Tracking of dendritic cell migration into lymph nodes using molecular imaging with sodium iodide symporter and enhanced firefly luciferase genes.
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ABSTRACT:
SUBMITTER: Lee HW
PROVIDER: S-EPMC4431315 | biostudies-literature | 2015 May
REPOSITORIES: biostudies-literature
Publications
Tracking of dendritic cell migration into lymph nodes using molecular imaging with sodium iodide symporter and enhanced firefly luciferase genes.
Lee Ho Won HW Yoon Seung Yun SY Singh Thoudam Debraj TD Choi Yoon Ju YJ Lee Hong Je HJ Park Ji Young JY Jeong Shin Young SY Lee Sang-Woo SW Ha Jeoung-Hee JH Ahn Byeong-Cheol BC Jeon Yong Hyun YH Lee Jaetae J
Scientific reports 20150514
We sought to evaluate the feasibility of molecular imaging using the human sodium iodide symporter (hNIS) gene as a reporter, in addition to the enhanced firefly luciferase (effluc) gene, for tracking dendritic cell (DCs) migration in living mice. A murine dendritic cell line (DC2.4) co-expressing hNIS and effluc genes (DC/NF) was established. For the DC-tracking study, mice received either parental DCs or DC/NF cells in the left or right footpad, respectively, and combined I-124 PET/CT and biol ...[more]
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Project description:BackgroundRecently, 18F-tetrafluoroborate (TFB) was used as a substrate for the human sodium/iodide symporter (hNIS) reporter gene. This study evaluated the feasibility of performing molecular-genetic imaging by using the new radiotracer (18F-TFB) for the hNIS gene, to track dendritic cell (DC) migration in live mice. A murine dendritic cell line (DC2.4) co-expressing the hNIS and effluc genes (DC/NF) was established. To confirm the functional cellular expression of both effluc and NIS in the inoculated DC/NF cells by bio-medical imaging, combined bioluminescence imaging (BLI) and 18F-TFB positron emission tomography/computed tomography (PET/CT) imaging was performed after intramuscular injection with parental DCs and DC/NF cells. For DC-tracking, parental DCs or DC/NF cells were injected in the left or right mouse footpad, respectively, and 18F-TFB PET/CT and BLI were performed to monitor these cells in live mice.ResultsIn vivo PET/CT and BLI showed a clear signal in DC/NF injection sites but not in parental DC injection sites. The signal intensity in DC/NF cells was correlated with time. In vivo 18F-TFB PET/CT imaging showed higher radiotracer activity in the draining popliteal lymph nodes (DPLNs) in DC/NF injection sites than those in DC injection sites on day 2. BLI also showed DC/NF cell migration to the DPLNs on day 2 after the injection.ConclusionsMigration of DCs to the lymph nodes was successfully monitored using 18F-TFB PET/CT imaging of the NIS gene and optical imaging of the effluc gene in live mice. These data support the feasibility of using 18F-TFB as a substrate for hNIS reporter gene imaging to track the migration of DCs to the lymph nodes in live animals. The use of 18F-TFB may facilitate enhanced PET imaging of the hNIS reporter gene in small animals and humans in future studies.
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Project description:Background: The ability of thyroid follicular epithelial cells to accumulate iodide via the sodium/iodide symporter (NIS) is exploited to successfully treat most thyroid cancers, although a subset of patients lose functional NIS activity and become unresponsive to radioiodide therapy, with poor clinical outcome. Our knowledge of NIS regulation remains limited, however. While numerous membrane proteins are functionally regulated via dimerization, there is little definitive evidence of NIS dimerization, and whether this might impact upon radioiodide uptake and treatment success is entirely unknown. We hypothesized that NIS dimerizes and that dimerization is a prerequisite for iodide uptake. Methods: Coimmunoprecipitation, proximity ligation, and Förster resonance energy transfer (FRET) assays were used to assess NIS:NIS interaction. To identify residues involved in dimerization, a homology model of NIS structure was built based on the crystal structure of the dimeric bacterial protein vSGLT. Results: Abundant cellular NIS dimerization was confirmed in vitro via three discrete methodologies. FRET and proximity ligation assays demonstrated that while NIS can exist as a dimer at the plasma membrane (PM), it is also apparent in other cellular compartments. Homology modeling revealed one key potential site of dimeric interaction, with six residues <3Å apart. In particular, NIS residues Y242, T243, and Q471 were identified as critical to dimerization. Individual mutation of residues Y242 and T243 rendered NIS nonfunctional, while abrogation of Q471 did not impact radioiodide uptake. FRET data show that the putative dimerization interface can tolerate the loss of one, but not two, of these three clustered residues. Conclusions: We show for the first time that NIS dimerizes in vitro, and we identify the key residues via which this happens. We hypothesize that dimerization of NIS is critical to its trafficking to the PM and may therefore represent a new mechanism that would need to be considered in overcoming therapeutic failure in patients with thyroid cancer.
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Project description:Molecular imaging is useful for longitudinal assessment of engraftment. However, it is not known which factors, other than cell number, can influence the molecular imaging signal obtained from reporter genes.The effects of cell dissociation/suspension on cellular bioenergetics and the signal obtained by firefly luciferase and human sodium-iodide symporter labeling of cardiosphere-derived cells were investigated.(18)Fluorodeoxyglucose uptake, ATP levels, (99m)Tc-pertechnetate uptake, and bioluminescence were measured in vitro in adherent and suspended cardiosphere-derived cells. In vivo dual-isotope single-photon emission computed tomography/computed tomography imaging or bioluminescence imaging (BLI) was performed 1 hour and 24 hours after cardiosphere-derived cell transplantation. Single-photon emission computed tomography quantification was performed using a phantom for signal calibration. Cell loss between 1 hour and 24 hours after transplantation was quantified by quantitative polymerase chain reaction and ex vivo luciferase assay. Cell dissociation followed by suspension for 1 hour resulted in decreased glucose uptake, cellular ATP, (99m)Tc uptake, and BLI signal by 82%, 43%, 42%, and 44%, respectively, compared with adherent cells, in vitro. In vivo (99m)Tc uptake was significantly lower at 1 hour compared with 24 hours after cell transplantation in the noninfarct (P<0.001; n=3) and infarct (P<0.001; n=4) models, despite significant cell loss during this period. The in vivo BLI signal was significantly higher at 1 hour than at 24 hours (P<0.01), with the BLI signal being higher when cardiosphere-derived cells were suspended in glucose-containing medium compared with saline (PBS).Adhesion is an important determinant of cellular bioenergetics, (99m)Tc-pertechnetate uptake, and BLI signal. BLI and sodium-iodide symporter imaging may be useful for in vivo optimization of bioenergetics in transplanted cells.
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