Gayatri Gowrishankar

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.

Investigation of 6-[18F]-fluoromaltose as a novel PET tracer for imaging bacterial infection

Despite advances in the field of nuclear medicine, the imaging of bacterial infections has remained a challenge. The existing reagents suffer from poor sensitivity and specificity. In this study we investigate the potential of a novel PET (positron emission tomography) tracer that overcomes these limitations. Methods 6-[18F]-fluoromaltose was synthesized. Its behavior in vitro was evaluated in bacterial and mammalian cultures. Detailed pharmacokinetic and biodistribution profiles for the tracer were obtained from a murine model. Results 6-[18F]-fluoromaltose is taken up by multiple strains of pathogenic bacteria. It is not taken up by mammalian cancer cell lines. 6-[18F]-fluoromaltose is retained in infected muscles in a murine model of bacterial myositis. It does not accumulate in inflamed tissue. Conclusion We have shown that 6-[18F]-fluoromaltose can be used to image bacterial infection in vivo with high specificity. We believe that this class of agents will have a significant impact on the clinical management of patients.

GLUT 5 is not over-expressed in breast cancer cells and patient breast cancer tissues.

F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues.

Imaging Target mRNA and siRNA-Mediated Gene Silencing In Vivo with Ribozyme-Based Reporters

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans‐splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme‐mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180‐fold, and by ten‐fold for the stably expressed target. siRNA‐mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme‐based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.

Detection of mRNA in mammalian cells with a split ribozyme reporter

The detection of mRNA expression in vivo can reveal essential information about basic biology and disease processes. Current methods primarily involve the use of labeled (with fluorophores or radioactive isotopes) antisense oligomers, based on a one-to-one receptor–ligand type of interaction without robust signal amplification, such as molecular beacons. Given that the copy number of a particular mRNA target per cell is generally ~50–1000, it is challenging to detect such a small number of mRNA in vivo with current methods. Here we introduce a new strategy with a mechanism of signal amplification for sensing target mRNAs in mammalian cells. This strategy utilizes the Tetrahymena group I intron ribozyme as an RNA sensor. Group I introns of the ciliated protozoan Tetrahymena thermophila have been shown to catalyze trans-splicing of mRNA molecules in mammalian cells in which the ribozyme splices an attached 3’ exon to a designated splice site on any chosen target RNA. This property has been variously exploited for ribozyme-mediated repairing of mutant mRNA transcripts. We were interested in exploiting this splicing activity to detect mRNAs in mammalian cells because it can potentially achieve robust signal amplification through enzymatic catalysis. Splitreporter technology based on protein (intein) splicing has been demonstrated with Renilla luciferase and green fluorescent protein in detecting protein–protein interactions. The splicing activity of the Tetrahymena ribozyme may be similarly applied to develop a biosensor for mRNA detection. We previously reported a cis-splicing ribozyme construct, Rz156, in which the Tetrahymena group I intron ribozyme was inserted into the coding sequence of the cDNA of the nonsecreted TEM-1 b-lactamase (Bla). Based on this cis-splicing ribozyme, we devised a split-reporter strategy for the detection of target mRNA in cells (Scheme 1). In this design, Rz156 was split at the L1 loop into a pair of plasmid constructs—TRzL and TRzR—each carrying a part of the coding sequence of the Bla reporter and a fragment of the Tetrahymena ribozyme. Both TRzL and TRzR also carried an antisense sequence complementary to a target mRNA, and a six-nucleotide-long linker

The Exosome Total Isolation Chip

Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4-1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the devices broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10-100 μL) of blood.

PET imaging of tumor glycolysis downstream of hexokinase through noninvasive measurement of pyruvate kinase M2

A PET imaging reagent targeting PKM2 allows noninvasive assessment of glycolysis in glioblastoma multiforme, distinguishing it from normal brain tissue. A new view of brain tumors Tumor cells are well known to have metabolic abnormalities that are not present in normal cells, and positron emission tomography (PET) imaging relies on these abnormalities to identify tumors within a patient’s body. Unfortunately, the most common type of PET imaging is based on detection of cells that most actively use glucose, and thus, it cannot detect tumors in the brain, where even the normal cells require large amounts of glucose. Now, Witney et al. have developed a new PET imaging reagent, which detects abnormalities of glycolysis that are specifically associated with brain tumors but not normal brain, allowing a clear differentiation of the two in mouse models. Cancer cells reprogram their metabolism to meet increased biosynthetic demands, commensurate with elevated rates of replication. Pyruvate kinase M2 (PKM2) catalyzes the final and rate-limiting step in tumor glycolysis, controlling the balance between energy production and the synthesis of metabolic precursors. We report here the synthesis and evaluation of a positron emission tomography (PET) radiotracer, [11C]DASA-23, that provides a direct noninvasive measure of PKM2 expression in preclinical models of glioblastoma multiforme (GBM). In vivo, orthotopic U87 and GBM39 patient-derived tumors were clearly delineated from the surrounding normal brain tissue by PET imaging, corresponding to exclusive tumor-associated PKM2 expression. In addition, systemic treatment of mice with the PKM2 activator TEPP-46 resulted in complete abrogation of the PET signal in intracranial GBM39 tumors. Together, these data provide the basis for the clinical evaluation of imaging agents that target this important gatekeeper of tumor glycolysis.

Synthesis of [18F]-labelled Maltose Derivatives as PET Tracers for Imaging Bacterial Infection

PurposeTo develop novel positron emission tomography (PET) agents for visualization and therapy monitoring of bacterial infections.ProceduresIt is known that maltose and maltodextrins are energy sources for bacteria. Hence, 18F-labelled maltose derivatives could be a valuable tool for imaging bacterial infections. We have developed methods to synthesize 4-O-(α-D-glucopyranosyl)-6-deoxy-6-[18F]fluoro-D-glucopyranoside (6-[18F]fluoromaltose) and 4-O-(α-D-glucopyranosyl)-1-deoxy-1-[18F]fluoro-D-glucopyranoside (1-[18F]fluoromaltose) as bacterial infection PET imaging agents. 6-[18F]fluoromaltose was prepared from precursor 1,2,3-tri-O-acetyl-4-O-(2′,3′,-di-O-acetyl-4′,6′-benzylidene-α-D-glucopyranosyl)-6-deoxy-6-nosyl-D-glucopranoside (5). The synthesis involved the radio-fluorination of 5 followed by acidic and basic hydrolysis to give 6-[18F]fluoromaltose. In an analogous procedure, 1-[18F]fluoromaltose was synthesized from 2,3, 6-tri-O-acetyl-4-O-(2′,3′,4′,6-tetra-O-acetyl-α-D-glucopyranosyl)-1-deoxy-1-O-triflyl-D-glucopranoside (9). Stability of 6-[18F]fluoromaltose in phosphate-buffered saline (PBS) and human and mouse serum at 37 °C was determined. Escherichia coli uptake of 6-[18F]fluoromaltose was examined.ResultsA reliable synthesis of 1- and 6-[18F]fluoromaltose has been accomplished with 4–6 and 5–8 % radiochemical yields, respectively (decay-corrected with 95 % radiochemical purity). 6-[18F]fluoromaltose was sufficiently stable over the time span needed for PET studies (∼96 % intact compound after 1-h and ∼65 % after 2-h incubation in serum). Bacterial uptake experiments indicated that E. coli transports 6-[18F]fluoromaltose. Competition assays showed that the uptake of 6-[18F]fluoromaltose was completely blocked by co-incubation with 1 mM of the natural substrate maltose.ConclusionWe have successfully synthesized 1- and 6-[18F]fluoromaltose via direct fluorination of appropriate protected maltose precursors. Bacterial uptake experiments in E. coli and stability studies suggest a possible application of 6-[18F]fluoromaltose as a new PET imaging agent for visualization and monitoring of bacterial infections.

Specific Imaging of Bacterial Infection Using 6″-18F-Fluoromaltotriose: A Second-Generation PET Tracer Targeting the Maltodextrin Transporter in Bacteria

6″-18F-fluoromaltotriose is a PET tracer that can potentially be used to image and localize most bacterial infections, much like 18F-FDG has been used to image and localize most cancers. However, unlike 18F-FDG, 6″-18F-fluoromaltotriose is not taken up by inflammatory lesions and appears to be specific to bacterial infections by targeting the maltodextrin transporter that is expressed in gram-positive and gram-negative strains of bacteria. Methods: 6″-18F-fluoromaltotriose was synthesized with high radiochemical purity and evaluated in several clinically relevant bacterial strains in cultures and in living mice. Results: 6″-18F-fluoromaltotriose was taken up in both gram-positive and gram-negative bacterial strains. 6″-18F-fluoromaltotriose was also able to detect Pseudomonas aeruginosa in a clinically relevant mouse model of wound infection. The utility of 6″-18F-fluoromaltotriose to help monitor antibiotic therapies was also evaluated in rats. Conclusion: 6″-18F-fluoromaltotriose is a promising new tracer that has significant diagnostic utility, with the potential to change the clinical management of patients with infectious diseases of bacterial origin.

Imaging B cells in a mouse model of multiple sclerosis using 64Cu-Rituximab-PET

B lymphocytes are a key pathologic feature of multiple sclerosis (MS) and are becoming an important therapeutic target for this condition. Currently, there is no approved technique to noninvasively visualize B cells in the central nervous system (CNS) to monitor MS disease progression and response to therapies. Here, we evaluated 64Cu-rituximab, a radiolabeled antibody specifically targeting the human B cell marker CD20, for its ability to image B cells in a mouse model of MS using PET. Methods: To model CNS infiltration by B cells, experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice that express human CD20 on B cells. EAE mice were given subcutaneous injections of myelin oligodendrocyte glycoprotein fragment1–125 emulsified in complete Freund adjuvant. Control mice received complete Freund adjuvant alone. PET imaging of EAE and control mice was performed 1, 4, and 19 h after 64Cu-rituximab administration. Mice were perfused and sacrificed after the final PET scan, and radioactivity in dissected tissues was measured with a γ-counter. CNS tissues from these mice were immunostained to quantify B cells or were further analyzed via digital autoradiography. Results: Lumbar spinal cord PET signal was significantly higher in EAE mice than in controls at all evaluated time points (e.g., 1 h after injection: 5.44 ± 0.37 vs. 3.33 ± 0.20 percentage injected dose [%ID]/g, P < 0.05). 64Cu-rituximab PET signal in brain regions ranged between 1.74 ± 0.11 and 2.93 ± 0.15 %ID/g for EAE mice, compared with 1.25 ± 0.08 and 2.24 ± 0.11 %ID/g for controls (P < 0.05 for all regions except striatum and thalamus at 1 h after injection). Similarly, ex vivo biodistribution results revealed notably higher 64Cu-rituximab uptake in the brain and spinal cord of huCD20tg EAE, and B220 immunostaining verified that increased 64Cu-rituximab uptake in CNS tissues corresponded with elevated B cells. Conclusion: B cells can be detected in the CNS of EAE mice using 64Cu-rituximab PET. Results from these studies warrant further investigation of 64Cu-rituximab in EAE models and consideration of use in MS patients to evaluate its potential for detecting and monitoring B cells in the progression and treatment of this disease. These results represent an initial step toward generating a platform to evaluate B cell–targeted therapeutics en route to the clinic.