Joo Hyun Kang

Molecular-genetic imaging based on reporter gene expression.

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.

RGD Peptide–Conjugated Multimodal NaGdF4:Yb3+/Er3+ Nanophosphors for Upconversion Luminescence, MR, and PET Imaging of Tumor Angiogenesis

Multimodal nanoparticles have been extensively studied for target-specific imaging and therapy of various diseases, including cancer. In this study, radiolabeled arginine-glycine-aspartic acid (RGD)–functionalized Er3+/Yb3+ co-doped NaGdF4 upconversion nanophosphors (UCNPs) were synthesized and evaluated as a multimodal PET/MR/optical probe with tumor angiogenesis–specific targeting properties. Methods: A dimeric cyclic RGDyk ((cRGDyk)2) peptide was conjugated to polyacrylic acid–coated NaGdF4:Yb3+/Er3+ UCNPs along with polyethylene glycol molecules and was consecutively radiolabeled with 124I. In vitro cytotoxicity testing was performed for 3 d. Upconversion luminescence imaging of (cRGDyk)2-UCNP was performed on U87MG cells with a laboratory-made confocal microscope. In vivo small-animal PET and clinical 3-T T1-weighted MR imaging of 124I-labeled RGD–functionalized UCNPs was acquired with or without blocking of cyclic RGD peptide in a U87MG tumor model. Inductively coupled plasma mass spectrometry and biologic transmission electron microscopy were done to evaluate gadolinium concentration and UCNP localization, respectively. Results: Polymer-coated UCNPs and dimeric RGD–conjugated UCNPs were monodispersely synthesized, and those of hydrodynamic size were 30 ± 8 nm and 32 ± 9 nm, respectively. (cRGDyk)2-UCNPs have a low cytotoxic effect on cells. Upconversion luminescence signals of (cRGDyk)2-UCNP were specifically localized on the surface of U87MG cells. 124I-c(RGDyk)2-UCNPs specifically accumulated in U87MG tumors (2.8 ± 0.8 vs. 1.3 ± 0.4 percentage injected dose per gram in the blocking experiment), and T1-weighted MR images showed significant positive contrast enhancement in U87MG tumors. Tumor localization of 124I-c(RGDyk)2-UCNPs was confirmed by inductively coupled plasma mass spectrometry and biologic transmission electron microscopy analysis. Conclusion: These results suggest that 124I-labeled RGD–functionalized UCNPs have high specificity for αvβ3 integrin–expressing U87MG tumor cells and xenografted tumor models. Multimodal UCNPs can be used as a platform nanoparticle with multimodal imaging for cancer-specific diagnoses.

Doxorubicin enhances the expression of transgene under control of the CMV promoter in anaplastic thyroid carcinoma cells

We investigated the effect of doxorubicin on transgene expression and evaluated the mechanism of enhanced transgene expression by doxorubicin in transfected human anaplastic thyroid cancer cells (ARO cells). Methods: ARO cells were transfected with plasmid vectors or adenoviral vectors expressing human sodium/iodide symporter (hNIS) or luciferase (Luc) under the control of cytomegalovirus (CMV) promoter. After treating transfected and control ARO cells with doxorubicin, iodide uptake assay and luciferase assay were performed. Reversed-phase polymerase chain reaction analysis for hNIS and Western blot analysis for IκB protein were executed. Electrophoretic mobility-shift assay (EMSA) was performed to evaluate nuclear factor-κB (NF-κB) binding activity induced by doxorubicin. Scintigraphic and bioluminescent images were acquired and quantitated before and after doxorubicin in a tumor-bearing mouse model. Results: Radioiodide uptake in ARO cells transfected with the NIS gene under the CMV promoter was remarkably enhanced by doxorubicin, and this corresponded to a significant increase in NIS messenger RNA. In addition, luciferase gene upregulation by doxorubicin was also observed in luciferase gene transfected ARO cells. These results were verified by in vivo imaging in a tumor-bearing mouse model. Moreover, transgene expressional enhancement by doxorubicin was observed after transfecting ARO cells with adenoviral vector or plasmid vector, when transgenes were under the control of a CMV promoter. In addition, NF-κB, activated by doxorubicin, induced transgene transcription under the control of the CMV promoter, which possesses an NF-κB binding site. Conclusion: These findings indicate that doxorubicin enhances transgene expression under the control of the CMV promoter and that doxorubicin might be used as an adjuvant to radioiodine therapy by NIS gene transfer in anaplastic thyroid carcinoma.

Sodium Iodide Symporter and the Radioiodine Treatment of Thyroid Carcinoma

Since the specific accumulation of iodide in thyroid was found in 1915, radioiodine has been widely applied to diagnose and treat thyroid cancer. Iodide uptake occurs across the membrane of the thyroid follicular cells and cancer cells through an active transporter process mediated by the sodium iodide symporter (NIS). The NIS coding genes were cloned and identified from rat and human in 1996. Evaluation of the NIS gene and protein expression is critical in the management of thyroid cancer, and several approaches have been tried to increase NIS levels. Identification of the NIS gene has provided a means of expanding its role in the radionuclide gene therapy of nonthyroidal cancers as well as thyroid cancer. In this article, we explain the relationship between NIS expression and the treatment of thyroid carcinoma with I-131, and we include a review of the results of our experimental and clinical trials.

In vivo long-term imaging and radioiodine therapy by sodium-iodide symporter gene expression using a lentiviral system containing ubiquitin C promoter

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 I-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 μM 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.

Immune response to firefly luciferase as a naked DNA

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 1 day to 7 days post-injection when pcDNA3.1-Fluc was intradermally injected into ear-pinnae. To observe induced immune response, the percentages of CD8+IFN-γ+ 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+IFN-γ+ 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, IFN-γ protein levels in the pcDNA3.1-Fluc group were higher than 3 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 pre-clinical 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.

Establishment of animal model for the analysis of cancer cell metastasis during radiotherapy

BackgroundΓ-Ionizing radiation (IR) therapy is one of major therapeutic tools in cancer treatment. Nevertheless, γ-IR therapy failed due to occurrence of metastasis, which constitutes a significant obstacle in cancer treatment. The main aim of this investigation was to construct animal model which present metastasis during radiotherapy in a mouse system in vivo and establishes the molecular mechanisms involved.Materials and methodsThe C6L transfectant cell line expressing firefly luciferase (fLuc) was treated with γ-IR, followed by immunoblotting, zymography and invasion assay in vitro. We additionally employed the C6L transfectant cell line to construct xenografts in nude mice, which were irradiated with γ-IR. Irradiated xenograft-containing mice were analyzed via survival curves, measurement of tumor size, and bioluminescence imaging in vivo and ex vivo. Metastatic lesions in organs of mice were further assessed using RT-PCR, H & E staining and immunohistochemistry.Resultsγ-IR treatment of C6L cells induced epithelial-mesenchymal transition (EMT) and increased cell invasion. In irradiated xenograft-containing mice, tumor sizes were decreased dramatically and survival rates extended. Almost all non-irradiated xenograft-containing control mice had died within 4 weeks. However, we also observed luminescence signals in about 22.5% of γ-IR-treated mice. Intestines or lungs of mice displaying luminescence signals contained several lesions, which expressed the fLuc gene and presented histological features of cancer tissues as well as expression of EMT markers.ConclusionsThese findings collectively indicate that occurrences of metastases during γ-IR treatment accompanied induction of EMT markers, including increased MMP activity. Establishment of a murine metastasis model during γ-IR treatment should aid in drug development against cancer metastasis and increase our understanding of the mechanisms underlying the metastatic process.

Prognostic Implications of Tumor-Infiltrating Lymphocytes in Association With Programmed Death Ligand 1 Expression in Early-Stage Breast Cancer.

BACKGROUND 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. PATIENTS AND METHODS 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). RESULTS 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. CONCLUSION 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.

In vivo Imaging of Adenovirus Transduction and Enhanced Therapeutic Efficacy of Combination Therapy with Conditionally Replicating Adenovirus and Adenovirus-p27

Gene therapy is hampered by poor gene transfer to the tumor mass. We previously proposed a combination adenoviral gene therapy containing a conditionally replicating adenovirus (CRAD) expressing mutant E1 (delta24RGD) and a replication-defective E1-deleted adenovirus to enhance the efficiency of gene transfer. Mutant E1 expressed by delta24RGD enables the replication of replication-defective adenoviruses in tumors when cancer cells are co-infected with both viruses. In this study, gene transfer rates in xenografts tumors were monitored by bioluminescence in cells infected with the replication-defective adenovirus-luciferase (ad-luc). Tumor masses treated with CRAD + ad-luc showed dramatically stronger and more prolonged luciferase expression than ad-luc-treated tumors and this expression spread through the entire tumor mass without significant systemic spread. Transduction with CRAD + replication-defective adenovirus-p27 increased the expression of p27 by 24-fold versus transduction with ad-p27 alone. Treatment of a lung cancer cell line and of established lung cancer xenografts with CRAD + adenovirus-p27 also induced stronger growth suppression than treatment with either virus alone. These findings confirm the selective replication of E1-deleted adenovirus containing a therapeutic gene due to the presence of mutant E1 produced by delta24RGD in tumors. Moreover, this replication increased the therapeutic gene transfer rate and enhanced its antitumor effects.

Visualization of Hypoxia-Inducible Factor-1 Transcriptional Activation in C6 Glioma Using Luciferase and Sodium Iodide Symporter Genes

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor of hypoxic response in cancer cells and is associated with tumor progression, angiogenesis, metastasis, and resistance to therapy. We assessed whether the human sodium iodide symporter (NIS) reporter systems can be used to visualize transcriptional activation of HIF-1 in C6 glioma. Methods: Two types of plasmid-expressing human NIS or luciferase (Luc) genes, controlled by 5 copies of hypoxia response element (5HRE), were constructed: p5HRE-NIS or p5HRE-Luc. C6 glioma cells were stably transfected with p5HRE-NIS or p5HRE-Luc plasmids (C6-5HRE-NIS or C6-5HRE-Luc). Hypoxic conditions were modeled by exposing culture medium to desferrioxamine (DFO) or a low oxygen atmosphere (<1% O2) in a hypoxic chamber. HIF-1 transcription activity was assessed by measuring cellular 125I uptake and luminescent intensities. Reverse-transcription polymerase chain reaction and Western blotting were performed to observe the messenger RNA and protein levels of reporter and target genes under hypoxic or normoxic conditions. C6, C6-cytomegalovirus (CMV)-NIS, or C6-CMV-Luc and C6-5HRE-NIS or C6-5HRE-Luc cells were injected subcutaneously into nude mice (the NIS and Luc groups, respectively). Two weeks after tumor challenge, bioluminescence and 99mTc scintigraphic images were acquired before and after intraperitoneal DFO administration. Natural hypoxia in tumors was induced by growing tumors for 3 wk. Ex vivo studies, such as biodistribution, immunohistochemistry, and 99mTc autoradiography, were performed. Results: Time- and concentration-dependent increases of 125I uptake and bioluminescence were observed in hypoxically stressed reporter cells. Also, messenger RNA and protein levels of reporter and target genes increased under hypoxic conditions. 99mTc uptake and bioluminescence signals from C6-5HRE-NIS and C6-5HRE-Luc tumors increased during hypoxia. In the biodistribution study, a larger amount of 99mTc accumulated in C6-5HRE-NIS tumors than in the other tumors not containing 5HRE (P < 0.005). In the Luc group, immunostaining showed similar distribution patterns for luciferase and pimonidazole, and in the NIS group, autoradiography of C6-5HRE-NIS tumors showed a distribution similar to that observed for pimonidazole immunostaining. Conclusion: The transcriptional activation of HIF-1 induced by hypoxia or DFO was visualized by both bioluminescence and scintigraphic reporter gene systems.