The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (mCT) for interpretation. In this study, the non-pathogenic commensal bacteria E.coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (IV) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post IV-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and mCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations. Citation: Cronin M, Akin AR, Collins SA, Meganck J, Kim J-B, et al. (2012) High Resolution In Vivo Bioluminescent Imaging for the Study of Bacterial Tumour Targeting. PLoS ONE 7(1): e30940. doi:10.1371/journal.pone.0030940 Editor: Efstathios Karathanasis, Case Western Reserve University, United States of America Received September 26, 2011; Accepted December 26, 2011; Published January 25, 2012 Copyright: 2012 Cronin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors wish to acknowledge support relevant to this manuscript from the European Commission Seventh Framework Programme (PIOF-GA-2009255466) and the Irish Health Research Board (HRA_POR/2010/138). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: AA, JM, J-BK, NZ and KF are employees of Caliper Life Sciences. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: m.tangney@ucc.ie

Abstract Whole animal non-invasive imaging contributes significantly to understand tumor behavior. It also plays a critical role in drug discovery and development. Optical imaging is convenient because it does not require radioactive materials for imaging. Especially in preclinical applications, optical imaging can be a very useful tool because genetic modification is feasible. The most popular optical imaging is using bioluminescence. We introduced various cancer cell lines that express firefly luciferase. These cells have stable expression of light emission for prolonged cell culture situation. This enables for researchers to implant the cells into animals and to monitor tumor development and metastasis. In addition, these tumor cells can be detected using fluorescent agents that target tumor cells. As a consequence, one can co-register both bioluminescent and fluorescent images. Cells also can be labeled both bioluminescent and fluorescent markers such as luciferases and fluorescent proteins. Although there are many different types of cell lines available for different tumor types, studying metastasis can be challenging. That is mainly because most popular cell lines show delayed metastasis when implanted in the animal. To expedite the metastasis, intravenous injection or intracardiac injection is performed to generate secondary tumors in the animal. However, these methods do not represent true metastasis from originated organs. One of the most popular breast cancer cell line is MDA-MB-231. When these cells are implanted into mammary fat pads of female nude mice, it typically takes more than 90 days to detect metastasis in the secondary sites. Therefore, to study the tumor behavior or to examine the drug efficacy, one should wait for a long time to see the metastasis. Here, we generated tumor cell lines that were derived from MDA-MB-231 originated cells. We took MDA-MB-231 cells that were labeled with either luciferase (MDA-MB-231-luc2) or luciferase & tdTomato fluorescent protein (MDA-MB-231-luc2-tdTomato). These cells were implanted into mammary fat pads of nude mice and secondary tumors were isolated from lymph nodes. Tissues were dissociated to single cells and clonal cell lines were established (MDA-MB-231-luc2-LN and MDA-MB-231-luc2-tdTomato-LN). The growth patterns of these cells were compared to their corresponding parental cells. To find out the metastasis patterns of these cells, we implanted new cell lines orthotopically into nude mice. Our results showed that these cell lines showed faster metastases than parental cell lines. Moreover, we examined biomarker expression patterns with multiplexing multispectral microscopy. These cells can be used to study tumor metastasis and drug discovery using non-invasive in vivo imaging. Citation Format: Jae Beom Kim, Kenneth Wong, Konnie Urban. Generation of invasive breast cancer cell lines for in vivo imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3908. doi:10.1158/1538-7445.AM2013-3908

Abstract Cyclooxygenase-2 (Cox-2) is a biomarker for tumor progression and inflammatory diseases. It is known that normal tissues express minimal amounts of Cox-2 proteins while inflammatory and malignant tumor cells express high levels. Thus, inhibition of Cox-2 is a strategy in the treatment of tumor progression and inflammatory diseases. Recently, many different kinds of molecular imaging reagents for Cox-2 have been developed. Among them, an indomethacin-based Cox-2 probe, fluorocoxib, has been shown to bind to Cox-2 in vitro and in vivo. The binding specificity to Cox-2 was shown using cell culture and tumor xenograph models. In our present study, in order to optimize fluorocoxib imaging conditions, we utilized HCT116 and HT29 cells as low and high Cox-2 expressers, respectively. First, cultured cells were grown overnight on cover slips. Cells were then fixed with paraformaldehyde and incubated with fluorocoxib probe. Multispectral images were taken using a camera equipped with liquid crystal tunable filters. Spectral unmixing was applied to these images to enhance fluorocoxib signals. Our results indicate that HCT116 cells express fair amounts of Cox-2 protein. We also transplanted HCT116 and HT29 cells into the flank regions of nu/nu mice subcutaneously. After tumors grew to sufficient size (∼100 mm3) we injected fluorocoxib intravenously and monitored probe binding to the tumors. Whole animal in vivo imaging indicated that fluorocoxib binds to HT29 tumors after 3 hours. All the probes were eliminated from the animals by 24 hours. On the other hand, HCT116 tumors did not show strong binding of fluorocoxib as expected. However, HCT116 tumors did show some weak binding at the 3 and 6 hr time points. Once there was no longer any probe detected in vivo, we dissected the primary tumors from the HCT116 and HT29 implantations. Tissue sections were made with paraffin embedded tumors. After deparaffinization, tissue sections were incubated with fluorocoxib and fluorescent images were taken using multispectral imaging technology. Spectral unmixing data confirmed that HCT116 tumor sections showed fair amounts of Cox-2 probe expression. The amount of binding was also quantitated using a corresponding software package. Our results demonstrated that fluorescent biomarker probes can be imaged in vitro and in vivo non-invasively. Furthermore, we showed that the expression level can be quantitated using spectral unmixing imaging technology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4292. doi:1538-7445.AM2012-4292

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v.) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

Advances in the detection and quantification for 3D optical tomography of bioluminescent and fluorescent reporters to quantify in terms of either cell number or absolute pmol concentration will be discussed. These methods include enhancing the detected signal levels using slight compression which reduces the amount of tissue light propagates through. Calibration techniques to improve signal location by reducing the excitation light artifacts, the amount of detected autofluorescence and techniques to quantify 3D reconstruction results in terms of biological activity will be demonstrated.

The effect of BaSnO3 on the phase formation and the dielectric properties of Ba2Ti9O20 was investigated as a function of the amount of BaSnO3 in the temperature range of 20 °C to 80 °C at 7 GHz. In the reaction of 2BaCO3 + 9TiO2 → Ba2Ti9O20 + 2CO2↑, the batch with the addition of BaSnO3 enhanced the reactivity compared to the batch with the addition of SnO2. The enhancement of reactivity caused single phase Ba2Ti9O20 to form effectively with less amounts of BaSnO3. As the amount of BaSnO3 increased up to 0.03 mol, the unloaded Q increased due to an increase of the Ba2Ti9O20 phase; for further addition of BaSnO3 over 0.3 mol, the unloaded Q decreased due to the increase of rutile and BaTi4O9 phases. The dielectric constant increased with increasing BaSnO3. As single phase Ba2Ti9O20 was present in the specimen, the Q·0 value, the dielectric constant, and the TCF were approximately 37,900, 38.7, and 1.7 ppm/°C, respectively.

Abstract Conventional fluorescence probes are monovalent molecules that interact with cellular or extra-cellular target molecules through affinity binding. To improve the binding affinity and sensitivity of detection of the fluorescence probe, we describe a novel strategy for the synthesis designed to contain multiple targeting moieties and a near infrared fluorescent dye Iflour750 for optimal in vivo detection. In this study, we applied several multivalent probes to in vivo tumor targeting and monitored the targeting process using fluorescence imaging. Deoxyglucose based fluorescence probes have been applied to tumor labeling as the high metabolic rate of the tumor cells causes preferential labeling of the tumor mass. With a multivalent 2-deoxy-D- glucose Iflour750 (2-DG-IF750) probe, we demonstrated sensitive in vivo targeting as compared to a control mono-valent 2DG probe in several tumor models, including PC3M-luc2 prostate tumor cells, MDA-MB-231-luc2 mammary fat tumor cells as well as LL/2-luc and H460-luc2 lung cancer cells. Distinct tumor signal was visualized within a few hours after intravenous delivery and reached the peak of signal/background ratio between 6-24 hours. With a multivalent cyclooxygenase (COX) binding fluorescence probe Indomethacin-IF750, we observed an interesting targeting of COX2 negative colon tumor cells HCT116-luc2, but not the COX2 positive HT29-luc2, hypothetically due to selective binding to COX1. We also tested a multivalent RGD probe for targeting integrin avb3, the expression of which is associated with tumor angiogenesis and metastasis. With our iFlur750-(RGD)4 probe, we demonstrated in vivo targeting of U87-MG-luc2, HCT116-luc2 and HT29-luc2 tumor cells. Finally, we demonstrated that optical imaging was conveniently applied to evaluating the PK/PD profile of the probes. Our multivalent probes showed a fast clearance with a half life of less than 1 hour, which is a desirable feature for achieving maximal tumor/background contrast. In summary, we have developed a new class of multi-valent fluorescence probes with improved specificity and sensitivity of tumor targeting as compared to mono-valent analogs. Utility of this class of multivalent probes is likely to pass beyond pre-clinical models, as imaging guided tumor surgery is on the verge of clinical applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5288A. doi:10.1158/1538-7445.AM2011-5288A

Abstract We previously engineered tumor cell lines with bioluminescent markers and fluorescent proteins for whole animal imaging. In addition to conventional non-invasive tumor growth detection, these cells when harvested from animals can also be used to analyze cell-cell interactions by immunohistochemistry. Moreover, fluorescent cells can be isolated using fluorescent activated cell sorting (FACS) for gene expression profiling. In this study, we used a mouse mammary gland tumor cell line 4T1 that was labeled with firefly luciferase and tdTomato fluorescent protein (4T1-luc2-tdTomato). The generated cell line was tested for in vitro signal stability with respect to bioluminescence and fluorescence prior to injection into the animals. Tumor cells were implanted subcutaneously and monitored for tumor development by acquisition of bioluminescent and fluorescent images. In a separate study, 4T1-luc2-tdTomato cells were implanted orthotopically into mammary fat pads. Tumor growth was monitored and signals from the implantation sites were reconstructed into three dimensional images. In order to non-invasively detect bone metastases by 4T1-luc2-tdTomato cells, we applied both microCT and optical imaging. For this, an interchangeable imaging platform between an optical imager and microCT machine was developed. 4T1-luc2-tdTomato cells were injected into nu/nu mice by intracardiac injection. Bioluminescent imaging was performed immediately to verify the injection of cells into the left ventricle. Fluorescent signals were not detected, likely due to the small number of cells in the secondary tumor sites. Animals demonstrating substantial whole body bioluminescence were selected for further monitoring until post injection day 9. We found that bioluminescent signals were located in the knee joint regions. For these animals, bioluminescent and X-ray images were also taken without changing the positions of the animals using the interchangeable platform. Bioluminescent images in the knee joints were reconstructed using a diffused luminescence imaging tomography algorithm. These images were then co-registered with microCT images. The results showed that bioluminescent signals were colocalized within the joint area obtained by microCT. Moreover, high resolution images of the joints from these mice revealed bone erosion in the tibia induced by 4T1-luc2-tdTomato cells. These findings demonstrate that multimodal imaging can pinpoint the tumor lesions non-invasively. In addition, low dose microCT imaging can be used to acquire high resolution skeletal images longitudinally without inducing adverse effects on the animals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5228. doi:10.1158/1538-7445.AM2011-5228

NF-κB activation is a critical signaling event in the inflammatory response and has been implicated in a number of pathological lung diseases. To enable the assessment of NF-κB activity in the lungs, we transfected a luciferase based NF-κB reporter into the lungs of mice or into Raw264.7 cells in culture. The transfected mice showed specific luciferase expression in the pulmonary tissues. Using these mouse models, we studied the kinetics of NF-κB activation following exposure to lipopolysaccharide (LPS). The Raw264.7 cells expressed a dose-dependent increase in luciferase following exposure to LPS and the NF-κB reporter mice expressed luciferase in the lungs following LPS challenge, establishing that bioluminescence imaging provides adequate sensitivity for tracking the NF-κB activation pathway. Interventions affecting the NF-κB pathway are promising clinical therapeutics, thus we further examined the effect of IKK-2 inhibition by MLN120B and glycogen synthase kinase 3 beta inhibition by TDZD-8 on NF-κB activation. Pre-treatment with either MLN120B or TDZD-8 attenuated NF-κB activation in the pulmonary tissues, which was accompanied with suppression of pro-inflammatory chemokine MIP-1ß and induction of anti-inflammatory cytokine IL-10. In summary, we have established an imaging based approach for non-invasive and longitudinal assessment of NF-κB activation and regulation during acute lung injury. This approach will potentiate further studies on NF-κB regulation under various inflammatory conditions.

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.

Abstract Whole animal optical imaging provides new ways to detect tumor cells and other biological activities in the system. Bioluminescent imaging has advantages over fluorescent imaging since animal tissues have less autoluminescence. Due to high signal to background ratio, bioluminescent imaging can detect subtle changes of light emission in the animals. Luciferases are the most popular enzymes for optical imaging. Lights are produced by enzymatic reactions in the presence of substrates. Commonly used luciferases were cloned from firefly (Photinus pyralis) and Sea Pansy (Renilla reniformis). Firefly and Renilla luciferase uses D-luciferin and coelenterazine as a substrate, respectively. Since these luciferases use different kinds of substrates, they can be used in a reporter assay simultaneously. Bioluminescent reporter assays can be achieved by conjugating transcription factor responding elements and a luciferase reporter gene. Upon signals from the outside of the cells, luciferase expression can be regulated. Using multiple responding elements for unique transcription factors, one can identify signaling pathways that are responsive to drug treatments. We have tested 10 different lentivirus reporters that carry destabilized firefly luciferase along with basal promoter element joined to tandem repeats of distinct transcription responding element. PC3M human prostate cancer cells were permanently transfected and stable cell lines were generated. As a control, constitutively active Renilla luciferase reporter was co-transfected to each reporter line. In order to identify signaling pathways that are responding to drug treatment, we plated equal number of reporter cells in well plates and treated cells with various compounds. Cisplatin, lipopolysaccaride (LPS), Paclitaxel, PMA, SB203580 or PD98059 was used for different duration. Bioluminescent images were taken at multiple time points and light emissions were quantitated. Among the compound tested, LPS generated more than 50 fold increase of bioluminescence in NF-kB luciferase (NFkB-luc) reporter cell line in vitro. In addition, the assay showed dose-dependent response. Renilla luciferase activities remained same in all reporter cell lines. To validate in vivo response, PC3M NFkB-luc cells were implanted into male nu/nu mice subcutaneously. As controls, we also implanted a non-responding reporter upon LPS treatment (TGFb-luc) and negative control without responding elements (Neg-luc). Animals were administered with LPS and in vivo bioluminescence images were taken using a cooled CCD camera. Our results showed that animals implanted with NFkB-luc mice showed 6 fold increase of luciferase activity while TGFb-luc or Neg-luc implanted animals showed little change in luciferase activity. Present study demonstrates that bioluminescent reporter assays can be applied to elucidate the mode of action of a drug. 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 5228.

Whole animal in vivo imaging has contributed significantly to the detection of disease progression and drug efficacy for the past several years (1). With the introduction of sensitive imaging instruments, applications in fluorescent imaging have expanded to monitoring tumor cell growth and gene expression. The new fluorescent proteins have improved brightness, photostability and better tissue penetration of signals at a far red wavelength. In addition, there has been an explosion of innovation in a variety of fluorescent proteins developed from existing red fluorescent proteins. Previously, we developed brighter bioluminescent cancer cell lines using enhanced firefly luciferase 2 (luc2) (2). Because 4T1-luc2 cells are so bright, we were able to detect a single bioluminescent cell in vivo. To expand the applications of genetically labeled fluorescent tumor cells, we have developed single and dual labeled 4T1, PC3M and MDA-MB-231 cell lines using tdTomato fluorescent protein and luc2. First, we engineered a vector encoding the tdTomato protein in which expression is under the control of human ubiquitin C promoter (3). Parental cells were stably transfected with the tdTomato vector and antibiotic resistant cells were selected. In parallel, luc2 labeled cells were transfected with the tdTomato vector to develop dual labeled cell lines (4T1-luc2-tdTomato, PC3M-luc2-tdTomato and MDA-MB-231-luc2-tdTomato). Our initial analyses showed that the expression level of tdTomato protein was unstable over time in both single and dual labeled cells. Therefore, we attempted single cell cloning and isolated individual clones of both single and dual labeled cells. Positive clones were subjected to fluorescent activated cell sorting (FACS). Once cells were isolated, luciferase expression was characterized in the dual labeled cell lines. Cultures were maintained to test the stability of tdTomato and luc2 expression levels over a 4 week time period. The results indicated that the cloned populations were stably expressing both reporters. We confirmed that the growth rates of both single and dually labeled cells were comparable to those of the parental cell line. To monitor tumor growth in vivo, cells were implanted subcutaneously and orthotopically into nu/nu mice. Tumor progressions were monitored non-invasively and in real time using an enclosed imaging system containing a cooled CCD camera. Our data demonstrates that we could follow primary tumor growth and metastases in vivo using fluorescent and bioluminescent imaging. These dual reporter systems enable the monitoring of pathway specific signaling in vivo with exquisite sensitivity using both bioluminescence and fluorescence. Furthermore, histological sections can later be interrogated with fluorescent detection.

Effects of BaO/WO3 ratio on the loss quality of complex perovskite Ba(Mg1/3Ta2/3)O-3 were investigated as a function of the amount of (1 - y)BaO-yWO(3)(y = 0.56, 0.8) from 0.3 wt.7.0 to 7.0 wt.% at 10 GHz. The specimens with 1.5 wt.% of y = 0.56 and/or with 1.0 wt.% of y = 0.8 showed a maximum value of loss quality, respectively, and then decreased with an increase of additive content. The increase of loss quality with (1 - y)BaO-yWO(3), is resulted that the W6+ ions substitution of Ta5+ ions is enhanced the B-site ordering of Ba(Mg1/3Ta2/3)O-3 The dielectric constant and temperature coefficient of resonant frequency (TCF) were decreased slightly with additives, which are due to the increase of BaWO4 phase as a secondary phase. The effects of additives on the intrinsic microwave losses of the specimens were investigated by the infrared reflectivity spectra from 50 to 4000 cm(-1).