Amnon Bar-Shir^1,2, Guanshu Liu^1,3, Nirbhay N Yadav^1,3, Yoshinori Kato¹, Galit Pelled^1,3, Piotr Walczak^1,2, Michael T McMahon^1,3, Martin G Pomper¹, Marc M Greenberg⁴, Peter C van Zijl^1,3, Jeff W Bulte^1,2, and Assaf A Gilad^1,2
1Department of Radiology, Johns Hopkins University, Baltimore, Maryland, United States, ²Cellular Imaging Section, Johns Hopkins University, Baltimore, Maryland, United States, ³F.M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute, Baltimore, Maryland, United States, ⁴Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, United States

By modifying a substrate for herpes simplex virus type-1 thymidine kinase (HSV1-tk), an existing PET reporter gene, we successfully transformed it into a CEST-MRI reporter gene. Thymidine analogues were synthesized with higher pKa values for imino protons, thus reducing their exchange rate to an optimum range for CEST detection. The substrate 5-methyl-5,6-dihydrothymidine provided the highest contrast after saturation at 5 ppm from the water protons. It is efficiently phosphorylated by HSV1-tk but not mammalian thymidine kinase, making it a specific reporter. Transplanted cells expressing HSV1-tk were easily detected in vivo following i.v. administration of 5-methyl-5,6-dihydrothymidine.

Thomas Christian Basse-Lüsebrink^1,2, Stephanie Weibel², Elisabeth Hofmann², Johanna Langbein², Volker Jörg Friedrich Sturm¹, Thomas Kampf¹, Peter Michael Jakob¹, and Aladár Szalay^2,3
1Experimental Physics 5, University of Würzburg, Würzburg, Bavaria, Germany, ²Biochemistry, University of Würzburg, Würzburg, Bavaria, Germany, ³Genelux Corporation, San Diego, CA, United States

In recent years, it was shown that oncolytic virus strains such as attenuated vaccinia virus have a promising therapeutic potential in the treatment of cancer. Importantly, the infection of tumors with viral agents often induces massive inflammation within the tumor microenvironment. In the past, ¹⁹F markers have shown their potential for visualizing inflammation in vivo using ¹⁹F MRI. Thus, the purpose of the present study was to monitor in vivo inflammation by employing ¹⁹F 3D MRI in a vaccinia virus treated melanoma model and to compare the spatial ¹⁹F patterns to non-infected controls. Furthermore, ex vivo ¹⁹F MRI and immunohistochemistry were performed to verify in vivo results.

Stephen Patrick¹, Tiago B. Rodrigues^1,2, Scott K. Lyons², Mikko I. Kettunen^1,2, and Kevin M. Brindle^1,2
1Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom, ²Cancer Research UK, Cambridge, United Kingdom

A new MRI reporter gene system is described based on TIM2, which is a receptor protein for the heavy-subunit of ferritin and which mediates its endocytosis. Ferritin, which produces negative contrast in T₂ weighted images, can also be emptied of iron and filled with manganese, making it an effective T₁ contrast agent as well. We demonstrate here that expression of TIM2 results in detectable changes in image contrast in vitro and in vivo following addition of ferritin.

Philipp Böhm-Sturm¹, Markus Aswendt¹, Laura Breucker¹, Nadine Henn¹, Luam Mengler¹, Joanna Adamczak¹, Eberhard D. Pracht¹, Annette Tennstaedt¹, Laura Mezzanotte², Clemens Löwik², and Mathias Hoehn^1
1In-Vivo-NMR, Max-Planck-Institute for Neurological Research, Cologne, Germany, ²Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, Netherlands

Stem cells are a promising candidate for new therapeutic approaches of acute and degenerative disorders of the brain. MRI of prelabeled cells allows to follow their spatio-temporal dynamics after implantation, however this provides little information on cell graft function. We present here a novel bimodal approach of bioluminescence imaging (BLI) and 19F MRI to track 19F labeled, luciferase+ stem cells in the mouse brain. As the luciferase is only expressed in viable cells, BLI gives information on cell graft vitality in addition to the purely structural information given by 19F MRI.

Sara Figueiredo^1,2, Silvia Rizzitelli¹, Juan Carlos Cutrin³, João Nuno Moreira⁴, Carlos F. G. C. Geraldes², Silvio Aime¹, and Enzo Terreno^1
1Department of Chemistry IFM and Molecular & Preclinical Imaging Centers, University of Turin, Turin, Turin, Italy, ²Department of Life Sciences, FCTUC and Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Coimbra, Portugal, ³Department of Clinical and Biological Sciences, University of Turin, Turin, Turin, Italy, ⁴Laboratory of Pharmaceutical Technology Faculty of Pharmacy and CNC, University of Coimbra, Coimbra, Coimbra, Portugal

Yeast cell wall particles (YCWPs) are a promising class of nature-inspired biocompatible microcarriers that can be used for the delivery of amphipathic/lipophilic imaging reporters. The new loading procedure applied to YCWPS yields the highest longitudinal relaxivity per particle ever reported for Gd-based systems. Furthermore, these particle display high affinity towards antigen presenting cells, namely macrophages and dendritic cells, potentiating their use in cell tracking experiments.

Stacey M. Cromer Berman¹, Kshitiz Gupta², C. Joanne Wang², Inema Orukari¹, Andre Levchenko², Piotr Walczak¹, and Jeff W.M. Bulte^1
1Dept. of Radiology, Johns Hopkins University, Baltimore, MD, United States, ²Dept. of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, United States

Time-lapse microscopy of cell division and random cellular motility assays were performed on SPIO-labeled neural stem cells. Both the maximum and average speed of NSCs were reduced as compared to unlabeled controls. Following transplantation into mouse brain, rapid exocytosis of SPIO by live (as determined by BLI) cells was observed as early as 48 hours post-engraftment, with SPIO-depleted cells showing the farthest migration distance. As label dilution is negligible at this early time point, we conclude that MRI underestimation of cell migration can occur as a result of reduced cell motility, which is mitigated following SPIO exocytosis.

Brenda Robledo¹, Xiaoyong Zhang¹, Steve Harris¹, and Xiaoping Hu^1
1Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States

The gene mms6, originally identified in magnetotactic bacteria, expresses a protein thought to initiate magnetite crystal formation inside specialized organelles. Genetic loss-of-function studies suggest the protein also regulates the size of the iron-oxide crystals. We hypothesized that cells transfected with mms6 would store increased levels of iron and thereby produce MR contrast. In vitro, we found that mms6 positive glioma cells stored 2.8 times more iron than control cells and also showed a 90.6% increase in transverse relaxivity at 9.4 Tesla compared to control cells. Our results suggest that mms6 may function as an MR reporter gene in cancer studies.

Bistra Iordanova^1,2, T K Hitchens², C S Robison¹, L K Pusateri^1,2, and Eric T Ahrens^1,2
1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, United States, ²Pittsburgh NMR Center for Biomedical Research, Pittsburgh, PA, United States

The blood-brain barrier presents a unique challenge for introducing therapeutic molecules. The olfactory epithelium in mammals circumvents this barrier and is a suitable target for therapeutic agents. Here, we report the design of improved MRI reporter gene. We modify mitochondrial ferritin to localize to the cell cytoplasm. We demonstrate its high iron loading efficiency in mammalian cells. We use this new reporter to image gene expression in native olfactory sensory neurons in the mouse epithelium. This MRI reporter can facilitate the study of molecular mechanisms of olfaction as well as monitoring the success of intranasal gene therapy in live animals.