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(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

ABSTRACT: To develop a clinically translatable method of cell labeling with zirconium 89 ((89)Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy.This study was approved by the institutional animal care committee. (89)Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of (89)Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444-555 kBq/[5 × 10(6)] cells, n = 5) and CTLs (185 kBq/[5 × 10(6)] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 10(6)] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses.(89)Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%-43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. (89)Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P < .05 on day 7).We have developed a (89)Zr-oxine complex cell tracking technique for use with PET that is applicable to a broad range of cell types and could be a valuable tool with which to evaluate various cell-based therapies.

SUBMITTER: Sato N

PROVIDER: S-EPMC4456181 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

Sato Noriko N Wu Haitao H Asiedu Kingsley O KO Szajek Lawrence P LP Griffiths Gary L GL Choyke Peter L PL

Radiology 20150220 2

<h4>Purpose</h4>To develop a clinically translatable method of cell labeling with zirconium 89 ((89)Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy.<h4>Materials and methods</h4>This study was approved by the institutional animal care committee. (89)Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cel ...[more]

PMID: 25706654

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Project description:The encapsulation of Glucocorticoids (GCs) into long-circulating liposomes (LCLs) is a proven strategy to reduce the side effects of glucocorticoids and improve the treatment of inflammatory diseases, such as rheumatoid arthritis (RA). With the aim of supporting the development of GC-loaded LCLs, and potentially predict patient response to therapy clinically, we evaluated a direct PET imaging radiolabelling approach for preformed GC-LCLs in an animal model of human inflammatory arthritis. Methods: A preformed PEGylated liposomal methylprednisolone hemisuccinate (NSSL-MPS) nanomedicine was radiolabelled using [89Zr]Zr(oxinate)4 (89Zr-oxine), characterised and tracked in vivo using PET imaging in a K/BxN serum-transfer arthritis (STA) mouse model of inflammatory arthritis and non-inflamed controls. Histology and joint size measurements were used to confirm inflammation. The biodistribution of 89Zr-NSSL-MPS was compared to that of free 89Zr in the same model. A therapeutic study using NSSL-MPS using the same time points as the PET/CT imaging was carried out. Results: The radiolabelling efficiency of NSSL-MPS with [89Zr]Zr(oxinate)4 was 69 ± 8 %. PET/CT imaging of 89Zr-NSSL-MPS showed high uptake (3.6 ± 1.5 % ID; 17.4 ± 9.3 % ID/mL) at inflamed joints, with low activity present in non-inflamed joints (0.5 ± 0.1 % ID; 2.7 ± 1.1 % ID/mL). Importantly, a clear correlation between joint swelling and high 89Zr-NSSL-MPS uptake was observed, which was not observed with free 89Zr. STA mice receiving a therapeutic dose of NSSL-MPS showed a reduction in inflammation at the time points used for the PET/CT imaging compared with the control group. Conclusions: PET imaging was used for the first time to track a liposomal glucocorticoid, showing high uptake at visible and occult inflamed sites and a good correlation with the degree of inflammation. A subsequent therapeutic response matching imaging time points in the same model demonstrated the potential of this radiolabeling method as a theranostic tool for the prediction of therapeutic response - with NSSL-MPS and similar nanomedicines - in the treatment of inflammatory diseases.

Project description:BackgroundThere is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [89Zr]Zr-oxine (8-hydroxyquinoline) and [89Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers.ProceduresSeveral parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. The cellular retention of 89Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining.ResultsThe optimised synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1-8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells' proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling.ConclusionsOur study demonstrates that [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [89Zr]Zr-oxine was superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.

Project description:BackgroundDe novo or acquired resistance in breast cancer leads to treatment failures and disease progression. In human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer, Src, a non-receptor tyrosine kinase, is identified as a major mechanism of trastuzumab resistance, with its activation stabilizing aberrant HER2 signaling, thus making it an attractive target for inhibition. Here, we explored the causal relationship between Src and HER2 by examining the potential of 89Zr-trastuzumab as a surrogate imaging marker of Src activity upon inhibition with dasatinib in HER2+ breast cancer.MethodsHER2+ primary breast cancer cell lines BT-474 and trastuzumab-resistant JIMT-1 were treated with dasatinib and assessed for expression and localization of HER2, Src, and phosphorylated Src (pSrc) (Y416) through western blots and binding assays. Mice bearing BT-474 or JIMT-1 tumors were treated for 7 or 14 days with dasatinib. At the end of each treatment, tumors were imaged with 89Zr-trastuzumab. The results of 89Zr-trastuzumab positron emission tomography (PET) was compared against tumor uptake of fluorodeoxyglucose (18F-FDG) obtained the day before in the same group of mice. Ex vivo western blots and immunohistochemical staining (IHC) were performed for validation.ResultsIn BT-474 and JIMT-1 cells, treatment with dasatinib resulted in a decrease in internalized 89Zr-trastuzumab. Confirmation with immunoblots displayed abrogation of pSrc (Y416) signaling; binding assays in both cell lines demonstrated a decrease in cell surface and internalized HER2-bound tracer. In xenograft models, dasatinib treatment for 7 days (BT-474, 11.05 ± 2.10 % injected dose per gram of tissue %(ID)/g; JIMT-1, 3.88 ± 1.47 %ID/g)) or 14 days (BT-474, 9.20 ± 1.85 %ID/g; JIMT-1, 4.45 ± 1.23 %ID/g) resulted in a significant decrease in 89Zr-trastuzumab uptake on PET compared to untreated control (BT-474, 17.88 ± 2.18 %ID/g; JIMT-1, 8.04 ± 1.47 %ID/g). No difference in 18F-FDG uptake was observed between control and treated cohorts. A parallel decrease in membranous HER2 and pSrc (Y416) staining was observed in tumors post treatment on IHC. Immunoblots further validated the 89Zr-trastuzumab-PET readout. Positive correlation was established between 89Zr-trastuzumab tumor uptake versus tumor regression, pSrc and pHER2 expression.Conclusions89Zr-trastuzumab can potentially assess tumor response to dasatinib in HER2+ breast cancer and could be used as a surrogate tool to monitor early changes in Src signaling downstream of HER2.

Project description:PurposeProgrammed cell death-1 receptor (PD-1) and its ligand (PD-L1) are the targets for immunotherapy in many cancer types. Although PD-1 blockade has therapeutic effects, the efficacy differs between patients. Factors contributing to this variability are PD-L1 expression levels and immune cells present in tumors. However, it is not well understood how PD-1 expression in the tumor microenvironment impacts immunotherapy response. Thus, imaging of PD-1-expressing immune cells is of interest. This study aims to evaluate the biodistribution of Zirconium-89 (89Zr)-labeled pembrolizumab, a humanized IgG4 kappa monoclonal antibody targeting PD-1, in healthy cynomolgus monkeys as a translational model of tracking PD-1-positive immune cells.ProceduresPembrolizumab was conjugated with the tetrafluorophenol-N-succinyl desferal-Fe(III) ester (TFP-N-sucDf) and subsequently radiolabeled with 89Zr. Four cynomolgus monkeys with no previous exposure to humanized monoclonal antibodies received tracer only or tracer co-injected with pembrolizumab intravenously over 5 min. Thereafter, a static whole-body positron emission tomography (PET) scan was acquired with 10 min per bed position on days 0, 2, 5, and 7. Image-derived standardized uptake values (SUVmean) were quantified by region of interest (ROI) analysis.Results89Zr-N-sucDf-pembrolizumab was synthesized with high radiochemical purity (>?99 %) and acceptable molar activity (>?7 MBq/nmol). In animals dosed with tracer only, 89Zr-N-sucDf-pembrolizumab distribution in lymphoid tissues such as mesenteric lymph nodes, spleen, and tonsils increased over time. Except for the liver, low radiotracer distribution was observed in all non-lymphoid tissue including the lung, muscle, brain, heart, and kidney. When a large excess of pembrolizumab was co-administered with a radiotracer, accumulation in the lymph nodes, spleen, and tonsils was reduced, suggestive of target-mediated accumulation.Conclusions89Zr-N-sucDf-pembrolizumab shows preferential uptake in the lymphoid tissues including the lymph nodes, spleen, and tonsils. 89Zr-N-sucDf-pembrolizumab may be useful in tracking the distribution of a subset of immune cells in non-human primates and humans.Trial registrationClinicalTrials.gov Identifier: NCT02760225.

Dataset Information

(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

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(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

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