic adenocarcinoma, due to intratumoral hypoxia and vascular endothelial growth factor A. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A37.

Integrin α(v)β(3) expression is upregulated during tumor growth and invasion in newly formed endothelial cells in tumor neovasculature and in some tumor cells. A tetrameric RGD‐based peptide, regioselectively addressable functionalized template‐(cyclo‐[RGDfK])4 (RAFT‐RGD), specifically targets integrin α(v)β(3) in vitro and in vivo. When labeled with indium‐111, the RAFT‐RGD is partially reabsorbed and trapped in the kidneys, limiting its use for further internal targeted radiotherapy and imaging investigations. We studied the effect of Gelofusine on RAFT‐RGD renal retention in tumor‐bearing mice. Mice were imaged using single photon emission computed tomography and optical imaging 1 and 24 h following tracer injection. Distribution of RAFT‐RGD was further investigated by tissue removal and direct counting of the tracer. Kidney sections were analyzed by confocal microscopy. Gelofusine significantly induced a >50% reduction of the renal reabsorption of (111) In‐DOTA‐RAFT‐RGD and A700‐RAFT‐RGD, without affecting tumor uptake. Injection of Gelofusine significantly reduced the renal retention of labeled RAFT‐RGD, while increasing the tumor over healthy tissue ratio. These results will lead to the development of future therapeutic approaches. (C ancer S ci 2012; 103: 1105–1110)

[Purpose]: In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma. [Methods]: We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated. [Results]: In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart. [Conclusion]: This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects. © Springer-Verlag 2010., This work was supported by grants from Cancer Research UK, the Medical Research Council, INSERM, la Ligue Nationale Contre le Cancer, the “Paris scientifiques régionaux” scheme of the Région Pays de la Loire, and the French National Cancer Institute (INCa, grant 0607-3D1615-66/AO INSERM).

International audience; Angiogenesis plays a central role in tumor growth and metastasis. Quantification or evaluation of angiogenesis is crucial for antiangiogenic therapeutic strategies. Since integrin alpha(v)beta(3) overexpression appears specific of angiogenesis at the adult stage, it became a target of choice over the past decade, and labeled RGD-based compounds, therefore, constitute promising agents for noninvasive tumor visualization and targeting. We evaluated the chemical and biologic properties of a new tetrameric RGD-based tracer named RAFT-RGD. RAFT-RGD was radiolabeled with indium-111, using the chelating agent [(1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid] (DOTA). Labeling reaction parameters, such as time, temperature, solvent, or molar ratio, were investigated in order to optimize the final properties of the labeled RGD peptide. A 97.7% +/- 0.7% binding efficiency was achieved. (111)In-DOTA-RAFT-RGD was injected intravenously in a cohort of alpha(v)beta(3)-positive tumor-bearing nude mice. We noninvasively visualized the in vivo distribution of the tracer, using a small-animal gamma camera. In vivo distribution and stability were also studied after organ removal. In vivo, the radiolabeled peptide showed rapid blood clearance and tumor uptake. Whole-body noninvasive planar imaging allowed tumor visualization from 1 hour postinjection. However, renal uptake must be reduced to increase the therapeutic potential of RAFT-RGD.

International audience; Monitoring p53 transcriptional activity to identify genotoxic damages induced by drugs has been proposed and validated in vitro. However, this methodology is by design limited to the cell line tested. In this study, we have fully validated a luciferase-based p53-reporter system in vitro and in vivo. We generated a mouse transgenic line to monitor non-invasively p53 activation in response to chemically induced DNA damage. Doxorubicin was used as a drug of known toxicity to validate our model. Reporter gene expression was measured using bioluminescence imaging. In females, a weak p53 luciferase activity driven by a p53-responsive promoter was detectable in the oral cavity region after doxorubicin treatment. In males, the signal increased in the lower abdominal region. Imaging of various organs revealed that the luciferase activity was mainly generated from the testes. Immunohistology demonstrated that the cells in the seminiferous tubules were damaged by the drug and confirmed that they were luciferase and p53 positive. Therefore, these transgenic mice could provide a powerful tool to predict, map and characterize at the organ and cellular levels the toxicity of compounds and help to develop new therapeutic agents in humans.

International audience; Purpose: Insulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state and it has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats with 125I-6-Deoxy-6-Iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo. Methods: Two protocols were performed, a hyperinsulinaemic-euglycaemic clamp and a normoinsulinaemic normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood were assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs. Results: Insulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady-state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p

Gene therapy is a very attractive strategy in experimental cancer therapy. Ideally, the approach aims to deliver therapeutic genes selectively to cancer cells. However, progress in the improvement of gene therapy formulations has been hampered by difficulties in measuring transgene delivery and in quantifying transgene expression in vivo. In clinical trials, endpoints rely almost exclusively on the analysis of biopsies by molecular and histopathological methods, which provide limited information. Therefore, to ensure the rational development of gene therapy, a crucial issue is the utilisation of technologies for the non-invasive monitoring of spatial and temporal gene expression in vivo upon administration of a gene delivery vector. Such imaging technologies would allow the generation of quantitative information about gene expression and the assessment of cancer gene therapy efficacy. In the past decade, progress has been made in the field of in vivo molecular imaging. This review highlights the various methods currently being developed in preclinical models.

Many pathologies are associated with abnormalities of glucose metabolism or with perturbations of its transport (type 2 diabetes or insulin-resistance). The pre-diabetic state is characterised by a state of insulin-resistance, in others words a defect of glucose transport in insulin-sensible tissues, such as muscles and adipose tissues. The mathematical modelling of experimental data can be an excellent method to explore the mechanisms implied in the studied biological phenomenon. Thus, starting from a symbolic formulation like the compartmental modelling, it can be possible to develop a theoretical basis for the observation and to consider the best-adapted experiments for the study. We showed with mathematical models that [123I]-6-deoxy-6-iodo-D-glucose (6-DIG), shown as a tracer of glucose transport in vitro, could point out this transport abnormality. To quantify the insulin resistance, we estimated the fractional transfer coefficients of 6-DIG from the blood to the organs. We realised many studies to lead to a satisfying model; special attention has been paid to the precision of the parameter to select the best model. The results showed that by associating experimental data obtained with 6-DIG activities and an adapted mathematical model, discriminating parameters (in and out fractional transfer coefficients) between the two groups (control and insulin-resistant rats) could be pointed out.

Background: Oncolytic virotherapy is a promising approach to treat cancers resistant to conventional therapies. Vaccinia virus, an enveloped DNA virus from the poxvirus family, has been used as a vector for cancer vaccines and oncolytic viral gene therapy. Wild type vaccinia virus has a natural tropism for cancer cells both in vitro and in vivo due to their increased proliferation, abnormalities in epidermal growth factor and interferon signalling, in addition to other unknown mechanisms. We have recently demonstrated that the anti-tumor efficacy of the Lister strain vaccinia virus, unlike some replicating oncolytic vectors, is not attenuated by hypoxia and even augmented 20 fold in some pancreatic ductal adenocarcinoma (PDAC) cells lines (CT Hiley et al. Gene Therapy 2009. In press). The underlying mechanism is not fully understood. Methods: Vascular Endothelial Growth Factor (VEGF) was detected by an enzyme-linked immunosorbent assay. The effect of VEGF on vaccinia virus infection in human cancer cells was investigated by manipulation of VEGF expression using siRNAs or gene over-expression. Normal human bronchial epithelial cells (NHBE) were used to assess the effect of VEGF on non-transformed cells. Results: Induction of VEGF by hypoxia was found in the supernatants of cancer cell lines showing sensitisation to vaccinia virus under hypoxia. Replication of vaccinia virus was correlated with the level of VEGF expression in a panel of human cancer cell lines. Knockdown of VEGF expression in Suit2 cells by siRNAs reduced transgene expression over 7 fold at 24 and 48 hours post infection (PI) (P Conclusion: This novel finding suggests that VEGF expression by cancer cells is at least partly responsible for some of the tumour tropism of wild type Lister vaccinia virus and highlights its suitability for use in poor prognosis tumour types, notably those with high levels of hypoxia and VEGF expression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 589.

International audience; Several bone conditions, eg, bone cancer, osteoporosis, and rheumatoid arthritis (RA), are associated with a risk of developing persistent pain. Increased osteoclast activity is often the hallmark of these bony pathologies and not only leads to bone remodeling but is also a source of pronociceptive factors that sensitize the bone-innervating nociceptors. Although historically bone loss in RA has been believed to be a consequence of inflammation, both bone erosion and pain can occur years before the symptom onset. Here, we have addressed the disconnection between inflammation, pain, and bone erosion by using a combination of 2 monoclonal antibodies isolated from B cells of patients with RA. We have found that mice injected with B02/B09 monoclonal antibodies (mAbs) developed a long-lasting mechanical hypersensitivity that was accompanied by bone erosion in the absence of joint edema or synovitis. Intriguingly, we have noted a lack of analgesic effect of naproxen and a moderate elevation of few inflammatory factors in the ankle joints suggesting that B02/B09-induced pain-like behavior does not depend on inflammatory processes. By contrast, we found that inhibiting osteoclast activity and acid-sensing ion channel 3 signaling prevented the development of B02/B09-mediated mechanical hypersensitivity. Moreover, we have identified secretory phospholipase A2 and lysophosphatidylcholine 16:0 as critical components of B02/B09-induced pain-like behavior and shown that treatment with a secretory phospholipase A2 inhibitor reversed B02/B09-induced mechanical hypersensitivity and bone erosion. Taken together, our study suggests a potential link between bone erosion and pain in a state of subclinical inflammation and offers a step forward in understanding the mechanisms of bone pain in diseases such as RA.

International audience