Iodine-124 Based Dual Positron Emission Tomography and Fluorescent Labeling Reagents for In vivo Cell Tracking.

Understanding the in vivo behavior of experimental therapeutic cells is fundamental to their successful development and clinical translation. Iodine-124 has the longest half-life (4.2 days) among the clinically used positron emitters. Consequently, this isotope offers the longest possible tracking time for directly labeled cells using positron emission tomography (PET). Herein, we have radiosynthesized and evaluated two iodine-124/fluorescein-based dual PET and fluorescent labeling reagents; namely 124I-FIT-Mal and 124I-FIT-(PhS)2Mal for cell surface thiol bioconjugation. 124I-FIT-(PhS)2Mal labeled cells significantly more effectively than 124I-FIT-Mal. It conjugated to various cell lines in 22%-62% labeling efficiencies with prolonged iodine-124 retention. 124I-FIT-(PhS)2Mal was mainly conjugated on the cell membrane confirmed by high-resolution fluorescence imaging. The migration of 124I-FIT-(PhS)2Mal labeled Jurkat cells was visualized in NSG mice with excellent target-to-background contrast using PET/CT over 7 days. These data demonstrate that 124I-FIT-(PhS)2Mal can dynamically track cell migration in vivo using PET/CT over a clinically relevant timeframe.


■ INTRODUCTION
Emerging as the fourth pillar of healthcare, cell-based therapies have shown great promise in cancer treatment, 1 stem cell regenerative medicine, 2 and immune tolerance in organ transplantation. 3 For example, adoptive transfer of chimeric antigen receptor (CAR)-engineered T-cells is a novel immunotherapy that utilizes the patient's own immune system to treat cancer. 4 One fundamental challenge in both medical research and clinical applications of cell therapies is to understand the in vivo behavior of the infused cells. Imaging studies can dynamically track the migration, proliferation, and final fate of the administered cells providing early insight into their safety, mechanism of action, and efficacy. 5,6 Therefore, it is essential to incorporate tracking studies at the earliest stage of clinical development in order to monitor the in vivo location and persistence of the cells on a patient-by-patient basis. 5,6 To detect the initial distribution and migration of the infused cells, various direct cell labeling methods have been developed by which the therapeutic cells are labeled with a contrast reagent in vitro. Once inoculated into the experimental animals or patients, the movement of the labeled cells can be monitored using the corresponding imaging modality. 7−10 Positron emission tomography (PET) is a noninvasive imaging technique that can produce real-time images of radiolabeled cells in vivo and monitor their whole-body migration. 11 Currently, the most successful direct cell labeling method for PET involves the use of lipophilic radiometal complex, 89 Zr-(oxine) 4 to deposit 89 Zr intracellularly. 12,13 Owning to the 3.3 days half-life of 89 Zr, this method has been employed to track a variety of cells for several days in vivo. Alternatively, an 89 Zrdesferrioxamine-isothiocyanate bioconjugation reagent was reported to label cells through the amine groups in cell surface proteins, allowing the assessment of the distribution of the labeled cells in vivo for days. 14 However, when applying both of the above 89 Zr based cell tracking methods to monitor the experimental therapeutic cells in preclinical settings, the 89 Zr leaks gradually from the labeled cells in vivo and deposits in bones, complicating the interpretation of PET images. 10,11,13,14 Iodine-124 has the longest half-life (t 1/2 = 4.2 d) among the clinically used PET radioisotopes. 15 In principle, it can provide the longest possible tracking time for directly labeled cells with PET. In addition, the thyroid and stomach uptake of any free iodine-124 generated through the catabolism of the labeling reagent can be readily blocked by pretreatment with potassium iodide. Consequently, this approach provides a low background that will improve the sensitivity and accuracy to detect the labeled cells. However, the major challenge with the use of iodine-124 based reagents for direct cell labeling is how to overcome various intracellular oxido-reductase-mediated deiodination. 15 We envisage that the deiodination can be minimized by coupling the iodine-124 on the cell surface. For this purpose, we choose thiols present in cell membrane proteins for labeling because they have been proven to be ideal functional groups in various bioconjugation applications. 16,17 To achieve both effective cell conjugation and minimum passive diffusion of labeling reagents into cells, we hypothesize that the labeling reagents should be amphiphiles in nature. In this context, such reagents will be equipped with a lipophilic warhead to reach and react with thiols in the lipid surrounded cell surface proteins. They will also contain a negatively charged hydrophilic moiety that will repulse the negatively charged phosphate heads of the membrane lipids to minimize the labeling reagents diffusing into cells. In addition, a fluorophore will also be employed to confirm effective cell surface conjugation with fluorescence imaging. To achieve these goals, we selected two thiol-targeting moieties, maleimide and dithiophenolmaleimide, for cell surface conjugation. The maleimide reacts irreversibly with the free thiols on the cell surface as demonstrated by others. 16,17 The dithiophenolmaleimide spontaneously reacts with both thiols from the reduced disulfide bridge and preserves the bridge structure in antibodies and peptides. 18,19 Although never having been applied to cell labeling before, we envisaged that these characteristics of dithiophenolmaleimide would enable it to conjugate to the cell surface proteins effectively with minimal adverse effects. We selected fluorescein as the fluorescent reporter for this application since it is used clinically in man and thus should exert negligible toxicity. Moreover, the carboxylic acid group of this hydrophilic dye is deprotonated and becomes negatively charged under physiological pH, which would retard the labeling reagent from entering cells.
Herein, using a copper-mediated one-pot three-component radioiodination reaction, 20−22 we synthesized two trifunctional dual PET and fluorescent bioconjugation reagents, 124 I-FIT-Mal [1] and 124 I-FIT-(PhS)2Mal [2]. These are equipped with (i) iodine-124 for longitudinal cell tracking with PET; (ii) a hydrophilic fluorescein moiety to balance lipophilicity and enable fluorescence cell imaging; and (iii) a maleimide or dithiophenolmaleimide moiety for cell membrane protein thiol bioconjugation.
In Vivo Cell Tracking with PET Imaging and Biodistribution Study. Subsequently, we carried out a tracking study of 124 I-FIT-(PhS) 2 Mal [2] labeled Jurkat cells (∼0.5−1.0 × 10 7 cells, ∼0.5−1.0 MBq) in NSG mice (n = 3) by five sequential PET/CT scans at 4, 24 h, 2, 5, and 7 days postintravenous (IV) injection. Radioactivity accumulated mainly in the lungs, liver, and bladder in the 4 and 24 h PET/CT images. The radioactivity gradually migrated from the lungs to the liver and cleared from the bladder in the NSG mice in the day 2, 5, and 7 PET/CT images ( Figure 4A). After completion of PET/CT imaging on day 7, all three animals were euthanized for a biodistribution study. Radioactivity uptake of 7.90 ± 0.88%, 2.94 ± 0.28%, and 2.38 ± 0.74% injected dose (ID)/g was detected in lungs, liver, and spleen, respectively (mean ± SD, n = 3). Radioactivity uptake in other organs such as blood, bone, etc. was all less than 1% ID/g ( Figure 5A and Table S1). In the control group, NSG mice (n = 4) received the dual labeling reagent, 124 I-FIT-(PhS) 2 Mal [2] IV, and underwent a PET/CT scan after 24 h. Most of the radioactivity had cleared from the animals, and only very weak radioactivity signals were observed in the large intestine and bladder, as shown in the 24 h PET/CT image ( Figure 4B). All four animals were euthanized after PET/CT imaging for a biodistribution study. Minimal radioactivity was detected in the lungs (0.88 ± 0.20% ID/g), liver (0.63 ± 0.09% ID/g), and spleen (0.44 ± 0.05% ID/g) of these animals ( Figure 5B and Table S2).
Ex Vivo Immunohistochemistry Study. Finally, we undertook immunohistology staining of liver and lung tissues harvested from NSG mice that were inoculated with 124 I-FIT-(PhS) 2 Mal [2] labeled Jurkat cells in the PET imaging study. As controls, the liver and lungs from three NSG mice received either PBS or nonlabeled Jurkat cells (∼1.0 × 10 7 ), were collected for immunohistology staining 7 days post IV injection. All tissue sections were sequentially incubated with anti-human CD3 as the primary antibody and polymer-  used. This is because thioureas are susceptible to copper(II) oxidation. 25 In terms of radiosynthesis, a longer reaction time of 18 h at RT was required to prepare 124 I-FIT-Mal [1] in both excellent RCYs and molar activity. When radiolabeling was attempted at elevated temperature, the formation of both radioactive and nonradioactive side products was observed, which significantly reduced the RCYs and molar activity of 124 I-FIT-Mal [1]. As iodine-124 has a half-life of 4.2 days, we decided to adopt the longer reaction time for the preparation of 124 I-FIT-Mal [1]. In contrast, 124 I-FIT-(PhS) 2 Mal [2] was formed in both excellent RCYs and molar activity in 90 min which allows its production, cell labeling, and subsequent biological evaluation in a clinically relevant time frame. Control    In the proof of concept in vivo tracking study, the migration of 124 I-FIT-(PhS) 2 Mal [2] labeled Jurkat cells from the lungs to the liver was clearly visualized in NSG mice using five consecutive PET scans over 7 days. Spleen is a major cell migration organ we expected to observe in PET. However, the high liver radioactivity uptake and the anatomical proximity between liver and spleen completely obscured the spleen in the PET images. It is worth noting that in contrast to the high bone uptake of 89 Zr-based cell tracking reagents, 10,11,13,14 no bone uptake was observed in all five PET images from 4 h until 7 days post IV injection of 124 I-FIT-(PhS) 2 Mal [2] labeled Jurkat cells. This illustrates the superiority of this iodine-124 based strategy over the current zirconium-89 methods for direct cell labeling and long-term cell tracking with PET. As expected, thyroid and stomach uptake of iodine-124 were completely blocked by treating the animals with potassium iodide, generating a clear background for visualizing cell migration. Moreover, the initial dissociation of the labeling reagent from the Jurkat cells was observed in the 4 and 24 h PET images, as indicated by the accumulation of radioactivity in the bladder. However, little radioactivity was observed in the bladder at later time points (2, 5, and 7 days), indicating that the labeling reagent was still associated with the Jurkat cells. labeled Jurkat cells also provided strong evidence that the radioactivity was still concentrated in the organs to which the cells migrated. These include the lungs, liver, and spleen, with little radioactivity detected in other organs at this late time point. In contrast, in the control group that received 124 I-FIT-(PhS) 2 Mal [2], both PET/CT imaging and a biodistribution study confirmed that most of the radioactivity was cleared from the NSG mice within 24 h post IV injection. Moreover, the presence of Jurkat cells in the lungs and liver of the NSG mice that received 124 I-FIT-(PhS) 2 Mal [2] labeled Jurkat cells from the PET imaging was further confirmed using anti-human CD3 antibody in an ex vivo immunohistology staining study. The distribution patterns of the 124 I-FIT-(PhS) 2 Mal [2] labeled Jurkat cells in the lung and liver tissues were similar to the nonlabeled Jurkat cells in the positive control group shown by the immunohistology staining study, which hints that the in vivo movability of Jurkat cells was reserved post 124 I-FIT-(PhS) 2 Mal [2] labeling.
One possible concern is the low radioactivity dose of 124 I-FIT-(PhS) 2 Mal [2] (∼100 KBq/10 6 cells) used in the in vivo cell tracking study, which might be a limitation to translate this new method to clinical applications. Although there is no clinical PET imaging data to indicate the optimal radioactivity dose for directly labeled cell tracking in human, one ongoing clinical trial employs 7.4−18.5 MBq of 89 Zr-labeled leukocytes to track peripheral immune cell infiltration of the brain in patients with central inflammatory disorders over 6 days. 27 Moreover, in a recent 89 Zr-labeled natural killer (NK) cell (∼1.5 × 10 8 ) tracking study in rhesus macaques (∼6.6 kg), clear PET images were acquired with only 2.0 MBq of radioactivity over 7 days. 28 Given the very similar positron abundance of iodine-124 (22.5%) and zirconium-89 (22.7%), longer half-life of iodine-124 (4.2 days) than zirconium-89 (3.3 days), and excellent iodine-124 retention of 124 I-FIT-(PhS) 2 Mal [2] labeled cells, it is a reasonable estimation that this novel iodine-124 based cell labeling method could provide a sufficient radioactivity dose (∼20 MBq in 2 × 10 8 cells) for directly labeled cell tracking with PET in clinical trials.
Additionally, the latest ultrasensitive total-body PET scanner requires a very low radioactivity dose to achieve the same signal-to-noise ratio comparing to the current clinical PET scanners. For example, in the first human PET imaging study with the EXPLORER Total-Body PET Scanner, high quality full-body PET images were acquired by using only 25 MBq of 18 F-FDG (1/15 of the normal radioactivity dose) in 10 min. 29 Therefore, we anticipate that the radioactivity dose will not be a barrier to translate 124 I-FIT-(PhS) 2 Mal [2] to clinical cell tracking.

■ CONCLUSION
Two dual PET and fluorescent labeling reagents, 124 I-FIT-Mal   3.49, s and 1.09, s), and coupling constants (J) are given in Hertz. HPLC analysis was performed with an Agilent 1200 HPLC system equipped with a 1200 series diode array detector. Radio-HPLC analysis was performed with an Agilent 1200 HPLC system equipped with a series diode array detector and Raytest GABI Star radioactivity detector. The radiochemical purity of 124 I-FIT-Mal and 124 I-FIT-(PhS) 2 Mal was determined using ra-dioHPLC, and both were greater than 95% (Figures S2 and  S4). Reductant free [ 124 I]NaI was purchased from PerkinElmer (product number NEZ309) in 0.02 M NaOH (pH 14) aqueous solution. All reagents were purchased from Sigma-Aldrich and were used without further purification.

124
I-FIT-(PhS) 2 Mal [2]. After incubation at 60°C for 90 min, the reaction mixture was quenched with DMSO (100 μL) followed by water/ MeOH (4:1, 1.0 mL) The identity of both dual labeling reagents was confirmed by coeluting with their corresponding nonradioactive reference compounds (Figures S2 and S4). Formulation: the HPLC eluent containing the dual labeling reagent was diluted to 15% MeOH in water and was loaded on either a preconditioned Waters C18 light Sep-Pak cartridge for 124 I-FIT-Mal [1] or Waters t-C18 Sep-Pak cartridge for 124 I-FIT-(PhS) 2 Mal [2]. After washing with water (5 mL), the dual labeling reagent was released using EtOH. The EtOH was removed by a stream of N 2 , and then the dual labeling reagent was redissolved in DMSO for further use.
Log (1 μL, ∼ 2 KBq) in DMSO was added to a mixture of PBS (200 μL) and noctanol (200 μL) in a 1.5 mL Eppendorf vial (n = 6). The mixture was vigorously agitated at RT for 5 min and then centrifuged at 3000 g for 10 min. A 100 μL aliquot from each layer was drawn for measurement using a gamma counter.
Cell Culture. T-cells were isolated from peripheral blood samples taken from healthy volunteers aged 18−45 (KCL ethics approval: HR-18/19-8846) via Ficoll separation. Blood was slowly added to a 50 mL Falcon tube containing 15 mL of Ficoll-Paque (GE Healthcare) solution and spun in a centrifuge at 500 g for 25 min. The peripheral blood mononuclear cell layer was extracted, and the cells were washed twice with PBS and prepared for activation. Cells were resuspended in RPMI 1640 cell growth medium at a concentration of 3 × 10 6 cells/mL and plated on a 6-well plate (4 mL per well). T-cells were activated with 5 μg/mL of phytohemagglutinin (PHA-L, Sigma-Aldrich). IL-2 (100 U/ mL, PeproTech) and fresh medium were added every 2−3 days.
Jurkat and murine myeloma 5T33 (generous gift from Dr. Yolanda Calle-Patino) cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 200 U/L penicillin, 0.1 g/L streptomycin, and 2 mM L-glutamine. The cell concentration was maintained in 1 × 10 5 −1 × 10 6 cells/mL in a humidified chamber containing 5% CO 2 at 37°C. . After incubation at 37°C for another 30 min, the cells were centrifuged (1200 rpm, 5 min), and the supernatants were transferred to new Eppendorf tubes. The pellets were further rinsed with PBS (2 × 0.5 mL) before being transferred to new Eppendorf tubes and recultivated in complete medium. To determine the cell labeling efficiency, the supernatants and the washes for each sample were combined. The pellets, supernatants, and Eppendorf tubes used for cell labeling were measured by a Capintec CRC-25R dose calibrator (Capintec Inc., USA).
For the nonradioactive reference compound of 124 I-FIT-(PhS) 2 Mal [2]: suspension cells (2 × 10 6 , n = 3) were treated with TCEP (1.0 mL, 1.0 mM) in PBS and incubated at RT for 15 min before being washed with PBS (3 × 1.0 mL) to remove TCEP. The cells were then resuspended in PBS (1.0 mL), and the nonradioactive reference compound of 124 I-FIT-(PhS) 2 Mal [2] in DMSO (0.5 μL, 2.0 mM) was added to achieve the final concentration of 1.0 μM. The cells were incubated at 37°C for another 30 min. Hoechst 33342 in DMSO (2 μL, 250 μM) was added to the cells 5 min before the end of incubation. The cells were centrifuged (1200 rpm, 5 min), the supernatants were removed, and the pellets were further rinsed with PBS (3 × 1.0 mL). The sample was then fixed with 4% paraformaldehyde at RT for 10 min in darkness. Following the fixation step, the cells were washed with PBS (3 × 1.0 mL) and resuspended in PBS (50 μL). The cell sample (10 μL) was mounted on a microscopic glass slide, covered with a coverslip, and sealed with transparent nail enamel. High-resolution confocal fluorescence images were obtained with a Leica TCS SP5 II confocal microscope (Leica Microsystems Ltd.) system and processed using LAS AF Lite (ver 2.  Preclinical PET/CT images were acquired using a Nano-Scan PET/CT (Mediso, Budapest, Hungary) scanner with mice under 2% isoflurane in oxygen anesthesia. Drinking water containing potassium iodide (0.1%, w/v) was provided to 6−7 week old male NSG mice (n = 7) for 1 week before and throughout the PET/CT imaging experiments. On day eight, they were randomly divided into two groups and received either the 124 I-FIT-(PhS) 2  injection followed by a 15 min CT scan. All PET/CT data were reconstructed with the Monte Carlo-based full-3D iterative algorithm Tera-Tomo (Mediso Medical Imaging Systems, Budapest, Hungary). Raw PET data were reconstructed into 60 min bins using reconstruction settings (4 iterations, 6 subsets, 0.4 × 0.4 × 0.4 mm 3 voxel size) as well as intercrystal scatter correction. All reconstructed data were analyzed with VivoQuant software (v3.0, inviCRO, LLC, Boston, USA). All animals were euthanized by cervical dislocation at the end of the last PET/CT scan. The major thoracoabdominal organs, brain, blood, urine, left femur, and thigh muscle were harvested, weighed, and gamma-counted. The radioactivity in each organ was expressed as % ID/g. The total injected dose was defined as the sum of the whole body counts excluding the tail.
Immunohistochemistry. Three cryosections (10 μm thick) were prepared from the liver and lung of NSG mice (n = 3) 1 week after receiving either 124 I-FIT-(PhS) 2 Mal [2] labeled Jurkat cells from the above PET imaging study, PBS, or nonlabeled Jurkat cells (∼1.0 × 10 7 ). These tissue sections were fixed with acetone and dried in air. The tissue sections were then sequentially incubated with anti-CD3 primary antibody (clone LN10, Leica #CD3-565-L-CE) and Polymersupported peroxidase and anti-Mouse IgG followed by DAB treatment (MaxVision2 HRP-Polymer anti-Mouse IHC Kit) and finally stained with hematoxylin. The tissue sections were then observed under an Olympus DP73 digital microscope.