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Oncology

Rousseau J, Escriou V, Perrot P, Picarda G, Charrier C, Scherman D, Heymann D, Rédini F, Trichet V.: Advantages of bioluminescence imaging to follow siRNA or chemotherapeutic treatments in osteosarcoma preclinical models Cancer Gene Therapy January (2010)

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Abstract
Osteosarcoma is the most common malignant primary bone tumor for which pertinent preclinical models are still needed to develop new therapeutic strategies. As osteosarcoma growth is strongly supported by bone resorption, previous studies have inhibited the cytokine receptor activator of nuclear factor-kappaB ligand using antibodies or recombinant proteins. However, its expression has not yet been inhibited using genetic approaches using small interfering RNA. To optimize the delivery of small interfering RNA to its cellular target and demonstrate their efficiency in vivo, two new osteosarcoma models expressing the firefly luciferase enzyme were developed. These luciferase-expressing osteosarcomas showed conserved osteolytic and osteogenic activities in mice and were detectable by in vivo bioluminescence imaging. In comparison with measurement of tumor volume, bioluminescence analysis enabled earlier tumor detection and revealed extensive cell death in response to ifosfamide treatment. Finally, by targeting the luciferase expression into osteosarcoma, we established a protocol for in vivo administration of small interfering RNA combined with cationic liposome.

Kuroda J, Kuratsu J, Yasunaga M, Koga Y, Kenmotsu H, Sugino T, Matsumura Y: Antitumor effect of NK012, a 7-ethyl-10-hydroxycamptothecin-incorporating polymeric micelle, on U87MG orthotopic glioblastoma in mice compared with irinotecan hydrochloride in combination with bevacizumab Clin Cancer Res 16(2):521-9 January (2010)

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Abstract
Purpose: To clarify the effect of bevacizumab on NK012 therapy in mice bearing U87MG glioblastoma orthotopic xenografts in comparison with the combination therapy of irinotecan hydrochloride (CPT-11) with bevacizumab. Experimental Design: NK012 at 7-ethyl-10-hydroxycamptothecin (SN-38) equivalent dose of 30 mg/kg was administered intravenously three times every 4 days with or without bevacizumab. CPT-11 at 66.7 mg/kg was administered intravenously three times every 4 days or CPT-11 at 40 mg/kg/d over 5 consecutive days with or without bevacizumab. Bevacizumab was administered intraperitoneally six times every 4 days in each experiment. In vivo antitumor effects were evaluated by bioluminescence imaging, histopathologic evaluation, and immunohistochemistry. To evaluate interaction with bevacizumab, free SN-38 concentra- tion in tumor tissues was examined by high-performance liquid chromatography. Results: CPT-11 in combination with bevacizumab showed significantly more potent antitumor activ- ity and longer survival than CPT-11 monotherapy (P < 0.05). However, there was no difference between NK012 monotherapy and NK012 in combination with bevacizumab. Concentration of free SN-38 re- leased from NK012 in tumor tissue decreased in combination with bevacizumab (P = 0.027). NK012 monotherapy or NK012 with bevacizumab showed potent antitumor activity and longer survival than any dosing method of CPT-11 in combination with bevacizumab (P < 0.05). Orthotopic tumors treated with NK012 showed decreased tumor cellularity and lower Ki-67 index (P < 0.001) relative to those trea- ted with CPT-11/bevacizumab. Conclusions: The present study using orthotopic glioblastoma model in mice may warrant further preclinical evaluation of NK012 before conducting the clinical trial of the drug, because the antitumor activity of NK012 monotherapy was superior to the combination therapy of CPT-11 with bevacizumab.

Saito Y, Yasunaga M, Kuroda JI, Koga Y, Matsumura Y: Antitumour activity of NK012, SN-38-incorporating polymeric micelles, in hypovascular orthotopic pancreatic tumour. Eur J Cancer 46 (2010) 650 – 658 December (2009)

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Abstract
Human pancreatic cancer is refractory to chemotherapy partly because of blockage to penetration of anticancer agents. This issue must be taken into account particularly for the drug delivery system (DDS). The aim of the present study is to investigate how NK012 (SN-38-incorporating polymeric micelles) categorised as DDS exerts its antitumour effect in an orthotopic pancreatic tumour model compared with gemcitabine and irinotecan hydrochloride (CPT-11), a low-molecular-weight prodrug of a 7-ethyl-10-hydroxy-camptothecin (SN-38). The maximum tolerated doses (MTDs) of NK012 (30mg/kg/d), CPT-11 (66.7mg/kg/d) and gemcitabine (16.5mg/kg/d) were administered to mice bearing human pancreatic cancer cell (SUIT-2) xenografts implanted orthotopically. Antitumour effects of these compounds were evaluated. Drug distribution within the tumour was examined by fluorescence microscopy and high performance liquid chromatography (HPLC). NK012 exerted potent antitumour effects compared with CPT-11 and gemcitabine. A high concentration of NK012 and SN-38 released from NK012 had been observed until 192h. On the other hand, SN-38 converted from CPT-11 was detected only 1h postinjection. Fluorescence from NK012 was detected up to 48h, whereas that from CPT-11 almost disappeared by 24h postinjection. NK012 appeared to exert potent antitumour activity against intractable stroma-rich orthotopic pancreatic tumour xenografts due to its sufficient accumulation followed by the effective sustained release of SN-38 from NK012.

Shibata MA, Shibata E, Morimoto J, Eid NAS, Tanaka Y, Watanabe M, Otsuki Y: An Immunocompetent Murine Model of Metastatic Mammary Cancer Accessible to Bioluminescence Imaging Anticancer Research 29: 4389-4396 November (2009)

> Abstract

Abstract
Background: Many areas of research, including gene and pharmacological therapeutics, would benefit from longitudinal in vivo monitoring methodologies. To investigate the feasibility of one such methodology, we developed a murine mammary cancer model amenable to sequential bioluminescent imaging of tumor growth and metastasis in living animals. Materials and Methods: Metastatic mouse mammary carcinoma BJMC3879 cells were transfected to stably express firefly luciferase and inoculated into immunocompetent female BALB/c mice. Results: Sequential analysis using bioluminescent imaging showed increasing photon counts correlated to expanding mammary tumor volumes; in addition, strong signals from axillary, mandibular, femoral, thoracic and abdominal regions in mice were histopathologically determined to be due to metastases, the majority of which occurred in lymph nodes and lungs. Conclusion: The bioluminescent mouse mammary cancer model we established provides a method for quantifiable longitudinal in vivo imaging that can be used in gene and pharmacological therapy applications.

Reijmers RM, Groen RW, Rozemuller H, Kuil A, de Haan-Kramer A, Csikos T, Martens AC, Spaargaren M, Pals ST: Targeting EXT-1 reveals a crucial role for heparan sulfate in the growth of multiple myeloma Blood November (2009)

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Abstract
Expression of the heparan sulfate proteoglycan (HSPG) syndecan-1 is a hallmark of both normal and multiple myeloma (MM) plasma cells. Syndecan-1 could affect plasma cell fate by strengthening integrin-mediated adhesion via its core protein and/or by accommodating and presenting soluble factors via its HS side-chains. Here, we show that inducible RNAi-mediated knockdown of syndecan-1 in human MM cells leads to reduced growth rates and a strong increase of apoptosis. Importantly, knockdown of EXT-1, a co-polymerase critical for HS-chain biosynthesis, had similar effects. By employing an innovative myeloma xenotransplant model in Rag-2(-/-)gamma(c)(-/-) mice, we demonstrate that induction of EXT-1 knockdown in vivo dramatically suppresses the growth of bone marrow localized myeloma. Our findings provide direct evidence that the HS-chains of syndecan-1 are crucial for the growth and survival of MM cells within the bone marrow environment, and indicate the HS biosynthesis machinery as a potential treatment target in MM.

Kang MR, Kang JS, Han SB, Kim JH, Kim DM, Lee K, Lee CW, Lee KH, Lee CH, Han G, Kang JS, Kim HM, Park SK: A novel δ-lactam-based histone deacetylase inhibitor, KBH-A42, induces cell cycle arrest and apoptosis in colon cancer cells Biochem Pharmacol. 78(5):486-94 September (2009)

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Abstract
In this study, we investigated the anti-tumor activity of KBH-A42 [N-hydroxy-3-(2-oxo-1-(3-phenylpropyl)-1,2,5,6-tetrahydropyridin-3-yl)propanamide], a novel synthetic histone deacetylase (HDAC) inhibitor. KBH-A42 inhibited a variety of HDAC isoforms in enzyme assays and suppressed growth of various cancer cell lines. Among the cell lines examined, colon cancer cells, including SW620, SW480 and HCT-15, were the cell types most sensitive to KBH-A42. KBH-A42 inhibition of cancer cell growth was comparable to or stronger than that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor approved by the FDA to treat cutaneous T cell lymphomas. In SW620 cells, KBH-A42 increased the acetylation of histones, mediated cell cycle arrest (G1 arrest at low doses and G2 arrest at high doses), and induced apoptosis. The cell cycle arrest and apoptosis induced by KBH-A42 might be mediated through up-regulation of p21Waf1 and activation of caspases, respectively. In addition, KBH-A42 inhibited SW620 tumor growth in a human tumor xenograft model. Taken together, our results indicate that KBH-A42 exerts an anti-tumor activity in vitro and in vivo and is a promising therapeutic candidate to treat human cancers.

Graphical abstract

Keywords: HDAC; KBH-A42; Colon cancer; Cell cycle arrest; Apoptosis

Serganova I, Moroz E, Vider J, Gogiberidze G, Moroz M, Pillarsetty N, Doubrovin M, Minn A, Thaler HT, Massague J, Gelovani J, Blasberg R: Multimodality imaging of TGF {beta} signaling in breast cancer metastases FASEB J. 23(8):2662-72 August (2009)

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Abstract
The skeleton is a preferred site for breast cancer metastasis. We have developed a multimodality imaging approach to monitor the transforming growth factor beta (TGFbeta) signaling pathway in bone metastases, sequentially over time in the same animal. As model systems, two MDA-MB-231 breast cancer cells lines with different metastatic tropisms, SCP2 and SCP3, were transduced with constitutive and TGFbeta-inducible reporter genes and were tested in vitro and in living animals. The sites and expansion of metastases were visualized by bioluminescence imaging using a constitutive firefly luciferase reporter, while TGFbeta signaling in metastases was monitored by microPET imaging of HSV1-TK/GFP expression with [(18)F]FEAU and by a more sensitive and cost-effective bioluminescence reporter, based on nonsecreted Gaussia luciferase. Concurrent and sequential imaging of metastases in the same animals provided insight into the location and progression of metastases, and the timing and course of TGFbeta signaling. The anticipated and newly observed differences in the imaging of tumors from two related cell lines have demonstrated that TGFbeta signal transduction pathway activity can be noninvasively imaged with high sensitivity and reproducibility, thereby providing the opportunity for an assessment of novel treatments that target TGFbeta signaling.

Kuroda J, Kuratsu J, Yasunaga M, Koga Y, Saito Y, Matsumura Y.: Potent antitumor effect of SN-38-incorporating polymeric micelle, NK012, against malignant glioma Int J Cancer. 1;124(11):2505-11 June (2009)

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Abstract
Recent published reports on clinical trials of CPT-11 indicate the effectiveness of this compound, a prodrug of SN-38, against malignant glioma in combination with anti-vascular endothelial growth factor antibody. Here, we determined if NK012, and SN-38 incorporating micelle, can be an appropriate formulation for glioblastoma treatment compared with CPT-11. In vitro cytotoxicity was evaluated against several glioma lines with NK012, CPT-11, SN-38, ACNU, CDDP and etoposide. For the in vivo test, a human glioma line (U87MG) transfected with the luciferase gene was inoculated into nude mice brain for pharmacokinetic analysis by fluorescence microscopy and high-performance liquid chromatography after intravenous injection of NK012 and CPT-11. In vivo antitumor activity of NK012 and CPT-11 was evaluated by bioluminescence image and Kaplan-Meier analyses. The growth-inhibitory effects of NK012 were 34- to 444-fold more potent than those of CPT-11. Markedly enhanced and prolonged distribution of free SN-38 in the xenografts was observed after NK012 injection compared with CPT-11. NK012 showed significantly potent antitumor activity against an orthotopic glioblastoma multiforme xenograft and significantly longer survival rate than CPT-11 (p = 0.0014). This implies that NK012 can pass through the blood brain tumor barrier effectively. NK012, which combines enhanced distribution with prolonged sustained release, may be ideal for glioma treatment. Currently, a phase I study of NK012 is almost complete in Japan and the US. The present translational study warrants the clinical phase II study of NK012 in patients with malignant glioma.

Lecointre A, Vianaa B, LeMasne Q, Bessière A, Chanéac C, Gourier D: Red long-lasting luminescence in clinoenstatite Journal of Luminescence May (2009)

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Lecointre A, Vianaa B, LeMasne Q, Bessière A, Chanéac C, Gourier D: Red long-lasting luminescence in clinoenstatite Journal of Luminescence May (2009)

Abstract
A long-lasting phosphor Ca0.2Zn0.9Mg0.9Si2O6:Eu2+, Dy3+, Mn2+ was prepared by a sol–gel method. Nanoparticles crystallizing in a clinoenstatite structure were obtained. Long persistent phosphorescence in the red has been observed with persistence time over one hour at 680 nm and was attributed to Mn2+ emission. The persistent luminescence is suggested to involve Eu2+ as a sensitizer, Dy3+ or Dy3+-related defect as a trap center and Mn2+ as the luminescent center. However, the details of the mechanism are still under further investigation.

Tavitian B, Duconge F, Boisgard R, Dollé F: Membrane-Type 4 Matrix Metalloproteinase (MT4-MMP) induces lung metastasis by alteration of primary breast tumor vascular architecture J Cell Mol Med. May (2009)

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Abstract
The present study aims at investigating the mechanism by which MT4-MMP, a membrane-anchored MMP expressed by human breast tumor cells promotes the metastatic dissemination into lung. We applied experimental (intravenous) and spontaneous (subcutaneous) models of lung metastasis using human breast adenocarcinoma MDA-MB-231 cells overexpressing or not MT4-MMP. We found that MT4-MMP does not affect lymph node colonization nor extravasation of cells from the bloodstream, but increases the intravasation step leading to metastasis. Ultrastructural and fluorescent microscopic observations coupled with automatic computer-assisted quantifications revealed that MT4-MMP expression induces blood vessel enlargement and promotes the detachment of mural cells from the vascular tree, thus causing an increased tumor vascular leak. On this basis, we propose that MT4-MMP promotes lung metastasis by disturbing the tumor vessel integrity and thereby facilitating tumor cell intravasation.

Moroz E, Carlin S, Dyomina K, Burke S, Thaler HT, Blasberg R, Serganova I.: Real-time imaging of HIF-1alpha stabilization and degradation. PLoS ONE 4(4):e5077 April (2009)

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Moroz E, Carlin S, Dyomina K, Burke S, Thaler HT, Blasberg R, Serganova I.: Real-time imaging of HIF-1alpha stabilization and degradation. PLoS ONE 4(4):e5077 April (2009)

Abstract
HIF-1a is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1a chimeric reporter systems, HIF-1a/FLuc and HIF-1a(?ODDD)/FLuc, to investigate the tightly controlled level of HIF-1a protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1a in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1a/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1a in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1a was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1a/FLuc protein degradation and quantify the half-life of HIF-1a fusion proteins. The rapid clearance component (t1/2 ~4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ~200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1a/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1a.

Matsumura Y, Kataoka K: Preclinical and clinical studies of anticancer agent-incorporating polymer micelles Cancer Sci. 100(4):572-9 April (2009)

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Matsumura Y, Kataoka K: Preclinical and clinical studies of anticancer agent-incorporating polymer micelles Cancer Sci. 100(4):572-9 April (2009)

Abstract
The size of anticancer agent-incorporating micelles can be controlled within the diameter range of 20-100 nm to ensure that they do not penetrate normal vessel walls. With this development, it is expected that the incidence of drug-induced side-effects may be decreased owing to the reduced drug distribution in normal tissue. Micelle systems can also evade non-specific capture by the reticuloendothelial system because the outer shell of a micelle is covered with polyethylene glycol. Consequently, a polymer micelle carrier can be delivered selectively to a tumor by utilizing the enhanced permeability and retention effect. Moreover, a water-insoluble drug can be incorporated into polymer micelles. Presently, several anticancer agent-incorporating micelle carrier systems are under preclinical and clinical evaluation. Furthermore, nucleic acid-incorporating micelle carrier systems are also being developed.

Shibata MA, Morimoto J, Shibata E, Otsuki Y: Combination therapy with short interfering RNA vectors against VEGF-C and VEGF-A suppresses lymph node and lung metastasis in a mouse immunocompetent mammary cancer model Cancer Gene Ther. 15(12):776-86 December (2008)

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Abstract
Cancer metastasis contributes significantly to cancer mortality and is facilitated by lymphangiogenesis and angiogenesis. Vascular endothelial growth factor-C (VEGF-C) and VEGF-A are involved in lymphangiogenesis and angiogenesis. To inhibit metastasis, combination therapy with vector-based small interfering RNA (siRNA) against VEGF-C and/or VEGF-A was conducted on murine metastatic mammary cancer. Syngeneic, inoculated, metastatic mammary cancers received direct intratumoral injection of plasmid siRNA vector targeting VEGF-C (psiRNA-VEGF-C), VEGF-A (psiRNA-VEGF-A), both VEGF-C and VEGF-A (both psiRNA-VEGF-C and psiRNA-VEGF-A vectors injected, referred to as the psiRNA-VEGF-C+A group) or a scrambled sequence (psiRNA-SCR) as control, once a week for 8 weeks. Gene electrotransfer was performed on the tumors after each injection. Tumor volume was significantly lower in the psiRNA-VEGF-A and the psiRNA-VEGF-C+A groups throughout the study. Lymph node metastasis was significantly less frequent in all therapeutic groups, whereas the multiplicity of lung metastases was significantly lower in the psiRNA-VEGF-C+A group only. All siRNA therapeutic groups showed a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells and microvessel density. Our data suggest that specific silencing of the VEGF-C or VEGF-A gene alone can inhibit lymph node metastasis. However, combination siRNA therapy targeting both VEGF-C and VEGF-A inhibits both lymph node and lung metastasis, rendering this combined therapy more beneficial than either alone. The observed anti-metastatic activity of siRNA-expressing vectors targeting VEGF-C or VEGF-A may be of high clinical significance in the treatment of metastatic breast cancer.

Keyaerts M, Verschueren J, Bos TJ, Tchouate-Gainkam LO, Peleman C, Breckpot K, Vanhove C, Caveliers V, Bossuyt A, Lahoutte T: Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D-luciferin: effect on intensity, time kinetics and repeatability of photon emission Eur J Nucl Med Mol Imaging 35(5):999-1007 May (2008)

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Abstract
In vivo bioluminescence imaging (BLI) is a promising technique for non-invasive tumour imaging. d-luciferin can be administrated intraperitonealy or intravenously. This will influence its availability and, therefore, the bioluminescent signal. The aim of this study is to compare the repeatability of BLI measurement after IV versus IP administration of d-luciferin and assess the correlation between photon emission and histological cell count both in vitro and in vivo.
Materials and methods: Fluc-positive R1M cells were subcutaneously inoculated in nu/nu mice. Dynamic BLI was performed after IV or IP administration of d-luciferin. Maximal photon emission (PEmax) was calculated. For repeatability assessment, every acquisition was repeated after 4 h and analysed using Bland–Altman method. A second group of animals was serially imaged, alternating IV and IP administration up to 21 days. When mice were killed, PEmax after IV administration was correlated with histological cell number.
Results: The coefficients of repeatability were 80.2% (IV) versus 95.0% (IP). Time-to-peak is shorter, and its variance lower for IV (p < 0.0001). PEmax was 5.6 times higher for IV. A trend was observed towards lower photon emission per cell in larger tumours.
Conclusion: IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden.

Molecular Imaging

Kruttwig K, Brueggemann C, Kaijzel E, Vorhagen S, Hilger T, Löwik C, Hoehn M: Development of a Three-Dimensional In Vitro Model for Longitudinal Observation of Cell Behavior: Monitoring by Magnetic Resonance Imaging and Optical Imaging Molecular Imaging and Biology December (2009)

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Abstract
Purpose The aim of this study is the development of a three-dimensional multicellular spheroid cell culture model for the longitudinal comparative and large-scale screening of cancer cell proliferation with noninvasive molecular imaging techniques under controlled and quantifiable conditions.
Procedures The human glioblastoma cell line Gli36-delta-EGFR was genetically modified to constitutively express the fluorescence protein mCherry, and additionally labeled with iron oxide nanoparticles for high-field MRI detection. The proliferation of aggregates was longitudinally monitored with fluorescence imaging and correlated with aggregate size by light microscopy, while MRI measurements served localization in 3D space. Irradiation with ?-rays was used to detect proliferational response.
Results Cell proliferation in the stationary three-dimensonal model can be observed over days with high accuracy. A linear relationship of fluorescence intensity with cell aggregate size was found, allowing absolute quantitation of cells in a wide range of cell amounts. Glioblastoma cells showed pronounced suppression of proliferation for several days following high-dose gamma-irradiation.
Conclusions Through the combination of two-dimensional optical imaging and 3D MRI, the position of individual cell aggregates and their corresponding light emission can be detected. This allows an exact quantification of cell proliferation, with a focus on very small cell amounts (below 100 cells) using high resolution noninvasive techniques as a well-controlled basis for further cell transplantation studies.
Key words Fluorescence imaging - Magnetic resonance imaging - Generation of mCherry expressing cells - Human glioblastoma cell line - Quantification of cells by fluorescence intensity - Proliferation dynamics of cell aggregates - Multimodality imaging - X-ray treatment of cancer cells

Wu C, Mino K, Akimoto H, Kawabata M, Nakamura K, Ozaki M, Ohmiya Y: In vivo far-red luminescence imaging of a biomarker based on BRET from Cypridina bioluminescence to an organic dye PNAS 106:37 15599-15603 September (2009)

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Abstract
We aimed to develop a far-red luminescence imaging technology for visualization of disease specific antigens on cell surfaces in a living body. First, we conjugated a far-red fluorescent indocyanine derivative to biotinylated Cypridina luciferase. This conjugate produced a bimodal spectrum that has long-wavelength bioluminescence emission in the far-red region as a result of bioluminescence resonance energy transfer. To generate a far-red luminescent probe with targeting and imaging capabilities of tumors, we then linked this conjugate to an anti-human Dlk-1 monoclonal antibody via the biotin-avidin interaction. This far-red luminescent probe enabled us to obtain high-resolution microscopic images of live, Dlk-1-expressing Huh-7 cells without an external light source, and to monitor the accumulation of this probe in tumor-bearing mice. Thus this far-red luminescent probe is a convenient analytical tool for the evaluations of monoclonal antibody localization in a living body.

Tavitian B, Duconge F, Boisgard R, Dollé F: In Vivo Imaging of Oligonucleotidic Aptamers Nucleic Acid and Peptide Aptamers (Methods in Molecular Biology) 535 April (2009)

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Tavitian B, Duconge F, Boisgard R, Dollé F: In Vivo Imaging of Oligonucleotidic Aptamers Nucleic Acid and Peptide Aptamers (Methods in Molecular Biology) 535 April (2009)

Abstract
In this chapter we present the methods developed in our laboratory for in vivo imaging of oligonucleotidic aptamers. These methods relate to (i) the labelling of aptamers with fluorine-18, a positron emitter, (ii) Positron Emission Tomography imaging of laboratory animals with [18F]aptamers and (iii) labelling with fluorescent dyes and optical imaging of aptamers in mice.

Viel T, Boisgard R, Kuhnast B, Jego B, Siquier-Pernet K, Hinnen F, Dolle F, Tavitian B: Molecular Imaging Study on In Vivo Distribution and Pharmacokinetics of Modified Small Interfering RNAs (siRNAs) Oligonucleotides September (2008)

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Abstract
Molecular imaging was used to study the biodistribution, pharmacokinetics, and activity of naked small interfering RNAs (siRNAs). siRNAs with riboses chemically modified in the 2' position were compared with unmodified siRNA. In vitro, replacement of the 2'-hydroxyl (2'OH) group of certain nucleotides in an siRNA sequence by a fluorine atom (2'F) on both antisense (AS) and sense (S) strands [2'F(AS/S)], or by a methoxy group (2'OMe) on the S strand [2'OH(AS)/2'OMe(S)], was compatible with RNA interference. Different siRNAs [2'F(AS/S), 2'OH(AS)/2'OMe(S), and 2'OH(AS/S)] were labeled with fluorine-18 (conjugation with [18F]FPyBrA), and comparative dynamic and quantitative imaging was performed with positron emission tomography. After intravenous injections of [18F]siRNAs in rodents, total radioactivity was rapidly eliminated by the kidneys and the liver. Tissue distribution of the different siRNAs were similar, and their bioavailability (as judged from blood persistence and stability) increased in the order 2'OH(AS/S) = 2'OH(AS)/2'OMe(S) lower than 2'F(AS/S). However, in our in vivo model, the 2'F(AS/S) siRNA, despite its higher bioavailability, was not able to induce a higher interference effect with respect to the 2'OH(AS/S) siRNA. Molecular imaging approaches, applied in the present work to both natural and chemically modified siRNAs, can contribute to the development of these macromolecules as therapeutic agents.

Roncali E, Savinaud M, Levrey 0, Rogers KL, Maitrejean S, Tavitian B: New device for real-time bioluminescence imaging in moving rodents J Biomed Opt October (2008)

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Roncali E, Savinaud M, Levrey 0, Rogers KL, Maitrejean S, Tavitian B: New device for real-time bioluminescence imaging in moving rodents J Biomed Opt October (2008)

Abstract
Bioluminescence imaging (BLI) allows detection of biological functions in genetically modified cells, bacteria, or animals expressing a luciferase (i.e., firefly, Renilla, or aequorin). Given the high sensitivity and minimal toxicity of BLI, in vivo studies on molecular events can be performed noninvasively in living rodents. To date, detection of bioluminescence in living animals has required long exposure times that are incompatible with studies on dynamic signaling pathways or nonanaesthetised freely moving animals. Here we develop an imaging system that allows: 1. bioluminescence to be recorded at a rate of 25 images/s using a third generation intensified charge-coupled device (CCD) camera running in a photon counting mode, and 2. coregistration of a video image from a second CCD camera under infrared lighting. The sensitivity of this instrument permits studies with subsecond temporal resolution in nonanaesthetized and unrestrained mice expressing firefly luciferase and imaging of calcium signaling in transgenic mice expressing green fluorescent protein (GFP) aequorin. This imaging system enables studies on signal transduction, tumor growth, gene expression, or infectious processes in nonanaesthetized and freely moving animals.

Rogers KL, Picaud S, Roncali E, Boisgard R, Colasante C, Stinnakre J, Tavitian B, Brulet P: Non-invasive in vivo imaging of calcium signaling in mice PLoS ONE 2(10): e974 October (2007)

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Rogers KL, Picaud S, Roncali E, Boisgard R, Colasante C, Stinnakre J, Tavitian B, Brulet P: Non-invasive in vivo imaging of calcium signaling in mice PLoS ONE 2(10): e974 October (2007)

Abstract
Rapid and transient elevations of Ca2+ within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca2+ concentration ([Ca2+]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca2+-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca2+] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca2+] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca2+] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca2+ signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies.

le Masne de Chermont Q, Chaneac C, Seguin J, Pelle F, Maitrejean S, Jolivet JP, Gourier D, Bessodes M, Scherman D: Nanoprobes with near-infrared persistent luminescence for in vivo imaging Proc Natl Acad Sci USA. 104(22), 9266-9271 May (2007)

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Abstract
Fluorescence is increasingly used for in vivo imaging and has provided remarkable results. Yet this technique presents several limitations, especially due to tissue autofluorescence under external illumination and weak tissue penetration of low wavelength excitation light. We have developed an alternative optical imaging technique by using persistent luminescent nanoparticles suitable for small animal imaging. These nanoparticles can be excited before injection, and their in vivo distribution can be followed in real-time for more than 1 h without the need for any external illumination source. Chemical modification of the nanoparticles surface led to lung or liver targeting or to long-lasting blood circulation. Tumor mass could also be identified on a mouse model.

Neurosciences

Akimoto H, Kwon HJ, Ozaki M, Yasuda K, Honma K, Ohmiya Y.: In vivo bioluminescence imaging of bone marrow-derived cells in brain inflammation Biochem Biophys Res Commun. April (2009)

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Akimoto H, Kwon HJ, Ozaki M, Yasuda K, Honma K, Ohmiya Y.: In vivo bioluminescence imaging of bone marrow-derived cells in brain inflammation Biochem Biophys Res Commun. April (2009)

Abstract
It has been accepted that bone marrow cells infiltrate the brain and play important roles in neuroinflammation. However, there is no good tool for the visualization of these cells in living animals. In this study, we generated mice that were transplanted with GFP- or luciferase-expressing bone marrow cells, and performed in vivo fluorescence imaging (FLI) and in vivo bioluminescence imaging (BLI) to visualize the infiltrated cells. Brain inflammation was induced by intrahippocampal injection of lipopolysaccharide (LPS). Immunohistochemical investigation demonstrated an increase in the infiltration of bone marrow cells into the hippocampus because of the LPS injection and differentiation of the infiltrated cells into microglia, but not into neurons or astrocytes. BLI, but not FLI, successfully detected an increase in signal intensity with the LPS injection, and the increase of BLI coincided with that of luciferase activity in hippocampus. BLI could quantitatively and continuously monitor bone marrow-derived cells in vivo.

Bergwerf I, De Vocht N, Tambuyzer B, Verschueren J, Reekmans K, Daans J, Ibrahimi A, Van Tendeloo V, Chatterjee S, Goossens H, Jorens PG, Baekelandt V, Ysebaert D, Van Marck E, Berneman ZN, Linden AV, Ponsaerts P: Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice. BMC Biotechnol. January (2009)

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Abstract
Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.
In this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at ifferent time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-?-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.
We here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.

Infectious Diseases

Sugawara I, Udagawa T, Aoki T, Mizuno S: Establishment of a guinea pig model of latent tuberculosis with GFP-introduced Mycobacterium tuberculosis Tohoku J Exp Med. 219(3):257-62 November (2009)

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Abstract
There exists latent tuberculosis, in which small numbers of tubercle bacilli remain viable in the host without visible granulomatous lesions. As few data exist on the mechanisms of latent tuberculosis, it is important to examine latent tuberculosis in terms of pathogenesis and efficacy of chemotherapy. As a first step, we used green fluorescent protein (GFP)-introduced H37Rv Mycobacterium tuberculosis to establish latent tuberculosis in the guinea pig that provides one of the best animal models of tuberculosis. We inoculated the guinea pigs subcutaneously with 100 or 1,000 colony-forming unit (CFU) of tubercle bacilli. During the 300-day follow-up period after infection, there were no clinical signs of disease, suggesting a lack of visible granulomatous lesions. In fact, upon necropsy, no macroscopic tuberculous lesions were recognized, but histopathological examination of the lung, spleen and liver revealed microgranulomas consisting of epithelioid macrophages and lymphocytes without central necrosis. Importantly, photon imaging visualized granulomatous lesions corresponding to these histologically apparent microgranulomas. Tuberculin skin testing of infected guinea pigs showed strong positivity (> or = 10 mm induration) until the end of the experiments. Real-time PCR analysis showed a significant increase in the expression levels of interferon-gamma, tumor necrosis factor-alpha, interleukin-12, and inducible nitric oxide synthase mRNAs in infected lung tissues after 300 days (P < 0.01). As human samples are hardly available to study latent tuberculosis, our guinea pig model would be useful for examining the pathogenesis and molecular mechanisms of latent tuberculosis as well as for monitoring the results of chemotherapy with green fluorescence emission of tubercle bacilli.

Giroud C, Ottones F, Coustou V, Dacheux D, Biteau N, Miezan B, Van Reet N, Carrington M, Doua F, Baltz T: Murine Models for Trypanosoma brucei gambiense Disease Progression-From Silent to Chronic Infections and Early Brain Tropism PLoS Negl Trop Dis. 3(9):e509 September (2009)

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Abstract
BACKGROUND: Human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense remains highly prevalent in west and central Africa and is lethal if left untreated. The major problem is that the disease often evolves toward chronic or asymptomatic forms with low and fluctuating parasitaemia producing apparently aparasitaemic serological suspects who remain untreated because of the toxicity of the chemotherapy. Whether the different types of infections are due to host or parasite factors has been difficult to address, since T. b. gambiense isolated from patients is often not infectious in rodents thus limiting the variety of isolates. METHODOLOGY/PRINCIPAL FINDINGS: T. b. gambiense parasites were outgrown directly from the cerebrospinal fluid of infected patients by in vitro culture and analyzed for their molecular polymorphisms. Experimental murine infections showed that these isolates could be clustered into three groups with different characteristics regarding their in vivo infection properties, immune response and capacity for brain invasion. The first isolate induced a classical chronic infection with a fluctuating blood parasitaemia, an invasion of the central nervous system (CNS), a trypanosome specific-antibody response and death of the animals within 6-8 months. The second group induced a sub-chronic infection resulting in a single wave of parasitaemia after infection, followed by a low parasitaemia with no parasites detected by microscope observations of blood but detected by PCR, and the presence of a specific antibody response. The third isolate induced a silent infection characterised by the absence of microscopically detectable parasites throughout, but infection was detectable by PCR during the whole course of infection. Additionally, specific antibodies were barely detectable when mice were infected with a low number of this group of parasites. In both sub-chronic and chronic infections, most of the mice survived more than one year without major clinical symptoms despite an early dissemination and growth of the parasites in different organs including the CNS, as demonstrated by bioluminescent imaging. CONCLUSIONS/SIGNIFICANCE: Whereas trypanosome characterisation assigned all these isolates to the homogeneous Group I of T. b. gambiense, they clearly induce very different infections in mice thus mimicking the broad clinical diversity observed in HAT due to T. b. gambiense. Therefore, these murine models will be very useful for the understanding of different aspects of the physiopathology of HAT and for the development of new diagnostic tools and drugs.

Wiles S, Robertson BD, Frankel G, Kerton A: Bioluminescent monitoring of in vivo colonization and clearance dynamics by light-emitting bacteria Methods Mol Biol. 574:137-53 August (2009)

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Wiles S, Robertson BD, Frankel G, Kerton A: Bioluminescent monitoring of in vivo colonization and clearance dynamics by light-emitting bacteria Methods Mol Biol. 574:137-53 August (2009)

Abstract
Bioluminescence is an excellent reporter system for analysing bacterial colonization and clearance dynamics in vivo. Many bacterial species have been rendered bioluminescent, allowing the sensitive detection of bacterial burden and metabolic activity in real-time and in situ in living animals. In this chapter we describe the protocols for characterizing in vivo infection models using bioluminescent bacteria: from real-time imaging in living animals by bioluminescence imaging (BLI) to ex vivo BLI of harvested organs and tissues and, finally, to quantification of bacterial numbers in organ and tissue homogenates by luminometry and viable counts. While the lux operon from Photorhabdus luminescens is ideally suited for use in such models, there may be times when alternative luciferases, such as those from the firefly (luc) or marine copepods (Gluc), may be more appropriate. Here we describe the protocols required to monitor colonization and clearance dynamics using bioluminescent bacteria that are lux-, luc-, or Gluc-positive.

Costes B, Raj VS, Michel B, Fournier G, Thirion M, Gillet L, Mast J, Lieffrig F, Bremont M, Vanderplasschen A.: The major portal of entry of koi herpesvirus in Cyprinus carpio is the skin. J Virol. 83(7):2819-30 April (2009)

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Abstract
Koi herpesvirus (KHV), recently designated Cyprinid herpesvirus 3, is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp by using bioluminescence imaging. Taking advantage of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between open reading frame (ORF) 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid, the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain, including a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 h postinfection, while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different times postinfection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish covering the fins and also the body is the major portal of entry for KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system, nicknamed 'U-tube', to perform percutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin, and not the gills, is the major portal of entry for KHV in carp.

Raaben M, Prins HJ, Martens AC, Rottier PJ, de Haan CA: Non-invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo Cell Microbiol. February (2009)

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Abstract
Bioluminescence imaging (BLI) is a powerful new method to study virus dissemination in the live animal. Here we used this method to monitor the spatial and temporal progression of mouse hepatitis coronavirus (MHV) infection in mice using luciferase-expressing viruses. Upon intranasal inoculation, virus replication could initially be observed in the nasal cavity and the cervical lymph nodes, after which the infection spread to the brain and frequently to the eyes. The kinetics of virus spread to and clearance from the brain appeared to depend on the inoculation dose. After intraperitoneal inoculation, virus replication was predominantly observed in the liver and occasionally in the intestines, but interestingly also in the tail and paws. BLI thus elucidated new anatomic locations of virus replication. Furthermore, MHV dissemination was shown to be critically depended on the viral spike protein, but also on the mouse strain used. Widespread dissemination was observed in mice lacking a functional type I interferon response. The importance of the type I interferon system in limiting viral spread was also demonstrated by the administration of type I interferons to mice. Our results provide new insights in coronavirus pathogenesis and demonstrate the potential of BLI to study coronavirus-host interactions in vivo.

Sugawara I, Mizuno S, Tatsumi T, Taniyama T: Imaging of Pulmonary Granulomas Using a Photon Imager Japanese Journal of Infectious Disease Vol 59, 332-333 July (2006)

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Sugawara I, Mizuno S, Tatsumi T, Taniyama T: Imaging of Pulmonary Granulomas Using a Photon Imager Japanese Journal of Infectious Disease Vol 59, 332-333 July (2006)

Abstract
To clarify the location of pulmonary granulomas in vivo, we prepared a Mycobacterium tuberculosis H37Rv mutant in which the gene for a green fluorescent protein (GFP) (GFP-H37Rv) was introduced. Five weeks after aerosol infection with GFP-H37Rv, the infected lungs from guinea pigs and mice were subjected to imaging using a photon imager. Pulmonary granulomas more than 1 mm in diameter were localized clearly by the photon imager. Therefore, if a method for binding a dye (GFP, fluorescein isothiocyanate [FITC], etc.) specifically to M. tuberculosis can be developed, it will be possible to visualize granulomas using a photon imager.

Cardiovascular Research

Holopainen T, Huang H, Chen C, Kim KE, Zhang L, Zhou F, Han W, Li C, Yu J, Wu J, Koh GY, Alitalo K, He Y: Angiopoietin-1 overexpression modulates vascular endothelium to facilitate tumor cell dissemination and metastasis establishment Cancer Res. 1;69(11):4656-64 June (2009)

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Abstract
The angiopoietin-1 (Ang1)/Tie2 signaling pathway is known to play an important role in the regulation of vascular maturation and maintenance of vessel integrity. In this study, we have investigated the effect of systemic Tie2 activation or inhibition on tumor growth and metastasis. We found that treatment with Ang1 delivered via an adenoviral vector promoted s.c. implanted tumor metastasis to the lungs. Ang1 treatment did not significantly increase vascular density in the tumors but induced enlargement of blood vessels in both the tumor and normal tissues, which increased tumor cell dissemination into the blood circulation. Ang1 also enhanced the formation of metastatic foci in the lungs when tumor cells were injected into the circulation via the tail vein. The effect of Ang1 on metastasis was validated by a simultaneous treatment with a soluble form of Tie2 (sTie2), which led to the suppression of Ang1-induced increase of tumor metastasis. Furthermore, using a highly metastatic tumor model, we confirmed that systemic treatment with sTie2 suppressed tumor metastasis to the lungs and lymph nodes, whereas tumor-associated angiogenesis and lymphangiogenesis were not significantly affected. This suggests that the Ang1/Tie2 signals contribute to tumor progression by increasing vascular entry and exit of tumor cells to facilitate tumor dissemination and establishment of metastases.

Ozaki M, Haga S, Remington J, Morita N, Terui K: Hepatic ischemia induced immediate oxidative stress after reperfusion and determined the severity of the reperfusion-induced damage Antioxid Redox Signal. June (2009)

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Abstract
Non-invasive evaluation of organ redox states provides invaluable information in many clinical settings. We evaluated a newly developed reduction/oxidation-sensitive green fluorescent protein (roGFP) probe which reports cellular redox potentials and their dynamic changes in live cells. On hypoxia/reoxygenation (H/R) of AML12 liver cells, roGFP indicated mild reduction during hypoxia, but immediate transient oxidation after reoxygenation. The roGFP probe confirmed the anti-oxidative effects of N-acetyl cysteine, catalase, redox factor-1 and Mn-SOD/CuZn-SOD against H/R-induced cellular oxidative stress (OS). In a mouse liver ischemia/reperfusion (I/R) model, roGFP transduced using an adenoviral vector revealed immediate reduction of the liver under ischemia, and two distinct peaks of OS: 1) early, observed within 60 minutes post-reperfusion similar to the in vitro study; 2) later, at 24 hours. The early peak levels paralleled the ischemic time up to 75 minutes and the post-ischemic liver injury (sGOT/GPT/LDH) in the later phase (6 and 24 hours post-I/R). The roGFP probe successfully indicated post-ischemic OS of the liver in living mice, accurately predicting post-ischemic liver injury. This probe may represent an effective OS marker indicating organ redox states and also predicting the damage/function.

Tissue Engineering

Liu J, Barradas A, Fernandes H, Janssen F, Papenburg B, Stamatialis D, Martens AC, van Blitterswijk CA, De Boer J: In vitro and in vivo bioluminescence imaging of hypoxia in tissue engineered grafts Tissue Eng Part C Methods August (2009)

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Abstract
Survival and growth of cellular grafts in tissue engineering (TE) are limited by the rate of oxygen and nutrient diffusion. As such, monitoring the levels of nutrients and oxygen available to the cells is essential to assess the physiology of the cells and to evaluate strategies aiming at improving nutrient availability. In this manuscript, a reporter system containing the luciferase gene driven by a hypoxia responsive promoter was used to monitor cellular hypoxia in a tissue engineering context. We report that luciferase activity correlates with the oxygen tension in cell culture medium. When transgenic cells were seeded onto scaffolds and implanted in immune-deficient mice subcutaneously, luciferase activity was detected. To validate the response to oxygen levels of this reporter system, two tissue-engineering-culturing models were investigated. Wwe cultured transgenic cells on biomaterials in a flow perfusion bioreactor and observed that cells in the bioreactor displayed a drastically lower luciferase activity compared to conventional static culture, and that higher luciferase activity is observed in the interior of a TE tissue engineered construct, illustrating the uneven oxygen distribution in 3D constructs under conventional static culture. We conclude that this reporter system is a versatile tool to investigate cellular oxygen availability in tissue engineering both in vitro and in vivo.

Gastroenterology

Baeyens L, Bonne S, Bos T, Rooman I, Peleman C, Lahoutte T, German M, Heimberg H, Bouwens L: Notch Signaling as Gatekeeper of Rat Acinar-to-beta-Cell Conversion in Vitro Gastroenterology January (2009)

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Abstract

Background and Aims

Exocrine acinar cells in the pancreas are highly differentiated cells that retain a remarkable degree of plasticity. After isolation and an initial phase of dedifferentiation in vitro, rodent acinar cells can convert to endocrine beta-cells when cultured in the presence of appropriate factors. The mechanisms regulating this phenotypic conversion are largely unknown.

Methods

Using rat acinar cell cultures, we studied the role of Notch signaling in a model of acinar-to-beta-cell conversion.

Results

We report a novel lectin-based cell labeling method to demonstrate the acinar origin of newly formed insulin-expressing beta-cells. This method allows for specific tracing of the acinar cells. We demonstrate that growth factor-induced conversion of adult acinar cells to beta-cells is negatively regulated by Notch1 signaling. Activated Notch1 signaling prevents the reexpression of the proendocrine transcription factor Neurogenin-3, the key regulator of endocrine development in the embryonic pancreas. Interfering with Notch1 signaling allows modulating the acinar cell susceptibility to the differentiation-inducing factors. Its inhibition significantly improves ß-cell neoformation with approximately 30% of acinar cells that convert to ß-cells. The newly formed ß-cells mature when transplanted ectopically and are capable of restoring normal blood glycemia in diabetic recipients.

Conclusions

We report for the first time an efficient way to reprogram one third of the acinar cells to beta-cells by adult cell type conversion. This could find application in cell replacement therapy of type 1 diabetes, provided that it can be translated from rodent to human models.

Drug discovery

Eglen RM, Reisine T.: Photoproteins: important new tools in drug discovery. Assay Drug Dev Technol. 6(5):659-72 October (2008)

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Eglen RM, Reisine T.: Photoproteins: important new tools in drug discovery. Assay Drug Dev Technol. 6(5):659-72 October (2008)

Abstract
The G protein-coupled receptor (GPCR) family is a major target for drug discovery, and most, if not all, GPCRs can couple to Ca2+ signaling. Consequently, there are a number of cellbased, primary, high-throughput screening (HTS) assays used for drug discovery that assess changes in intracellular Ca2+ as a functional readout of GPCR activation. Historically, changes in intracellular Ca2+ levels have been readily detected using fluorescent dyes that emit light in proportion to changes in intracellular Ca2+ concentration. An alternative approach to indirectly measure changes in Ca2+ concentrations involves the use of recombinantly expressed biosensor photoproteins, of which aequorin is a prototypic example. These biosensors have the advantage that they provide an intense luminescent signal in response to elevations in intracellular Ca2+. This exquisite sensitivity, the high signal-to-noise ratios, and the ability to target expression to discrete subcellular sites (in order to detect Ca2+ microdomains) have made photoproteins a principal choice in a wide range of GPCR drug discovery programs. Photoproteins are also finding increasing use in detecting activation of other molecular target classes such as ligand-gated ion channels and transporters. This review focuses upon the use of calcium photoproteins principally for use in GPCR drug discovery and HTS.

Gene Therapy

Marie C, Richard M, Vandermeulen G, Quiviger M, Préat M, Scherman D: pFAR plasmids: New Eukaryotic Expression Vectors for Gene Therapy, devoid of Antibiotic Resistance Markers precedings.nature.com October (2008)

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Abstract
Efficient production of eukaryotic expression vectors requires the selection of plasmid-containing bacteria. To avoid the risk of dissemination of antibiotic resistance markers, we developed a new system to produce a family of plasmids Free of Antibiotic Resistance genes, called pFARs. The strategy is based on the suppression of a chromosomal nonsense mutation by a plasmid-borne function. The amber mutation was introduced into the Escherichia coli thyA gene that encodes a thymidylate synthase required for dTMP synthesis, resulting in thymidine auxotrophy. In parallel, a small plasmid vector that carries an amber suppressor t-RNA gene was entirely synthesised. The introduction of pFAR plasmids into an optimised thyA mutant restored normal growth to the auxotrophic strain, and led to an efficient production of monomeric supercoiled plasmids, as required for clinical trials. Luciferase activities measured after intramuscular injection and electrotransfer of LUC-encoding pFAR vector were similar to those obtained with a commercial vector containing the same expression cassette. Interestingly, whereas luciferase activities decreased within three weeks after intradermal electrotransfer of conventional expression vectors, sustained levels were observed with the pFAR derivative. Thus, pFAR plasmids represent a novel family of biosafe eukaryotic expression vectors, suitable for gene therapy.

Workshops and symposiums

Savinaud M., Sotiras A., Maitrejean S., Paragios N.: Bioluminescence enhancement through fusion of optical imaging and cinematic video flow 'IEEE ISBI'10' April (2010)

> Abstract

Savinaud M., Sotiras A., Maitrejean S., Paragios N.: Bioluminescence enhancement through fusion of optical imaging and cinematic video flow 'IEEE ISBI'10' April (2010)

Abstract
Optical imaging is an efficient mean to measure biological signal. However, it can suffer from low spatial and temporal resolution while animal deformable displacements could also degrade significantly the localization of the measurements. In this paper, we propose a novel approach to perform fusion of cinematic flow and optical imaging towards enhancement of the biological signal. To this end, fusion is reformulated as a population (all vs. all) registration problem where the two (being spatially aligned) signals are registered in time using the same deformation field. Implicit silhouette and landmark matching are considered for the cinematic images and are combined with global statistical congealing-type measurements of the optical one. The problem is reformulated using a discrete MRF, where optical imaging costs are expressed in singleton (global) potentials, while smoothness constraints as well as cinematic measurements through pair-wise potentials. Promising experimental results demonstrate the potentials of our approach

Savinaud M., Paragios N., Maitrejean S.: Motion-Based Enhancement of Optical Imaging 'IEEE ISBI'09' June (2009)

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Savinaud M., Paragios N., Maitrejean S.: Motion-Based Enhancement of Optical Imaging 'IEEE ISBI'09' June (2009)

Abstract
Optical imaging offers the possibility to study and image biology at molecular scale in small animal with applications such as oncology or gene expression studies. New device enable cinematic acquisitions but in some cases signals could suffer from weak spatial information. The aim of this paper is to improve localization of the luminescent data for a freely moving animal using motion information obtained with an additional camera. To this end, we propose an approach that performs a non-rigid registration in the scene video and use the corresponding motion vectors to filter the optical one. Due to the lack of contrast and texture, our method introduces silhouette constraints and landmarks on the mouse skin within a variation framework. Motion is represented using thin-platesplines and the objective function is optimized using a gradient conjugate descent approach. Experimental results demonstrate the potentials of the proposed framework.

Pestourie C, Gombert K, Cerchia L, Herin L, Theze B, Boulay J, Aissouni Y, de Franciscis V, Libri D, Tavitian B, Duconge F: Sélection d'aptamères pour l'imagerie moléculaire [French] Médecine Nucléaire 31(9):485 September (2007)

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Abstract
Les aptamères sont des structures oligonucléotidiques sélectionnées par une technique dite de Selex^TM selon leur aptitude à se lier à une cible donnée. En comparaison avec les anticorps, les aptamères présentent une faible immunogénicité, une taille réduite, une synthèse chimique possible et un faible coût. Le Selex^TM permet de cribler des banques de 10¹³ à 10¹⁵ oligonucléotides par réitération d'étapes de sélection et d'amplification. Ce processus d'évolution in vitro permet d'identifier les séquences présentant le plus d'affinité pour une cible choisie. Généralement, le Selex^TM est réalisé contre des cibles purifiées dans la limite où celles-ci conservent leur structure native. Cependant, certaines protéines, telles les protéines membranaires, ont des structures difficiles à conserver hors de leur environnement cellulaire naturel.

Press articles and blogs

In Actio article & discussion from a neuroendocrinologist (Feb. 2009) on ResearchBlogging.org
Calcium imaging is a technique that is definitely coming to age, and fancier and fancier genetically encoded indicators are constantly being developed [...]. The approach recently described in PlOS One by Rogers and colleagues is completely non invasive. They generated a transgenic mouse line expressing the calcium sensitive protein GFP-aequorin in an inducible manner through the use of a... Read more

Getting the whole picture: glowing, conscious, and free: New bioluminescence system performs whole-body imaging of moving rodents. Online news published by Analytical Chemical on November 20, 2008. (link)