Alternative medicine: therapeutic effects on gastric original signet ring carcinoma via ascorbate and combination with sodium alpha lipoate.

BACKGROUND: Gastric signet ring cell carcinoma (SRCC) is an aggressive gastric adenocarcinoma with a poor prognosis when diagnosed at an advanced stage. As alternative medicine, two natural supplements (ascorbate (AA) and sodium alpha lipoate (LA)) have been shown to inhibit various cancers with mild side effects. METHODS: These two natural supplements and a series of combinations (AA&LA, AA+LA and LA + AA) were incubated with non-SRCC cells (GPM-1), patient-derived gastric origin SRCC (GPM-2), gastric-origin SRCCs (HSC-39 and KATO-3), human pancreatic (MIA PaCa-2) and ovarian (SKOV-3) cells for evaluating their therapeutic effects. Moreover, these treatments were applied in 3D-cultured organoids to reveal the feasibility of these approaches for in vivo study. RESULTS: Analyzing their antioxidant capabilities and dose-response curves, we observed that all four gastric cell lines, including three patient-derived cell lines were sensitive to ascorbate (~ 10 mM). The influence of ascorbate incubation time was studied, with a 16-h incubation found to be optimal for in vitro studies. Moreover, a simultaneous combination of AA and LA (AA&LA) did not significantly inhibit cell proliferation, while prior LA treatment increased the growth inhibition of AA therapy (LA + AA). Anti-cancer efficacy of AA was further confirmed in 3D-cultured SRCC (KATO-3) organoids. CONCLUSIONS: This study highlights the potential of AA and LA + AA in treating gastric origin SRCC, and demonstrates the influence of order in which the drugs are administered.

Minicircles for a two-step blood biomarker and PET imaging early cancer detection strategy.

Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection reagent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection reagent. Secreted GLuc correlated with HSV-1 sr39TK expression with R2 = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.

PET Reporter Gene Imaging and Ganciclovir-Mediated Ablation of Chimeric Antigen Receptor T Cells in Solid Tumors.

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[18F]-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. SIGNIFICANCE: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells.

Synthesis and Characterization of 9-(4-[18F]Fluoro-3-(hydroxymethyl)butyl)-2-(phenylthio)-6-oxopurine as a Novel PET Agent for Mutant Herpes Simplex Virus Type 1 Thymidine Kinase Reporter Gene Imaging.

PURPOSE: [18F]FHBG has been used as a positron emission tomography (PET) imaging tracer for the monitoring of herpes simplex virus type 1 thymidine kinase (HSV1-tk), a reporter gene for cell and gene therapy in humans. However, this tracer shows inadequate blood-brain barrier (BBB) penetration and, therefore, would be limited for accurate quantification of reporter gene expression in the brain. Here, we report the synthesis and evaluation of 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)-2(phenylthio)-6-oxopurine ([18F]FHBT) as a new PET tracer for imaging reporter gene expression of HSV1-tk and its mutant HSV1-sr39tk, with the aim of improved BBB penetration. PROCEDURES: [18F]FHBT was prepared by using a tosylate precursor and [18F]KF. The cellular uptake of [18F]FHBT was performed in HSV1-sr39tk-positive (+) or HSV1-sr39tk-negative (-) MDA-MB-231 breast cancer cells. The specificity of [18F]FHBT to assess HSV1-sr39tk expression was evaluated by in vitro blocking studies using 1 mM of ganciclovir (GCV). Penetration of [18F]FHBT and [18F]FHBG across the BBB was assessed by dynamic PET imaging studies in normal mice. RESULTS: The tosylate precursor reacted with [18F]KF using Kryptofix2.2.2 followed by deprotection to give [18F]FHBT in 10 % radiochemical yield (decay-corrected). The uptake of [18F]FHBT in HSV1-sr39tk (+) cells was significantly higher than that of HSV1-sr39tk (-) cells. In the presence of GCV (1 mM), the uptake of [18F]FHBT was significantly decreased, indicating that [18F]FHBT serves as a selective substrate of HSV1-sr39TK. PET images and time-activity curves of [18F]FHBT in the brain regions showed similar initial brain uptakes (~ 12.75 min) as [18F]FHBG (P > 0.855). Slower washout of [18F]FHBT was observed at the later time points (17.75 - 57.75 min, P > 0.207). CONCLUSIONS: Although [18F]FHBT showed no statistically significant improvement of BBB permeability compared with [18F]FHBG, we have demonstrated that the 2-(phenylthio)-6-oxopurine backbone can serve as a novel scaffold for developing HSV1-tk/HSV1-sr39tk reporter gene imaging agents for additional research in the future.

The Characterization of 18F-hGTS13 for Molecular Imaging of xC- Transporter Activity with PET.

The aim of this study was development of an improved PET radiotracer for measuring xC- activity with increased tumor uptake and reduced uptake in inflammatory cells compared with (S)-4-(3-18F-fluoropropyl)-l-glutamate (18F-FSPG). Methods: A racemic glutamate derivative, 18F-hGTS13, was evaluated in cell culture and animal tumor models. 18F-hGTS13 was separated into C5 epimers, and the corresponding 18F-hGTS13-isomer1 and 18F-hGTS13-isomer2 were evaluated in H460 tumor-bearing rats. Preliminary studies investigated the cellular uptake of 18F-hGTS13-isomer2 in multiple immune cell populations and states. Results:18F-hGTS13 demonstrated excellent H460 tumor visualization with high tumor-to-background ratios, confirmed by ex vivo biodistribution studies. Tumor-associated radioactivity was significantly higher for 18F-hGTS13 (7.5 ± 0.9 percentage injected dose [%ID]/g, n = 3) than for 18F-FSPG (4.6 ± 0.7 %ID/g, n = 3, P = 0.01). 18F-hGTS13-isomer2 exhibited excellent H460 tumor visualization (6.3 ± 1.1 %ID/g, n = 3) and significantly reduced uptake in multiple immune cell populations relative to 18F-FSPG. 18F-hGTS13-isomer2 exhibited increased liver uptake relative to 18F-FSPG (4.6 ± 0.8 vs. 0.7 ± 0.01 %ID/g), limiting its application in hepatocellular carcinoma. Conclusion:18F-hGTS13-isomer2 is a new PET radiotracer for molecular imaging of xC- activity that may provide information on tumor oxidation states. 18F-hGTS13-isomer2 has potential for clinical translation for imaging cancers of the thorax because of the low background signal in healthy tissue.

Positron emission tomography reporter gene strategy for use in the central nervous system.

There is a growing need for monitoring or imaging gene therapy in the central nervous system (CNS). This can be achieved with a positron emission tomography (PET) reporter gene strategy. Here we report the development of a PET reporter gene system using the PKM2 gene with its associated radiotracer [18F]DASA-23. The PKM2 reporter gene was delivered to the brains of mice by adeno-associated virus (AAV9) via stereotactic injection. Serial PET imaging was carried out over 8 wk to assess PKM2 expression. After 8 wk, the brains were excised for further mRNA and protein analysis. PET imaging at 8 wk post-AAV delivery showed an increase in [18F]DASA-23 brain uptake in the transduced site of mice injected with the AAV mice over all controls. We believe PKM2 shows great promise as a PET reporter gene and to date is the only example that can be used in all areas of the CNS without breaking the blood-brain barrier, to monitor gene and cell therapy.

Engineered immune cells as highly sensitive cancer diagnostics.

Endogenous biomarkers remain at the forefront of early disease detection efforts, but many lack the sensitivities and specificities necessary to influence disease management. Here, we describe a cell-based in vivo sensor for highly sensitive early cancer detection. We engineer macrophages to produce a synthetic reporter on adopting an M2 tumor-associated metabolic profile by coupling luciferase expression to activation of the arginase-1 promoter. After adoptive transfer in colorectal and breast mouse tumor models, the engineered macrophages migrated to the tumors and activated arginase-1 so that they could be detected by bioluminescence imaging and luciferase measured in the blood. The macrophage sensor detected tumors as small as 25-50 mm3 by blood luciferase measurements, even in the presence of concomitant inflammation, and was more sensitive than clinically used protein and nucleic acid cancer biomarkers. Macrophage sensors also effectively tracked the immunological response in muscle and lung models of inflammation, suggesting the potential utility of this approach in disease states other than cancer.

Assessment of Tumor Redox Status through (S)-4-(3-[18F]fluoropropyl)-L-Glutamic Acid PET Imaging of System xc - Activity.

The cell's endogenous antioxidant system is vital to maintenance of redox homeostasis. Despite its central role in normal and pathophysiology, no noninvasive tools exist to measure this system in patients. The cystine/glutamate antiporter system xc - maintains the balance between intracellular reactive oxygen species and antioxidant production through the provision of cystine, a key precursor in glutathione biosynthesis. Here, we show that tumor cell retention of a system xc --specific PET radiotracer, (S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), decreases in proportion to levels of oxidative stress following treatment with a range of redox-active compounds. The decrease in [18F]FSPG retention correlated with a depletion of intracellular cystine resulting from increased de novo glutathione biosynthesis, shown through [U-13C6, U-15N2]cystine isotopic tracing. In vivo, treatment with the chemotherapeutic doxorubicin decreased [18F]FSPG tumor uptake in a mouse model of ovarian cancer, coinciding with markers of oxidative stress but preceding tumor shrinkage and decreased glucose utilization. Having already been used in pilot clinical trials, [18F]FSPG PET could be rapidly translated to the clinic as an early redox indicator of tumor response to treatment. SIGNIFICANCE: [18F]FSPG PET imaging provides a sensitive noninvasive measure of tumor redox status and provides an early marker of tumor response to therapy.See related commentary by Lee et al., p. 701.

[18F]FSPG-PET reveals increased cystine/glutamate antiporter (xc-) activity in a mouse model of multiple sclerosis.

BACKGROUND: The cystine/glutamate antiporter (xc-) has been implicated in several neurological disorders and, specifically, in multiple sclerosis (MS) as a mediator of glutamate excitotoxicity and proinflammatory immune responses. We aimed to evaluate an xc-specific positron emission tomography (PET) radiotracer, (4S)-4-(3-[18F]fluoropropyl)-L-glutamate ([18F]FSPG), for its ability to allow non-invasive monitoring of xc- activity in a mouse model of MS. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG35-55) peptide in complete Freund's adjuvant (CFA) followed by pertussis toxin. Control mice received CFA emulsion and pertussis toxin without MOG peptide, while a separate cohort of naïve mice received no treatment. PET studies were performed to investigate the kinetics and distribution of [18F]FSPG in naïve, control, pre-symptomatic, and symptomatic EAE mice, compared to 18F-fluorodeoxyglucose ([18F]FDG). After final PET scans, each mouse was perfused and radioactivity in dissected tissues was measured using a gamma counter. Central nervous system (CNS) tissues were further analyzed using ex vivo autoradiography or western blot. [18F]FSPG uptake in human monocytes, and T cells pre- and post-activation was investigated in vitro. RESULTS: [18F]FSPG was found to be more sensitive than [18F]FDG at detecting pathological changes in the spinal cord and brain of EAE mice. Even before clinical signs of disease, a small but significant increase in [18F]FSPG signal was observed in the spinal cord of EAE mice compared to controls. This increase in PET signal became more pronounced in symptomatic EAE mice and was confirmed by ex vivo biodistribution and autoradiography. Likewise, in the brain of symptomatic EAE mice, [18F]FSPG uptake was significantly higher than controls, with the largest changes observed in the cerebellum. Western blot analyses of CNS tissues revealed a significant correlation between light chain of xc- (xCT) protein levels, the subunit of xc- credited with its transporter activity, and [18F]FSPG-PET signal. In vitro [18F]FSPG uptake studies suggest that both activated monocytes and T cells contribute to the observed in vivo PET signal. CONCLUSION: These data highlight the promise of [18F]FSPG-PET as a technique to provide insights into neuroimmune interactions in MS and the in vivo role of xc- in the development and progression of this disease, thus warranting further investigation.

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 device's 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.

A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant.

A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-ß-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR.

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.

AshwaMAX and Withaferin A inhibits gliomas in cellular and murine orthotopic models.

Glioblastoma multiforme (GBM) is an aggressive, malignant cancer Johnson and O'Neill (J Neurooncol 107: 359-364, 2012). An extract from the winter cherry plant (Withania somnifera ), AshwaMAX, is concentrated (4.3 %) for Withaferin A; a steroidal lactone that inhibits cancer cells Vanden Berghe et al. (Cancer Epidemiol Biomark Prev 23: 1985-1996, 2014). We hypothesized that AshwaMAX could treat GBM and that bioluminescence imaging (BLI) could track oral therapy in orthotopic murine models of glioblastoma. Human parietal-cortical glioblastoma cells (GBM2, GBM39) were isolated from primary tumors while U87-MG was obtained commercially. GBM2 was transduced with lentiviral vectors that express Green Fluorescent Protein (GFP)/firefly luciferase fusion proteins. Mutational, expression and proliferative status of GBMs were studied. Intracranial xenografts of glioblastomas were grown in the right frontal regions of female, nude mice (n = 3-5 per experiment). Tumor growth was followed through BLI. Neurosphere cultures (U87-MG, GBM2 and GBM39) were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 µM equivalent respectively and by Withaferin A with IC50 of 0.31, 0.28 and 0.25 µM respectively. Oral gavage, every other day, of AshwaMAX (40 mg/kg per day) significantly reduced bioluminescence signal (n = 3 mice, p < 0.02, four parameter non-linear regression analysis) in preclinical models. After 30 days of treatment, bioluminescent signal increased suggesting onset of resistance. BLI signal for control, vehicle-treated mice increased and then plateaued. Bioluminescent imaging revealed diffuse growth of GBM2 xenografts. With AshwaMAX, GBM neurospheres collapsed at nanomolar concentrations. Oral treatment studies on murine models confirmed that AshwaMAX is effective against orthotopic GBM. AshwaMAX is thus a promising candidate for future clinical translation in patients with GBM.

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

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, [(11)C]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.

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

UNLABELLED: 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.

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.

Positron emission tomography of 64Cu-DOTA-Rituximab in a transgenic mouse model expressing human CD20 for clinical translation to image NHL.

PURPOSE: This study aims to evaluate (64)Cu-DOTA-rituximab (PETRIT) in a preclinical transgenic mouse model expressing human CD20 for potential clinical translation. PROCEDURES: (64)Cu was chelated to DOTA-rituximab. Multiple radiolabeling, quality assurance, and imaging experiments were performed. The human CD20 antigen was expressed in B cells of transgenic mice (CD20TM). The mice groups studied were: (a) control (nude mice, n = 3) that received 7.4 MBq/dose, (b) with pre-dose (CD20TM, n = 6) received 2 mg/kg pre-dose of cold rituximab prior to PETRIT of 7.4 MBq/dose, and (c) without pre-dose (CD20TM, n = 6) PETRIT alone received 7.4 MBq/dose. Small animal PET was used to image mice at various time points (0, 1, 2, 4, 24, 48, and 72 h). The OLINDA/EXM software was used to determine the human equivalent dose for individual organs. RESULTS: PETRIT was obtained with a specific activity of 545 ± 38.91 MBq/nmole, radiochemical purity >95%, and immunoreactivity >75%. At 24 h, spleenic uptake of PETRIT%ID/g (mean ± STD) with and without pre-dose was 1.76 ± 0.43% and 16.5 ± 0.45%, respectively (P value = 0.01). Liver uptake with and without pre-dose was 0.41 ± 0.51% and 0.52 ± 0.17% (P value = 0.86), respectively. The human equivalents of highest dose organs with and without pre-dose are osteogenic cells at 30.8 ± 0.4 µSv/MBq and the spleen at 99 ± 4 µSv/MBq, respectively. CONCLUSIONS: PET imaging with PETRIT in huCD20 transgenic mice provided human dosimetry data for eventual applications in non-Hodgkins lymphoma 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.

Reproducibility study of [(18)F]FPP(RGD)2 uptake in murine models of human tumor xenografts.

PURPOSE: An (18)F-labeled PEGylated arginine-glycine-aspartic acid (RGD) dimer {[(18)F]FPP(RGD)(2)} has been used to image tumor α(v)ß(3) integrin levels in preclinical and clinical studies. Serial positron emission tomography (PET) studies may be useful for monitoring antiangiogenic therapy response or for drug screening; however, the reproducibility of serial scans has not been determined for this PET probe. The purpose of this study was to determine the reproducibility of the integrin α(v)ß(3)-targeted PET probe, [(18)F]FPP(RGD)(2,) using small animal PET. METHODS: Human HCT116 colon cancer xenografts were implanted into nude mice (n = 12) in the breast and scapular region and grown to mean diameters of 5-15 mm for approximately 2.5 weeks. A 3-min acquisition was performed on a small animal PET scanner approximately 1 h after administration of [(18)F]FPP(RGD)(2) (1.9-3.8 MBq, 50-100 µCi) via the tail vein. A second small animal PET scan was performed approximately 6 h later after reinjection of the probe to assess for reproducibility. Images were analyzed by drawing an ellipsoidal region of interest (ROI) around the tumor xenograft activity. Percentage injected dose per gram (%ID/g) values were calculated from the mean or maximum activity in the ROIs. Coefficients of variation and differences in %ID/g values between studies from the same day were calculated to determine the reproducibility. RESULTS: The coefficient of variation (mean±SD) for %ID(mean)/g and %ID(max)/g values between [(18)F]FPP(RGD)(2) small animal PET scans performed 6 h apart on the same day were 11.1 ± 7.6% and 10.4 ± 9.3%, respectively. The corresponding differences in %ID(mean)/g and %ID(max)/g values between scans were -0.025 ± 0.067 and -0.039 ± 0.426. Immunofluorescence studies revealed a direct relationship between extent of α(ν)ß(3) integrin expression in tumors and tumor vasculature with level of tracer uptake. Mouse body weight, injected dose, and fasting state did not contribute to the variability of the scans; however, consistent scanning parameters were necessary to ensure accurate studies, in particular, noting tumor volume, as well as making uniform: the time of imaging after injection and the ROI size. Reanalysis of ROI placement displayed variability for %ID(mean)/g of 6.6 ± 3.9% and 0.28 ± 0.12% for %ID(max)/g. CONCLUSION: [(18)F]FPP(RGD)(2) small animal PET mouse tumor xenograft studies are reproducible with relatively low variability.

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.