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The molecular imaging techniques allow monitoring of the transplanted cells in the same individuals over time, from early localization to the survival, migration, and differentiation. Generally, there are two methods of stem cell labeling: direct and indirect labeling methods. The direct labeling method introduces a labeling agent into the cell, which is stably incorporated or attached to the cells prior to transplantation. Direct labeling of cells with radionuclides is a simple method with relatively fewer adverse events related to genetic responses. However, it can only allow short-term distribution of transplanted cells because of the decreasing imaging signal with radiodecay, according to the physical half-lives, or the signal becomes more diffuse with cell division and dispersion. The indirect labeling method is based on the expression of a reporter gene transduced into the cell before transplantation, which is then visualized upon the injection of an appropriate probe or substrate. In this review, various imaging strategies to monitor the survival and behavior change of transplanted stem cells are covered. Taking these new approaches together, the direct and indirect labeling methods may provide new insights on the roles of in vivo stem cell monitoring, from bench to bedside.

// Jae-Wook Lee 1, * , Yun-Kyoung Ryu 1, * , Young-Hoon Ji 2 , Joo Hyun Kang 3 , Eun-Yi Moon 1 1 Department of Bioscience and Biotechnology, Sejong University, Seoul 143–747, Korea 2 Research Center for Radiotherapy, Korea Institute of Radiological and Medical Science, Seoul 139–709, Korea 3 Molecular Imaging Research Center, Korea Institute of Radiological and Medical Science, Seoul 139–709, Korea * These authors have contributed equally to this work Correspondence to: Eun-Yi Moon, e-mail: eunyimoon@sejong.ac.kr , eunyimoon@gmail.com Keywords: thymosin beta-4, Rac1-GTPase, Rap1-GTPase, cancer cell migration, tumor metastasis Received: September 28, 2014 Accepted: January 26, 2015 Published: March 23, 2015 ABSTRACT Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tβ4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo , and H/R-experienced cancer cell migration in vitro , more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

Gastrin‑releasing peptide receptor (GRPR) is overexpressed by a variety of human tumors and in particular, identified to be upregulated in prostate cancers. The current study aimed to develop clinically translatable BBN analogue‑based radioligands for positron emission tomography (PET) of GRPR‑positive tumors. We developed radiolabeled BBN analogues and modified radiolabeled galacto‑BBN analogues and then investigated their tumor‑targeting efficacy in vivo. The chelator 1,4,7‑triazacyclononane, 1‑glutaric acid‑4,7 acetic acid (NODAGA) was used to radiolabel the peptides with 64Cu. The peptides were evaluated by measuring cell‑based receptor‑binding affinities. Biodistribution experiments and small animal imaging using PET were performed in nude mice bearing subcutaneous PC3 human prostate cancer xenografts. The conjugates were radiolabeled with yields >99%. The stability assay showed that [64Cu]NODAGA‑BBN and [64Cu]NODAGA‑galacto‑BBN remained stable in both human and mouse serum for 1 h at 37˚C. PET images of PC3 tumor‑bearing nude mice were acquired at 1, 3, 24, 48 and 72 h after injection. [64Cu]NODAGA‑galacto‑BBN showed retention in tumors for 72 h, low liver uptake, and rapid renal clearance. PET imaging results were also confirmed by biodistrubution 1 and 3 h after injection. [64Cu]NODAGA‑BBN and [64Cu]NODAGA‑galacto‑BBN are promising new PET probes for GRPR‑positive prostate cancer.

This study aims to monitor how the change of cell survival of transplanted adipose-derived stem cells (ADSCs) responds to myocardial infarction (MI) via the hexadecyl-4- 124 I-iodobenzoate ( 124 I-HIB) mediated direct labeling method in vivo. Stem cells have shown the potential to improve cardiac function after MI. However, monitoring of the fate of transplanted stem cells at target sites is still unclear. Rat ADSCs were labeled with 124 I-HIB, and radiolabeled ADSCs were transplanted into the myocardium of normal and MI model. In the group of 124 I-HIB-labeled ADSC transplantation, in vivo imaging was performed using small-animal positron emission tomography (PET)/computed tomography (CT) for 9 days. Twenty-one days post-transplantation, histopathological analysis and apoptosis assay were performed. ADSC viability and differentiation were not affected by 124 I-HIB labeling. In vivo tracking of the 124 I-HIB-labeled ADSCs was possible for 9 and 3 days in normal and MI model, respectively. Apoptosis of transplanted cells increased in the MI model compared than that in normal model. We developed a direct labeling agent, 124 I-HIB, and first tried to longitudinally monitor transplanted stem cell to MI. This approach may provide new insights on the roles of stem cell monitoring in living bodies for stem cell therapy from pre-clinical studies to clinical trials.

The aim of the present study was to evaluate functional changes of mGluR5 expression in advanced Alzheimer's disease (AD) using positron emission tomography (PET) with an mGluR5 specific radiotracer ([18F]FPEB) in 5xFAD AD model. Subsequently, in the same animal, mGluR5 expression was quantified by immunoassay techniques. The non-displaceable binding potential values for mGluR5 was estimated by the Logan's graphical analysis. Brain PET imaging revealed that radioactivities in the hippocampus and the striatum were significantly lower in 5xFAD mice compared to control animals. Binding values were also significantly lowered in 5xFAD mice. This decline was validated by immunoblotting of protein isolates from brain tissues, as the mean band density for 5xFAD mice had a lower mGluR5 intensity than for wild type mice. These results indicated that mGluR5 levels in 5xFAD mice were down regulated in the limbic system.

Cancer-associated microRNAs have been stably detected in blood. The objective of this study was to identify a panel of circulating microRNAs with the potential to serve as biomarkers for estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2 (HER2)− breast cancer. We used microarray-based expression profiling to compare the levels of circulating microRNAs in blood samples from 11 ER+/HER2− advanced breast cancer patients plus 5 age-matched controls. MicroRNA levels were validated by reverse transcription quantitative polymerase chain reaction in 40 control subjects, 187 early breast cancer patients, and 45 metastatic breast cancer patients. Then, we assessed the association between the levels of microRNA and clinical outcomes of ER+/HER2− metastatic breast cancer. Initially, we found that miR-1280, miR-1260, and miR-720 were up-regulated in blood from breast cancer patients (P < 0.05). In validation, miR-1280 levels significantly increased in breast cancer patients and reflected tumor status (control<

This study was to evaluate the anti-atherosclerotic effect of Korean red ginseng (KRG) in apolipoprotein E knockout (ApoE KO) mice. Female ApoE KO mice were divided into 2 groups and were treated via intragastric inoculation for 21 weeks. The body weight increased gradually in both the KRG extract inoculation group and the control group inoculated with saline. Plaque lesion areas in the aortic sinus were reduced largely in the KRG inoculation group than in the control group. Triglyceride and inflammatory cytokines such as monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) were significantly reduced in the KRG inoculation group. Additionally, the KRG inoculation group showed significantly lower cardiovascular inflammation by 18F-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography compared to that in the control group. Taken together, KRG alleviates atherosclerotic lesions through MCP-1 and TNF-α suppression. These results suggest that KRG could be a valuable therapeutic strategy for the treatment of atherosclerosis.

The Siemens Biograph TruePoint TrueV (B-TPTV) positron emission tomography (PET) scanner performs 3D PET reconstruction using a system matrix with point spread function (PSF) modeling (called the True X reconstruction). PET resolution was dramatically improved with the True X method. In this study, we assessed the spatial resolution and image quality on a B-TPTV PET scanner. In addition, we assessed the feasibility of animal imaging with a B-TPTV PET and compared it with a microPET R4 scanner. Spatial resolution was measured at center and at 8 cm offset from the center in transverse plane with warm background activity. True X, ordered subset expectation maximization (OSEM) without PSF modeling, and filtered back-projection (FBP) reconstruction methods were used. Percent contrast (% contrast) and percent background variability (% BV) were assessed according to NEMA NU2-2007. The recovery coefficient (RC), non-uniformity, spill-over ratio (SOR), and PET imaging of the Micro Deluxe Phantom were assessed to compare image quality of B-TPTV PET with that of the microPET R4. When True X reconstruction was used, spatial resolution was

Purpose The expression of glucose transporter-1 (Glut-1) gene and those of major thyroid-specific genes were examined in papillary carcinoma tissues, and the expressions of these genes were compared with cancer differentiation grades. Materials and Methods Twenty-four human papillary carcinoma tissues were included in this study. The expressions of Glut-1- and thyroid-specific genes [sodium/iodide symporter (NIS), thyroid peroxidase, thyroglobulin, TSH receptor and pendrin] were analyzed by RT-PCR. Expression levels were expressed as ratios versus the expression of beta-actin. Pathologic differentiation of papillary carcinoma was classified into a relatively well-differentiated group (n = 13) and relatively less differentiated group (n = 11). Results Glut-1 gene expression was significantly higher in the less differentiated group (0.66 ± 0.04) than in the well-differentiated group (0.59 ± 0.07). The expression levels of the NIS, PD and TG genes were significantly higher in the well-differentiated group (NIS: 0.67 ± 0.20, PD: 0.65 ± 0.21, TG: 0.74 ± 0.16) than in the less differentiated group (NIS: 0.36 ± 0.05, PD: 0.49 ± 0.08, TG: 0.60 ± 0.11), respectively. A significant negative correlation was found between Glut-1 and NIS expression, and positive correlations were found between NIS and TG, and between NIS and PD. Conclusion The NIS, PD and TG genes were highly expressed in well-differentiated thyroid carcinomas, whereas the Glut-1 gene was highly expressed in less differentiated thyroid carcinomas. These findings provide a molecular rationale for the management of papillary carcinoma, especially in the selection of FDG PET or radioiodine whole-body scan and I-131-based therapy.

18F -FP-CIT PET image was useful for diagnosis of Parkinson's disease (PD). Although spatial normalization step is necessary in SPM analysis, it was difficult to apply the spatial normalization on 18F-FP-CIT PET due to the lack of reference for spatial normalization. Therefore, additional data acquisition such as MRI or FDG PET scans is needed. In this study, we developed the voxel based statistical analysis method for 18F-FP-CIT PET using early phase of 18F-FP-CIT. 18F-FP-CIT PET data was acquired from 7 normal and 1 PD model rat brain. Emission PET data was collected for 1 h at the start of 1 mCi of 18F-FP-CIT. We used the early phase uptake image (from 0 to 1200 s) including perfusion phase as referential information for spatial normalization. Additionally, 18F-FDG PET was acquired as referential data to validate the substitution of 18F-FP-CIT. To validate early phase 18F-FP-CIT PET data for spatial normalization, correlation coefficient between early phase 18F-FP-CIT and 18F-FDG were assessed. Using our proposed method, SPM for 18F-FP-CIT PET was successfully implemented. Early phase 18F-FP-CIT PET corresponding early phase and 18F-FDG PET were highly correlated. When we applied our developed method for the comparison between normal and PD, we can identify the difference of 18F -FP-CIT between the normal and PD in the striatum (P 0). The SPM method for 18F -FP-CIT PET data could provide high localization accuracy in PD analysis. This method may be useful for assessment of PD.

Cell-tracking methods with molecular-imaging modality can monitor the biodistribution of cells. In this study, the direct-labeling method with ⁶⁴Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (⁶⁴Cu-PTSM), indirect cell-labeling methods with herpes simplex virus type 1-thymidine kinase (HSV1-tk)-mediated ¹²⁴I-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil (¹²⁴I-FIAU) were comparatively investigated in vitro and in vivo for tracking of human chronic myelogenous leukemia cells. K562-TL was established by retroviral transduction of the HSV1-tk and firefly luciferase gene in the K562 cell. K562-TL cells were labeled with ⁶⁴Cu-PTSM or ¹²⁴I-FIAU. Cell labeling efficiency, viability, and radiolabels retention were compared in vitro. The biodistribution of radiolabeled K562-TL cells with each radiolabel and small-animal positron emission tomography imaging were performed. Additionally, in vivo and ex vivo bioluminescence imaging (BLI) and tissue reverse transcriptase-polymerase chain reaction (RT-PCR) analysis were used for confirming those results. K562-TL cells were efficiently labeled with both radiolabels. The radiolabel retention (%) of ¹²⁴I-FIAU (95.2%±1.1%) was fourfold higher than ⁶⁴Cu-PTSM (23.6%±0.7%) at 24 hours postlabeling. Viability of radiolabeled cells was statistically nonsignificant between ¹²⁴I-FIAU and ⁶⁴Cu-PTSM. The radioactivity of each radiolabeled cells was predominantly accumulated in the lungs and liver at 2 hours. Both the radioactivity of ⁶⁴Cu-PTSM- and ¹²⁴I-FIAU-labeled cells was highly accumulated in the liver at 24 hours. However, the radioactivity of ¹²⁴I-FIAU-labeled cells was markedly decreased from the body at 24 hours. The K562-TL cells were dominantly localized in the lungs and liver, which also verified by BLI and RT-PCR analysis at 2 and 24 hours postinjection. The ⁶⁴Cu-PTSM-labeled cell-tracking method is more efficient than ¹²⁴I-FIAU-labeled cell tracking, because of markedly decrease of radioactivity and fast efflux of ¹²⁴I-FIAU in vivo. In spite of a high labeling yield and radiolabel retention of ¹²⁴I-FIAU in vitro, the in vivo cell-tracking method using ⁶⁴Cu-PTSM could be a useful method to evaluate the distribution and targeting of various cell types, especially, stem cells and immune cells.

The purpose of this study was to enhance the effect in the PET image quality according to event bootstrap of small animal PET data. In order to investigate the time difference condition, realigned sinograms were generated from randomly sampled data set using bootstrap. List-mode data was obtained from small animal PET scanner for Ge-68 30 sec, Y-90 20 min and Y-90 60 min. PET image was reconstructed by Ordered Subset Expectation Maximization(OSEM) 2D with the list-mode format. Image analysis was investigated by Signal to Noise Ratio(SNR) of Ge-68 and Y-90 image. Non-parametric resampled PET image SNR percent change for the Ge-68 30 sec, Y-90 60 min, and Y-90 20 min was 1.69 %, 7.03 %, and 4.78 %, respectively. SNR percent change of non-parametric resampled PET image with time difference condition was 1.08 % for the Ge-68 30 sec, 6.74 % for the Y-90 60 min and 10.94 % for the Y-90 29 min. The result indicated that the bootstrap with time difference condition had a potential to improve a noisy Y-90 PET image quality. This method should be expected to reduce Y-90 PET measurement time and to enhance its accuracy.

There is a need for non-invasive monitoring the differentiation of transplanted cell in stem cell therapy. In this study, we evaluated the usefulness of sodium iodide symporter (NIS) gene as molecular imaging reporter gene during cardiomyogenic differentiation of bone marrow derived mesenchymal stem cells (BMSCs). In a previous study, we showed that a transgenic mouse model expressing NIS driven by the α-myosin heavy chain (α-MHC) promoter was developed to image cardiomyocytes in vivo with a γ-camera and micro positron emission tomography (microPET). BMSCs were found to express stem cell specific surface markers including stem cell antigen-1 (Sca-1) and CD44. After treatment with all-trans retinoic acid (ATRA), BMSCs morphologically changed and acquired a cardiomyocyte-like shape. The cardiomyogenic differentiation of BMSCs resulted in upregulated expression of cardiac specific genes including α-MHC and ventricular myosin light chain 2 (MLC-2v) and decreased expression of the stem cell specific marker, Sca-1. The ATRA treated BMSCs exhibited higher 125I uptake than that of nontreated cells, and this result suggested that the cardiomyogenic differentiation upregulated NIS gene activity as reporter in vitro. We demonstrated that, under the control of the α-MHC promoter, NIS activity reflected cardiomyogenic differentiation of BMSCs in vitro, and an imaging system based on NIS, the reporter gene, may be a useful tool for monitoring in the lineage specific differentiation of stem cells.

This study aimed to develop a gene expression targeting method for specific imaging and therapy of alpha-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) cells, using an adenovirus vector containing the human sodium/iodide symporter (hNIS) gene driven by an AFP enhancer/promoter.The recombinant adenovirus vector, AdAFPhNIS (containing the hNIS gene driven by human AFP enhancer/promoter) was prepared. After in vitro infection by the adenovirus, hNIS gene expression in AFP-producing cells and in AFP-nonproducing cells was investigated using (125)I uptake assay and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The killing effect of (131)I on AdAFPhNIS-infected HCC cells was studied using an in vitro clonogenic assay. In addition, tumor-bearing mice were intravenously injected with the adenovirus, and scintigraphic images were obtained.The expression of hNIS was efficiently demonstrated by (125)I uptake assay in AFP-producing cells, but not in AFP-nonproducing cells. AFP-producing HCC-targeted gene expression was confirmed at the mRNA level. Furthermore, in vitro clonogenic assay showed that hNIS gene expression induced by AdAFPhNIS infection in AFP-producing cells caused more sensitivity to (131)I than that in AFP-nonproducing cells. Injected intravenously in HuH-7 tumor xenografts mice by adenovirus, the functional hNIS gene expression was confirmed in tumor by in vivo scintigraphic imaging.An AFP-producing HCC was targeted with an adenovirus vector containing the hNIS gene using the AFP enhancer/promoter in vitro and in vivo. These findings demonstrate that AFP-producing HCC-specific molecular imaging and radionuclide gene therapy are feasible using this recombinant adenovirus vector system.

Background The in vivo molecular imaging method is a useful tool for monitoring carcinogenesis in various hepatocellular carcinoma (HCC) models, such as xenografted-, chemical induced- and transgenic mice. The tumor-specific gene expression strategy, such as transcriptional targeting, is essential for achieving a lower toxicity for normal liver tissue in therapy and the monitoring of tumor progression in diagnosis, respectively. The present study aimed to visualize spontaneously developing a-fetoprotein (AFP)-producing HCC through targeted gene expression in tumors using recombinant adenoviral vector. Methods The recombinant adenovirus vector, AdAFPfLuc (containing firefly luciferase gene driven by human AFP enhancer/promoter) was prepared. After in vitro infection by adenovirus, gene expression was confirmed using the luciferase assay, semi-quantitative reverse transcriptase-polymerase chain reaction and western blotting in AFP-producing and nonproducing cells. Tumor-bearing mice were intravenously injected with adenovirus, and bioluminescent images were obtained. Results The expression of fLuc was efficiently demonstrated by the luciferase assay in AFP-producing cells but not in AFP-nonproducing cells. AFP-producing HCC targeted gene expression was confirmed at the mRNA and protein levels. After being injected intravenously in HuH-7 xenografts and HCC-bearing diethylnitrosamine-treated mice using adenovirus, functional reporter gene expression was confirmed in tumors by in vivo bioluminescent imaging (BLI). Conclusions The recombinant adenovirus vector system can be used to monitor spontaneously developing AFP-producing HCC and to evaluate targeted gene expression in tumors by in vivo BLI in a small animal model. Copyright © 2012 John Wiley & Sons, Ltd.

The importance of noninvasive imaging methods to bacterial infections is widely recognized. To obtain bacterial infection imaging with radioisotope-labeled nucleosides, bacterial thymidine kinase (tk) activities of Salmonella typhimurium with [(125)I]5-iodo-1-(2'-fluoro-2'-deoxy-β-d-arabinofuranosyl)uracil ([(125)I]FIAU) or 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) were measured. The infection model in BALB/c mice was imaged with [(125)I]FIAU or [(18)F]FLT using small-animal Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET), respectively. The accumulated radioactivity of [(125)I]FIAU or [(18)F]FLT in the two strains showed a linearly increased pattern with increasing incubation time or bacterial numbers. The image clearly demonstrated a high uptake of [(125)I]FIAU and [(18)F]FLT in the bacterial infection site. [(18)F]FLT uptake in the infection site of was 7.286±2.405, whereas that in the uninfected site was 0.519±0.561. The relative activity ratio of the infected region in relation to the uninfected region was 2.98 at 4h after an injection with [(125)I]FIAU determined by biodistribution data. In conclusion, the bacterial tk activity was confirmed by the cellular uptake and imaging with [(125)I]FIAU or [(18)F]FLT. Therefore, a localized bacterial infection in living mice can be monitored using radioisotope-labeled nucleosides with a nuclear medicine imaging modality.

Purpose : Multimodality imaging contributes to the activation of translational research by compensating for its weak points. Herein, we developed a noninvasive dual-reporter gene system for nuclear and optical imaging. Materials and methods : We constructed a fusion reporter vector concurrently expressing the human sodium/iodide symporter (hNIS) and monomeric red fl uorescent protein (mCherry), and evaluated the function of this fusion reporter system under in vitro and in vivo conditions. Results : The expression of hNIS/mCherry fusion gene was confi rmed in transfected cells using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. As the numbers of cells increased, the fl uorescence and 125 I uptake increased in the hNIS/mCherry-transfected cells, and a high correlation between fl uorescence intensity and radioactivity was noted. The fl uorescence intensities and radioactivity signals were also well-correlated in HT-29-hNIS/mCherry tumors (R 2 0.9304) in in vivo fl uorescence and gamma camera imaging. Conclusions : The dual-reporter imaging method using hNIS and mCherry genes refl ected tumor extent as well as viable cell numbers, and correlated well with one another. This suggests that the hNIS/mCherry dual-reporter system can be a useful tool for multi-modal imaging.

In this study, the radioiodination and biodistribution of quantum dots (QDs) using Bolton-Hunter reagent were investigated. Radioiodination yield was 33.4 ± 2.0%. Fluorescent intensity of radioiodinated QDs decreased to 75.4% of the maximum prior to radioiodination. In biodistribution and ex vivo fluorescence imaging, radioiodinated QDs were highly accumulated in reticuloendothelial system (liver and spleen) and had low level bone uptakes and slow clearance from body. These results suggest that the radioiodination method of nanoparticles using Bolton-Hunter reagent could be easily used in the biodistribution and quantification of nanoparticles in vivo.

Lentiviral vector containing the HSV1-tk and firefly luciferase (fLuc) gene was infected into C6 and C6-TL expressing HSV1-tk and fLuc gene was generated. C6-TL showed higher [ 125 I]IVDU uptake than C6. The survival rate of C6-TL decreased more rapidly with increasing GCV dose and was well correlated with fLuc activity. The images of microPET clearly demonstrated higher uptake of [ 18 F]FHBG into the C6-TL tumor. Inhibition of tumor growth was observed in C6-TL tumor-bearing mice treated with GCV through tumor size measurement and bioluminescence imaging. The therapeutic effect of HSV1-tk/GCV system can be monitored using bioluminescent imaging and tumor size measurement.

Angiogenesis is induced by soluble factors such as vascular endothelial growth factor (VEGF) released from tumor cells in hypoxia. It enhances solid tumor growth and provides an ability to establish metastasis at peripheral sites by tumor cell migration. Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether angiogenesis and tumor metastasis are dependent on hypoxia conditioning-induced TB4 expression in B16F10 melanoma cells. TB4 expression in B16F10 cells was increased by hypoxia conditioning in a time-dependent manner. In addition, we found an increase of angiogenesis and HIF-1α expression in TB4-transgenic (Tg) mice as compared to wildtype mice. When wound healing assay was used to assess in vitro tumor cell migration, hypoxia conditioning for 1 h enhanced B16F10 cell migration. When TB4 expression in B16F10 cells was inhibited by the infection with small hairpin (sh) RNA of TB4 cloned in lentiviral vector, tumor cell migration was retarded. In addition, hypoxia conditioning-induced tumor cell migration was reduced by the infection of lentiviral shRNA of TB4. HIF-1α stabilization and the expression of VEGF isoform 165 and 121 in hypoxia were also reduced by the infection of lentiviral shRNA of TB4 in B16F10 cells. We also found an increase of tumor growth and lung metastasis count in TB4-Tg mice as compared to wildtype mice. Collectively, hypoxia conditioning induced tumor cell migration by TB4 expression-dependent HIF-1α stabilization. It suggests that TB4 could be a hypoxia responsive regulator to control tumor cell migration in angiogenesis and tumor metastasis.

We determined the cytotoxic properties of cytochrome P450 4B1 (CYP4B1) activated 4-ipomeanol (4-ipo) and 2-aminoanthracene (2-AA) in rat glioma to verify the CYP4B1/4-ipo or 2-AA system for prodrug-activating gene therapy. The cyp4B1 cDNA was cloned into pcDNA3.1/Hygro from rabbit lung total RNA (pcDNA-cyp4B1). Lentiviral vector encoding firefly luciferase (fLuc) was infected into C6 (rat glioma), and the fLuc-expressing cell was selected (C6-L). After transfection with pcDNA-cyp4B1 vector into C6-L, the single clone expressing cyp4B1 gene was selected (C6-CL). Prodrug for various concentrations of 4-ipo or 2-AA was treated for 72 h and 96 h. The cell survival rate of C6-CL was determined using MTT assay and trypan-blue dye exclusion methods. By RT-PCR analysis, fLuc and CYP4B1 expression was detected in C6-CL, but not in C6. MTT assay and trypan-blue dye exclusion showed that IC50 of C6-CL was 0.3 mM and

123 I-FP-CIT and 18 F-FP-CIT are radiotracers which are widely used to diagnose Parkinson's disease (PD). However, to our knowledge, no studies to date have made head-to-head comparisons between 123 I-FP-CIT and 18 F-FP-CIT. Therefore, in this study, 123 I-FP-CIT SPECT/CT was compared with 18 F-FP-CIT PET/CT in the same cohort of subjects. Patients with PD and essential tremor (ET) underwent 123 I-FP-CIT SPECT/CT and 18 F-FP-CIT PET/CT. Visual and semiquantitative analyses were conducted. The specific binding ratio (SBR) and putamen to caudate ratio (PCR) were compared between subjects who underwent 123 I-FP-CIT SPECT/CT and 18 F-FP-CIT PET/CT. Visual analysis showed that the striatal uptake of both radiotracers was decreased in the PD group, whereas striatal uptake was intact in the ET group. The SBR between 123 I-FP-CIT SPECT/CT and 18 F-FP-CIT PET/CT showed a positive correlation (r = .78, p < .01). However, the mean SBRs on 18 F-FP-CIT PET/CT were higher than those on 123 I-FP-CIT SPECT/CT (2.19 ± .87 and 1.22 ± .49, respectively; p < .01). The PCRs in these two modalities were correlated with each other (r = .71, p < .01). The mean PCRs on 18 F-FP-CIT PET/CT were not significantly higher than those on 123 I-FP-CIT SPECT/CT (1.31 ± .19 and 0.98 ± .06, respectively; p = .06). These preliminary results indicate that the uptake of both 123 I-FP-CIT and 18 F-FP-CIT was decreased in the PD group when compared with the ET controls. Visual analyses using both methods did not affect the diagnostic accuracy in this study. However, semiquantitative analysis indicated a better contrast of 18 F-FP-CIT PET/CT relative to 123 I-FP-CIT SPECT/CT.

To evaluate the role of p53 in radionuclide gene therapy, we investigated the cytotoxic effect of (131)I and (188)Re following cotransfection of the sodium iodide symporter (NIS) and wild-type p53 (wt-p53) genes into cancer cells.The NIS gene was transfected to human anaplastic thyroid carcinoma cells (ARO) expressing mutant p53 (mt-p53) using liposomes. The uptakes of (125)I and (188)Re were measured in the transfected (ARO-N) and wild-type cell lines (ARO). A recombinant adenovirus-5 vector containing a CMV promoter and wt-p53 cDNA, called Ad-p53, was established and transduced to ARO and ARO-N cells. After incubating cells with (131)I and (188)Re, the survival rate of each cell line was measured using a clonogenic assay. For radionuclide gene therapy in an animal model, Ad-p53 was injected directly into ARO and ARO-N tumours which were transplanted to nude mice. Two days later, (188)Re or saline was injected intraperitoneally into the mice, and the tumours were measured using a calliper for 4 weeks.In ARO-N cells, the uptakes of (125)I and (188)Re were 505.16+/-21.30 pmol/10(6) cells and 13,875.20+/-504.85 cpm/10(6) cells at 30 min, respectively. There was no difference between the survival rates of ARO cells and ARO-N cells after incubation with (131)I or (188)Re. When Ad-p53 was transduced to ARO-N cells, the survival rate of wt-p53-expressing ARO-N cells incubated with (131)I (18.5 MBq/5 ml) and (188)Re (18.5 MBq/5 ml) decreased to 48.8+/-18.4% and 32.6+/-23.5%, respectively. In the nude mice experiment, ARO and ARO-N tumours gradually grew up to six to eight times larger than the initial volume. ARO and ARO-N tumours transduced with Ad-p53 continued to grow. However, the ARO-N tumours treated with Ad-p53 and 185 MBq of (188)Re regressed to 20% of the initial volume.Growth of ARO-N tumour treated with (131)I or (188)Re was significantly inhibited by Ad-p53 transduction in vivo as well as in vitro. Transfection of the NIS gene into human anaplastic thyroid cancer induced the accumulation of beta-emitter radionuclides, and cotransfection with a wt-p53 gene enhanced the cytotoxic effect.

Introduction The goal of this study was to compare the glucose analog, 2-[ 18 F]fluoro-2-deoxy-d-glucose ([ 18 F]-FDG), the amino acid analog, o -(2-[ 18 F]fluoroethyl)-l-tyrosine ([ 18 F]-FET) and nucleoside analog, 3′-[ 18 F]fluoro-3′-deoxythymidine ([ 18 F]-FLT) with regard to their feasibility for differentiating tumors from inflammation. Methods In Fisher rat models bearing both 9L tumor and inflammation, the biodistributions and positron emission tomography (PET) images of [ 18 F]-FDG, [ 18 F]-FET and [ 18 F]-FLT at 60 min post injection were compared. Pretreatment with thymidine phosphorylase before injection of [ 18 F]-FLT was performed. Results The tumor-to-blood (T/B) and tumor-to-muscle (T/M) ratios of [ 18 F]-FDG were significantly higher than those of [ 18 F]-FET and [ 18 F]-FLT ( P 18 F]-FDG [1.23±0.52 percent injected dose per gram of tissue (%ID/g)] in inflammation was also elevated. T/B and T/M ratios of [ 18 F]-FET (2.3±0.5 and 2.2±0.5) were higher than those of [ 18 F]-FLT (1.6±0.6 and 1.6±0.5), and inflammation uptake of those tracers was very low (0.63±0.19 and 0.27±0.16 %ID/g, respectively). [ 18 F]-FET and [ 18 F]-FLT showed higher selectivity indices (tumor-to-inflammation ratio corrected background) than [ 18 F]-FDG. In PET images, [ 18 F]-FDG was found to be accumulated in both tumor and inflammation, but [ 18 F]-FET and [ 18 F]-FLT selectively localized in tumor. Conclusion Our data confirm the result of previous studies that [ 18 F]-FET and [ 18 F]-FLT are superior to [ 18 F]-FDG in differentiating tumor from inflammation.

The antimicrobial peptide, lactoferricin, is generated upon the gastric pepsin cleavage of lactoferrin and has many basic and hydrophobic amino acid residues essential for its biological activity. To investigate the structure-antimicrobial activity relationships, the basic amino acid-rich region of bovine lactoferricin (BLFC), RRWQWRMKKLG, was selected. Using chemically synthesized BLFC and its substituted peptides, the antimicrobial activities of the peptides were tested by determining the minimal inhibitory concentration (MIC) of Escherichia coli and Bacillus subtilis and the disruption of the outer cell membrane of E. coli, and the peptide's toxicities were assayed by hemolysis. The short peptide (B3) composed of only 11 residues had similar antimicrobial activities while losing most of the hemolytic activities as compared with the 25 residue-long ones (B1 and B2). The short peptides (B3, B5 and B7) with double arginines at the N-termini had more potent antimicrobial activity than those (B4 and B6) with lysine. However, no antimicrobial and hemolytic activities were found in B8, in which all basic amino acids were substituted with glutamic acid, and in B9, in which all hydrophobic amino acids were substituted with alanine. The circular dichroism (CD) spectra of the short peptides in 30 mM SDS were correlated with their antimicrobial activities. These results suggested that the 11-residue peptide of BLFC is involved in the interaction with bacterial phospholipid membranes and plays an important role in antimicrobial activity with little or no hemolytic activity.

The membrane-disrupting properties of cecropin A (1–8)-magainin 2 (1–12) hybrid peptide, which has higher antitumor with less hemolytic activities than cecropin A (1–8)-melittin (1–12), and its analogues were assessed by measuring the induced release of vesicle-entrapped fluorescence probes. A model membrane was made of zwitterionic phospholipid (phosphatidylcholine) or the mixture of negatively and zwitterionic phospholipids (phosphatidylcholine and phosphatidylserine). The extent of leakage of the aqueous content of the phospholipid vesicles was found to have remarkable discrepancies according to the amphipathic nature of each analogue peptide. The entrapped high molecular weight solute (fluorescein-labeled immunoglobulin G, 55 kDa) also was released by the analogue which had the largest hydrophobic region and the highest amphipathic score among peptides tested. As the result of the determination of the relationships between the membrane-disrupting properties and the hydrophobicity values of peptides, it was found that the membrane-disrupting activity increased according to increasing the hydrophobicity of the peptide. The tryptophan fluorescence emission spectra and CD spectra showed that on interaction with the phospholipid vesicle, the peptide acquired the ordered structure and α-helical conformation by moving a tryptophan residue into the nonpolar environment of the phospholipid vesicle. These results suggest that the breakdown of the lipid bilayer was mediated by the α-helical amphipathic structure of the peptide interacting with the lipid bilayers as well as the by the hydrophobicity of the peptide.

We conducted a molecular imaging and gene therapy method in alpha-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) by tumor-specific expression of the human sodium/iodide symporter (hNIS) using an AFP promoter.The tumor-specific expression of hNIS gene by the AFP enhancer/promoter was constructed as pcDNA3-AFP/hNIS. The pcDNA3-AFP/hNIS was stably transfected to human HCC (Huh-7/AN) and rat glioma cells (C6/AN). Functional hNIS expression was confirmed by radioiodine uptake. The mRNA and protein-expression level of hNIS were measured. Biodistribution of 131I was evaluated, and scintigraphic images of 99mTc were obtained in xenografted mice. A clonogenic assay was performed by 131I. And, the in vivo therapeutic effect of 131I was evaluated in xenografted mice.In Huh-7/AN cells, iodine was highly accumulated and completely blocked by perchlorate. The protein and mRNA expression levels were correlated with iodine uptake. Radioiodine uptake in Huh-7/AN tumors was higher than those of control tumors and clearly visualized. The survival rate was significantly decreased in Huh-7/AN cells by 131I. Moreover, a growth of Huh-7/AN tumors was inhibited by 131I in mice.AFP-producing hepatoma can be targeted and treated with radionuclides and hNIS, using AFP enhancer/promoter. This targeted hNIS gene therapy and molecular imaging have the potential to be used in the management of AFP-producing HCC.

Molecular imaging includes proteomic, metabolic, cellular biologic process, and genetic imaging. In a narrow sense, molecular imaging means genetic imaging and can be called molecular-genetic imaging. Imaging reporter genes play a leading role in molecular-genetic imaging. There are 3 major methods of molecular-genetic imaging, based on optical, MRI, and nuclear medicine modalities. For each of these modalities, various reporter genes and probes have been developed, and these have resulted in successful transitions from bench to bedside applications. Each of these imaging modalities has its unique advantages and disadvantages. Fluorescent and bioluminescent optical imaging modalities are simple, less expensive, more convenient, and more user friendly than other imaging modalities. Another advantage, especially of bioluminescence imaging, is its ability to detect low levels of gene expression. MRI has the advantage of high spatial resolution, whereas nuclear medicine methods are highly sensitive and allow data from small-animal imaging studies to be translated to clinical practice. Moreover, multimodality imaging reporter genes will allow us to choose the imaging technologies that are most appropriate for the biologic problem at hand and facilitate the clinical application of reporter gene technologies. Reporter genes can be used to visualize the levels of expression of particular exogenous and endogenous genes and several intracellular biologic phenomena, including specific signal transduction pathways, nuclear receptor activities, and protein-protein interactions. This technique provides a straightforward means of monitoring tumor mass and can visualize the in vivo distributions of target cells, such as immune cells and stem cells. Molecular imaging has gradually evolved into an important tool for drug discovery and development, and transgenic mice with an imaging reporter gene can be useful during drug and stem cell therapy development. Moreover, instrumentation improvements, the identification of novel targets and genes, and imaging probe developments suggest that molecular-genetic imaging is likely to play an increasingly important role in the diagnosis and therapy of cancer.

Small animal positron emission tomography (PET) with 18 F-FDG has been increasingly used for tumor imaging in the murine model. The aim of this study was to establish the anesthesia condition for imaging of lung metastasis tumor using small animal 18 F-FDG PET. Methods To determine the impact of anesthesia on 18 F-FDG distribution in normal mice, five groups were studied under the following conditions: no anesthesia, ketamine and xylazine (Ke/Xy), 0.5% isoflurane (Iso 0.5), 1% isoflurane (Iso 1) and 2% isoflurane (Iso 2). The ex vivo counting, standard uptake value (SUV) image and glucose SUV of 18 F-FDG in various tissues were evaluated. The 18 F-FDG images in the lung metastasis tumor model were obtained under no anesthesia, Ke/Xy and Iso 0.5, and registered with CT image to clarify the tumor region. Results Blood glucose concentration and muscle uptake of 18 F-FDG in the Ke/Xy group markedly increased more than in the other groups. The Iso 2 group increased 18 F-FDG uptake in heart compared with the other groups. The Iso 0.5 anesthesized group showed the lowest 18 F-FDG uptake in heart and chest wall. The small size of lung metastasis tumor (2 mm) was clearly visualized by 18 F-FDG image with the Iso 0.5 anesthesia. Conclusion Small animal 18 F-FDG PET imaging with Iso 0.5 anesthesia was appropriate for the detection of lung metastasis tumor. To acquire 18 F-FDG PET images with small animal PET, the type and level of anesthetic should be carefully considered to be suitable for the visualization of target tissue in the experimental model.

To establish stable and long-term gene expression in vitro and in vivo, we developed a lentiviral vector system carrying sodium iodide symporter (hNIS) gene under UbC promoter, and transfected this into a colon cancer cell line. The in vitro and in vivo kinetics of radioiodine and [99mTc]-pertechnetate were then investigated, and the therapeutic effect of 1-131 was evaluated in this system. The hNIS gene was transferred into CT26 cells using lentivirus containing UbC promoter. In vitro iodide uptake and efflux were measured in CT26-hNIS cells at various time points. In addition, scintigraphic images were acquired at 30 min after injecting [99mTc]-pertechnetate i.p. into Balb/C mice for 27 days after CT26-hNIS induction. Biodistribution studies were performed at 10 and 30 min and at 1.5, 6 and 24 h after [99mTc]-pertechnetate injections, and the therapeutic effects of radioiodine were investigated by measuring tumor size using a caliper or by quantifying tumor radioactivity levels in scintigraphic images. The iodide uptakes of CT26-hNIS tumors were 10-fold greater than those of CT26 tumors. In addition, iodide uptake was completely blocked by 100 microM potassium perchlorate. The accumulation of [99mTc]-pertechnetate in hNIS expressing tumor cells was found to be positively related to tumor growth. In biodistribution studies, the %ID/g values of CT26-hNIS were 84.0 +/- 4.5 at 1.5 h and 40.8 +/- 3.9 at 24 h and these were approximately 60 times greater than those of CT26 at these time points. Tumor growth in mice treated with 131I was retarded until 46 days post-tumor challenge. The devised lentiviral vector system carrying hNIS controlled by UbC promoter was found to be suitable for the long-term monitoring and radionuclide therapy of cancer in living organism.

Firefly luciferase (Fluc) has been widely used as a reporter gene. The aim of this study was to investigate immune response to luciferase protein after an intradermal injection of pcDNA3.1-Fluc in immunocompetent BALB/c mice. We observed bioluminescence at injection sites from one to seven days post-injection when pcDNA3.1-Fluc was intradermally injected into ear-pinnae. To observe induced immune response, the percentages of CD8+IFNgamma+ cells in the draining lymphoid cells of immunocompetent BALB/c mice immunized by pcDNA3.1-Fluc were measured. And the tumor growths of CT26/Fluc in pcDNA3.1-Fluc group were monitored by observing bioluminescent signals and measuring tumor mass, and these were compared with those of the pcDNA3.1 group in immunocompetent BALB/c mice and immunodeficient Nu/Nu mice. In the immunocompetent BALB/c mice, percentages of CD8+IFNgamma+ cells in the pcDNA3.1-Fluc group were higher than those in the pcDNA3.1 group. Ten days after tumor inoculation, tumor growth inhibition was found in the pcDNA3.1-Fluc group, but not in the pcDNA3.1 group in the immunocompetent BALB/c mice. No significant difference in tumor growth inhibition was observed when CT26/Fluc was injected into immunodeficient Nu/Nu mice. In terms of cytokine profiles of draining lymphoid cells of immunized mice, IFNgamma protein levels in the pcDNA3.1-Fluc group were higher than in pcDNA3.1 group animals among the immunocompetent BALB/c mice. In conclusion, Fluc induced a Th1 immune response to Fluc protein delivered by injecting pcDNA3.1-Fluc into immunocompetent BALB/c mice. We suggest that immune response to the Fluc gene is cautionary in preclinical or clinical trials involving the Fluc gene, and that the immunologic potential of firefly luciferase as a naked DNA may be useful in cancer immunotherapy.

For noninvasive monitoring of cellular status by dual reporters, a dual membrane protein reporter system was developed and its in vivo applicability was examined. Human sodium iodide symporter (hNIS) and mutant dopamine D(2) receptor (D(2)R) transgenes were chosen considering their complementarity.pIRES-hNIS/D(2)R containing NIS and D(2)R linked with an internal ribosomal entry site (IRES) was constructed and transfected into human hepatoma SK-Hep1 and rat glioma C6 cells. The cell lines stably expressing hNIS and D(2)R (named SK-ND and C6-ND) were produced, which was confirmed by messenger RNA expression of reporter genes. The functional activities of hNIS and D(2)R were measured by (125)I uptake assay and (3)H-spiperone receptor-binding assays. A biodistribution study was performed on SK-ND tumor-bearing mice using (99m)Tc-pertechnetate and (3)H-spiperone. In vivo hNIS expression was examined using (99m)Tc-pertechnetate gamma-camera imaging and, D(2)R expression was examined using a (3)H-spiperone autoradiographic study.(125)I uptake of SK-ND and C6-ND cell lines showed a maximum 97-fold and 43-fold increase, respectively, which were completely inhibited by KClO(4). Specific (3)H-spiperone binding to SK-ND and C6-ND cell homogenates was observed, which were completely inhibited by (+)-butaclamol. Among the dual reporter gene-expressing cell lines, the activities of both reporters were inversely correlated with each other. Competition assay of hNIS-expressing cells by D(2)R vector transfection and D(2)R-expressing cells by hNIS vector transfection showed a dose-dependent decrease of hNIS and D(2)R activities, respectively. In the biodistribution study, (99m)Tc-pertechnetate accumulated 10-fold and (3)H-spiperone accumulated 4-fold more in SK-ND tumors than that in parental SK tumors. In vivo imaging of (99m)Tc-pertechnetate persisted until 5 wk after the cell graft in SK-ND tumors. Autoradiographic study of brain tissues from these mice also revealed an accumulation of (3)H-spiperone in SK-ND tumors.We developed a dual membrane-bound positron and gamma-imaging reporter system of hNIS and D(2)R. We observed its reporting capability in vitro and in vivo and elucidated that these 2 membrane protein reporters competed with each other in their expression. Although we expect that hNIS and D(2)R transgenes can complement each other as a dual reporter system, we suggest that one needs to validate the ratio of expression of the 2 membrane protein reporter transgenes for cellular status tracking.

Human sodium iodide symporter (hNIS) is a transmembrane protein that actively transports iodide ions into thyroid cells. hNIS is over-expressed in some cases of the thyroid cancers compared with the surrounding normal tissues and has been considered to be an attractive target for immunotherapy. The aim of this study is to determine the feasibility of utilizing the hNIS antigenic protein in enhanced-antigen-associated immunotherapy using image analysis with a gamma counter. To accomplish this, minimalistic immunogenically defined gene expression (MIDGE), either plain or coupled to a nuclear localization signal (NLS) peptide, was used as a vector system. Vaccination with MIDGE/hNIS, MIDGE/hNIS-NLS and pcDNA3.1/hNIS produced a significant increase in the number of hNIS-associated IFN-gamma-secreting CD8(+) T cells, with MIDGE/hNIS having the strongest effect. In addition, immunization with the hNIS encoding vectors induced antigen-mediated antitumor activity against NIS-expressing CT26 tumors in vivo, with the highest tumor free rate (100%) and lowest tumor growth being observed up to 40 days after the CT26/NIS tumor challenge with MIDGE/hNIS than those resulting from other immunization groups. Tumor progression could be followed noninvasively and repetitively by monitoring levels of hNIS gene expression in the tumors using scintigraphic image analysis. Overall, hNIS has a potential use as an antigen for immunization approaches, and vaccination with MIDGE/hNIS vectors is an effective means of generating hNIS-associated immune responses in mice.

Immuno-PET provides valuable information about tumor location, phenotype, susceptibility to therapy, and treatment response, especially to targeted radioimmunotherapy. In this study, we prepared antiepidermal growth factor receptor (EGFR) antibody via identical chelator, 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-trience-3,6,9,-triacetic acid (PCTA), labeled with (64)Cu or (177)Lu to evaluate the EGFR expression levels using immuno-PET and the feasibility of radioimmunotherapy in an esophageal squamous cell carcinoma (ESCC) model.Cetuximab was conjugated with p-SCN-Bn-PCTA and radiolabeled with (64)Cu or (177)Lu. In vitro EGFR expression levels were determined and compared using flow cytometry and cell binding assay. In vivo EGFR expression levels were evaluated via immuno-PET imaging of (64)Cu-cetuximab and biodistribution analysis. Micro-SPECT/CT imaging, biodistribution, and radioimmunotherapy studies of (177)Lu-cetuximab were performed in the ESCC model. Therapeutic responses were monitored using (18)F-FDG PET and immunohistochemical staining.(64)Cu- or (177)Lu-labeled antibodies showed high radiolabeling yield (98%), stability (90%), and favorable immunoreactivity. In vitro EGFR status measured by cell binding assay was correlated with the flow cytometry data. Immuno-PET, micro-SPECT/CT, and biodistribution demonstrated specific uptake in ESCC tumors depending on the EGFR expression levels. Tumor accumulation of (64)Cu- and (177)Lu-cetuximab was peaked at 48 and 120 h, respectively. Radioimmunotherapy with (177)Lu-cetuximab showed significant inhibition of tumor growth (P0.01) and marked reduction of (18)F-FDG SUV compared with that of control (P0.05). Terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity and Ki-67 staining indices increased and decreased, respectively, in the radioimmunotherapy group compared with other groups (P0.01).(64)Cu-cetuximab immuno-PET represented EGFR expression levels in ESCC tumors, and (177)Lu-cetuximab radioimmunotherapy effectively inhibited the tumor growth. The diagnostic and therapeutic convergence radiopharmaceutical (64)Cu-/(177)Lu-PCTA-cetuximab may be useful as a diagnostic tool in patient selection and a potent radioimmunotherapy agent in EGFR-positive ESCC tumors.

This study was aimed to evaluate the effect of (124)I-labeling with hexadecyl-4-iodobenzoate (HIB) on gene expression related to cell cycle, DNA repair, transcription, proliferation and differentiation of adipose-derived stem cells (ADSCs). [(124)I]HIB showed high labeling efficiency with ADSCs (51.3±1.3%, 0.3-2.0 Bq/cell) and there is no morphological change of ADSCs. In the microarray analysis of gene expression pattern, differences were not observed between non-labeled and [(124)I]HIB-labeled ADSCs. We demonstrated that (124)I-labeling with HIB did not affect the biological properties of ADSCs.

The immune system might influence breast cancer (BC) prognosis. However, the relationship between programmed death ligand 1 (PD-L1) and tumor-infiltrating lymphocyte (TIL) profiles remains unclear with respect to BC subtypes.We investigated the relationship between TIL profiles for CD8+ and forkhead box P3-positive (FOXP3+) and PD-L1 expression in primary tumor tissue using immunohistochemistry and the clinical outcomes in 2 patient cohorts at the National Cancer Center: 256 patients diagnosed with early-stage BC from January 2001 to December 2005 and 77 hormone receptor (HR)-negative BC patients diagnosed from January 2006 to December 2008. Clinical data were collected, including HR status, human epidermal growth factor receptor 2 expression, disease-free survival, and overall survival (OS).The median patient age was 47 years (range, 28-78), and the median follow-up period was 9.8 years. Of the 333 patients, 186 (55.9%) had HR-positive and 125 (37.5%) had node-positive BC. We found a strong positive correlation between CD8+ TILs and FOXP3+ TILs (P.001). CD8+ TILs were more abundant in tumors with low PD-L1 expression (P.001), although no association was found between FOXP3+ TILs and PD-L1 expression. More CD8+ TILs were present in HR-negative than in HR-positive BC (P.001), and PD-L1 expression was more frequent in HR-positive BC (P.001). A greater number of CD8+ TILs (increase in quartile) was strongly associated with OS (hazard ratio, 0.61; 95% confidence interval, 0.39-0.95; P = .03) only in HR-negative BC when adjusted for various clinical factors. PD-L1 expression and FOXP3+ TILs did not exhibit such associations.Higher CD8+ lymphocyte infiltration was related to lower PD-L1 expression and higher FOXP3+ TIL infiltration in BC. Higher CD8+ TIL expression was associated with prolonged survival only in those with HR-negative BC.

Tumor-specific enhancer/promoter is applicable for targeting gene expression in tumors and helpful for tumor-targeting imaging and therapy. We aimed to acquire α-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) specific images using adenovirus containing HSV1-tk gene controlled by AFP enhancer/promoter and evaluate in vivo ganciclovir (GCV)-medicated therapeutic effects on AFP-targeted HSV1-tk expression with (18)F-FDG positron emission tomography (PET). Recombinant adenovirus expressing HSV1-tk under AFP enhancer/promoter was produced (AdAFP-TK) and the expression levels were evaluated by RT-PCR and (125)I-IVDU uptake. GCV-mediated HSV1-tk cytotoxicity was determined by MTT assay. After the mixture of AdAFP-fLuc and AdAFP-TK was administrated, bioluminescent images (BLIs) and (18)F-FHBG PET images were obtained in tumor-bearing mice. In vivo therapeutic effects of AdAFP-TK and GCV in the HuH-7 xenograft model were monitored by (18)F-FDG PET. When infected with AdAFP-TK, cell viability in HuH-7 was reduced, but those in HT-29 and SK-Hep-1 were not significantly decreased at any GCV concentration less than 100 μM. AFP-targeted fLuc and HSV1-tk expression were clearly visualized by BLI and (18)F-FHBG PET images in AFP-producing HCC, respectively. In vivo GCV-mediated tumor growth inhibition by AFP-targeted HSV1-tk expression was monitored by (18)F-FDG PET. Recombinant AdAFP-TK could be applied for AFP-targeted HCC gene therapy and imaging in AFP-producing HCC.

RT is commonly used to treat malignant tumors. However, tumor regrowth is a major limitation to RT as an antitumor treatment. In the present study, we investigated the tumor-promoting effects of high-dose (or ablative) RT treatments on tumor-bearing mice. We focused on the role of macrophages that interact with IR-CCs in the TME, which cause tumor regrowth. We observed that CT26(H-2d) tumor growth was enhanced by i.v. injection of IR-CT26 cells compared with NR control CT26 cells. The levels of iNOS gene expression and NO production from RAW264.7 macrophages (H-2d) in response to the interaction with IR-CT26 cells were higher than with NR-CT26 cells. When CT26 tumor-bearing mice were treated i.v. with L-NMMA, a NOS inhibitor, the reduction in in vivo tumor growth was higher in the IR-CT26-injected group compared with the NR-CT26-injected control group. In vivo CT26 tumor growth was decreased after transplanting PEM extracted from L-NMMA-treated, tumor-bearing mice. Although iNOS activity was reduced by inhibiting TLR1 expression with TLR1-siRNA, it was enhanced by TLR1 overexpression. Transcriptional activation and protein expression levels of iNOS were also decreased in the presence of TLR1-siRNA but increased as a result of TLR1 overexpression. These results demonstrate that postradiotherapeutic tumor regrowth may be caused by interaction of IR-CCs with macrophages that induce TLR1-mediated iNOS expression and NO production. Our data suggest that iNOS in macrophages could be a useful target to regulate postradiotherapeutic responses in hosts and subsequently limit tumor regrowth.

The purpose of this study was to evaluate (64)Cu-labeled hexadecyl-1,4,7,10-tetraazacyclododecane-tetraacetic acid-benzoate ((64)Cu-DOTA-HB) (1) as positron emission tomography (PET) radiotracer for stem cell imaging. Hexadecyl-DOTA-benzoate (DOTA-HB) (2) was efficiently labeled with (64)Cu (99%), and cell labeling efficiency with adipose-derived stem cells (ADSCs) was over 50%. Labeling with 1 did not compromise cell viability. In the PET imaging, intramuscularly transplanted 1-labeled ADSCs were monitored for 18 h in normal rat heart. These results indicate that 1 can be utilized as a promising radiotracer for monitoring of transplanted stem cells.

Background Oestrogen receptors are ligand-dependent transcription factors whose activity is modulated either by oestrogens or by an alternative signalling pathway. Oestrogen receptors interact via a specific DNA-binding domain, the oestrogen responsive element (ERE), in the promoter region of sensitive genes. This binding leads to an initiation of gene expression and hormonal effects. Objective To determine the transcriptional activity of the oestrogen receptor, we developed a molecular imaging system using sodium iodide symporter (NIS) as a reporter gene. Methods The NIS reporter gene was placed under the control of an artificial ERE derived from pERE-TA-SEAP and named as pERE-NIS. pERE-NIS was transferred to MCF-7, human breast cancer cells, which highly expressed oestrogen receptor-alpha with lipofectamine. Stably expressing cells were generated by selection with G418 for 14 days. After treatment of 17beta-oestradiol and tamoxifen with serial doses, the (125)I uptake was measured for the determination of NIS expression. The inhibition of NIS activity was performed with 50 micromol x l(-1) potassium perchlorate. Results The MCF7/pERE-NIS treated with 17beta-oestradiol accumulated (125)I up to 70-80% higher than did non-treated cells. NIS expression was increased according to increasing doses of 17beta-oestradiol. MCF7/pERE-NIS treated with tamoxifen also accumulated (125)I up to 50% higher than did non-treated cells. Potassium perchlorate completely inhibited (125)I uptake. When MDA-MB231 cells, the oestrogen receptor-negative breast cancer cells, were transfected with pERE-NIS, (125)I uptake of MDA-MB-231/pERE-NIS did not increase. Conclusion This pERE-NIS reporter system is sufficiently sensitive for monitoring transcriptional activity of the oestrogen receptor. Therefore, cis-enhancer reporter systems with ERE will be applicable to the development of a novel selective oestrogen receptor modulator with low toxicity and high efficacy.

The administration of naked DNA into animals is increasing as a research tool to develop DNA vaccine. To monitor the distribution and duration of gene expression of a DNA vaccine in living organisms, we used the naked DNA encoding firefly luciferase (F luc ) as an imaging reporter gene, and evaluated in vivo bioluminescent images in a murine model. We observed bioluminescence at the injection site and at inguinal lymph node from 10 h to 24 h post-injection when DNA vaccine encoding F luc (pcDNA3.1-F luc ) was injected into the bilateral posterior flanks in mice. F luc gene expressions at injection sites and unilateral posterior flank inguinal lymph node were also confirmed by RT-PCR. However, when pcDNA3.1-F luc was injected into the mid-dorsum bioluminescent signals were observed at the injection site for up to 14 days post-injection, but no bioluminescent signals were detected in inguinal lymph nodes. Concurrent mRNA expressions of F luc gene at injection sites but not at inguinal lymph nodes were confirmed by RT-PCR. These findings suggest that optical imaging using F luc could be useful for monitoring the location, intensity and duration of gene expression of naked DNA vaccines in living animals non-invasively and repetitively.

The promyelocytic leukemia zinc finger (PLZF) protein has been described as a transcriptional repressor of the BTB-domain/zinc-finger family, and shown to regulate the expression of Hox genes during embryogenesis and the expression of cyclin A in the cell cycle progression. Here, a 45-kDa isoform of PLZF without a BTB domain was identified via yeast two-hybrid screening using the C-terminal region of ATP7B as bait in our determination of the biological roles of the Wilson disease protein outside of its copper-binding domain. Our immunoprecipitation experiments showed that the hepatocytic isoform of PLZF could specifically interact with the C-terminal region of ATP7B. The immunostaining of HepG2 cells revealed that the ATP7B and PLZF proteins were apparently colocalized into the trans-Golgi complexes. It was also determined that disruption of PLZF expression in the HepG2 cells affected an attenuation of ERK activity in a dose-dependent manner. The hepatocytic activities of ERK kinase were found to be enhanced as the result of PLZF or ATP7B expression, but this enhancement was abrogated by the deletion of the C-terminal region of ATP7B. Furthermore, a transgenic Drosophila strain that ectopically expressed the hepatocytic ΔBTB-PLZF exhibited phenotypic changes in eye and wing development, and these alterations were fully recovered as the result of ATP7B expression, indicating the obvious in vivo interaction between the two proteins. Those PLZF-induced abnormalities were attributed to the enhancement of ERK signaling, as was shown by phenotypic reversions with loss-of-function mutations in ERK signal transduction in Drosophila. These data suggest the existence of a mechanism that regulates ERK signaling via the C-terminus of ATP7B and the ATP7B-interacting hepatocytic PLZF. J. Cell. Biochem. 99: 719–734, 2006. © 2006 Wiley-Liss, Inc.

Retinoic acids are natural derivatives of vitamin A, and play important roles in modulating tumor cell growth by regulating differentiation, thus suggesting the potential use of these derivatives in cancer therapy and prevention. To visualize the intranuclear responses of functional retinoic acid receptors, we have developed a dual-imaging reporter gene system based on the use of sodium/iodide symporter (NIS) and luciferase in cancer cell lines. NIS and luciferase genes were linked with an internal ribosome entry site, and placed under the control of an artificial cis -acting retinoic acid responsive element (pRARE/NL). After retinoic acid treatment, I-125 uptake by pRARE/NL transfected cells was found to have increased by up to about five times that of nontreated cells. The bioluminescence intensity of pRARE/NL transfected cells showed dose-dependency. In vivo luciferase images showed higher intensity in retinoic acid treated SK-RARE/NL tumors, and scintigraphic images of SK-RARE/NL tumors showed increased Tc-99m uptake after retinoic acid treatment. The NIS/luciferase imaging reporter system was sufficiently sensitive to allow the visualization of intranuclear retinoic acid receptor activity. This cis -enhancer imaging reporter system may be useful in vitro and in vivo for the evaluation of retinoic acid responses in such areas as cellular differentiation and chemoprevention.