Diagnosis of Melanoma with 61Cu-Labeled PET Tracer.

Until the recent years, substances containing radioactive 61Cu were strongly considered as potential positron-emitting radiopharmaceuticals for use in positron emission tomography (PET) applications; however, due to their suitably long half-life, and generator-independent and cost-effective production, they seem to be economically viable for human imaging. Since malignant melanoma (MM) is a major public health problem, its early diagnosis is a crucial contributor to long-term survival, which can be achieved using radiolabeled α-melanocyte-stimulating hormone analog NAPamide derivatives. Here, we report on the physicochemical features of a new CB-15aneN5-based Cu(II) complex ([Cu(KFTGdiac)]-) and the ex vivo and in vivo characterization of its NAPamide conjugate. The rigid chelate possesses prompt complex formation and suitable inertness (t1/2 = 18.4 min in 5.0 M HCl at 50 °C), as well as excellent features in the diagnosis of B16-F10 melanoma tumors (T/M(SUVs) (in vivo): 12.7, %ID/g: 6.6 ± 0.3, T/M (ex vivo): 22).

Positron emission tomography guided synergistic treatment of melanoma using multifunctional zirconium-hematoporphyrin nanosonosensitizers.

Sonodynamic therapy (SDT) has emerged as a useful approach for tumor treatment. However, its widespread application is impeded by poor pharmacokinetics of existing sonosensitizers. Here we developed a metal-organic nanoplatform, wherein a small-molecule sonosensitizer (hematoporphyrin monomethyl ether, HMME) was ingeniously coordinated with zirconium, resulting in a multifunctional nanosonosensitizer termed Zr-HMME. Through post-synthetic modifications involving PEGylation and tumor-targeting peptide (F3) linkage, a nanoplatform capable of homing on melanoma was produced, which could elicit robust immune responses to suppress tumor lung metastasis in the host organism. Importantly, after seamless incorporation of positron-emitting 89Zr into this nanosonosensitizer, positron emission tomography (PET) could be used to monitor its in vivo pharmacokinetics. PET imaging studies revealed that this nanoplatform exhibited potent tumor accumulation and strong in vivo stability. Using intrinsic fluorescence from HMME, a dual-modal diagnostic capability (fluorescence and PET) was confirmed for this nanosonosensitizer. In addition, the mechanisms of how this nanoplatform interacted with immune system were also investigated. The collective data proved that the coordination structure between small-molecule drug cargos and metals may enhance the functions of each other while mitigating their weaknesses. This straightforward approach can expand the potential applications of suitable drug molecules.

Investigating Effects of IR-780 in Animal Models of B16-F10 Melanoma: New Approach in Lung Metastasis.

IR-780 is a fluorescent marker, photostable and non-toxic, and is widely used in tumor targeting; however, studies on the impact of IR-780 in animal models of B16-F10 melanoma are scarce in the literature. Therefore, this study aims to analyze behavior of this marker in melanoma cells using in vitro and in vivo analyses with fluorescence microscopy to conduct an analysis of cell culture, and an in vivo imaging system for an analysis of cell culture, tumor targeting on animals, and organ examination. In vitro analysis showed that B16-F10 cells at a concentration of 2 × 105 cells.plate-1 allowed a better visualization using 20 µM of IR-780. Furthermore, the location of IR-780 accumulation was confirmed by its fluorescence microscopy. Through in vivo studies, fluorescence was not observed in subcutaneous nodules, and it was found that animals that received intraperitoneal injection of B16-F10 cells presented ascites and did not absorb IR-780. Additionally, animals exhibiting lung metastasis showed fluorescence in ex vivo lung images. Therefore, use of the IR-780 marker for evaluating the progression of tumor growth did not demonstrate efficiency; however, it was effective in diagnosing pulmonary metastatic tumors. Although this marker presented limitations, results of evaluating pulmonary involvement through ex vivo fluorescence imaging were determined based on intensity of fluorescence.

In Vivo evaluation of newly synthesized 213Bi-conjugated alpha-melanocyte stimulating hormone (α-MSH) peptide analogues in melanocortin-1 receptor (MC1-R) positive experimental melanoma model.

Given the rising pervasiveness of melanocortin-1 receptor (MC1-R) positive melanoma malignum (MM) and pertinent metastases, radiolabelled receptor-affine alpha-melanocyte stimulating hormone-analogue (α-MSH analogue) imaging probes would be of crucial importance in timely tumor diagnostic assessment. Herein we aimed at investigating the biodistribution and the MM targeting potential of newly synthesized 213Bi-conjugated MC1-R specific peptide-based radioligands with the establishment of MC1-R overexpressing MM preclinical model. DOTA-conjugated NAP, -HOLD, -FOLD, -and MARSamide were labelled with 213Bi. Ex vivo biodistribution studies were conducted post-administration of 3.81 ± 0.32 MBq [213Bi]Bi-DOTA conjugated deriva-tives into twenty B16-F10 tumor-bearing C57BL/6 J and healthy mice. Organ Level Internal Dose Assessment (OLINDA) and IDAC-Dose were used to calculate translational data-based absorbed radiation dose in human organs. Moderate or low %ID/g uptake of [213Bi]Bi-DOTA conjugated NAP, -HOLD, -and MARSamide and significantly increased [213Bi]Bi-DOTA-FOLDamide accumulation was observed in the thoracic and abdominal organs (p ≤ 0.01). High [213Bi]Bi-DOTA-NAP (%ID/g:3.76 ± 0.96), -and FOLDamide (%ID/g:3.28 ± 0.95) tumor tracer activity confirmed their MC1-R-affinity. The bladder wall received the highest radiation absorbed dose followed by the kidneys (bladder wall: 1.95·10-2 and 8.97·10-2 mSv/MBq; kidneys: 7.47·10-3 vs. 5.88·10-2 mSv/MBq measured by IDAC and OLINDA; respectively) indicating the suitability of the NAPamide derivative for clinical use. These novel [213Bi]Bi-DOTA-linked peptide probes displaying meaningful MC1-R affinity could be promising molecular probes in MM imaging.

Synthesis and Preclinical Evaluation of a 68Ga-Labeled Pyridine-Based Benzamide Dimer for Malignant Melanoma Imaging.

Benzamide (BZA), a small molecule that can freely cross cell membranes and bind to melanin, has served as an effective targeting group for melanoma theranostics. In this study, a novel pyridine-based BZA dimer (denoted as H-2) was labeled with 68Ga ([68Ga]Ga-H-2) for positron emission tomography (PET) imaging of malignant melanomas. [68Ga]Ga-H-2 was obtained with high radiochemical yield (98.0 ± 2.0%) and satisfactory radiochemical purity (>95.0%). The specificity and affinity of [68Ga]Ga-H-2 were confirmed in melanoma B16F10 cells and in vivo PET imaging of multiple tumor models (B16F10 tumors, A375 melanoma, and lung metastases). Monomeric [68Ga]Ga-H-1 was prepared as a control radiotracer to verify the effects of the molecular structure on pharmacokinetics. The values of the lipid-water partition coefficient of [68Ga]Ga-H-2 and [68Ga]Ga-H-1 demonstrated hydrophilicity with log P = -2.37 ± 0.07 and -2.02 ± 0.09, respectively. PET imaging and biodistribution showed a higher uptake of [68Ga]Ga-H-2 in B16F10 primary and metastatic melanomas than that in A375 melanomas. However, the relatively low uptake of monomeric [68Ga]Ga-H-1 in B16F10 tumors and high accumulation in nontarget organs resulted in poor PET imaging quality. This study demonstrates the synthesis and preclinical evaluation of the novel pyridine-based BZA dimer [68Ga]Ga-H-2 and indicates that the dimer tracer has promising applications in malignant melanoma-specific PET imaging because of its high uptake and long-time retention in malignant melanoma.

61Cu-Labelled radiodiagnostics of melanoma with NAPamide-targeted radiopharmaceutical.

Malignant melanoma is a major public health problem with an increasing incidence and mortality in the Caucasian population due to its significant metastatic potential. The early detection of this cancer type by imaging techniques like positron emission tomography acts as an important contributor to the long-term survival. Based on literature data, the radio labelled alpha-MSH analog NAPamide molecule is an appropriate diagnostic tool for the detection of melanoma tumors. Inspired by these facts, a new radiotracer, the [61Cu]Cu-KFTG-NAPamide has been synthesized to exploit the beneficial features of the positron emitter 61Cu and the melanoma specificity of the NAPamide molecule. In this work, we report a new member of the CB-15aneN5 ligand family (KFTG) as the chelator for 61Cu(II) complexation. On the basis of the thorough physico-chemical characterization, the rigid [Cu(KFTG)]+ complex exhibits fast complex formation (t1/2 = 155 s at pH 5.0 and 25 °C) and high inertness (t1/2 = 2.0 h in 5.0 M HCl at 50 °C) as well as moderate superoxide dismutase activity (IC50 = 2.3 µM). Furthermore, the [61Cu]Cu-KFTG-NAPamide possesses outstanding features in the diagnostics of B16-F10 melanoma tumors by PET imaging: (T/M(SUVs) (in vivo): appr. 14, %ID/g: 7 ± 1 and T/M (ex vivo): 315 ± 24 at 180 min).

Investigation of the Distribution of Magnetic Nanoparticles in Tumor Tissues by the Method of Scanning Magnetic Force Nanotomography.

The development of effective biomedical technologies using magnetic nanoparticles (MNPs) for the tasks of oncotherapy and nanodiagnostics requires the development and implementation of new methods for the analysis of micro- and nanoscale distributions of MNPs in the volume of cells and tissues. The paper presents a new approach to three-dimensional analysis of MNP distributions - scanning magnetic force nanotomography as applied to the study of tumor tissues. Correlative reconstruction of MNP distributions and nanostructure features of the studied tissues made it possible to quantitatively estimate the parameters of three-dimensional distributions of composite nanoparticles based on silicon and iron oxide obtained by femtosecond laser ablation and injected intravenously and intratumorally into tumor tissue samples of B16/F1 mouse melanoma. The developed technology based on the principles of scanning probe nanotomography is applicable for studying the features of three-dimensional micro- and nanoscale distributions of magnetic nanoparticles in biomaterials, cells and tissues of various types.

Maximizing response to intratumoral immunotherapy in mice by tuning local retention.

Direct injection of therapies into tumors has emerged as an administration route capable of achieving high local drug exposure and strong anti-tumor response. A diverse array of immune agonists ranging in size and target are under development as local immunotherapies. However, due to the relatively recent adoption of intratumoral administration, the pharmacokinetics of locally-injected biologics remains poorly defined, limiting rational design of tumor-localized immunotherapies. Here we define a pharmacokinetic framework for biologics injected intratumorally that can predict tumor exposure and effectiveness. We find empirically and computationally that extending the tumor exposure of locally-injected interleukin-2 by increasing molecular size and/or improving matrix-targeting affinity improves therapeutic efficacy in mice. By tracking the distribution of intratumorally-injected proteins using positron emission tomography, we observe size-dependent enhancement in tumor exposure occurs by slowing the rate of diffusive escape from the tumor and by increasing partitioning to an apparent viscous region of the tumor. In elucidating how molecular weight and matrix binding interplay to determine tumor exposure, our model can aid in the design of intratumoral therapies to exert maximal therapeutic effect.

Phototheranostic Metal-Phenolic Networks with Antiexosomal PD-L1 Enhanced Ferroptosis for Synergistic Immunotherapy.

Tumor-derived exosome can suppress dendritic cells (DCs) and T cells functions. Excessive secretion of exosomal programmed death-ligand 1 (PD-L1) results in therapeutic resistance to PD-1/PD-L1 immunotherapy and clinical failure. Restored T cells by antiexosomal PD-L1 tactic can intensify ferroptosis of tumor cells and vice versa. Diminishing exosomal suppression and establishing a nexus of antiexosomal PD-L1 and ferroptosis may rescue the discouraging antitumor immunity. Here, we engineered phototheranostic metal-phenolic networks (PFG MPNs) by an assembly of semiconductor polymers encapsulating ferroptosis inducer (Fe3+) and exosome inhibitor (GW4869). The PFG MPNs elicited superior near-infrared II fluorescence/photoacoustic imaging tracking performance for a precise photothermal therapy (PTT). PTT-augmented immunogenic cell death relieved exosomal silencing on DC maturation. GW4869 mediated PD-L1 based exosomal inhibition revitalized T cells and enhanced the ferroptosis. This novel synergy of PTT with antiexosomal PD-L1 enhanced ferroptosis evoked potent antitumor immunity in B16F10 tumors and immunological memory against metastatic tumors in lymph nodes.

The Effect of Albumin-Binding Moiety on Tumor Targeting and Biodistribution Properties of 67Ga-Labeled Albumin Binder-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides.

Background: The purpose of this study was to examine the effect of 4-p-(tolyl)butyric acid as an albumin-binding (ALB) moiety on tumor targeting and biodistribution properties of 67Ga-labeled albumin binder-conjugated alpha-melanocyte-stimulating hormone peptides. Materials and Methods: DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(ALB)-Gly/GlyGly/GlyGlyGly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} were synthesized with 4-p-(tolyl)butyric acid serving as an ALB moiety. The melanocortin-1 receptor (MC1R)-binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of 67Ga-DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex was examined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 67Ga-DOTA-Lys(ALB)-GGNle-CycMSHhex {67Ga-ALB-G2} were determined on B16/F10 melanoma-bearing C57 mice. Results: The IC50 value of DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {ALB-G1, ALB-G2, ALB-G3} was 0.67 ± 0.07, 0.5 ± 0.09 and 0.51 ± 0.03 nM on B16/F10 cells, respectively. 67Ga-ALB-G2 was further evaluated as a lead peptide because of its higher tumor uptake (30.25 ± 3.24%ID/g) and lower kidney uptake (7.09 ± 2.22%ID/g) than 67Ga-ALB-G1 and 67Ga-ALB-G3 at 2 h postinjection. The B16/F10 melanoma uptake of 67Ga-ALB-G2 was 15.64 ± 4.55, 30.25 ± 3.24, 26.76 ± 3.23, and 10.71 ± 1.21%ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 67Ga-ALB-G2 as an imaging probe at 2 h postinjection. Conclusions: The introduction of 4-p-(tolyl)butyric acid as an ALB moiety increased the blood retention, and resulted in higher tumor/kidney ratio of 67Ga-ALB-G2 as compared with its counterpart without an albumin binder. However, the resulting high uptake of 67Ga-ALB-G2 in blood and liver need to be further reduced to facilitate its therapeutic application when replacing 67Ga with therapeutic radionuclides.

Chromatic-aberration-free multispectral optical-resolution photoacoustic microscopy using reflective optics and a supercontinuum light source.

A supercontinuum (SC) light source enables multispectral photoacoustic imaging at excitation wavelengths in the visible-to-near-infrared range. However, for such a broad optical wavelength range, chromatic aberration is non-negligible. We developed a multispectral optical-resolution photoacoustic microscopy (MS-OR-PAM) setup with a nanosecond pulsed SC light source and a reflective objective lens to avoid chromatic aberration. Chromatic aberrations generated by reflective and conventional objective lenses were compared, and the images acquired using the reflective objective were not affected by chromatic aberration. Hence, MS-OR-PAM with the reflective objective was used to distinguish red blood cells from melanoma cells via spectral subtraction processing.

Photodynamic Therapy of Melanoma B16 with Chlorin E6 Conjugated with a PSMA-Ligand.

The efficacy of a new photosensitizer of chlorin E6 conjugated with a prostate-specific membrane antigen (PSMA) in photodynamic therapy of murine melanoma B16 was studied in in vivo experiments. The dynamics of photosensitizer accumulation in the tumor and surrounding tissues was evaluated and antitumor efficacy of photodynamic therapy was assessed by parameters of regression and morphological characteristics of experimental transplanted melanoma B16. The inhibitory effect of photodynamic therapy on melanoma was evaluated by complete regression of the tumor, absolute tumor growth coefficient in animals with continuation of tumor growth, and the increase in life span in comparison with the control; the criterion of cure was the absence of signs of tumor recurrence in mice within 90 days after therapy. The therapeutic potential of photodynamic therapy was determined by devitalization of tumor cells (histological examination of the zones of laser exposure on day 21 after treatment). The photosensitizer with PSMA-ligand exhibited high antitumor activity in photodynamic therapy for melanoma B16. Photodynamic therapy carried out at the optimum time after photosensitizer injection with experimentally determined parameters of laser exposure allows achieving the maximum inhibitory effect on melanoma. Pathomorphological study in the zones of exposure detected no survived tumor cells.

In vitro and in vivo comparison of MRI chemical exchange saturation transfer (CEST) properties between native glucose and 3-O-Methyl-D-glucose in a murine tumor model.

D-Glucose and 3-O-Methyl-D-glucose (3OMG) have been shown to provide contrast in magnetic resonance imaging-chemical exchange saturation transfer (MRI-CEST) images. However, a systematic comparison between these two molecules has yet to be performed. The current study deals with the assessment of the effect of pH, saturation power level (B1 ) and magnetic field strength (B0 ) on the MRI-CEST contrast with the aim of comparing the in vivo CEST contrast detectability of these two agents in the glucoCEST procedure. Phosphate-buffered solutions of D-Glucose or 3OMG (20 mM) were prepared at different pH values and Z-spectra were acquired at several B1 levels at 37°C. In vivo glucoCEST images were obtained at 3 and 7 T over a period of 30 min after injection of D-Glucose or 3OMG (at doses of 1.5 or 3 g/kg) in a murine melanoma tumor model (n = 3-5 mice for each molecule, dose and B0 field). A markedly different pH dependence of CEST response was observed in vitro for D-Glucose and 3OMG. The glucoCEST contrast enhancement in the tumor region following intravenous administration (at the 3 g/kg dose) was comparable for both molecules: 1%-2% at 3 T and 2%-3% at 7 T. The percentage change in saturation transfer that resulted was almost constant for 3OMG over the 30-min period, whereas a significant increase was detected for D-Glucose. Our results show similar CEST contrast efficiency but different temporal kinetics for the metabolizable and the nonmetabolizable glucose derivatives in a tumor murine model when administered at the same doses.

Development of Radiofluorinated Nicotinamide/Picolinamide Derivatives as Diagnostic Probes for the Detection of Melanoma.

Regarding the increased incidence and high mortality rate of malignant melanoma, practical early-detection methods are essential to improve patients' clinical outcomes. In this study, we successfully prepared novel picolinamide-benzamide (18F-FPABZA) and nicotinamide-benzamide (18F-FNABZA) conjugates and determined their biological characteristics. The radiochemical yields of 18F-FPABZA and 18F-FNABZA were 26 ± 5% and 1 ± 0.5%, respectively. 18F-FPABZA was more lipophilic (log P = 1.48) than 18F-FNABZA (log P = 0.68). The cellular uptake of 18F-FPABZA in melanotic B16F10 cells was relatively higher than that of 18F-FNABZA at 15 min post-incubation. However, both radiotracers did not retain in amelanotic A375 cells. The tumor-to-muscle ratios of 18F-FPABZA-injected B16F10 tumor-bearing mice increased from 7.6 ± 0.4 at 15 min post-injection (p.i.) to 27.5 ± 16.6 at 3 h p.i., while those administered with 18F-FNABZA did not show a similarly dramatic increase throughout the experimental period. The results obtained from biodistribution studies were consistent with those derived from microPET imaging. This study demonstrated that 18F-FPABZA is a promising melanin-targeting positron emission tomography (PET) probe for melanotic melanoma.

Tumor perfusion evaluation using dynamic contrast-enhanced ultrasound after electrochemotherapy and IL-12 plasmid electrotransfer in murine melanoma.

Electrochemotherapy with bleomycin (ECT BLM) is an effective antitumor treatment already used in clinical oncology. However, ECT alone is still considered a local antitumor therapy because it cannot induce systemic immunity. When combined with adjuvant gene electrotransfer of plasmid DNA encoding IL-12 (GET pIL-12), the combined therapy leads to a systemic effect on untreated tumors and distant metastases. Although the antitumor efficacy of both therapies alone or in combination has been demonstrated at both preclinical and clinical levels, data on the predictors of efficacy of the treatments are still lacking. Herein, we evaluated the results of dynamic contrast-enhanced ultrasound (DCE-US) as a predictive factor for ECT BLM and GET pIL-12 in murine melanoma. Melanoma B16F10 tumors grown in female C57Bl/6NCrl mice were treated with GET pIL-12 and ECT BLM. Immediately after therapy, 6 h and 1, 3, 7 and 10 days later, tumors were examined by DCE-US. Statistical analysis was performed to inspect the correlation between tumor doubling time (DT) and DCE-US measurements using semilinear regression models and Bland-Altman plots. Therapeutic groups in which DCE-US showed reduced tumor perfusion had longer tumor DTs. It was confirmed that the DCE-US parameter peak enhancement (PE), reflecting relative blood volume, had predictive value for the outcome of therapy: larger PE correlated with shorter DT. In addition, perfusion heterogeneity was also associated with outcome: tumors that had more heterogeneous perfusion had faster growth, i.e., shorter DTs. This study demonstrates that DCE-US can be used as a method to predict the efficacy of electroporation-based treatment.

Calcium Carbonate Core-Shell Particles for Incorporation of 225Ac and Their Application in Local α-Radionuclide Therapy.

Actinium-225 (225Ac) radiolabeled submicrometric core-shell particles (SPs) made of calcium carbonate (CaCO3) coated with biocompatible polymers [tannic acid-human serum albumin (TA/HSA)] have been developed to improve the efficiency of local α-radionuclide therapy in melanoma models (B16-F10 tumor-bearing mice). The developed 225Ac-SPs possess radiochemical stability and demonstrate effective retention of 225Ac and its daughter isotopes. The SPs have been additionally labeled with zirconium-89 (89Zr) to perform the biodistribution studies using positron emission tomography-computerized tomography (PET/CT) imaging for 14 days after intratumoral injection. According to the PET/CT analysis, a significant accumulation of 89Zr-SPs in the tumor area is revealed for the whole investigation period, which correlates with the direct radiometry analysis after intratumoral administration of 225Ac-SPs. The histological analysis has revealed no abnormal changes in healthy tissue organs after treatment with 225Ac-SPs (e.g., no acute pathologic findings are detected in the liver and kidneys). At the same time, the inhibition of tumor growth has been observed as compared with control samples [nonradiolabeled SPs and phosphate-buffered saline (PBS)]. The treatment of mice with 225Ac-SPs has resulted in prolonged survival compared to the control samples. Thus, our study validates the application of 225Ac-doped core-shell submicron CaCO3 particles for local α-radionuclide therapy.

Towards Enhanced MRI Performance of Tumor-Specific Dimeric Phenylboronic Contrast Agents.

It is known that phenylboronic acid (PBA) can target tumor tissues by binding to sialic acid, a substrate overexpressed by cancer cells. This capability has previously been explored in the design of targeting diagnostic probes such as Gd- and 68Ga-DOTA-EN-PBA, two contrast agents for magnetic resonance imaging (MRI) and positron emission tomography (PET), respectively, whose potential has already been demonstrated through in vivo experiments. In addition to its high resolution, the intrinsic low sensitivity of MRI stimulates the search for more effective contrast agents, which, in the case of small-molecular probes, basically narrows down to either increased tumbling time of the entire molecule or elevated local concentration of the paramagnetic ions, both strategies resulting in enhanced relaxivity, and consequently, a higher MRI contrast. The latter strategy can be achieved by the design of multimeric GdIII complexes. Based on the monomeric PBA-containing probes described recently, herein, we report the synthesis and characterization of the dimeric analogues (GdIII-DOTA-EN)2-PBA and (GdIII-DOTA-EN)2F2PBA. The presence of two Gd ions in one molecule clearly contributes to the improved biological performance, as demonstrated by the relaxometric study and cell-binding investigations.

In Vivo Molecular Imaging of the Efficacy of Aminopeptidase N (APN/CD13) Receptor Inhibitor Treatment on Experimental Tumors Using 68Ga-NODAGA-c(NGR) Peptide.

INTRODUCTION: The aminopeptidase N (APN/CD13) receptor plays an important role in the neoangiogenic process and metastatic tumor cell invasion. Clinical and preclinical studies reported that bestatin and actinonin are cytotoxic to APN/CD13-positive tumors and metastases due to their APN/CD13-specific inhibitor properties. Our previous studies have already shown that 68Ga-labeled NGR peptides bind specifically to APN/CD13 expressing tumor cells. The APN/CD13 specificity of 68Ga-NGR radiopharmaceuticals enables the following of the efficacy of antiangiogenic therapy with APN/CD13-specific inhibitors using positron emission tomography (PET). The aim of this in vivo study was to assess the antitumor effect of bestatin and actinonin treatment in subcutaneous transplanted HT1080 and B16-F10 tumor-bearing animal models using 68Ga-NODAGA-c(NGR). MATERIALS AND METHODS: Three days after the inoculation of HT1080 and B16-F10 cells, mice were treated with intraperitoneal injection of bestatin (15 mg/kg) or actinonin (5 mg/kg) for 7 days. On the 5th and 10th day, in vivo PET scans and ex vivo biodistribution studies were performed 90 min after intravenous injection of 5.5 ± 0.2 MBq68Ga-NODAGA-c(NGR). RESULTS: Control-untreated HT1080 and B16-F10 tumors were clearly visualized by the APN/CD13-specific 68Ga-NODAGA-c(NGR) radiopharmaceutical. The western blot analysis also confirmed the strong APN/CD13 positivity in the investigated tumors. We found significantly (p ≤ 0.05) lower radiopharmaceutical uptake after bestatin treatment and higher radiotracer accumulation in the actinonin-treated HT1080 tumors. In contrast, significantly lower (p ≤ 0.01) 68Ga-NODAGA-c(NGR) accumulation was observed in both bestatin- and actinonin-treated B16-F10 melanoma tumors compared to the untreated-control tumors. Bestatin inhibited tumor growth and 68Ga-NODAGA-c(NGR) uptake in both tumor models. CONCLUSION: The bestatin treatment is suitable for suppressing the neoangiogenic process and APN/CD13 expression of experimental HT1080 and B16-F10 tumors; furthermore, 68Ga-NODAGA-c(NGR) is an applicable radiotracer for the in vivo monitoring of the efficacy of the APN/CD13 inhibition-based anticancer therapies.

Multiple Applications of a Novel Biarsenical Imaging Probe in Fluorescence and PET Imaging of Melanoma.

A new fluorescent biarsenical peptide labeling probe was synthesized and labeled with the radioactive isotopes 11C and 18F. The utility of this probe was demonstrated by installing each of these isotopes into a melanocortin 1 receptor (MC1R) binding peptide, which targets melanoma tumors. Its applicability was further showcased by subsequent in vitro imaging in cells as well as in vivo imaging in melanoma xenograft mice by fluorescence and positron emission tomography.