Background-Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities. Methods and Results-Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (PϭNS). Afterward, 1ϫ10 7 of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (nϭ20). Control animals received nontransduced ES cells (nϭ6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7ϫ10 7 Ϯ5.8ϫ10 6 photons · s Ϫ1 · cm Ϫ2 per steradian (sr) and 0.08Ϯ0.03% injected dose/g, respectively (PϽ0.05 versus control). Both signals increased progressively from week 1 to week 4, which indicated ES cell survival and proliferation in the host. Histological analysis demonstrated the formation of intracardiac and extracardiac teratomas. Finally, animals (nϭ4) that were treated with intraperitoneal injection of ganciclovir (50 mg/kg) did not develop teratomas when compared with control animals (nϭ4) treated with saline (1 mL/kg). Conclusion-This is the first study to characterize ES cells that stably express fluorescence, bioluminescence, and positron emission tomography reporter genes and monitor the kinetics of ES cell survival, proliferation, and migration. This versatile imaging platform should have broad applications for basic research and clinical studies on stem cell therapy.

DNA Ligase IV is responsible for sealing of double-strand breaks (DSBs) during nonhomologous end-joining (NHEJ). Inhibiting Ligase IV could result in amassing of DSBs, thereby serving as a strategy toward treatment of cancer. Here, we identify a molecule, SCR7 that inhibits joining of DSBs in cell-free repair system. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding but not that of T4 DNA Ligase or Ligase I. SCR7 inhibits NHEJ in a Ligase IV-dependent manner within cells, and activates the intrinsic apoptotic pathway. More importantly, SCR7 impedes tumor progression in mouse models and when coadministered with DSB-inducing therapeutic modalities enhances their sensitivity significantly. This inhibitor to target NHEJ offers a strategy toward the treatment of cancer and improvement of existing regimens.

Imaging reporter gene expression in living subjects with various imaging modalities is a rapidly accelerating area of research. Applications of these technologies to cancer research, gene therapy, and transgenic models are rapidly expanding. We report construction and testing of several triple fusion reporter genes compatible with bioluminescence, fluorescence and positron emission tomography (

We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of proteinprotein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-B promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-B promoter through tumor necrosis factor ␣. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein-protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein-protein interactions in living subjects. The approaches validated should have important implications for the study of protein-protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein-protein interactions.

http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11090563/-/DC1.

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, D-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase ( fl)] both in cell culture and in living mice revealed that mtfl possessed 6-to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes ( jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 F 0.1 versus 1.9 F 0.1) Â (10 6 p/s/cm 2 /sr)] but similar level of fluorine-18-labeled 2 ¶-fluoro-2 ¶-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 F 0.15 versus 1.37 F 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk-expressing tumors compared with the fl-mrfp1-wttkexpressing tumors. Both tumors showed 4-to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18-labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques. [Cancer Res 2007;67(7):3085-93]

BackgroundIncreasing efforts and financial resources are being invested in early cancer detection research. Blood assays detecting tumor biomarkers promise noninvasive and financially reasonable screening for early cancer with high potential of positive impact on patients' survival and quality of life. For novel tumor biomarkers, the actual tumor detection limits are usually unknown and there have been no studies exploring the tumor burden detection limits of blood tumor biomarkers using mathematical models. Therefore, the purpose of this study was to develop a mathematical model relating blood biomarker levels to tumor burden.Methods and FindingsUsing a linear one-compartment model, the steady state between tumor biomarker secretion into and removal out of the intravascular space was calculated. Two conditions were assumed: (1) the compartment (plasma) is well-mixed and kinetically homogenous; (2) the tumor biomarker consists of a protein that is secreted by tumor cells into the extracellular fluid compartment, and a certain percentage of the secreted protein enters the intravascular space at a continuous rate. The model was applied to two pathophysiologic conditions: tumor biomarker is secreted (1) exclusively by the tumor cells or (2) by both tumor cells and healthy normal cells. To test the model, a sensitivity analysis was performed assuming variable conditions of the model parameters. The model parameters were primed on the basis of literature data for two established and well-studied tumor biomarkers (CA125 and prostate-specific antigen [PSA]). Assuming biomarker secretion by tumor cells only and 10% of the secreted tumor biomarker reaching the plasma, the calculated minimally detectable tumor sizes ranged between 0.11 mm3 and 3,610.14 mm3 for CA125 and between 0.21 mm3 and 131.51 mm3 for PSA. When biomarker secretion by healthy cells and tumor cells was assumed, the calculated tumor sizes leading to positive test results ranged between 116.7 mm3 and 1.52 × 106 mm3 for CA125 and between 27 mm3 and 3.45 × 105 mm3 for PSA. One of the limitations of the study is the absence of quantitative data available in the literature on the secreted tumor biomarker amount per cancer cell in intact whole body animal tumor models or in cancer patients. Additionally, the fraction of secreted tumor biomarkers actually reaching the plasma is unknown. Therefore, we used data from published cell culture experiments to estimate tumor cell biomarker secretion rates and assumed a wide range of secretion rates to account for their potential changes due to field effects of the tumor environment.ConclusionsThis study introduced a linear one-compartment mathematical model that allows estimation of minimal detectable tumor sizes based on blood tumor biomarker assays. Assuming physiological data on CA125 and PSA from the literature, the model predicted detection limits of tumors that were in qualitative agreement with the actual clinical performance of both biomarkers. The model may be helpful in future estimation of minimal detectable ...

Taking advantage of the bioluminescence resonance energy transfer (BRET) phenomenon, we report the development of a highly photon‐efficient, self‐illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant Renilla reniformis luciferase (RLuc8). This new BRET fusion protein (BRET3) exhibits severalfold improvement in light intensity in comparison with existing BRET fusion proteins. BRET3 also exhibits the most red‐shifted light output (564‐nm peak wavelength) of any reported bioluminescent protein that utilizes its natural substrate coelenterazine, a benefit of which is demonstrated at various tissue depths in small animals. The imaging utility of BRET3 at the single‐cell level is demonstrated using an intramolecular sensor incorporating two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. With its increased photon intensity, red‐shifted light output, and good spectral resolution (~85 nm), BRET3 shows improved spatial and temporal resolution for measuring intracellular events in single cells and in living small animal models. The development of further BRET3‐based assays will allow imaging of protein‐protein interactions using a single assay directly scalable from intact living cells to small living subjects, allowing accelerated drug discovery.— De, A.,Ray, P., Loening, A. M., Gambhir, S. S. BRET3: a red‐shifted bioluminescence resonance energy transfer (BRET) based integrated platform for imaging protein‐protein interactions from single live cells and living animals. FASEBJ. 23, 2702–2709 (2009)