Srabani Bhaumik

Optical imaging of Renilla luciferase reporter gene expression in living mice

Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imaging research. Studies have validated the use of reporter genes with positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI, fluorescence with wild-type and mutants of green fluorescent protein, as well as bioluminescence using Firefly luciferase enzyme/protein (FL). In the current study, we validate for the first time the ability to image bioluminescence from Renilla luciferase enzyme/protein (RL) by injecting the substrate coelenterazine in living mice. A highly sensitive cooled charge-coupled device camera provides images within a few minutes of photon counting. Cells, transiently expressing the Rluc were imaged while located in the peritoneum, s.c. layer, as well as in the liver and lungs of living mice tail-vein injected with coelenterazine. Furthermore, d-luciferin (a substrate for FL) does not serve as a substrate for RL, and coelenterazine does not serve as a substrate for FL either in cell culture or in living mice. We also show that both Rluc and Fluc expression can be imaged in the same living mouse and that the kinetics of light production are distinct. The approaches validated will have direct applications to various studies where two molecular events need to be tracked, including cell trafficking of two cell populations, two gene therapy vectors, and indirect monitoring of two endogenous genes through the use of two reporter genes.

Uptake kinetics and biodistribution of 14C-d-luciferin—a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction: impact on bioluminescence based reporter gene imaging

PurposeFirefly luciferase catalyzes the oxidative decarboxylation of d-luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase reporter gene. Biokinetics and distribution of the substrate most likely have a significant impact on levels of light signal and therefore need to be investigated.MethodsBenzene ring 14C(U)-labeled d-luciferin was utilized. Cell uptake and efflux assays, murine biodistribution, autoradiography and CCD-camera based optical bioluminescence imaging were carried out to examine the in vitro and in vivo characteristics of the tracer in cell culture and in living mice respectively.ResultsRadiolabeled and unlabeled d-luciferin revealed comparable levels of light emission when incubated with equivalent amounts of the firefly luciferase enzyme. Cell uptake assays in pCMV-luciferase-transfected cells showed slow trapping of the tracer and relatively low uptake values (up to 22.9-fold higher in firefly luciferase gene-transfected vs. nontransfected cells, p = 0.0002). Biodistribution studies in living mice after tail-vein injection of 14C-d-luciferin demonstrated inhomogeneous tracer distribution with early predominant high radioactivity levels in kidneys (10.6% injected dose [ID]/g) and liver (11.9% ID/g), followed at later time points by the bladder (up to 81.3% ID/g) and small intestine (6.5% ID/g), reflecting the elimination routes of the tracer. Kinetics and uptake levels profoundly differed when using alternate injection routes (intravenous versus intraperitoneal). No clear trapping of 14C-d-luciferin in firefly luciferase-expressing tissues could be observed in vivo.ConclusionsThe data obtained with 14C-d-luciferin provide insights into the dynamics of d-luciferin cell uptake, intracellular accumulation, and efflux. Results of the biodistribution and autoradiographic studies should be useful for optimizing and adapting optical imaging protocols to specific experimental settings when utilizing the firefly luciferase and d-luciferin system.

Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice.

We have recently demonstrated that Renilla luciferase (Rluc) is a promising bioluminescence reporter gene that can be used for noninvasive optical imaging of reporter gene expression in living mice, with the aid of a cooled charged couple device (CCD) camera. In the current study, we explore the expression of a novel synthetic Renilla luciferase reporter gene (hRluc) in living mice, which has previously been reported to be a more sensitive reporter than native Rluc in mammalian cells. We explore the strategies of simultaneous imaging of both Renilla luciferase enzyme (RL) and synthetic Renilla luciferase enzyme (hRL):coelenterazine (substrate for RL/hRL) in the same living mouse. We also demonstrate that hRL:coelenterazine can yield a higher signal when compared to Firefly luciferase enzyme (FL): D-Luciferin, both in cell culture studies and when imaged from cells at the surface and from lungs of living mice. These studies demonstrate that hRluc should be a useful primary reporter gene with high sensitivity when used alone or in conjunction with other bioluminescence reporter genes for imaging in living rodents.

Noninvasive optical imaging of nitroreductase gene-directed enzyme prodrug therapy system in living animals.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising and emerging strategy that attempts to limit the systemic toxicity inherent to cancer chemotherapy by means of tumor-targeted delivery and expression of an exogenous gene whose product converts nontoxic prodrug(s) into activated cytotoxic agent(s). The bacterial nitroreductase (NTR) enzyme, coupled with its substrate prodrug 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), is a promising GDEPT strategy that has reached clinical trials. However, no strategy exists to visually monitor and quantitatively evaluate the therapeutic efficacy of NTR/CB1954 prodrug therapy in cells and imaging in living animals. As the success of any GDEPT is dependent upon the efficiency of transgene expression in vivo, we developed a safe, sensitive and reproducible noninvasive imaging method to monitor NTR transgene expression that would allow quantitative assessment of both therapeutic efficacy and diagnostic outcome of NTR/CB1954 prodrug therapy in the future. Here, we investigate the use of a novel fluorescent imaging dye CytoCy5S (a Cy5-labeled quenched substrate of NTR enzyme) on various cancer cell lines in vitro and in NTR-transfected tumor-bearing animals in vivo. CytoCy5S-labeled cells become fluorescent at ‘red-shifted’ wavelengths (638 nm) when reduced by cellular NTR enzyme and remains trapped within the cells for extended periods of time. The conversion and entrapment was dynamically recorded using a time-lapsed microscopy. Systemic and intratumoral delivery of CytoCy5S to NTR-expressing tumors in animals indicated steady and reproducible signals even 16 h after delivery (P<0.001). This is the first study to address visual monitoring and quantitative evaluation of NTR activity in small animals using CytoCy5S, and establishes the capability of NTR to function as an imageable reporter gene.

Whole-body, real-time preclinical imaging of quantum dot fluorescence with time-gated detection

We describe a wide-field preclinical imaging system optimized for time-gated detection of quantum dot fluorescence emission. As compared to continuous wave measurements, image contrast was substantially improved by suppression of short-lifetime background autofluorescence. Real-time (8 frames/s) biological imaging of subcutaneous quantum dot injections is demonstrated simultaneously in multiple living mice.

Compact instrument for fluorescence image-guided surgery

Fluorescence image-guided surgery (FIGS) is an emerging technique in oncology, neurology, and cardiology. To adapt intraoperative imaging for various surgical applications, increasingly flexible and compact FIGS instruments are necessary. We present a compact, portable FIGS system and demonstrate its use in cardiovascular mapping in a preclinical model of myocardial ischemia. Our system uses fiber optic delivery of laser diode excitation, custom optics with high collection efficiency, and compact consumer-grade cameras as a low-cost and compact alternative to open surgical FIGS systems. Dramatic size and weight reduction increases flexibility and access, and allows for handheld use or unobtrusive positioning over the surgical field.

Evaluation of the novel USPIO GEH121333 for MR imaging of cancer immune responses.

Tumor-associated macrophages (TAM) maintain a chronic inflammation in cancers, which is associated with tumor aggressiveness and poor prognosis. The purpose of this study was to: (1) evaluate the pharmacokinetics and tolerability of the novel ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) compound GEH121333; (2) assess whether GEH121333 can serve as a MR imaging biomarker for TAM; and (3) compare tumor MR enhancement profiles between GEH121333 and ferumoxytol. Blood half-lives of GEH121333 and ferumoxytol were measured by relaxometry (n = 4 each). Tolerance was assessed in healthy rats injected with high dose GEH121333, vehicle or saline (n = 4 each). Animals were monitored for 7 days regarding body weight, complete blood counts and serum chemistry, followed by histological evaluation of visceral organs. MR imaging was performed on mice harboring MMTV-PyMT-derived breast adenocarcinomas using a 7 T scanner before and up to 72 h post-injection (p.i.) of GEH121333 (n = 10) or ferumoxytol (n = 9). Tumor R1, R2* relaxation rates were compared between different experimental groups and time points, using a linear mixed effects model with a random effect for each animal. MR data were correlated with histopathology. GEH121333 showed a longer circulation half-life than ferumoxytol. Intravenous GEH121333 did not produce significant adverse effects in rats. All tumors demonstrated significant enhancement on T1, T2 and T2*-weighted images at 1, 24, 48 and 72 h p.i. GEH121333 generated stronger tumor T2* enhancement than ferumoxytol. Histological analysis verified intracellular compartmentalization of GEH121333 by TAM at 24, 48 and 72 h p.i. MR imaging with GEH121333 nanoparticles represents a novel biomarker for TAM assessment. This new USPIO MR contrast agent provides a longer blood half-life and better TAM enhancement compared with the iron supplement ferumoxytol. Copyright

Strategies to minimize background autofluorescence in live mice during noninvasive fluorescence optical imaging.

As small-animal fluorescence imaging becomes increasingly accessible to a broad spectrum of users, many lab animal researchers are just beginning to be exposed to its challenges. One setback to fluorescence imaging is background autofluorescence generated in animal tissue and in ingested food. The authors bring this issue into focus, and show how autofluorescence can be reduced in nude mice through selection of appropriate excitation wavelength and mouse diet.

Imaging Tumor Necrosis with Ferumoxytol

Objective Ultra-small superparamagnetic iron oxide nanoparticles (USPIO) are promising contrast agents for magnetic resonance imaging (MRI). USPIO mediated proton relaxation rate enhancement is strongly dependent on compartmentalization of the agent and can vary depending on their intracellular or extracellular location in the tumor microenvironment. We compared the T1- and T2-enhancement pattern of intracellular and extracellular USPIO in mouse models of cancer and pilot data from patients. A better understanding of these MR signal effects will enable non-invasive characterizations of the composition of the tumor microenvironment. Materials and Methods Six 4T1 and six MMTV-PyMT mammary tumors were grown in mice and imaged with ferumoxytol-enhanced MRI. R1 relaxation rates were calculated for different tumor types and different tumor areas and compared with histology. The transendothelial leakage rate of ferumoxytol was obtained by our measured relaxivity of ferumoxytol and compared between different tumor types, using a t-test. Additionally, 3 patients with malignant sarcomas were imaged with ferumoxytol-enhanced MRI. T1- and T2-enhancement patterns were compared with histopathology in a descriptive manner as a proof of concept for clinical translation of our observations. Results 4T1 tumors showed central areas of high signal on T1 and low signal on T2 weighted MR images, which corresponded to extracellular nanoparticles in a necrotic core on histopathology. MMTV-PyMT tumors showed little change on T1 but decreased signal on T2 weighted images, which correlated to compartmentalized nanoparticles in tumor associated macrophages. Only 4T1 tumors demonstrated significantly increased R1 relaxation rates of the tumor core compared to the tumor periphery (p<0.001). Transendothelial USPIO leakage was significantly higher for 4T1 tumors (3.4±0.9x10-3 mL/min/100cm3) compared to MMTV-PyMT tumors (1.0±0.9x10-3 mL/min/100 cm3). Likewise, ferumoxytol imaging in patients showed similar findings with high T1 signal in areas of tumor necrosis and low signal in areas of intracellularly compartmentalized iron. Conclusion Differential T1- and T2-enhancement patterns of USPIO in tumors enable conclusions about their intracellular and extracellular location. This information can be used to characterize the composition of the tumor microenvironment.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.