Imaging Of Reporter Gene Expression Research Articles

Synthesis and Characterization of 9-(4-[18F]Fluoro-3-(hydroxymethyl)butyl)-2-(phenylthio)-6-oxopurine as a Novel PET Agent for Mutant Herpes Simplex Virus Type 1 Thymidine Kinase Reporter Gene Imaging.

[18F]FHBG has been used as a positron emission tomography (PET) imaging tracer for the monitoring of herpes simplex virus type 1 thymidine kinase (HSV1-tk), a reporter gene for cell and gene therapy in humans. However, this tracer shows inadequate blood-brain barrier (BBB) penetration and, therefore, would be limited for accurate quantification of reporter gene expression in the brain. Here, we report the synthesis and evaluation of 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)-2(phenylthio)-6-oxopurine ([18F]FHBT) as a new PET tracer for imaging reporter gene expression of HSV1-tk and its mutant HSV1-sr39tk, with the aim of improved BBB penetration. [18F]FHBT was prepared by using a tosylate precursor and [18F]KF. The cellular uptake of [18F]FHBT was performed in HSV1-sr39tk-positive (+) or HSV1-sr39tk-negative (-) MDA-MB-231 breast cancer cells. The specificity of [18F]FHBT to assess HSV1-sr39tk expression was evaluated by in vitro blocking studies using 1mM of ganciclovir (GCV). Penetration of [18F]FHBT and [18F]FHBG across the BBB was assessed by dynamic PET imaging studies in normal mice. The tosylate precursor reacted with [18F]KF using Kryptofix2.2.2 followed by deprotection to give [18F]FHBT in 10% radiochemical yield (decay-corrected). The uptake of [18F]FHBT in HSV1-sr39tk (+) cells was significantly higher than that of HSV1-sr39tk (-) cells. In the presence of GCV (1mM), the uptake of [18F]FHBT was significantly decreased, indicating that [18F]FHBT serves as a selective substrate of HSV1-sr39TK. PET images and time-activity curves of [18F]FHBT in the brain regions showed similar initial brain uptakes (~ 12.75min) as [18F]FHBG (P > 0.855). Slower washout of [18F]FHBT was observed at the later time points (17.75 - 57.75 min, P >0.207). Although [18F]FHBT showed no statistically significant improvement of BBB permeability compared with [18F]FHBG, we have demonstrated that the 2-(phenylthio)-6-oxopurine backbone can serve as a novel scaffold for developing HSV1-tk/HSV1-sr39tk reporter gene imaging agents for additional research in the future.

Evaluating Nonintegrating Lentiviruses as Safe Vectors for Noninvasive Reporter-Based Molecular Imaging of Multipotent Mesenchymal Stem Cells.

Reporter gene-based molecular imaging can provide invaluable information on the fate of cellular therapies postimplantation. Integrating lentiviral vectors (ILVs) are commonly used for stably engineering cells; however, their potential for insertional mutagenesis poses a significant safety concern and barrier to widespread clinical adoption. In cells that slowly divide or are postmitotic in vivo, such as mesenchymal stem cells (MSCs), nonintegrating lentiviral vectors (NILVs) may be a safer alternative option because NILVs remain episomal and can provide prolonged expression profiles. Here, NILVs coexpressing fluorescence and bioluminescence imaging (BLI) reporters were engineered and used to longitudinally image the viability of human MSCs in vitro and in vivo in mice. In vitro, ILV-transduced cancer cells and MSCs maintained steady reporter gene expression over time, whereas NILV-transduced cells progressively lost signal. NILV reporter loss was accelerated in highly proliferative cancer cells compared with less proliferative MSCs. In vivo, ILV- and NILV-transduced MSCs were each detectable with BLI postintramuscular implantation, with significantly higher ILV-based signal compared with NILV-based signal. BLI signal was observed to similarly diminish over time for both cell populations, which was attributed to cell death. Despite the reduced signal intensity with NILVs and the minimal number of cells injected (40,000), live NILV-transduced MSCs were reliably visualized for up to 2 weeks. Safety is a concern for future clinical reporter gene applications. We present NILVs as a safe means of imaging reporter gene expression for slowly dividing or nondividing cells and showed effective tracking of the viability of a small number of live transplanted MSCs over time with optical imaging. Future work will evaluate improvements to episomal NILV reporter expression, explore sensitive clinically relevant reporters, and apply this approach to clinically relevant MSC applications in preclinical models. NILVs may have broad clinical applications for expression of imaging reporters and other gene products in MSC-based therapies.

Intravital Imaging of Human Melanoma Cells in the Mouse Ear Skin by Two-Photon Excitation Microscopy.

Noninvasive imaging of reporter gene expression by two-photon excitation (2PE) laser scanning microscopy is uniquely suited to perform dynamic and multidimensional imaging down to single-cell detection sensitivity in vivo in deep tissues. Here we used 2PE microscopy to visualize green fluorescent protein (GFP) as a reporter gene in human melanoma cells implanted into the dermis of the mouse ear skin. We first provide a step-by-step methodology to set up a 2PE imaging model of the mouse ear's skin and then apply it for the observation of the primary tumor and its associated vasculature in vivo. This approach is minimally invasive and allows repeated imaging over time and continuous visual monitoring of malignant growth within intact animals. Imaging fluorescence reporter gene expression in small living animals by 2PE provides a unique tool to investigate critical pathways and molecular events in cancer biology such as tumorigenesis and metastasis in vivo with high-spatial and temporal resolutions.

Preliminary results of a non-invasive method to measure tumor size and distribution in vivo.

Molecular imaging of reporter gene expression in cancer cells can provide rapid, sensitive and non-invasive monitoring of tumor behavior. The aim of the present study was to establish a non-invasive method to measure tumor size and distribution in vivo. Briefly, H-Ras-transformed cells were stably transfected with a plasmid containing the luciferase gene (Luc), designated as Ras/Luc. Ras/Luc cells were injected into the back or tail vein of nude BALB/cAnN-Foxn1nu/CrlNarl mice (age, 6–8 weeks). Mice were subsequently administered D-luciferin via intra-peritoneal injection, prior to image acquisition. Photons emitted from the mice were detected via an imaging system. Tumor size and distribution in vivo were quantified as photons/second. Andrographolide has demonstrated radiosensitization in previous in vitro and in vivo studies. In the present study, the potential effects of andrographolide cancer metastasis were investigated further, using an imaging system. Preliminary results of andrographolide combined with radiation indicated the inhibition of cancer metastasis. The present mechanistic study of andrographolide-mediated effects demonstrated that activated extracellular signal regulated kinase protein and H2O2 production levels were significantly increased by andrographolide. In summary, the present study established a non-invasive method to measure tumor size and distribution in vivo and indicated that andrographolide may be a potential therapeutic strategy in cancer therapy.

Spatially coordinated dynamic gene transcription in living pituitary tissue.

Transcription at individual genes in single cells is often pulsatile and stochastic. A key question emerges regarding how this behaviour contributes to tissue phenotype, but it has been a challenge to quantitatively analyse this in living cells over time, as opposed to studying snap-shots of gene expression state. We have used imaging of reporter gene expression to track transcription in living pituitary tissue. We integrated live-cell imaging data with statistical modelling for quantitative real-time estimation of the timing of switching between transcriptional states across a whole tissue. Multiple levels of transcription rate were identified, indicating that gene expression is not a simple binary 'on-off' process. Immature tissue displayed shorter durations of high-expressing states than the adult. In adult pituitary tissue, direct cell contacts involving gap junctions allowed local spatial coordination of prolactin gene expression. Our findings identify how heterogeneous transcriptional dynamics of single cells may contribute to overall tissue behaviour.

Design and use of transgenic reporter strains for detecting activity of signaling pathways in Xenopus

Embryos and larvae of vertebrate species with external development are ideal subjects for investigating the dynamic spatiotemporal activity of developmental signaling pathways. The availability of efficient transgene technologies in Xenopus and zebrafish and the translucency and/or transparency of their embryos and larvae make these two species attractive for direct in vivo imaging of reporter gene expression. In this article we describe the design of efficient signaling reporters, using the Wnt/β-catenin pathway as a representative example. We define methods for validating the reporter constructs and describe how they can be used to generate stable transgenic lines in Xenopus. We provide efficient methods used in our laboratory for raising the tadpoles and froglets rapidly to sexual maturity. We further discuss how the reporter lines can be used for delineating the dynamic activity of a signaling pathway and how modulators of the pathway can be scrutinized via chemical intervention and the micro-injection of synthetic RNAs or morpholinos. The strategic outline discussed in this paper provides a template for studying other developmental signaling pathways in Xenopus.

Abstract 4535: Molecular-genetic imaging of prostate cancer.

Abstract Cancer-specific promoters, such as the progression elevated gene-3 or the astrocyte elevated gene promoters, PEG-Prom and AEG-Prom, respectively, can be used as molecular genetic imaging instruments for tumor-selective imaging of cancer. We have previously shown this in experimental models of metastatic breast cancer and melanoma using bioluminescence imaging (BLI) and single photon emission computed tomography with computed tomography (SPECT/CT). Here we show that we can express PEG-Prom and AEG-Prom selectively in PC3-ML (human bone metastatic derived prostate cancer) cells and use these constructs for imaging prostate cancer in vivo. PC3-ML cells were transfected with firefly luciferase (FLuc) or the herpes simplex type I thymidine kinase (HSV1-tk), and validated for suitable transfection through bioluminescence and [125I]FIAU cell uptake assays, respectively. PC3-ML cells were administered through intra-cardiac injection to allow widespread metastases. A linear polyethyleneimine (l-PEI) nanoparticle was used for intravenous (IV) delivery of the promoter constructs to animals bearing metastases. PEG-Prom- and AEG-Prom-driven imaging reporter gene expression was monitored by small animal BLI and by SPECT/CT after IV administration of D-luciferin or [125I]FIAU, respectively. Both BLI and SPECT/CT imaging demonstrated tumor-specific PEG-Prom and AEG-Prom activity in the model of metastatic prostate cancer but not in control. Using the thoracic cavity and tibia as the regions of interest (ROI), quantification of the BLI data (n=6) at the 48 h time point after reporter gene delivery showed a range of 16- to 19-fold higher accumulation of FLuc in the metastasis group compared to control for PEG-Prom and AEG-Prom, respectively. BLI results were confirmed by histopathology, immunohistochemistry and western blot. Quantification of the SPECT/CT data (n=5) at the 56 h time point after reporter gene and 8 h after [125I]FIAU delivery showed a 3- to 11-fold higher accumulation of [125I]FIAU, depending on the tumor burden, in the group bearing metastases compared to control for AEG-Prom. Metastatic nodules detected by the SPECT/CT imaging were confirmed by pathology. The results demonstrate the potential of systemically delivered, PEG-Prom- and AEG-Prom-driven constructs for enabling the imaging of metastatic prostate cancer in vivo. Citation Format: Akrita Bhatnager, Kathleen Gabrielson, Paul B. Fisher, Martin G. Pomper. Molecular-genetic imaging of prostate cancer. [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 4535. doi:10.1158/1538-7445.AM2013-4535

Imaging Reporters and Multimodal Molecular Bio-Imaging: A Database of Available Probes for Multi-Modality Bio-Imaging of Reporter Gene Expression

The review is on molecular imaging reporter gene expression probes to generate the image contrast. These probes utilize the imaging technology specific to microscopic physical, physiological, metabolic events and changes to differentiate pathology from normal tissue. Reporter gene expression technique generates the specific information at micro- and nanoscopic scale with details of specific molecular or metabolic event. The present paper reviews a database of measurable imaging methods of transgene expression of maltose binding domain with resource of protein molecules in bioimaging. Molecular bioimaging is based on imaging the transgene expression products and offers multimodal real-time genomic kinetics. With this aim, gene expression probes combined with adjunct molecular imaging techniques are identified and their applications are defined for: 1. noninvasive in vivo imaging methods for specific molecular expression and interactions of protein-protein domains, 2. to visualize time-based molecular events in the tissues, 3. to visualize and follow-up trafficking and targeting of cells, 4. to optimize conditions of gene therapy and monitoring drug therapeutics, 5. to develop rapid drug chemosensitivity assay to image the drug effect at cellular and molecular level by multimodal radioimaging, optical and bioluminescence imaging. The feasibility of multimodal molecular imaging techniques on one platform is reported to visualize the cells in animal body after administration of binding domain ligand into animals through oral, intraperitoneal, intravenous, and intrathecal routes. The binding domain ligand acts as a secretory signal or plasma membrane trafficking signal domain. It can be visualized by positron emission tomography imaging. Today, the main challenge in developing multimodal multifunctional double labeled molecular imaging probes is to achieve goal of rapid, reproducible multimodal imaging (optical, physical, biosensor) or radiolabel mapping (PET, CT) in noninvasive and quantitative manner that can give information in time-dependent experimental, developmental, environmental and therapeutic influence on gene products and proteins in animals or patients. This patent review will be useful resource database of molecules as ‘bioinformatics of potential reporters’ and multimodal imaging methods on same platform resource in selecting molecules to develop imaging techniques of cells or molecular events during influence of drugs, metabolism, physiology, experimental, environmental and genetics in the body. Keywords: Proteins, MRI-PET, multimodal imaging, oligonucleotides, gene expression.

Theranostic Potential of Oncolytic Vaccinia Virus

Biological cancer therapies, such as oncolytic, or replication-selective viruses have advantages over traditional therapeutics as they can employ multiple different mechanisms to target and destroy cancers (including direct cell lysis, immune activation and vascular collapse). This has led to their rapid recent clinical development. However this also makes their pre-clinical and clinical study complex, as many parameters may affect their therapeutic potential and so defining reason for treatment failure or approaches that might enhance their therapeutic activity can be complicated. The ability to non-invasively image viral gene expression in vivo both in pre-clinical models and during clinical testing will considerably enhance the speed of oncolytic virus development as well as increasing the level and type of useful data produced from these studies. Further, subsequent to future clinical approval, imaging of reporter gene expression might be used to evaluate the likelihood of response to oncolytic viral therapy prior to changes in tumor burden. Here different reporter genes used in conjunction with oncolytic viral therapy are described, along with the imaging modalities used to measure their expression, while their applications both in pre-clinical and clinical testing are discussed. Possible future applications for reporter gene expression from oncolytic viruses in the phenotyping of tumors and the personalizing of treatment regimens are also discussed.

Bioluminescent imaging of reporter gene expression in the lungs of wildtype and model mice following the administration of PEG‐stabilized DNA nanoparticles

DNA nanoparticles (DNPs) formed by compacting DNA with polyethyleneglycolylated poly-L-lysine are a nonviral vector shown to be safe and efficacious in animals and humans. To extend our capabilities of assessing the efficacy and duration of expression achieved by DNPs, we tested the utility of bioluminescent imaging (BLI) of transgene expression in wildtype and cystic fibrosis (CF) mouse models. We tested the effect of route of administration, mouse coat color, anesthesia, dose, and promoter sequence on the level and duration of expression. Furthermore, we investigated the correlation between imaging and direct analysis of luciferase expression in lung homogenates. We found that intratracheal instillation, and the use of deep and prolonged anesthesia with avertin produced significantly higher expression compared with intranasal administration, and the use of lighter anesthesia with isoflurane. Although similar expression was observed for both dark and light coat animals, imaging signal intensity was attenuated in mice with dark fur. Furthermore, good correlation between imaging and direct homogenate analysis was observed for single dose (r = 0.96), and dose response studies in wildtype (r = 0.82) and CF mice (r = 0.87). Finally, we used imaging to track gene expression over a 56-day time course. We found that the human ubiquitin B promoter gives stable transgene expression up to 49 days following nanoparticle administration, while expression with the cytomegalovirus promoter diminished after 2 days and returned to background levels by day 14. Taken together, our results demonstrate that BLI is an effective and useful modality for measuring gene expression conferred by DNPs in the lung.

A simplified one-pot automated synthesis of [18F]FHBG for imaging reporter gene expression

9-(4-[F]fluoro-3-hydroxymethylbutyl) guanine ([F]FHBG) has been used as a reporter probe to image the expression of the herpes simplex virus type 1 thymidine kinase (TK) reporter gene in living organisms with positron emission tomography (PET). However, the routine production of [F]FHBG presents many challenging laboratory requirements. To develop a simple one-pot fully-automated synthesis procedure of [F]FHBG amenable for its routine use in reporter gene expression PET imaging studies. A TRACERlab FXF-N synthesizer was substantially modified and adapted to the synthesis of [F]FHBG through the two-step one-pot procedure. After the fluorination reaction of the tosylate precursor and the hydrolysis of the intermediate product in the same reaction vessel, the final product was purified by Sep-Pak cartridges instead of the high performance liquid chromatography system. The fully automated synthesis of [F]FHBG with Sep-Pak purification was performed within a short synthesis time. The decay-uncorrected radiochemical yield of [F]FHBG was 8-14% (n=10), the radiochemical purity was more than 99%, and the entire synthesis time was less than 40 min. In addition, the PET image of theTK-transfected nude mice model indicated a much higher uptake of [F]FHBG in the TK-transfected tumor region than in the control tumor region. The automated synthesis of [F]FHBG is very easy to carry out using one-pot reactions combined with Sep-Pak purification. The synthetic [F]FHBG can be used for PET imaging and monitoring of in vivo herpes simplex virus type 1 TK gene expression.

Abstract CN01-04: Imaging-guided therapy in different tumor models using positron emission tomography (PET) and optical imaging

Abstract Positron emission tomography (PET) allows non-invasive assessment of physiological, metabolic and molecular processes in vivo. With the achievements in detector technology, spatial resolution of PET has been improved (1–2mm) enabling investigations in small experimental animals such as mice. With the development in tracer technology a variety of endogenously expressed and exogenously introduced genes can be analyzed by PET. This opens up the exciting and rapidly evolving field of molecular imaging aiming towards the non-invasive localization of the dynamics of disease specific molecular alterations and biological processes in animal models and humans in vivo. The main and most intriguing advantage of molecular imaging is the kinetic analysis of a given molecular event in the same experimental subject over time. This allows non-invasive characterization of animal models of human disease at various disease stages and after therapeutic intervention. Optical imaging (OI) is a non-invasive molecular imaging technology that uses light to interrogate cellular and molecular function in the living body. Although OI technologies are still limited by light scattering and depth of light penetration they provide a low cost and rapid alternative to the more expensive and time-consuming PET imaging. By introducing multi-modality reporter constructs (PET, fluorescence and/or bioluminescence) drawbacks of each modality can be overcome and advantages will complement one another. Thus, allowing on the one side rapid, cost-effective optical reporter imaging and on the other side tomographic as well as quantitative imaging of reporter gene expression by PET. With PET being a modality well developed for imaging in human subjects as well as small animals it provides a link from bench-to-bedside and may facilitate translation into clinical applications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN01-04.

Converting Tumor-specific Markers Into Reporters of Oncolytic Virus Infection

Preferential killing of transformed cells, while keeping normal cells and organs unharmed, is the main goal of cancer gene therapy. Genetically engineered trackable markers and imaging reporters enable noninvasive monitoring of transduction efficiency and pharmacokinetics of anticancer virotherapeutics. However, none of these reporters can differentiate between infection in the targeted tumors and that in the normal tissue. Thus, we constructed oncolytic measles virus (MV) armed with a human light immunoglobulin chain reporter gene for the treatment of multiple myeloma (MM). Excessive production of monoclonal immunoglobulin is a key characteristic and marker for diagnostics of MM. Once expressed in infected target cells, vector-encoded lambda protein recombines with myeloma IgG-kappa immunoglobulin creating a unique IgG-kappa/lambda. A modified immunoassay technique allows precise quantification of converted marker molecules. Only antibody producing cells were able to assemble this chimeric immunoglobulin molecule, whereas other cells secreted only free lambda light chain. Human myeloma xenografts inoculated with lambda chain expressing MV secreted converted IgG-kappa/lambda in the plasma of tumor bearing animals and elevated reporter levels correlated with response to the therapy. This is the first report of a gene therapy vector engineered to discriminate between infection in malignant and normal cells by molecular modification of a tumor-specific protein.

Phase I Study of Noninvasive Imaging of Adenovirus-mediated Gene Expression in the Human Prostate

To monitor noninvasively potentially therapeutic adenoviruses for cancer, we have developed a methodology based on the sodium iodide symporter (NIS). Men with clinically localized prostate cancer were administered an intraprostatic injection of a replication-competent adenovirus, Ad5-yCD/utTK(SR39)rep-hNIS, armed with two suicide genes and the NIS gene. NIS gene expression (GE) was imaged noninvasively by uptake of Na(99 m)TcO(4) in infected cells using single photon emission-computed tomography (SPECT). The investigational therapy was safe with 98% of the adverse events being grade 1 or 2. GE was detected in the prostate in seven of nine (78%) patients at 1 x 10(12) virus particles (vp) but not at 1 x 10(11) vp. Volume and total amount of GE was quantified by SPECT. Following injection of 1 x 10(12) vp in 1 cm(3), GE volume (GEV) increased to a mean of 6.6 cm(3), representing, on average, 18% of the total prostate volume. GEV and intensity peaked 1-2 days after the adenovirus injection and was detectable in the prostate up to 7 days. Whole-body imaging demonstrated intraprostatic gene expression, and there was no evidence of extraprostatic dissemination of the adenovirus by SPECT imaging. The results demonstrate that noninvasive imaging of adenovirus-mediated gene therapy in humans is feasible and safe.

Cre‐loxP system as a versatile tool for conferring increased levels of tissue‐specific gene expression from a weak promoter

Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak compared with stronger but constitutively expressing viral promoters. In this study, we validated methods of enhancing the transcriptional activity of weak promoters using a Cre-loxP system in vitro and in vivo. We constructed a tester vector, pCTL, which carries a strong systemic cytomegalovirus enhancer/chicken beta-actin promoter (CAG), loxP-flanked CAT, and firefly luciferase (luc) cDNAs. Herpes simplex virus-thymidine kinase (HSV-tk) promoter was used as a weak and systemic promoter and ligated to Cre for construction of pTC. Luc activity was higher (about 10-fold enhancement) in co-transfected (with pCTL and pTC) than in singly (with HSV-tk promoter-driven luc expression vector pTL) transfected NIH3T3 cells. In vivo electroporation-mediated gene delivery of both pCTL and pTC into murine oviductal epithelium yielded results (about 16-fold enhancement) similar to those obtained with in vitro-transfected NIH3T3 cells. To evaluate tissue-specific enhancement of gene expression, podocyte (glomerular visceral epithelial cell)-specific nephrin promoter was ligated to the Cre gene or luc cDNA to create pNC and pNL, respectively. We achieved 2.4-fold improvement of luc gene expression in the mouse kidney in vivo when pCTL and pNC were co-transfected via the tail vein via the lipoplex method. The combination of a weak tissue-specific promoter with the Cre-loxP system could thus be used to enhance the strength of tissue-specific promoters in vitro and in vivo.

MRI detection of transcriptional regulation of gene expression in transgenic mice

Ferritin, the iron storage protein, was recently suggested to be a candidate reporter for the detection of gene expression by magnetic resonance imaging (MRI). Here we report the generation of TET:EGFP-HAferritin (tet-hfer) transgenic mice, in which tissue-specific inducible transcriptional regulation of expression of the heavy chain of ferritin could be detected in vivo by MRI. We show organ specificity by mating the tet-hfer mice with transgenic mice expressing tetracycline transactivator (tTA) in liver hepatocytes and in vascular endothelial cells. Tetracycline-regulated overexpression of ferritin resulted in specific alterations of the transverse relaxation rate (R(2)) of water. Transgene-dependent changes in R(2) were detectable by MRI in adult mice, and we also found fetal developmental induction of transgene expression in utero. Thus, the tet-hfer MRI reporter mice provide a new transgenic mouse platform for in vivo molecular imaging of reporter gene expression by MRI during both embryonic and adult life.

Monitoring of tumor growth and metastasis potential in MDA-MB-435s/ tk-luc human breast cancer xenografts

Molecular imaging of reporter gene expression provides a rapid, sensitive and non-invasive monitoring of tumor behaviors. In this study, we reported the establishment of a novel animal model for longitudinal examination of tumor growth kinetics and metastatic spreading in vivo. The highly metastatic human breast carcinoma MDA-MB-435s cell line was engineered to stably express herpes simplex virus type 1 thymidine kinase (HSV-1- tk) and luciferase ( luc). Both 131I-FIAU and D-luciferin were used as reporter probes. For orthotopic tumor formation, MDA-MB-435s/ tk-luc cells were implanted into the first nipple of 6-week-old female NOD/SCID mice. For metastatic study, cells were injected via the lateral tail vein. Mice-bearing MDA-MB-435s/ tk-luc tumors were scanned for tumor growth and metastatsis using Xenogen IVIS50 system. Gamma scintigraphy and whole-body autoradiography were also applied to confirm the tumor localization. The results of bioluminescence imaging as well as histopathological finding showed that tumors could be detected in femur, spine, ovary, lungs, kidney, adrenal gland, lymph nodes and muscle at 16 weeks post i.v. injection, and correlated photons could be quantified. This MDA-MB-435s/ tk-luc human breast carcinoma-bearing mouse model combined with multimodalities of molecular imaging may facilitate studies on the molecular mechanisms of cancer invasion and metastasis.

Molecular imaging: A new way to study molecular processes in vivo

Non-invasive imaging of reporter gene expression using different imaging modalities is increasing its role for the in vivo assessment of molecular processes. Multimodality imaging protocols overcome limitations to a single imaging modality and provide a thorough view of specific processes, often allowing a quantitative measurement and direct visualization of the process in a specific target organ or tissue. The use of the right reporter gene for the development of animal models and the characterization of its expression in different conditions and tissues is fundamental for basic, translational and future pharmacological applications of a given model. This paper summarizes the major steps in the development and evaluation of a specific animal model for in vivo molecular imaging studies and describes the first example of an animal model designed for the in vivo assessment of a specific receptor activity and its possible evolution towards multimodality imaging analysis.

High-Resolution Imaging of Bone Precursor Cells Within the Intact Bone Marrow Cavity of Living Mice

Bone precursor cells (BPCs) play a critical role in bone maintenance and regeneration. Currently, no tool exists to study BPCs or other bone marrow cell types directly within their complex microenvironment. Here, we describe in vivo magnetic resonance imaging (MRI) of anatomical structures inside the medullary cavity of the mouse femur. We demonstrate that BPCs passively labeled with iron oxide-containing particles can be monitored by MRI within the intact bone marrow at an in-plane resolution of 43x25 microm. Anatomical detail provided by MRI is complemented by functional optical imaging of reporter gene expression. Single-cell dual iron oxide-reporter gene labeling has potential for combined cell tracking and cell biology studies. In summary, we describe a versatile platform suitable for studying the biology of many bone marrow cell types in living bone.

Positron Emission Tomography Imaging of Adenoviral-Mediated Transgene Expression in Liver Cancer Patients

In gene-therapy protocols, imaging of gene expression is needed to evaluate the transduction efficiency of the vector, its tissue distribution, and the duration of transgene expression and to assess the feasibility of repeated vector administration. We have used positron emission tomography with a fluorine-18-labeled penciclovir analogue to monitor thymidine kinase gene expression after intratumoral injection of a first-generation recombinant adenovirus in patients with hepatocellular carcinoma. Patients were enrolled in a pilot clinical trial and treated with escalating doses of the vector. Two days after adenovirus inoculation, transgene expression was evaluated during the first hours after administration of the radiotracer both on the treated lesion and on a whole-body basis. Transgene expression in the tumor was dependent on the injected dose of the adenovirus and was detectable in all patients who received > or = 10(12) viral particles. However, when the study was repeated 9 days after vector injection, no expression could be observed. It is interesting to note that no specific expression of the transgene could be detected in distant organs or in the surrounding cirrhotic tissue in any of the cases studied. Our findings show the real possibility of imaging transgene expression in humans by using viral vectors. We show that hepatocarcinoma is a permissive tumor for adenoviral infection and that the nontumoral cirrhotic liver is spared from transduction when the vector is administered by intratumoral injection. These results show that positron emission tomography imaging may help in the design of gene-therapy strategies and in the clinical assessment of new-generation vectors.