In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters.

The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4(-/-) mice or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter constructs in somatotransgenic mice, respectively

Breast imaging technology: Recent advances in imaging endogenous or transferred gene expression utilizing radionuclide technologies in living subjects - applications to breast cancer

A variety of imaging technologies is being investigated as tools for studying gene expression in living subjects. Two technologies that use radiolabeled isotopes are single photon emission computed tomography (SPECT) and positron emission tomography (PET). A relatively high sensitivity, a full quantitative tomographic capability, and the ability to extend small animal imaging assays directly into human applications characterize radionuclide approaches. Various radiolabeled probes (tracers) can be synthesized to target specific molecules present in breast cancer cells. These include antibodies or ligands to target cell surface receptors, substrates for intracellular enzymes, antisense oligodeoxynucleotide probes for targeting mRNA, probes for targeting intracellular receptors, and probes for genes transferred into the cell. We briefly discuss each of these imaging approaches and focus in detail on imaging reporter genes. In a PET reporter gene system for in vivo reporter gene imaging, the protein products of the reporter genes sequester positron emitting reporter probes. PET subsequently measures the PET reporter gene dependent sequestration of the PET reporter probe in living animals. We describe and review reporter gene approaches using the herpes simplex type 1 virus thymidine kinase and the dopamine type 2 receptor genes. Application of the reporter gene approach to animal models for breast cancer is discussed. Prospects for future applications of the transgene imaging technology in human gene therapy are also discussed. Both SPECT and PET provide unique opportunities to study animal models of breast cancer with direct application to human imaging. Continued development of new technology, probes and assays should help in the better understanding of basic breast cancer biology and in the improved management of breast cancer patients

In Vivo, Multimodal Imaging of B Cell Distribution and Response to Antibody Immunotherapy in Mice

BACKGROUND: B cell depletion immunotherapy has been successfully employed to treat non-Hodgkin's lymphoma. In recent years, increasing attention has been directed towards also using B-cell depletion therapy as a treatment option in autoimmune disorders. However, it appears that the further development of these approaches will depend on a methodology to determine the relation of B-cell depletion to clinical response and how individual patients should be dosed. Thus far, patients have generally been followed by quantification of peripheral blood B cells, but it is not apparent that this measurement accurately reflects systemic B cell dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cellular imaging of the targeted population in vivo may provide significant insight towards effective therapy and a greater understanding of underlying disease mechanics. Superparamagnetic iron oxide (SPIO) nanoparticles in concert with near infrared (NIR) fluorescent dyes were used to label and track primary C57BL/6 B cells. Following antibody mediated B cell depletion (anti-CD79), NIR-only labeled cells were expeditiously cleared from the circulation and spleen. Interestingly, B cells labeled with both SPIO and NIR were not depleted in the spleen. CONCLUSIONS/SIGNIFICANCE: Whole body fluorescent tracking of B cells enabled noninvasive, longitudinal imaging of both the distribution and subsequent depletion of B lymphocytes in the spleen. Quantification of depletion revealed a greater than 40% decrease in splenic fluorescent signal-to-background ratio in antibody treated versus control mice. These data suggest that in vivo imaging can be used to follow B cell dynamics, but that the labeling method will need to be carefully chosen. SPIO labeling for tracking purposes, generally thought to be benign, appears to interfere with B cell functions and requires further examination

Herpesvirus saimiri-based vector biodistribution using noninvasive optical imaging

Herpesvirus saimiri (HVS) is capable of infecting a range of human cell types with high efficiency and the viral genome persists as high copy number, circular, nonintegrated episomes which segregate to progeny upon cell division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Here we assess the dissemination of HVS-based vectors in vivo following intravenous and intraperitoneal administration. Bioluminescence imaging of an HVS-based vector expressing luciferase demonstrates that the virus can infect and establish a persistent latent infection in a variety of mouse tissues. Moreover, the long-term in vivo maintenance of the HVS genome as a nonintegrated circular episome provided sustained expression of luciferase over a 10-week period. A particularly high level of transgene expression in the liver and the ability of HVS to infect and persist in hepatic stellate cells suggest that HVS-based vectors may have potential for the treatment of inherited and acquired liver diseases

Immediate <it>in vivo</it> target-specific cancer cell death after near infrared photoimmunotherapy

<p>Abstract</p> <p>Background</p> <p>Near infrared (NIR) photoimmunotherapy (PIT) is a new type of cancer treatment based on a monoclonal antibody (mAb)-NIR phthalocyanine dye, (IR700) conjugate. <it>In vitro</it> cancer-specific cell death occurs during NIR light exposure in cells previously incubated with mAb-IR700 conjugates. However, documenting rapid cell death <it>in vivo</it> is more difficult.</p> <p>Methods</p> <p>A luciferase-transfected breast cancer cell (epidermal growth factor receptor+, MDA-MB-468luc cells) was produced and used for both <it>in vitro</it> and <it>in vivo</it> experiments for monitoring the cell killing effect of PIT. After validation of cytotoxicity with NIR exposure up to 8 J/cm²<it>in vitro</it>, we employed an orthotopic breast cancer model of bilateral MDA-MB-468luc tumors in female athymic mice, which subsequently received a panitumumab-IR700 conjugate <it>in vivo</it>. One side was used as a control, while the other was treated with NIR light of dose ranging from 50 to 150 J/cm². Bioluminescence imaging (BLI) was performed before and after PIT.</p> <p>Results</p> <p>Dose-dependent cell killing and regrowth was successfully monitored by the BLI signal <it>in vitro</it>. Although tumor sizes were unchanged, BLI signals decreased by >95% immediately after PIT <it>in vivo</it> when light intensity was high (>100 J/cm²), however, in mice receiving lower intensity NIR (50 J/cm²), tumors recurred with gradually increasing BLI signal.</p> <p>Conclusion</p> <p>PIT induced massive cell death of targeted tumor cells immediately after exposure of NIR light that was demonstrated with BLI <it>in vivo</it>.</p