Utility of 99mTc-PEG4-E[PEG4-c(RGDfK)]2 in Posttherapy Surveillance of Patients with Reelevated Carcinoembryonic Antigen Levels

ObjectivesTo evaluate the efficacy of ^99mTc-PEG₄-E[PEG₄-c (RGDfK)]₂ (^99mTc-3PRGD2) single photon emission computed tomography (SPECT) in monitoring the recurrence of malignancies.Materials and Methods^99mTc-3PRGD2 SPECT was performed on 28 patients (10 females and 18 males; median age 49.2 years) suspected of recurrent malignancies due to an asymptomatically reelevated carcinoembryonic antigen level. The SPECT was performed 0.5 h after an intravenous injection of 11.1 MBq/kg (0.3 mCi/kg) of ^99mTc-3PRGD2. The SPECT and concurrent contrast-enhanced computed tomography (ceCT) findings were analyzed with reference to the histopathological findings and/or clinical follow-up data.ResultsRecurrences were identified in 20 out of the 28 patients (prevalence 71.4s%) with altogether 26 lesions. Fifteen lesions were confirmed by histopathological findings, and the other 11 lesions were confirmed by serial radiological or clinical follow-up. Of the 20 patients with recurrent malignancies, 12 (60s%) were correctly identified by ^99mTc-3PRGD2 SPECT. In the patient-based analysis, the sensitivity and specificity of ^99mTc-3PRGD2 SPECT were 60 and 100s%, respectively, and the positive and negative predictive values were 100 and 50s%, respectively. In the lesion-based evaluation, the sensitivity and specificity were 62 and 100s%, respectively. The sensitivity and specificity of the ceCT in the patient-based evaluation were 60 and 75s%, respectively, and the positive and negative predictive values were 86 and 40s%, respectively. In the lesion-based evaluation, the sensitivity and specificity of the ceCT were 70 and 84s%, respectively.Conclusions^99mTc-3PRGD2, as a new SPECT tracer targeting the integrin α_vβ₃ receptor, was more useful in distinguishing recurrences as compared to ceCT.Key Words: ^99mTc-PEG₄-E[PEG₄-c(RGDfK)]₂, Integrin α_vβ₃, Carcinoembryonic antigen, Recurrence, Single photon emission computed tomography     

Imaging VEGF Receptors and α<Subscript>v</Subscript>β<Subscript>3</Subscript> Integrins in a Mouse Hindlimb Ischemia Model of Peripheral Arterial Disease

Purpose

To compare targeted imaging of vascular endothelial growth factor (VEGF) receptors vs. α_vβ₃ integrins in a mouse hindlimb ischemia model of peripheral artery disease.

Procedures

Male wild-type (WT) C57BL/6 mice (8- to 10-week old) (n?=?24) underwent left femoral artery ligation. The right leg served as control. Five days later, mice were injected with either VEGF receptor targeting [^99mTc]DOTA-PEG-scVEGF ([^99mTc]scV) (n?=?8) or with α_vβ₃-targeting tracer [^99mTc]HYNIC-cycloRGD ([^99mTc]RGD) (n?=?8) and underwent single photon emission computed tomography (SPECT) x-ray computed tomography imaging. To assess non-specific [^99mTc]scV uptake, six additional mice received a mixture of [^99mTc]scV and 30-fold excess of targeting protein, scVEGF. Tracer uptake as %ID was measured using volumetric regions encompassing the hindlimb muscles and as %ID/g from harvested limb muscles. Double and triple immunofluorescent analysis on tissue sections established localization of α_vβ₃, VEGFR-1, VEGFR-2, as well as certain cell lineage markers.

Results

Tracer uptake, as %ID/g, was higher in ligated limbs of mice injected with [^99mTc]scV compared to ligated hindlimbs in mice injected with [^99mTc]RGD (p?=?0.02). The ratio of tracer uptake for ligated/control hindlimb was borderline higher for [^99mTc]scV than for [^99mTc]RGD (p?=?0.06). Immunofluorescent analysis showed higher prevalence of VEGFR-1, VEGFR-2, and α_vβ₃, in damaged vs. undamaged hindlimb tissue, but with little co-localization of these markers. Double immunofluorescent staining showed partial co-localization of VEGFR-1, VEGFR-2, and α_vβ_3, with endothelial cell marker FVIII, but not with CD31. Immunostaining for VEGFR-1 and VEGFR-2 additionally co-localized with lineage markers for endothelial progenitor cell and monocytes/macrophages, with a more diverse pattern of co-localization for VEGFR-2.

Conclusion

In a mouse hindlimb ischemia model of peripheral artery disease, [^99mTc]scV SPECT tracer-targeting VEGF receptors showed a more robust signal than [^99mTc]RGD tracer-targeting α_vβ₃. Immunofluorescent analysis suggests that uptake of [^99mTc]scV and [^99mTc]RGD in damaged tissue is due to non-overlapping cell populations and reflects different dynamic processes and that enhanced uptake of [^99mTc]scV may be due to the presence of VEGF receptors on additional cell types.

     

Tumor Cell Uptake of 99mTc-Labeled 1-Thio-β-d-Glucose and 5-Thio-d-Glucose in Comparison with 2-Deoxy-2-[18 F]Fluoro-d-Glucose In Vitro: Kinetics,Dependencies, Blockage and Cell Compartment of Accumulation

Purpose

This study was conducted to investigate the capacity of ^99mTc-labeled 1-thio-β-d-glucose (^99mTc-1-TG) and 5-thio-d-glucose (^99mTc-5-TG) to act as a marker for glucose metabolism in tumor cells in vitro.

Procedures

We investigated the cellular uptake of ^99mTc-1-TG, ^99mTc-5-TG, and 2-deoxy-2-[¹⁸?F]fluoro-d-glucose(¹⁸?F-FDG) in a human colorectal carcinoma and human lung adenocarcinoma cell line (HCT-116, A549) at different time points and varying glucose/insulin concentrations and under transporter blockage by cytochalasin-B and phloretin. Cell compartment analysis was performed.

Results

A significant uptake and time dependency thereof, a significant uptake dependency on glucose and insulin and a significant uptake inhibition by cytochalasin-B for ^99mTc-1-TG and ^99mTc-5-TG, was shown. Albeit substantial, the uptake was less pronounced in ^99mTc-1-TG and ^99mTc-5-TG compared with ¹⁸?F-FDG. ^99mTc-1-TG and ^99mTc-5-TG showed a higher accumulation in the cell membranes compared with ¹⁸?F-FDG.

Conclusion

Tc-1-TG and ^99mTc-5-TG showed an uptake in vitro with glucose analog characteristics but with membranous accumulation. Tumor imaging should be investigated in an animal model.     

SPECT Imaging with <Superscript>99m</Superscript>Tc-Labeled EGFR-Specific Nanobody for <Emphasis Type="Italic">In Vivo</Emphasis> Monitoring of EGFR Expression

Purpose

Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnostics and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody®, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice.

Methods

Fluorescence-activated cell sorting (FACS) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The Nanobody was then labeled with ^99mTc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of ^99mTc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU145) overexpression of EGFR.

Results

FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of ^99mTc-8B6 Nanobody is relatively fast (half-life, 1.5 h) and mainly via the kidneys. At 3 h postinjection, total kidney accumulation is high (46.6?±?0.9%IA) compared to total liver uptake (18.9?±?0.6%IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2?±?0.5%IA/cm³) of ^99mTc-8B6 Nanobody compared to DU145 xenografts (1.8?±?0.3%IA/cm³, p?

Conclusion

The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with ^99mTc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.

     

ImmunoPET Imaging of α<Subscript>v</Subscript>β<Subscript>6</Subscript> Expression Using an Engineered Anti-α<Subscript>v</Subscript>β<Subscript>6</Subscript> Cys-diabody Site-Specifically Radiolabeled with Cu-64: Considerations for Optimal Imaging with Antibody Fragments

Purpose

Increased expression of the α_vβ₆ integrin correlates with advanced tumor grade and poor clinical outcome, identifying α_vβ₆ as a prognostic indicator and an attractive target for molecular imaging. This work investigated the ability of a disulfide-stabilized [⁶⁴Cu]NOTA-α_vβ₆ cys-diabody to image α_vβ₆ expression in vivo using a nu/nu mouse model bearing human melanoma xenografts and positron-emission tomography.

Procedures

Small-animal positron emission tomography (PET) imaging, quantitative ROI analysis, and ex vivo biodistribution were conducted to ascertain tumor uptake and organ distribution of the [⁶⁴Cu]NOTA-α_vβ₆ cys-diabody. Immunohistochemical staining of tumors and mouse organs and immunoreactivity assays were utilized to correlate in vivo and ex vivo observations.

Results

PET imaging of the [⁶⁴Cu]NOTA-α_vβ₆ cys-diabody revealed low tumor uptake at 24 h p.i. in DX3Puroβ₆ tumors (2.69 ± 0.45 %ID/g) with comparable results found in the DX3Puro tumors (2.24 ± 0.15 %ID/g). Quantitative biodistribution confirmed that DX3Puroβ₆ tumor uptake was highest at 24 h p.i. (4.63 ± 0.18 %ID/g); however, uptake was also observed in the stomach (4.84 ± 2.99 %ID/g), small intestines (4.50 ± 1.69 %ID/g), large intestines (4.73 ± 0.97 %ID/g), gallbladder (6.04 ± 1.88 %ID/g), and lungs (3.89 ± 0.69 %ID/g).

Conclusions

Small-animal PET imaging was successful in visualizing α_vβ₆-positive tumor uptake of the [⁶⁴Cu]NOTA-α_vβ₆ cys-diabody. Cys-diabody cross-reactivity was observed between human and murine α_vβ₆ and immunohistochemical staining confirmed the presence of an endogenous α_vβ₆ antigen sink, which led to suboptimal tumor contrast in this mouse model. Future investigations will focus on dose escalation studies to overcome the endogenous antigen sink while increasing DX3Puroβ₆ tumor uptake.

     

MicroPET Imaging of Integrin ��v��3 Expressing Tumors Using 89Zr-RGD Peptides

Purpose

The dimeric transmembrane integrin, ??_v??₃, is a well-investigated target by different imaging modalities through suitably labeled arginine?Cglycine?Caspartic acid (RGD) containing peptides. In this study, we labeled four cyclic RGD peptides with or without PEG functional groups: c(RGDfK) (denoted as FK), PEG₃-c(RGDfK) (denoted as FK-PEG₃), E[c(RGDfK)]₂ (denoted as [FK]₂), and PEG₄-E[PEG₄-c(RGDfK)]₂ (denoted as [FK]₂-3PEG₄), with ⁸⁹Zr (t _1/2?=?78.4 h), using the chelator desferrioxamine-p-SCN (Df) for imaging tumor integrin ??_v??₃.

Methods

The Df conjugated RGD peptides were subjected to integrin ??_v??₃ binding assay in vitro using MDA-MB-435 breast cancer cells. The ⁸⁹Zr-labeled RGD peptides were then subjected to small animal positron emission tomography (PET) and direct tissue sampling biodistribution studies in an orthotopic MDA-MB-435 breast cancer xenograft model.

Results

All four tracers, ⁸⁹Zr-Df-FK, ⁸⁹Zr-Df-FK-PEG₃, ⁸⁹Zr-Df-[FK]₂, and ⁸⁹Zr-Df-[FK]₂-3PEG₄, were labeled in high radiochemical yield (89?±?4%) and high specific activity (4.07?C6 MBq/??g). Competitive binding assay with ¹²⁵I-echistatin showed that conjugation of the RGD peptides to the Df chelator did not have significant impact on their integrin ??_v??₃ binding affinity and the dimeric peptides were shown to be more potent than the monomers. In agreement with binding results, tumor uptake of ⁸⁹Zr-Df-[FK]₂ and ⁸⁹Zr-Df-[FK]₂-3PEG₄ was significantly higher (4.32?±?1.73%ID/g and 4.72?±?0.66%ID/g, respectively, at 2 h post-injection) than the monomers ⁸⁹Zr-Df-FK and ⁸⁹Zr-Df-FK-PEG₃ (1.97?±?0.38%ID/g and 1.57?±?0.49%ID/g, respectively, at 2 h post-injection). Out of the four labeled peptides, ⁸⁹Zr-Df-[FK]₂-3PEG₄ gave the highest tumor-to-background ratio (18.21?±?2.52 at 2 h post-injection and 19.69?±?3.99 at 4 h post-injection), with the lowest uptake in metabolic organs. Analysis of late time points biodistribution data revealed that the uptake in the tumor was decreased, along with increase in the bone, which implies decomplexation of ⁸⁹Zr-Df.

Conclusion

Efficient radiolabeling of peptides with an appropriate chelator such as Df-RGD with ⁸⁹Zr was observed. The ⁸⁹Zr radiolabeled peptides provided high-quality and high-resolution microPET images in xenograft models. ⁸⁹Zr-Df-[FK]₂-3PEG₄ demonstrated the highest tumor-to-background ratio of the compounds tested. Preparation of ⁸⁹Zr peptides to take advantage of the longer half-life is unwarranted due to the relatively rapid clearance from the tumor region of peptide tracers prepared for this study and the increased uptake in the bone of transchelated ⁸⁹Zr with time (2.0?±?0.36%ID/g, 24 h post-injection).     

Comparison of [<Superscript>99m</Superscript>Tc]3PRGD<Subscript>2</Subscript> Imaging and [<Superscript>18</Superscript>F]FDG PET/CT in Breast Cancer and Expression of Integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> in Breast Cancer Vascular Endothelial Cells

Purpose

This study aimed to investigate the value of ^99mtechnetium-three polyethylene glycol spacers-arginine-glycine-aspartic acid ([^99mTc]3PRGD₂) imaging in diagnosis and staging of breast cancer compared with 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) imaging, and to explore the expression of integrin α_vβ₃ in tumor vascular endothelial cells.

Procedures

Forty-two women with suspected breast cancer underwent both [^99mTc]3PRGD₂ imaging and [¹⁸F]FDG imaging. Visual analysis was used to assess primary breast lesion, axillary lymph node, and distant metastasis. The tumor-blood (T/B) ratios from [^99mTc]3PRGD₂ imaging and the maximum standardized uptake value (SUVmax) from [¹⁸F]FDG imaging were analyzed for breast lesions. Integrin α_vβ₃ was analyzed through immunohistochemistry.

Results

Forty-five breast lesions were found (malignant, n?=?38; benign, n?=?7). The sensitivity, specificity, and accuracy of [^99mTc]3PRGD₂ and [¹⁸F]FDG imaging in visual analysis for the breast lesion were 97.4, 87.5, and 95.6 % and 97.4, 71.4, and 93.3 %, respectively (P?>?0.05). For semi-quantitative analysis, no significant difference of the area under the curves (AUC) was found in the imaging using the two radiopharmaceuticals (0.880 and 0.955; Z?=?0.88, P?>?0.05). The sensitivity, specificity, and accuracy for axillary lymph node metastasis with [^99mTc]3PRGD₂ and [¹⁸F]FDG were 78.05, 99.36, and 94.92 % and 85.37, 98.72, and 95.64 %, respectively (P?>?0.05). Nine patients with distant metastases were all detected with the two radiopharmaceuticals. The expression of integrin α_vβ₃ was correlated with [^99mTc]3PRGD₂ uptake (r?=?0.582, P?=?0.001), which were significantly higher in the HER2-positive and stage III–IV patients (P?<?0.05).

Conclusions

The prospective study demonstrated that [^99mTc]3PRGD₂ imaging seems to be valuable for diagnosis of breast cancer and its staging. It may be less sensitive for detecting small lymph node metastatic lesions when compared with [¹⁸F]FDG imaging. Integrin α_vβ₃ in tumor microvessels was associated with the breast cancer subtype and its staging.

     

Efficient Construction of PET/Fluorescence Probe Based on Sarcophagine Cage: An Opportunity to Integrate Diagnosis with Treatment

Purpose

Due to the shortage of established platforms/methods for multimodality probe construction, in this study, we developed a heterofunctional chelator, BaAn(Boc)Sar, from sarcophagine cage as a general platform for dual-modality probe construction.

Procedures

A dual-modality probe for positron-emission tomography (PET) and fluorescence imaging was synthesized using the developed BaAn(Boc)Sar chelator. The c(RGDyK)₂ peptide (denoted as RGD₂) and fluorescence dye Cy5.5 were conjugated with BaAn(Boc)Sar to form BaAnSar-RGD₂-Cy5.5. Then, BaAnSar-RGD₂-Cy5.5 was labeled with ⁶⁴Cu in ammonium acetate buffer. PET and fluorescent imaging were carried out to evaluate ⁶⁴Cu-BaAnSar-RGD₂-Cy5.5 in nude mice bearing U87MG glioblastoma xenograft.

Results

The BaAnSar-RGD₂-Cy5.5 was labeled with ⁶⁴Cu very efficiently in 0.1?M NH₄OAc buffer within 10?min at 37?°C in the yield of 86.7?±?4.4?% (n?=?3). The specific activity of ⁶⁴Cu-BaBaSar-RGD₂ was controlled at 50?C200?mCi/??mol for the consideration of both PET and optical imaging. MicroPET quantification analysis shows that the U87MG tumor uptake is 6.41?±?0.28, 6.51?±?1.45, and 5.92?±?1.57?%ID/g at 1, 4, and 20?h postinjection, respectively. Good correlation was obtained between the tumor to muscle ratios measured by the radioactivity and fluorescence intensity. As a proof of concept, an animal surgery study demonstrated that this dual-modality probe would greatly benefit the patients because the PET moiety could be used for tumor detection, and the fluorescent moiety would allow image-guided surgery.

Conclusions

Our findings demonstrated the effectiveness and feasibility of preparing dual-modality imaging probes based on the sarcophagine scaffold. The resulting PET and fluorescent imaging probe also holds a great potential for clinical translation.     

Immunoglobulin G (IgG)-Based Imaging Probe Accumulates in M1 Macrophage-Infiltrated Atherosclerotic Plaques Independent of IgG Target Molecule Expression

Purpose

Vulnerable plaques are key factors for ischemic diseases. Thus, their precise detection is necessary for the diagnosis of such diseases. Immunoglobulin G (IgG)-based imaging probes have been developed for imaging biomolecules related to plaque formation for the diagnosis of atherosclerosis. However, IgG accumulates nonspecifically in atherosclerotic regions, and its accumulation mechanisms have not yet been clarified in detail. Therefore, we explored IgG accumulation mechanisms in atherosclerotic lesions and examined images of radiolabeled IgG for the diagnosis of atherosclerosis.

Procedures

Mouse IgG without specificity to biomolecules was labeled with technetium-99m via 6-hydrazinonicotinate to yield [^99mTc]IgG. ApoE^?/? or C57BL/6J mice were injected intravenously with [^99mTc]IgG, and their aortas were excised 24 h after injection. After radioactivity measurement, serial aortic sections were autoradiographically and histopathologically examined. RAW264.7 macrophages were polarized into M1 or M2 and then treated with [^99mTc]IgG. The radioactivities in the cells were measured after 1 h of incubation. [^99mTc]IgG uptake in M1 macrophages was also evaluated after the pretreatment with an anti-Fcγ receptor (FcγR) antibody. The expression levels of FcγRs in the cells were measured by western blot analysis.

Results

[^99mTc]IgG accumulation levels in the aortas were significantly higher in apoE^?/? mice than in C57BL/6J mice (5.1 ± 1.4 vs 2.8 ± 0.5 %ID/g, p < 0.05). Autoradiographic images showed that the accumulation areas highly correlated with the macrophage-infiltrated areas. M1 macrophages showed significantly higher levels of [^99mTc]IgG than M2 or M0 (nonpolarized) macrophages [2.2 ± 0.3 (M1) vs 0.5 ± 0.1 (M2), 0.4 ± 0.1 (M0) %dose/mg protein, p < 0.01] and higher expression levels of FcγRI and FcγRII. [^99mTc]IgG accumulation in M1 macrophages was suppressed by pretreatment with the anti-FcγR antibody [2.2 ± 0.3 (nonpretreatment) vs 1.2 ± 0.2 (pretreatment) %ID/mg protein, p < 0.01].

Conclusions

IgG accumulated in pro-inflammatory M1 macrophages via FcγRs in atherosclerotic lesions. Thus, the target biomolecule-independent imaging of active inflammation should be taken into account in the diagnosis of atherosclerosis using IgG-based probes.

     

Blood Clearance Kinetics,Biodistribution, and Radiation Dosimetry of a Kit-Formulated Integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript>-Selective Radiotracer <Superscript>99m</Superscript>Tc-3PRGD<Subscript>2</Subscript> in Non-Human Primates

Purpose  

^99mTc-3PRGD₂ is a ^99mTc-labeled dimeric cyclic RGD peptide with increased receptor binding affinity and improved kinetics for in vivo imaging of integrin α_vβ₃ expression in nude mouse model. To accelerate its clinical translation, we reported here the evaluation of the kit-formulated ^99mTc-3PRGD₂ in healthy cynomolgus primates for its blood clearance kinetics, biodistribution, and radiation dosimetry.     

In Vitro and In Vivo Structure–Property Relationship of 68Ga-Labeled Schiff Base Derivatives for Functional Myocardial PET Imaging

Purpose

SPECT (e.g., with ^99mTc-sestamibi) is routinely used for imaging myocardial damage, even though PET could offer a higher spatial resolution. Using the generator-gained isotope ⁶⁸Ga would allow a rapid supply of the tracer in the diagnostic unit. For this reason, the aim of the study was to develop ⁶⁸Ga-labeled PET tracers based on different Schiff base amines and to evaluate the cardiomyocyte uptake in vitro as well as the biodistribution of the tracers in vivo.

Procedures

Fifteen different Schiff bases (basing on 3 different backbones) were synthesized and labeled with ⁶⁸Ga. Lipophilicity varied between 0.87?±?0.24 and 2.72?±?0.14 (logD value). All tracers were positively charged and stable in plasma and apo-transferrin solution. In vitro uptake into cardiomyocytes was assessed in HL-1 cells in the absence and presence of the ionophor valinomycin. In vivo accumulation in the heart and in various organs was assessed by small animal PET imaging as well as by ex vivo biodistribution. The results were compared with ^99mTc-sestamibi and ¹⁸F-flurpiridaz.

Results

All cationic Schiff bases were taken up into cardiomyocytes but the amount varied by a factor of 10. When destroying the membrane potential, the cellular uptake was markedly reduced in most of the tracers, indicating the applicability of these tracers for identifying ischemic myocardium. PET imaging revealed that the in vivo myocardial uptake reached a constant value approximately 10 min after injection but the intracardial amount of the tracer varied profoundly (SUV 0.46 to 3.35). The most suitable tracers showed a myocardial uptake which was comparable to that of ^99mTc-sestamibi.

Conclusions

⁶⁸Ga-based Schiff bases appear suitable for myocardial PET images with uptake comparable to ^99mTc-sestamibi but offering higher spatial resolution. By systematical variation of the backbone and the side chains, tracers with optimal properties can be identified for further clinical evaluation.     

Positron Emission Tomography Imaging of Endometrial Cancer Using Engineered Anti-EMP2 Antibody Fragments

Purpose

As imaging of the cell surface tetraspan protein epithelial membrane protein-2 (EMP2) expression in malignant tumors may provide important prognostic and predictive diagnostic information, the goal of this study is to determine if antibody fragments to EMP2 may be useful for imaging EMP2 positive tumors.

Procedures

The normal tissue distribution of EMP2 protein expression was evaluated by immunohistochemistry and found to be discretely expressed in both mouse and human tissues. To detect EMP2 in tumors, a recombinant human anti-EMP2 minibody (scFv-hinge-C_H3 dimer; 80 kDa) was designed to recognize a common epitope in mice and humans and characterized. In human tumor cell lines, the antibody binding induced EMP2 internalization and degradation, prompting the need for a residualizing imaging strategy. Following conjugation to DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N′,N′″-tetraacetic acid), the minibody was radiolabeled with ⁶⁴Cu (t _1/2?=?12.7 h) and evaluated in mice as a positron emission tomography (PET) imaging agent for human EMP2-expressing endometrial tumor xenografts.

Results

The residualizing agent, ⁶⁴Cu-DOTA anti-EMP2 minibody, achieved high uptake in endometrial cancer xenografts overexpressing EMP2 (10.2?±?2.6, percent injected dose per gram (%ID/g) ± SD) with moderate uptake in wild-type HEC1A tumors (6.0?±?0.1). In both cases, precise tumor delineation was observed from the PET images. In contrast, low uptake was observed with anti-EMP2 minibodies in EMP2-negative tumors (1.9?±?0.5).

Conclusions

This new immune-PET agent may be useful for preclinical assessment of anti-EMP2 targeting in vivo. It may also have value for imaging of tumor localization and therapeutic response in patients with EMP2-positive malignancies.     

Radiolabeled RGD Tracer Kinetics Annotates Differential αvβ3 Integrin Expression Linked to Cell Intrinsic and Vessel Expression

Purpose

The purpose of this paper is to study the association between RGD binding kinetics and α_vβ₃ integrin receptor density in the complex tumor milieu.

Procedures

We assessed α_vβ₃ in vitro and by ⁶⁸Ga-DOTA-[c(RGDfK)]₂ positron emission tomography (PET) in tumors with varying α_vβ₃.

Results

Intrinsic α_vβ₃ expression decreased in the order of M21?>>>?MDA-MB-231?>?M21L in cells. Tumor volume of distribution by PET, V _T, was significantly higher in M21 compared to isogenic M21L tumors (0.40?±?0.01 versus 0.25?±?0.02; p?<?0.01) despite similar microvessel density (MVD) likely due to higher α_vβ₃. V _T for MDA-MB-231 (0.40?±?0.04) was comparable to M21 despite lower α_vβ₃ but in keeping with the higher MVD, suggesting superior tracer distribution.

Conclusions

This study demonstrates that radioligand binding kinetics of PET data can be used to discriminate tumors with different α_vβ₃ integrin expression—a key component of the angiogenesis phenotype—in vivo.     

Non-invasive PET Imaging of PARP1 Expression in Glioblastoma Models

Purpose

The current study presents [¹⁸F]PARPi as imaging agent for PARP1 expression.

Procedures

[¹⁸F]PARPi was generated by conjugating a 2H-phthalazin-1-one scaffold to 4-[¹⁸F]fluorobenzoic acid. Biochemical assays, optical in vivo competition, biodistribution analysis, positron emission tomography (PET)/X-ray computed tomography, and PET/magnetic resonance imaging studies were performed in subcutaneous and orthotopic mouse models of glioblastoma.

Results

[¹⁸F]PARPi shows suitable pharmacokinetic properties for brain tumor imaging (IC₅₀?=?2.8?±?1.1 nM; logP_CHI?=?2.15?±?0.41; plasma-free fraction?=?63.9?±?12.6 %) and accumulates selectively in orthotopic brain tumor tissue. Tracer accumulation in subcutaneous brain tumors was 1.82?±?0.21 %ID/g, whereas in healthy brain, the uptake was only 0.04?±?0.01 %ID/g.

Conclusions

[¹⁸F]PARPi is a selective PARP1 imaging agent that can be used to visualize glioblastoma in xenograft and orthotopic mouse models with high precision and good signal/noise ratios. It offers new opportunities to non-invasively image tumor growth and monitor interventions.

     

Initial Experience with the Radiotracer Anti-1-amino-3-[18F]Fluorocyclobutane-1-Carboxylic Acid (Anti-[18F]FACBC) with PET in Renal Carcinoma

Purpose

Assessment of renal masses with conventional imaging may be challenging. Anti-1-amino-3-[¹⁸F]fluorocyclobutane-1-carboxylic acid (anti-[18F]FACBC) is a synthetic l-leucine analog with relatively little renal excretion. The present study examines anti-[¹⁸F]FACBC positron emission tomography uptake in patients with renal masses.

Procedures

Six patients with seven renal lesions were imaged dynamically for 2 h after injection of 10–10.9 mCi (370–403 MBq) anti-[¹⁸F]FACBC. Lesions were evaluated qualitatively and quantitatively and correlated with histology.

Results

Four clear cell and one Rosai–Dorfman lesion were hypo/isointense to normal cortex; two papillary lesions in the same patient were hyperintense. Mean SUV_max?±?SD at 30 min was 2.8?±?0.24 for clear cell carcinomas and 4.5?±?1.7 for papillary cell lesions. Mean SUV_max/SUV_mean ratios?±?SD of lesion to normal cortex at 30 min was 1.15?±?0.19 for the clear cell carcinomas and 2.3?±?0.84 for papillary cell.

Conclusions

In this small patient sample, relative amino acid transport compared with renal cortex is elevated in renal papillary cell carcinoma but not in clear cell carcinoma.     

Pilot Study of a Novel 18F-labeled FSHR Probe for Tumor Imaging

Purpose

Follicle-stimulating hormone receptor (FSHR) is overexpressed in primary and metastatic tumor. Molecular imaging of FSHR is beneficial for prognosis and therapy of cancer. FSHβ(33–53) (YTRDLVYKDPARPKIQKTCTF), denoted as FSH1, is a FSHR antagonist. In the present study, maleimide-NOTA conjugate of FSH1 (NOTA-MAL-FSH1) was designed and labeled with [¹⁸F] aluminum fluoride. The resulting tracer, ¹⁸F-Al-NOTA-MAL-FSH1, was preliminarily evaluated in PET imaging of FSHR-positive tumor.

Procedures

NOTA-MAL-FSH1 was synthesized and radiolabeled with Al¹⁸F complex. The tumor-targeting potential and pharmacokinetic profile of the ¹⁸F-labeled compound were evaluated in vitro and in vivo using a PC3 human prostate tumor model.

Results

¹⁸F-Al-NOTA-MAL-FSH1 can be efficiently produced within 30 min with a non-decay-corrected yield of 48.6?±?2.1 % and a radiochemical purity of more than 95 %. The specific activity was at least 30 GBq/μmol. The radiotracer was stable in phosphate-buffered saline and human serum for at least 2 h. The IC₅₀ values of displacement ¹⁸F-Al-NOTA-MAL-FSH1 with FSH1 were 252?±?1.12 nM. The PC3 human prostate tumor xenografts were clearly visible with high contrast after injection of ¹⁸F-Al-NOTA-MAL-FSH1 via microPET. At 30, 60 and 120 min postinjection, the tumor uptakes were 2.98?±?0.29 % injected dose (ID)/g, 2.53?±?0.20 %ID/g and 1.36?±?0.12 %ID/g, respectively. Dynamic PET scanning showed that tumor uptake reached a plateau by about 6 min. Heart peaked earlier and then cleared quickly. Biodistribution studies confirmed that the normal organs except kidney uptakes were all below 1 %ID/g at 1 h p.i. The tumor-to-blood and tumor-to-muscle ratio at 10 min, 0.5, 1, and 2 h after injection were 1.64?±?0.36, 2.97?±?0.40, 9.31?±?1.06, and 13.59?±?2.33 and 7.05?±?1.10, 10.10?±?1.48, 16.17?±?3.29, and 30.88?±?4.67, respectively. The tracer was excreted mainly through the renal system, as evidenced by high levels of radioactivity in the kidneys. FSHR-binding specificity was also demonstrated by reduced tumor uptake of ¹⁸F-Al-NOTA-MAL-FSH1 after coinjection with an excess of unlabeled FSH1 peptide.

Conclusion

NOTA-MAL-FSH1 could be labeled rapidly and efficiently with ¹⁸F using one step method. Favorable preclinical data suggest that ¹⁸F-Al-NOTA-MAL-FSH1 may be a suitable radiotracer for the non-invasive visualization of FSHR positive tumor in vivo.     

In Vivo Imaging of Sentinel Nodes Using Fluorescent Silica Nanoparticles in Living Mice

Purpose

We examine the feasibility of fluorescent imaging system for sentinel lymph node detection by using functionalized silica nanoparticles.

Materials and Methods

We developed a functionalized RITC-SiO₂ nanoparticles containing fluorescent dye, C₂₈H₃₁N₂O₃Cl (rhodamine B isothiocyanate) inside, and subsequently synthesized ⁶⁸Ga-NOTA-RITC-SiO₂ nanoparticles.

Results

At 5 min after RITC-doped silica nanoparticles injection, fluorescent signals were shown in both right axillary lymph node (ALN) and injection site of living mice. Fluorescent signals were also observed at these locations in a biodistribution study. In addition, fluorescence was detected in frozen ALN sections microscopically. The percentages of doses injected per gram of tissue of axillary and brachial lymph nodes near footpad treated with ⁶⁸Ga-NOTA-RITC-SiO₂ nanoparticles were 308.3?±?3.4 and 41.5?±?6.1, respectively. Little ⁶⁸Ga radioactivity was found in other organs.

Conclusion

Our data provide strong evidence that functionalized silica nanoparticles has a promising potential as organic lymphatic tracer in biomedical imaging such as pre- and intraoperative surgical guidance.     

In Vivo Molecular Imaging of Apoptosisand Necrosis in Atherosclerotic PlaquesUsing MicroSPECT-CT and MicroPET-CT Imaging

Purpose

The purpose of this paper is to study molecular imaging of apoptosis and necrosis, two key players in atherosclerosis instability, using a multimodal imaging approach combining single photon emission computed tomography (SPECT), positron emission tomography (PET), and computed tomography (CT).

Procedures

Collar-induced carotid atherosclerosis ApoE knockout mice were imaged with ^99mTc-AnxAF568 SPECT-CT to study apoptosis and sequentially with PET-CT following ¹²⁴I-Hypericin (¹²⁴I-Hyp) injection to visualize necrosis.

Results

SPECT depicted increased ^99mTc-AnxAF568 uptake in both atherosclerotic carotid arteries, whereas our data suggest that this uptake is not merely apoptosis related. Although PET of ¹²⁴I-Hyp was hampered by the slow blood clearance in atherosclerotic mice, ¹²⁴I-Hyp was able to target necrosis in the atherosclerotic plaque.

Conclusion

Both ^99mTc-AnxAF568 and ¹²⁴I-Hyp uptake are increased in atherosclerotic carotid vasculature compared to control arteries. While apoptosis imaging remains challenging, necrosis imaging can be feasible after improving the biodistribution characteristics of the probe.     

Selective Imaging of Vascular Endothelial Growth Factor Receptor-1 and Receptor-2 in Atherosclerotic Lesions in Diabetic and Non-diabetic ApoE<Superscript>−/−</Superscript> Mice

Purpose

Plaque vulnerability is associated with inflammation and angiogenesis, processes that rely on vascular endothelial growth factor (VEGF) signaling via two receptors, VEGFR-1 and VEGFR-2. We have recently reported that enhanced uptake of scVEGF-PEG-DOTA/Tc-99m (scV/Tc) single photon emission computed tomography (SPECT) tracer that targets both VEGFR-1 and VEGFR-2, identifies accelerated atherosclerosis in diabetic relative to non-diabetic ApoE^?/? mice. Since VEGFR-1 and VEGFR-2 may play different roles in atherosclerotic plaques, we reasoned that selective imaging of each receptor can provide more detailed information on plaque biology.

Procedures

Recently described VEGFR-1 and VEGFR-2 selective mutants of scVEGF, named scVR1 and scVR2, were site-specifically derivatized with Tc-99m chelator DOTA via 3.4 kDa PEG linker, and their selectivity to the cognate receptors was confirmed in vitro. scVR1 and scVR2 conjugates were radiolabeled with Tc-99m to specific activity of 110 ± 11 MBq/nmol, yielding tracers named scVR1/Tc and scVR2/Tc. 34–40 week old diabetic and age-matched non-diabetic ApoE^?/? mice were injected with tracers, 2–3 h later injected with x-ray computed tomography (CT) contrast agent and underwent hybrid SPECT/CT imaging. Tracer uptake, localized to proximal aorta and brachiocephalic vessels, was quantified as %ID from. Tracer uptake was also quantified as %ID/g from gamma counting of harvested plaques. Harvested atherosclerotic arterial tissue was used for immunofluorescent analyses of VEGFR-1 and VEGFR-2 and various lineage-specific markers.

Results

Focal, receptor-mediated uptake in proximal aorta and brachiocephalic vessels was detected for both scVR1/Tc and scVR2/Tc tracers. Uptake of scVR1/Tc and scVR2/Tc was efficiently inhibited only by “cold” proteins of the same receptor selectivity. Tracer uptake in this area, expressed as %ID, was higher in diabetic vs. non- diabetic mice for scVR1/Tc (p = 0.01) but not for scVR2/Tc. Immunofluorescent analysis revealed enhanced VEGFR-1 prevalence in and around plaque area in diabetic mice.

Conclusions

Selective VEGFR-1 and VEGFR-2 imaging of atherosclerotic lesions may be useful to explore plaque biology and identify vulnerability.

     

Longitudinal PET Imaging of Doxorubicin-Induced Cell Death with 18F-Annexin V

Purpose

This study aims to apply longitudinal positron emission tomography (PET) imaging with ¹⁸?F-Annexin V to visualize and evaluate cell death induced by doxorubicin in a human head and neck squamous cell cancer UM-SCC-22B tumor xenograft model.

Procedures

In vitro toxicity of doxorubicin to UM-SCC-22B cells was determined by a colorimetric assay. Recombinant human Annexin V protein was expressed and purified. The protein was labeled with fluorescein isothiocyanate for fluorescence staining and ¹⁸?F for PET imaging. Established UM-SCC-22B tumors in nude mice were treated with two doses of doxorubicin (10?mg/kg each dose) with 1?day interval. Longitudinal ¹⁸?F-Annexin V PET was performed at 6?h, 24?h, 3?days, and 7?days after the treatment started. Following PET imaging, direct tissue biodistribution study was performed to confirm the accuracy of PET quantification.

Results

Two doses of doxorubicin effectively inhibited the growth of UM-SCC-22B tumors by inducing cell death including apoptosis. The cell death was clearly visualized by ¹⁸?F-Annexin V PET. The peak tumor uptake, which was observed at day 3 after treatment started, was significantly higher than that in the untreated tumors (1.56?±?0.23 vs. 0.89?±?0.31%ID/g, p?<?0.05). Moreover, the tumor uptake could be blocked by co-injection of excess amount of unlabeled Annexin V protein. At day 7 after treatment, the tumor uptake of ¹⁸?F-Annexin had returned to baseline level.

Conclusions

¹⁸?F-Annexin V PET imaging is sensitive enough to allow visualization of doxorubicin-induced cell death in UM-SCC-22B xenograft model. The longitudinal imaging with ¹⁸?F-Annexin will be helpful to monitor early response to chemotherapeutic anti-cancer drugs.