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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. [ABSTRACT FROM AUTHOR]

Purpose: Human pluripotency gene networks (PGNs), controlled in part by Oct4, are central to understanding pluripotent stem cells, but current fluorescent reporter genes (RGs) preclude noninvasive assessment of Oct4 dynamics in living subjects. Procedures: To assess Oc4 activity noninvasively, we engineered a mouse embryonic stem cell line which encoded both a pOct4-hrluc (humanized renilla luciferase) reporter and a pUbi-hfluc2-gfp (humanized firefly luciferase 2 fused to green fluorescent protein) reporter. Results: In cell culture, pOct4-hRLUC activity demonstrated a peak at 48 h (day 2) and significant downregulation by 72 h (day 3) ( p=0.0001). Studies in living subjects demonstrated significant downregulation in pOct4-hRLUC activity between 12 and 144 h ( p = 0.001) and between 12 and 168 h ( p = 0.0003). pOct4-hRLUC signal dynamics after implantation was complex, characterized by transient upregulation after initial downregulation in all experiments ( n = 10, p = 0.01). As expected, cell culture differentiation of the engineered mouse embryonic stem cell line demonstrated activation of mesendodermal, mesodermal, endodermal, and ectodermal master regulators of differentiation, indicating potency to form all three germ layers. Conclusions: We conclude that the Oct4-hrluc RG system enables noninvasive Oct4 imaging in cell culture and in living subjects. [ABSTRACT FROM AUTHOR]

Firefly 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. Benzene 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. Radiolabeled 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. The 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. [ABSTRACT FROM AUTHOR]