Anca Dragulescu-Andrasi

Bioluminescence resonance energy transfer (BRET) imaging of protein–protein interactions within deep tissues of living subjects

Identifying protein–protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular intermolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications.

Activatable oligomerizable imaging agents for photoacoustic imaging of furin-like activity in living subjects.

Photoacoustic (PA) imaging is continuing to be applied for physiological imaging and more recently for molecular imaging of living subjects. Owing to its high spatial resolution in deep tissues, PA imaging holds great potential for biomedical applications and molecular diagnostics. There is however a lack of probes for targeted PA imaging, especially in the area of enzyme-activatable probes. Here we introduce a molecular probe, which upon proteolytic processing is retained at the site of enzyme activity and provides PA contrast. The probe oligomerizes via a condensation reaction and accumulates in cells and tumors that express the protease. We demonstrate that this probe reports furin and furin-like activity in cells and tumor models by generating a significantly higher PA signal relative to furin-deficient and nontarget controls. This probe could report enzyme activity in living subjects at depths significantly greater than fluorescence imaging probes and has potential for molecular imaging in deep tumors.

A Selenium Analogue of Firefly D-Luciferin with Red-Shifted Bioluminescence Emission†

A selenium analogue of amino-D-luciferin, aminoseleno-D-luciferin, is synthesized and shown to be a competent substrate for the firefly luciferase enzyme. It has a red-shifted bioluminescence emission maximum at 600 nm and is suitable for bioluminescence imaging studies in living subjects.

A red-shifted Renilla luciferase for transient reporter-gene expression

To the Editor: The principal limitation when using Renilla reniformis luciferase (RLuc) as a reporter for bioluminescence imaging in small-animal models has been that, because of its blue-peaked (481-nm) emission spectrum and the preferential absorption of short-wavelength photons by biological tissues, RLuc has diminished sensitivity at non-superficial locations1. To overcome this, we previously developed the red-shifted variant RLuc8.6-535 (ref. 2), which, in addition to having enhanced enzymatic activity compared to the native luciferase, shows an additional threefold increase in sensitivity at non-superficial tissue depths because of its green-peaked (535nm) emission spectrum. RLuc8.6-535 was derived from the variant RLuc8 (ref. 3) and retains RLuc8’s stabilized phenotype (intracellular half-life >50 h). Though advantageous for following constitutive gene expression, this may obscure transient changes in gene expression. We have therefore now developed a red-shifted RLuc variant with intracellular stability comparable to that of the native enzyme. We carried out site-specific random mutagenesis and selection as previously described2 (Supplementary Methods). A full description of the mutagenesis screens is in Supplementary Results. Briefly, we used the destabilized variant RLuc/M185V/Q235A3 as starting template and performed ten rounds of mutagenesis at the residue pairs Asp162/Ile163, Val185/Leu186, Asp154/Glu155, Ile159/Leu163, Glu160/Glu161, Trp156/Pro157, Ala164/Leu165, Lys136/Ile137, Phe286/Ser287 and Pro220/Arg221. Asp162 and residues in close proximity were mutated because Asp162 substitutions have previously yielded large shifts in the emission spectrum2, as was the case here. We also identified Asp162 mutations leading to blue-shifts (Supplementary Fig. 1), but did not pursue these further. Mutations at residue 185 were probed because this residue lies at the top of the active site4 and prior substitutions have increased quantum yield and

In Vivo Bioluminescence Imaging of Furin Activity in Breast Cancer Cells Using Bioluminogenic Substrates

Furin, a proprotein convertases family endoprotease, processes numerous physiological substrates and is overexpressed in cancer and inflammatory conditions. Noninvasive imaging of furin activity will offer a valuable tool to probe furin function over the course of tumor growth and migration in the same animals in real time and directly assess the inhibition efficacy of drugs in vivo. Here, we report successful bioluminescence imaging of furin activity in xenografted MBA-MB-468 breast cancer tumors in mice with bioluminogenic probes. The probes are conjugates of furin substrate, a consensus amino acid motif R-X-K/R-R (X, any amino acid), with the firefly luciferase substrate D-aminoluciferin. In the presence of the luciferase reporter, the probes are unable to produce bioluminescent emission without furin activation. Blocking experiments with a furin inhibitor and control experiments with a scrambled probe showed that the bioluminescence emission in the presence of firefly luciferase is furin-dependent and specific. After furin activation, a 30-fold increase in the bioluminescent emission was observed in vitro, and on average, a 7-8-fold contrast between the probe and control was seen in the same tumor xenografts in mice. Direct imaging of furin activity may facilitate the study of furin function in tumorigenicity and the discovery of new drugs for furin-targeted cancer therapy.

Molecular Photoacoustic Imaging of Follicular Thyroid Carcinoma

Purpose: To evaluate the potential of targeted photoacoustic imaging as a noninvasive method for detection of follicular thyroid carcinoma. Experimental Design: We determined the presence and activity of two members of matrix metalloproteinase family (MMP), MMP-2 and MMP-9, suggested as biomarkers for malignant thyroid lesions, in FTC133 thyroid tumors subcutaneously implanted in nude mice. The imaging agent used to visualize tumors was MMP-activatable photoacoustic probe, Alexa750-CXeeeeXPLGLAGrrrrrXK-BHQ3. Cleavage of the MMP-activatable agent was imaged after intratumoral and intravenous injections in living mice optically, observing the increase in Alexa750 fluorescence, and photoacoustically, using a dual-wavelength imaging method. Results: Active forms of both MMP-2 and MMP-9 enzymes were found in FTC133 tumor homogenates, with MMP-9 detected in greater amounts. The molecular imaging agent was determined to be activated by both enzymes in vitro, with MMP-9 being more efficient in this regard. Both optical and photoacoustic imaging showed significantly higher signal in tumors of mice injected with the active agent than in tumors injected with the control, nonactivatable, agent. Conclusions: With the combination of high spatial resolution and signal specificity, targeted photoacoustic imaging holds great promise as a noninvasive method for early diagnosis of follicular thyroid carcinomas. Clin Cancer Res; 19(6); 1494–502. ©2013 AACR.

Combining SELEX Screening and Rational Design to Develop Light-Up Fluorophore−RNA Aptamer Pairs for RNA Tagging

We report here a new small molecule fluorogen and RNA aptamer pair for RNA labeling. The small-molecule fluorogen is designed on the basis of fluorescently quenched sulforhodamine dye. The SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure and fluorescence screening in E. coli have been applied to discover the aptamer that can specifically activate the fluorogen with micromolar binding affinity. The systematic mutation and truncation study on the aptamer structure determined the minimum binding domain of the aptamer. A series of rationally modified fluorogen analogues have been made to probe the interacting groups of fluorogen with the aptamer. These results led to the design of a much improved fluorogen ASR 7 that displayed a 33-fold increase in the binding affinity for the selected aptamer in comparison to the original ASR 1 and an 88-fold increase in the fluorescence emission after the aptamer binding. This study demonstrates the value of combining in vitro SELEX and E. coli fluorescence screening with rational modifications in discovering and optimizing new fluorogen-RNA aptamer labeling pairs.

Detecting cancers through tumor-activatable minicircles that lead to a detectable blood biomarker

Significance Blood-based cancer diagnosis is highly attractive, but current strategies suffer because they rely on the detection of endogenous molecules that often are secreted into the circulation by both malignant and nonmalignant cells. One solution to this problem is to avoid nonmalignant tissue expression by artificially engineering tumor cells to express a unique reporter not normally expressed by any tissue. This study shows that systemic administration of nonviral safe vectors we call “tumor-activatable minicircles” allows one to distinguish tumor-bearing from tumor-free subjects reliably and to assess tumor burden simply by measuring blood levels of such a reporter. Our system represents an alternative paradigm for improved cancer detection and could enable more timely interventions to combat this devastating disease. Earlier detection of cancers can dramatically improve the efficacy of available treatment strategies. However, despite decades of effort on blood-based biomarker cancer detection, many promising endogenous biomarkers have failed clinically because of intractable problems such as highly variable background expression from nonmalignant tissues and tumor heterogeneity. In this work we present a tumor-detection strategy based on systemic administration of tumor-activatable minicircles that use the pan-tumor–specific Survivin promoter to drive expression of a secretable reporter that is detectable in the blood nearly exclusively in tumor-bearing subjects. After systemic administration we demonstrate a robust ability to differentiate mice bearing human melanoma metastases from tumor-free subjects for up to 2 wk simply by measuring blood reporter levels. Cumulative change in reporter levels also identified tumor-bearing subjects, and a receiver operator-characteristic curve analysis highlighted this test’s performance with an area of 0.918 ± 0.084. Lung tumor burden additionally correlated (r2 = 0.714; P < 0.05) with cumulative reporter levels, indicating that determination of disease extent was possible. Continued development of our system could improve tumor detectability dramatically because of the temporally controlled, high reporter expression in tumors and nearly zero background from healthy tissues. Our strategy’s highly modular nature also allows it to be iteratively optimized over time to improve the test’s sensitivity and specificity. We envision this system could be used first in patients at high risk for tumor recurrence, followed by screening high-risk populations before tumor diagnosis, and, if proven safe and effective, eventually may have potential as a powerful cancer-screening tool for the general population.

Chemical Labeling of Protein in Living Cells

Fluorescence imaging of fluorophore-labeled proteins opens a window into cellular protein biochemistry and enables direct visualization of protein dynamics, localization, and interactions in single living cells. Green fluorescent protein (GFP) and its color variants are popular fluorescent tags because they can be genetically fused to any protein of interest with great specificity. 2] However, their relatively large size (~238 amino acids) can potentially alter the structure and/or function of the host proteins, such as mislocalization or misexpression. Alternatively, chemoselective labeling of a protein in the context of the native cellular environment with small-molecule fluorescent probes could offer an exciting opportunity to expand the utility of in vivo protein imaging. Several smallmolecule-based chemical labeling methods have been developed to address this great challenge. A commonly used chemical-labeling method takes advantage of the specific interaction between a receptor protein and its small-molecule ligand. Just like GFP, the receptor protein is genetically fused to the protein of interest, and the ligand is synthetically conjugated to a fluorophore (Scheme 1A). The binding of the ligand to the receptor protein directs the fluorophore label to the fusion protein. In this way, any fluorophore with desirable optical properties can, in principle, be introduced to the fusion protein. An incopmlete list of receptor proteins includes E. coli dihydrofolate reductase (DHFR), 7] FK506-binding protein (FKBP12), a mutant of human O-alkylguanine-DNA transferase (hAGT), 10] a mutated prokaryotic dehalogenase (Halo ag< protein), small dye-binding peptides (in this case, the ligand itself is the fluorescent tag), and lanthanidebinding tags. Depending on the nature of the interaction between the receptor protein and the ligand, the labeling can be covalent (e.g. , in the case of hAGT and HaloTag protein) or noncovalent (e.g. , DHFR, FKBP12). The fusion protein may be located intracellularly or at the membrane. This method allows the incorporation of a variety of small-molecule fluorophores with diverse spectral properties into the fusion protein, particularly fluorophores with infrared or near-infrared emission, which are not available with GFP mutants. Tags for other imaging modalities, such as MRI, may be incorporated as well with this method. However, this labeling flexibility comes with several drawbacks. The size of the receptor proteins varies, but is often comparable to that of GFPs. The fluorescent emission of the tag is not modulated by binding, and a washing procedure is generally required to remove unbound ligands and minimize the background. If an endogenous equivalent of the receptor protein [a] Dr. A. Dragulescu-Andrasi, Prof. J. Rao Molecular Imaging Program at Stanford Department of Radiology & Bio-X Program Cancer Biology Program Stanford University School of Medicine 1201 Welch Road Stanford, CA 94305-5484 (USA) Fax: (+1)650-736-7925 E-mail : jrao@stanford.edu Scheme 1. Chemical methods for in vivo protein labeling. A) Receptor-protein-based method. The fluorescent probe is linked to an affinity ligand that specifically binds to its receptor. B) Enzyme-mediated ACHTUNGTRENNUNGligation. The probe is linked to a substrate that can be ligated onto a peptide tag genetically fused to the host protein by a “third-party” enzyme. C) A fluorogenic probe binds to a peptide tag–protein fusion, which activates its fluorescence.

Improving Image Quality by Accounting for Changes in Water Temperature during a Photoacoustic Tomography Scan

The emerging field of photoacoustic tomography is rapidly evolving with many new system designs and reconstruction algorithms being published. Many systems use water as a coupling medium between the scanned object and the ultrasound transducers. Prior to a scan, the water is heated to body temperature to enable small animal imaging. During the scan, the water heating system of some systems is switched off to minimize the risk of bubble formation, which leads to a gradual decrease in water temperature and hence the speed of sound. In this work, we use a commercially available scanner that follows this procedure, and show that a failure to model intra-scan temperature decreases as small as 1.5°C leads to image artifacts that may be difficult to distinguish from true structures, particularly in complex scenes. We then improve image quality by continuously monitoring the water temperature during the scan and applying variable speed of sound corrections in the image reconstruction algorithm. While upgrading to an air bubble-free heating pump and keeping it running during the scan could also solve the changing temperature problem, we show that a software correction for the temperature changes provides a cost-effective alternative to a hardware upgrade. The efficacy of the software corrections was shown to be consistent across objects of widely varying appearances, namely physical phantoms, ex vivo tissue, and in vivo mouse imaging. To the best of our knowledge, this is the first study to demonstrate the efficacy of modeling temporal variations in the speed of sound during photoacoustic scans, as opposed to spatial variations as focused on by previous studies. Since air bubbles pose a common problem in ultrasonic and photoacoustic imaging systems, our results will be useful to future small animal imaging studies that use scanners with similarly limited heating units.