<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of a Bombesin Analogue Labeled with Ga-68 and Co-55/57

Purpose

The purpose of this study was to apply an analogue of bombesin, NOTA-AMBA, labeled with Co-55 or Ga-68, for preclinical imaging of prostate cancer.

Procedures

The peptide NOTA-AMBA was labeled with Ga-68 or Co-55 by microwave irradiation. Biodistribution in xenograft mice (PC3) was performed at 1, 4, and 24 h (only cobalt at 24 h) using a fixed amount of peptide. Four weeks post-inoculation, xenograft mice were positron emission tomography/X-ray computed tomography scanned after tail vein injection of [⁶⁸Ga]NOTA-AMBA or [⁵⁵Co]NOTA-AMBA.

Results

Labeling with Ga-68 and Co-55/57 was achieved in yields greater than 90 %. A radiochemical purity (RCP) of 95 and 90 % were obtained for Ga-68 and Co-55, respectively. Both radiopeptides showed high uptake in the intestines, stomach, pancreas, and in the tumor ([⁶⁸Ga]NOTA-AMBA, 10.3 %ID/g at 1 h to 6.4 %ID/g at 4 h; [⁵⁷Co]NOTA-AMBA, 8.2 %ID/g at 1 h to 5.3%ID/g at 24 h). Normal tissue cleared over time improving tumor-to-background ratios.

Conclusions

NOTA-AMBA was labeled in high yields and RCP with Ga-68 and Co-55/57. High tumor uptake in a subcutaneous mouse prostate cancer model was observed. At 24 h, [^55/57Co]NOTA-AMBA showed better tumor-to-organ ratios than [⁶⁸Ga]NOTA-AMBA at both 1 and 4 h post-injection. Hence, for imaging, [⁵⁵Co]NOTA-AMBA was found to be superior compared to [⁶⁸Ga]NOTA-AMBA.

     

Tumor Targeting <Emphasis Type="Italic">via</Emphasis> Sialic Acid: [<Superscript>68</Superscript>Ga]DOTA-en-pba as a New Tool for Molecular Imaging of Cancer with PET

Purpose

The aim of this study was to demonstrate the potential of Ga-68-labeled macrocycle (DOTA-en-pba) conjugated with phenylboronic vector for tumor recognition by positron emission tomography (PET), based on targeting of the overexpressed sialic acid (Sia).

Procedures

The imaging reporter DOTA-en-pba was synthesized and labeled with Ga-68 at high efficiency. Cell binding assay on Mel-C and B16-F10 melanoma cells was used to evaluate melanin production and Sia overexpression to determine the best model for demonstrating the capability of [⁶⁸Ga]DOTA-en-pba to recognize tumors. The in vivo PET imaging was done with B16-F10 tumor-bearing SCID mice injected with [⁶⁸Ga]DOTA-en-pba intravenously. Tumor, blood, and urine metabolites were assessed to evaluate the presence of a targeting agent.

Results

The affinity of [⁶⁸Ga]DOTA-en-pba to Sia was demonstrated on B16-F10 melanoma cells, after the production of melanin as well as Sia overexpression was proved to be up to four times higher in this cell line compared to that in Mel-C cells. Biodistribution studies in B16-F10 tumor-bearing SCID mice showed blood clearance at the time points studied, while uptake in the tumor peaked at 60 min post-injection (6.36?±?2.41 % ID/g). The acquired PET images were in accordance with the ex vivo biodistribution results. Metabolite assessment on tumor, blood, and urine samples showed that [⁶⁸Ga]DOTA-en-pba remains unmetabolized up to at least 60 min post-injection.

Conclusions

Our work is the first attempt for in vivo imaging of cancer by targeting overexpression of sialic acid on cancer cells with a radiotracer in PET.

     

Phage Display Selection, <Emphasis Type="Italic">In Vitro</Emphasis> Characterization,and Correlative PET Imaging of a Novel HER3 Peptide

Purpose

HER3 (ERBB3) is a receptor tyrosine kinase that is implicated in treatment resistance across multiple cancers, including those of the breast, lung, and prostate. Overexpression of HER3 following targeted therapy can occur rapidly and heterogeneously both within a single lesion and across sites of metastasis, making protein quantification by biopsy highly challenging. A global, non-invasive methodology such as positron emission tomography (PET) imaging can permit serial quantification of HER3, providing a useful approach to monitor HER3 expression across the entire tumor burden both prior to and following treatment. PET imaging of HER3 expression may permit a more personalized approach to targeted therapy by allowing for detection of HER3-mediated resistance, in addition to informing clinical trial patient selection for novel therapies targeting HER3.

Procedures

Phage display selection targeting the HER3 extracellular domain was performed in order to develop a peptide with optimal blood clearance and highly accurate HER3 quantification.

Results

The selection converged to a consensus peptide sequence that was subsequently found to bind HER3 with an affinity of 270 ± 151 nM. The peptide, termed HER3P1, was bound with high selectivity to HER3 over other similar receptor tyrosine kinases such as EGFR and HER2. Furthermore, HER3P1 was able to distinguish between high and low HER3-expressing cells in vitro. The peptide was radiolabeled with Ga-68 and demonstrated to specifically bind HER3 by in vivo PET imaging. Uptake of [⁶⁸Ga]HER3P1 was highly specific for HER3-positive tumors, with tumor-to-background ratios ranging from 1.59–3.32, compared to those of HER3-negative tumors, ranging from 0.84–0.93. The uptake of [⁶⁸Ga]HER3P1 also demonstrated high (P < 0.001) correlation with protein expression as quantified by Western blot and confirmed by biodistribution.

Conclusions

HER3P1 accurately quantifies expression of HER3 by PET imaging and has potential utility as a clinical imaging agent.

     

PET Imaging with <Emphasis Type="Italic">S</Emphasis>-[<Superscript>11</Superscript>C]Methyl-L-Cysteine and L-[<Emphasis Type="Italic">Methyl</Emphasis>-<Superscript>11</Superscript>C]Methionine in Rat Models of Glioma,Glioma Radiotherapy,and Neuroinflammation

Purpose

S-[¹¹C]-methyl-L-cysteine ([¹¹C]MCYS) has been claimed to offer higher tumor selectivity than L-[methyl- ¹¹C]methionine ([¹¹C]MET). We examined this claim in animal models.

Procedures

Rats with implanted untreated (n = 10) or irradiated (n = 7, 1 × 25 Gy, on day 8) orthotopic gliomas were scanned after 6, 9, and 12 days, using positron emission tomography. Rats with striatal injections of saline (n = 9) or bacterial lipopolysaccharide (n = 9) were scanned after 3 days.

Results

Uptake of the two tracers in untreated gliomas was similar. [¹¹C]MCYS was not accumulated in salivary glands, nasal epithelium, and healing wounds, in contrast to [¹¹C]MET, but showed 40 % higher accumulation in the healthy brain. Both tracers showed a reduced tumor uptake 4 days after irradiation and minor accumulation in inflamed striatum. [¹¹C]MCYS indicated higher lesion volumes than [¹¹C]MET (untreated tumor + 47 %; irradiated tumor up to + 500 %; LPS-inflamed striatum + 240 %).

Conclusions

[¹¹C]MCYS was less accumulated in some non-tumor tissues than [¹¹C]MET, but showed lower tumor-to-brain contrast.

     

Strain Differences Determine the Suitability of Animal Models for Noninvasive <Emphasis Type="Italic">In Vivo</Emphasis> Beta Cell Mass Determination with Radiolabeled Exendin

Purpose

Noninvasive beta cell mass (BCM) quantification is a crucial tool to understand diabetes development and progression. [¹¹¹In]exendin is a promising agent for in vivo beta cell imaging, but tracer testing has been hampered by the lack of well-defined rodent models.

Procedures

Biodistribution and pancreatic uptake of [¹¹¹In]exendin were compared in rats and mice. In selected models, the amount of [¹¹¹In]exendin accumulation in the pancreas and other organs was determined using a model of alloxan-induced beta cell loss. GLP-1R expression levels were analyzed by RT-PCR and immunohistochemistry.

Results

Namely Brown Norway rats showed beta-cell-specific tracer accumulation and favorable pancreas-to-background ratios for noninvasive BCM determination. Mice displayed receptor-mediated [¹¹¹In]exendin uptake in endocrine and exocrine pancreas, in spite of very low GLP-1R expression in exocrine tissue.

Conclusions

Rats display better characteristics for in vivo BCM determination than mice and are suggested as a more adequate model for humans.

     

Detection of Enzyme Activity and Inhibition during Studies in Solution, <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> with CatalyCEST MRI

Purpose

The detection of enzyme activities and evaluation of enzyme inhibitors have been challenging with magnetic resonance imaging (MRI). To address this need, we have developed a diamagnetic, nonmetallic contrast agent and a protocol known as catalyCEST MRI that uses chemical exchange saturation transfer (CEST) to detect enzyme activity as well as enzyme inhibition.

Procedures

We synthesized a diamagnetic MRI contrast agent that has enzyme responsive and enzyme unresponsive CEST signals. We tested the ability of this agent to detect the activity of kallikrein 6 (KLK6) in biochemical solutions, in vitro and in vivo, with and without a KLK6 inhibitor.

Results

The agent detected KLK6 activity in solution and also detected KLK6 inhibition by antithrombin III. KLK6 activity was detected during in vitro studies with HCT116 colon cancer cells, relative to the detection of almost no activity in a KLK6-knockdown HCT116 cell line and HCT116 cells treated with antithrombin III inhibitor. Finally, strong enzyme activity was detected within an in vivo HCT116 tumor model, while lower enzyme activity was detected in a KLK6 knockdown tumor model and in the HCT116 tumor model treated with antithrombin III inhibitor. In all cases, comparisons of the enzyme responsive and enzyme unresponsive CEST signals were critical for the detection of enzyme activity.

Conclusions

This study has established that catalyCEST MRI with an exogenous diaCEST agent can evaluate enzyme activity and inhibition in solution, in vitro and in vivo.

     

<Emphasis Type="Italic">L</Emphasis><Subscript><Emphasis Type="Italic">p</Emphasis></Subscript> Regularization for Bioluminescence Tomography Based on the Split Bregman Method

Purpose

Bioluminescence tomography (BLT) is a promising in vivo optical imaging technique in preclinical research at cellular and molecular levels. The problem of BLT reconstruction is quite ill-posed and ill-conditioned. In order to achieve high accuracy and efficiency for its inverse reconstruction, we proposed a novel approach based on L _p regularization with the Split Bregman method.

Procedures

The diffusion equation was used as the forward model. Then, we defined the objective function of L _p regularization and developed a Split Bregman iteration algorithm to optimize this function. After that, we conducted numerical simulations and in vivo experiments to evaluate the accuracy and efficiency of the proposed method.

Results

The results of the simulations indicated that compared with the conjugate gradient and iterative shrinkage methods, the proposed method is more accurate and faster for multisource reconstructions. Furthermore, in vivo imaging suggested that it could clearly distinguish the viable and apoptotic tumor regions.

Conclusions

The Split Bregman iteration method is able to minimize the L _p regularization problem and achieve fast and accurate reconstruction in BLT.

     

Synthesis and <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of [<Superscript>3</Superscript>H]LRRK2-IN-1 as a Novel Radioligand for LRRK2

Purpose

LRRK2 (leucine-rich repeat kinase 2) has recently been proven to be a promising drug target for Parkinson’s disease (PD) due to an apparent enhanced activity caused by mutations associated with familial PD. To date, there have been no reports in which a LRRK2 inhibitor has been radiolabeled and used for in in vitro or in vivo studies of LRRK2. In the present study, we radiolabeled the LRRK2 ligand, LRRK-IN-1, for the purposes of performing in vitro (IC₅₀, K _d , B _max, autoradiography) and in vivo (biodistribution, and blocking experiments) evaluations in rodents and human striatum tissues.

Procedures

[³H]LRRK2-IN-1 was prepared with high radiochemical purity (>99 %) and a specific activity of 41 Ci/mmol via tritium/hydrogen (T/H) exchange using Crabtree’s catalyst. For IC⁵⁰, K _d , and B _max determination, LRRK2-IN-1 was used as a competing drug for nonspecific binding assessment. The specific binding of the tracer was further evaluated via an in vivo blocking study in mice with a potent LRRK2 inhibitor, Pf-06447475.

Results

In vitro binding studies demonstrated a saturable binding site for [³H]LRRK2-IN-1 in rat kidney, rat brain striatum and human brain striatum with K _d of 26 ± 3 and 43 ± 8, 48 ± 2 nM, respectively. In rat, the density of LRRK2 binding sites (B _max) was higher in kidney (6.4 ± 0.04 pmol/mg) than in brain (2.5 ± 0.03 pmol/mg), however, in human brain striatum, the B _max was 0.73 ± 0.01 pmol/mg protein. Autoradiography imaging in striatum of rat and human brain tissues gave results consistent with binding studies. In in vivo biodistribution and blocking studies in mice, co-administration with Pf-06447475 (10 mg/kg) reduced the uptake of [³H]LRRK2-IN-1 (%ID/g) by 50–60% in the kidney or brain.

Conclusion

The high LRRK2 brain density observed in our study suggests the feasibility for positron emission tomography imaging of LRRK2 (a potential target) with radioligands of higher affinity and specificity.

     

Hybrid Light Imaging Using Cerenkov Luminescence and Liquid Scintillation for Preclinical Optical Imaging <Emphasis Type="Italic">In Vivo</Emphasis>

Purpose

Cerenkov luminescence imaging (CLI) has recently emerged as a molecular imaging modality for radionuclides emitting β-particles. The aim of this study was to develop a hybrid light imaging (HLI) technique using a liquid scintillator to assist CLI by increasing the optical signal intensity from both β-particle and γ-ray emitting radionuclides located at deep regions in vivo.

Procedures

A commercial optical imaging system was employed to collect all images by HLI and CLI. To investigate the performance characteristics of HLI with a commercially available liquid scintillator (Emulsifier-safe), phantom experiments were conducted for two typical β-particle and γ-ray emitters, sodium iodide (Na[¹³¹I]I) and 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG), respectively. To evaluate the feasibility of HLI for in vivo imaging, HLI was applied to a Na[¹³¹I]I injected nu/nu mouse and an [¹⁸F]FDG injected Balb-c mouse and compared with CLI alone.

Results

Measured HLI wavelength spectra with Emulsifier-safe showed higher signal intensities than for CLI at 500–600 nm. For material preventing light transmission of 12-mm thickness, CLI imaging provided quite low intensity and obscure signals of the source. However, despite degraded spatial resolution, HLI imaging provided sustained visualization of the source shape, with signal intensities 10–14 times higher than for CLI at 10-mm thickness. Furthermore, at 0, 4, and 8-mm material thicknesses, HLI showed a strong correlation between Na[¹³¹I]I or [¹⁸F]FDG radioactivity and signal intensity, as for CLI. In vivo studies also demonstrated that HLI could successfully visualize Na[¹³¹I]I uptake in the mouse thyroid gland in the prone position and [¹⁸F]FDG accumulation in the heart in the supine position, which were not observed with CLI.

Conclusion

Our preliminary studies suggest that HLI can provide enhanced imaging of a β-particle probe emitting together with γ-rays at deep tissue locations. HLI may be a promising imaging technique to assist with preclinical in vivo imaging using CLI.

     

Non-Invasive Glutamine PET Reflects Pharmacological Inhibition of BRAF<Superscript>V600E</Superscript><Emphasis Type="Italic">In Vivo</Emphasis>

Purpose

This study aimed to study whether cancer cells possess distinguishing metabolic features compared with surrounding normal cells, such as increased glutamine uptake. Given this, quantitative measures of glutamine uptake may reflect critical processes in oncology. Approximately, 10 % of patients with colorectal cancer (CRC) express BRAF ^V600E , which may be actionable with selective BRAF inhibitors or in combination with inhibitors of complementary signaling axes. Non-invasive and quantitative predictive measures of response to these targeted therapies remain poorly developed in this setting. The primary objective of this study was to explore 4-[¹⁸F]fluoroglutamine (4-[¹⁸F]F-GLN) positron emission tomography (PET) to predict response to BRAF^V600E-targeted therapy in preclinical models of colon cancer.

Procedures

Tumor microarrays from patients with primary human colon cancers (n = 115) and CRC liver metastases (n = 111) were used to evaluate the prevalence of ASCT2, the primary glutamine transporter in oncology, by immunohistochemistry. Subsequently, 4-[¹⁸F]F-GLN PET was evaluated in mouse models of human BRAF ^V600E -expressing and BRAF wild-type CRC.

Results

Approximately 70 % of primary colon cancers and 53 % of metastases exhibited positive ASCT2 immunoreactivity, suggesting that [¹⁸F]4-F-GLN PET could be applicable to a majority of patients with colon cancer. ASCT2 expression was not associated selectively with the expression of mutant BRAF. Decreased 4-[¹⁸F]F-GLN predicted pharmacological response to single-agent BRAF and combination BRAF and PI3K/mTOR inhibition in BRAF ^V600E -mutant Colo-205 tumors. In contrast, a similar decrease was not observed in BRAF wild-type HCT-116 tumors, a setting where BRAF^V600E-targeted therapies are ineffective.

Conclusions

4-[¹⁸F]F-GLN PET selectively reflected pharmacodynamic response to BRAF inhibition when compared with 2-deoxy-2[¹⁸F]fluoro-d-glucose PET, which was decreased non-specifically for all treated cohorts, regardless of downstream pathway inhibition. These findings illustrate the utility of non-invasive PET imaging measures of glutamine uptake to selectively predict response to BRAF-targeted therapy in colon cancer and may suggest further opportunities to inform colon cancer clinical trials using targeted therapies against MAPK activation.

     

<Emphasis Type="Italic">In vivo</Emphasis> Biodistribution of Radiolabeled Acoustic Protein Nanostructures

Purpose

Contrast-enhanced ultrasound plays an expanding role in oncology, but its applicability to molecular imaging is hindered by a lack of nanoscale contrast agents that can reach targets outside the vasculature. Gas vesicles (GVs)—a unique class of gas-filled protein nanostructures—have recently been introduced as a promising new class of ultrasound contrast agents that can potentially access the extravascular space and be modified for molecular targeting. The purpose of the present study is to determine the quantitative biodistribution of GVs, which is critical for their development as imaging agents.

Procedures

We use a novel bioorthogonal radiolabeling strategy to prepare technetium-99m-radiolabeled ([^99mTc])GVs in high radiochemical purity. We use single photon emission computed tomography (SPECT) and tissue counting to quantitatively assess GV biodistribution in mice.

Results

Twenty minutes following administration to mice, the SPECT biodistribution shows that 84 % of [^99mTc]GVs are taken up by the reticuloendothelial system (RES) and 13 % are found in the gall bladder and duodenum. Quantitative tissue counting shows that the uptake (mean ± SEM % of injected dose/organ) is 0.6 ± 0.2 for the gall bladder, 46.2 ± 3.1 for the liver, 1.91 ± 0.16 for the lungs, and 1.3 ± 0.3 for the spleen. Fluorescence imaging confirmed the presence of GVs in RES.

Conclusions

These results provide essential information for the development of GVs as targeted nanoscale imaging agents for ultrasound.

     

<Emphasis Type="Italic">In Vivo</Emphasis> Stabilized SB3, an Attractive GRPR Antagonist,for Pre- and Intra-Operative Imaging for Prostate Cancer

Purpose

The gastrin-releasing peptide receptor (GRPR), overexpressed on various tumor types, is an attractive target for receptor-mediated imaging and therapy. Another interesting approach would be the use of GRPR radioligands for pre-operative imaging and subsequent radio-guided surgery, with the goal to improve surgical outcome. GRPR radioligands were successfully implemented in clinical studies, especially Sarabesin 3 (SB3) is an appealing GRPR antagonist with high receptor affinity. Gallium-68 labeled SB3 has good in vivo stability, after labeling with Indium-111; however, the molecule shows poor in vivo stability, which negatively impacts tumor-targeting capacity. A novel approach to increase in vivo stability of radiopeptides is by co-administration of the neutral endopeptidase (NEP) inhibitor, phosphoramidon (PA). We studied in vivo stability and biodistribution of [¹¹¹In]SB3 without/with (?/+) PA in mice. Furthermore, SPECT/MRI on a novel, state-of-the-art platform was performed.

Procedures

GRPR affinity of SB3 was determined on PC295 xenograft sections using [¹²⁵I]Tyr⁴-bombesin with tracer only or with increasing concentrations of SB3. For in vivo stability, mice were injected with 200/2000 pmol [¹¹¹In]SB3 ?/+ 300 μg PA. Blood was collected and analyzed. Biodistribution and SPECT/MRI studies were performed at 1, 4, and 24 h postinjection (p.i.) of 2.5 MBq/200 pmol or 25 MBq/200 pmol [¹¹¹In]SB3 ?/+ 300 μg PA in PC-3-xenografted mice.

Results

SB3 showed high affinity for GRPR (IC₅₀ 3.5 nM). Co-administration of PA resulted in twice higher intact peptide in vivo vs [¹¹¹In]SB3 alone. Biodistribution studies at 1, 4, and 24 h p.i. show higher tumor uptake values with PA co-administration (19.7?±?3.5 vs 10.2?±?1.5, 17.6?±?5.1 vs 8.3?±?1.1, 6.5?±?3.3 vs 3.1?±?1.9 % ID/g tissue (P?<?0.0001)). Tumor imaging with SPECT/MRI clearly improved after co-injection of PA.

Conclusions

Co-administration of PA increased in vivo tumor targeting capacity of [¹¹¹In]SB3, making this an attractive combination for GRPR-targeted tumor imaging.

     

Detection of <Emphasis Type="Italic">Klebsiella. Pneumoniae</Emphasis> Infection with an Antisense Oligomer Against its Ribosomal RNA

Purpose

Previously, we demonstrated specific accumulation into bacteria of a 12-mer phosphorodiamidate morpholino (MORF) oligomer complementary to a ribosomal RNA (rRNA) segment found in all bacteria using the universal probe called Eub338 (Eub). Here, two MORF oligomers Eco and Kpn with sequences specific to the rRNA of Escherichia coli (Eco) and Klebsiella pneumoniae (Kpn) were investigated along with Eub and control (nonEub).

Procedures

To determine bacterial rRNA binding, oligomers were tagged with Alexa Fluor 633 (AF633) for fluorescence in situ hybridization (FISH) and fluorescence microscopy, and radiolabeled with technetium-99m (Tc-99m) for biodistribution and SPECT imaging in infected mice.

Results

By both FISH and fluorescence microscopy, Eub showed a positive signal in both E. coli and K. pneumoniae as expected, and Kpn showed significantly higher accumulation in K. pneumoniae with near background in E. coli (p?<?0.01). Conversely, Eco was positive in both E. coli and K. pneumoniae, hence nonspecific. As determined by biodistribution, the accumulation of [^99mTc]Kpn was higher in the thigh infected with live K. pneumoniae than with live E. coli (p?=?0.05), and significantly higher than with heat-killed K. pneumoniae (p?=?0.02) in the target thigh. By SPECT imaging, the accumulation of [^99mTc]Kpn was obviously higher in its specific target of K. pneumoniae compared to an E. coli infected thigh.

Conclusions

Kpn complementary to the rRNA of K. pneumoniae, labeled with Tc-99m or AF633, demonstrated specific binding to fixed and live K. pneumoniae in culture and in infected mice such that Tc-99m-labeled Kpn as the MORF oligomer may be useful for K. pneumoniae infection detection through imaging.

     

Tri-modal <Emphasis Type="Italic">In vivo</Emphasis> Imaging of Pancreatic Islets Transplanted Subcutaneously in Mice

Purpose

Transplantation of pancreatic islets (PIs) is a promising therapeutic approach for type 1 diabetes. The main obstacle for this strategy is that the outcome of islet engraftment depends on the engraftment site. It was our aim to develop a strategy for using non-invasive imaging techniques to assess the location and fate of transplanted PIs longitudinally in vivo.

Procedures

In order to overcome the limitations of individual imaging techniques and cross-validate findings by different modalities, we have combined fluorine magnetic resonance imaging (F-19 MRI), fluorescence imaging (FLI), and bioluminescent imaging (BLI) for studying subcutaneously transplanted PIs and beta cell-like cells (INS-1E cell line) in vivo. We optimized the transduction (using lentiviral vectors) and labeling procedures (using perfluoro crown ether nanoparticles with a fluorescence dye) for PIs and INS-1E cell imaging.

Results

The feasibility of using the proposed imaging methods for PI assessment was demonstrated both in vitro and in vivo. Our data suggested that F-19 MRI is suitable for high-resolution localization of transplanted cells and PIs; FLI is essential for confirmation of contrast localization by histology; and BLI is a reliable method to assess cell viability and survival after transplantation. No significant side effects on cell viability and function have been observed.

Conclusions

The proposed tri-modal imaging platform is a valuable approach for the assessment of engrafted PIs in vivo. It is potentially suitable for comparing different transplantation sites and evaluating novel strategies for improving PI transplantation technique in the future.

     

Imaging of <Emphasis Type="Italic">Sleeping Beauty</Emphasis>-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography

Purpose

We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells.

Procedures

We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19⁺ artificial antigen-presenting cells.

Results

After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19⁺ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2′-deoxy-2′-[¹⁸F]fluoro-5-ethyl-1-β-d-arabinofuranosyl-uracil ([¹⁸F]FEAU).

Conclusion

This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

     

<Emphasis Type="Italic">In</Emphasis><Emphasis Type="Italic">Vivo</Emphasis> Bioluminescence Imaging of Transplanted Mesenchymal Stromal Cells and Their Rejection Mediated by Intrahepatic NK Cells

Purpose

Mesenchymal stromal cells (MSCs) hold promise in the treatment of liver disease. However, short survival time of MSCs after intrahepatic transplantation limits their value; therefore, understanding the basis of MSCs survival and rejection may increase their utility. This study was aimed at determining the role of intrahepatic natural killer (NK) cells on MSCs survival and their retention in the liver shortly after transplant.

Procedures

Human MSCs were labeled with the Luc2-mKate2 dual-fusion reporter gene (MSCs-R), and the residence time and survival of MSCs-R xenografts after intrahepatic transplantation were evaluated by in vivo bioluminescence imaging (BLI). Coculture of MSCs and NK cells was performed to assess cytotoxicity. To evaluate the role of NK cells in rejection of the xenografted cells, the fates of transplanted MSCs-R were then assessed in vivo by BLI after activation of intrahepatic NK cells.

Results

We observed a linear correlation between luciferase activity from live MSCs-R and cell number in vitro (R ²?=?0.9956). In vivo, we observed a gradual decline in bioluminescent signals from transplanted MSCs-R over a region corresponding to the liver in both the control group and the NK-activated group. However, the survival time and retention of intrahepatic MSCs-R decreased more rapidly in the NK-activated group of mice compared to the control group. This indicated that activated NK cells accelerate the elimination of transplanted MSCs. Also, we found that the number of hepatic NK cells and the expression of NK activation markers significantly increased after intrahepatic delivery of MSCs. This suggested that resident NK cells, in a resting state, were activated by intrahepatic transplantation of human MSCs. Taken together, the data suggests that activated hepatic NK cells mediate, in part, rejection of the MSCs xenografts. Cytotoxicity assays showed that activated NK cells may inhibit the proliferation of MSCs and, to a certain extent, induce MSCs death.

Conclusion

Human MSCs could be followed dynamically in vivo by BLI, and the role of murine hepatic NK cells, especially activated NK cells, could be inferred from the loss of signals from MSCs. This finding may have practical clinical implications in MSCs transplantation in treating liver disease.

     

Comparison of clinical MRI liver iron content measurements using signal intensity ratios, <Emphasis Type="Italic">R</Emphasis><Subscript>2</Subscript> and <Emphasis Type="Italic">R</Emphasis><Subscript>2</Subscript>*

Purpose

To compare three types of MRI liver iron content (LIC) measurement performed in daily clinical routine in a single center over a 6-year period.

Methods

Patients undergoing LIC MRI-scans (1.5T) at our center between January 1, 2008 and December 31, 2013 were retrospectively included. LIC was measured routinely with signal intensity ratio (SIR) and MR-relaxometry (R ₂ and R ₂*) methods. Three observers placed regions-of-interest. The success rate was the number of correctly acquired scans over the total number of scans. Interobserver agreement was assessed with intraclass correlation coefficients (ICC) and Bland–Altman analysis, correlations between LIC_SIR, R ₂, R ₂*, and serum values with Spearman’s rank correlation coefficient. Diagnostic accuracies of LIC_SIR, R ₂ and serum transferrin, transferrin-saturation, and ferritin compared to increased R ₂* (≥44 Hz) as indicator of iron overload were assessed using ROC-analysis.

Results

LIC MRI-scans were performed in 114 subjects. SIR, R ₂, and R ₂* data were successfully acquired in 102/114 (89%), 71/114 (62%), and 112/114 (98%) measurements, with the lowest success rate for R ₂. The ICCs of SIR, R ₂, and R ₂* did not differ at 0.998, 0.997, and 0.999. R ₂ and serum ferritin had the highest diagnostic accuracies to detect elevated R ₂* as mark of iron overload.

Conclusions

SIR and R ₂* are preferable over R ₂ in terms of success rates. R ₂*’s shorter acquisition time and wide range of measurable LIC values favor R ₂* over SIR for MRI-based LIC measurement.

     

MAO-B Inhibitors Do Not Block <Emphasis Type="Italic">In Vivo</Emphasis> Flortaucipir([<Superscript>18</Superscript>F]-AV-1451) Binding

Purpose

Recent evidence suggests that the tau radiotracer [¹⁸F]THK-5351 displays high affinity for the monoamine oxidase type B (MAO-B) enzyme. Utilizing another tau-tracer, flortaucipir ([¹⁸F]AV-1451), we previously reported that non-demented Parkinson’s disease patients show off-target binding in subcortical structures, but no appreciable cortical uptake. However, 59 % of these patients were receiving MAO-B inhibitors at the time of their scan. Here, we retrospectively investigated if MAO-B inhibitors in clinical doses affect flortaucipir binding.

Procedures

We compared the standard uptake values of flortaucipir at regional and voxel levels in Parkinson’s disease patients who received MAO-B inhibitors with those who did not.

Results

Sixteen of 27 Parkinson’s disease patients received MAO-B inhibitors at the time of scan. We found no significant flortaucipir uptake differences between the groups at voxel or regional levels.

Conclusion

Use of MAO-B inhibitors at pharmaceutical levels did not significantly affect flortaucipir binding. Thus, MAO-B does not appear to be a significant binding target of flortaucipir.

     

Bioluminescence Imaging of Colonization and Clearance Dynamics of <Emphasis Type="Italic">Brucella Suis</Emphasis> Vaccine Strain S2 in Mice and Guinea Pigs

Purpose

The goal of this study was to develop a plasmid-based lux bio-reporter for use to obtain in vivo images of Brucella suis vaccine strain 2 (B.suis S2) infection with high resolution and good definition.

Procedures

The pBBR-lux (pBBR1MCS-2-lxCDABE) plasmid that carries the luxCDABE operon was introduced into B. suis S2 by electroporation yielding B. suis S2-lux. The spatial and temporal transit of B. suis S2 in mice and guinea pigs was monitored by bioluminescence imaging.

Results

The plasmid pBBR-lux is stable in vivo and does not appear to impact the virulence or growth of bacteria. This sensitive luciferase reporter could represent B. suis S2 survival in real time. B. suis S2 mainly colonized the lungs, liver, spleen, and uterus in mice and guinea pigs as demonstrated by bioluminescence imaging.

Conclusion

The plasmid-based lux bioreporter strategy can be used to obtain high resolution in vivo images of B. suis S2 infection in mice and guinea pigs.