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Elevated levels of superoxide anion radicals (O 2 ·- ) have been implicated in the pathogenesis of a variety of diseases, such as cancer, inflammatory diseases and autoimmune diseases. To determine the O 2 ·- concentration for assisting disease detection, a method based on surface-enhanced Raman scattering (SERS) combined with transparent polymer microneedles has been developed. Photocrosslinked NOA61 is used to prepare microneedles with sulfhydryl group, which can contribute to anchor gold nanoparticles (Au NPs) functionalized by p-mercaptobenzoic acid (PATP). This work successfully constructed SERS microneedles for in situ detection. A REDOX reaction occurred between PATP and O 2 ·- , resulting in the formation of dimethylaminoborane (DMAB) and a subsequent change in Raman signal. Based on the quantitative relationship between the change of peak area ratio at 1042 cm -1 and 1077 cm -1 and the concentration change of O 2 ·- , a standard curve with a linear range of 0-480 ng/mL was constructed. The SERS microneedles were effectively employed to track melanoma progression in mice, establishing a fundamental correlation between O2·- concentration and melanoma stage, as confirmed by ELISA. The benefits of this approach, including convenience, in situ applicability, and low cost, are anticipated to offer novel insights for non-invasive in situ detection, potentially enhancing disease monitoring and diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

A novel scaffold for in situ electrochemical detection of cell biomarkers was developed using electrospun nanofibers and commercial adhesive polymeric membranes. The electrochemical sensing of cell biomarkers requires the cultivation of the cells on/near the (bio)sensor surface in a manner to preserve an appropriate electroactive available surface and to avoid the surface passivation and sensor damage. This can be achieved by employing biocompatible nanofiber meshes that allow the cells to have a normal behavior and do not alter the electrochemical detection. For a better mechanical stability and ease of handling, nylon 6/6 nanofibers were collected on commercial polymeric membranes, at an optimal fiber density, obtaining a double-layered platform. To demonstrate the functionality of the fabricated scaffold, the screening of cellular stress has been achieved integrating melanoma B16-F10 cells and the (bio)sensor components on the transducer whereas the melanin exocytosis was successfully quantified using a commercial electrode. Either directly on the surface of the (bio)sensor or spatially detached from it, the integration of cell cultures in biosensing platforms based on electrospun nanofibers represents a powerful bioanalytical tool able to provide real-time information about the biomarker release, enzyme activity or inhibition, and monitoring of various cellular events., (© 2024. The Author(s).)

Purpose: Low frequency EPR can noninvasively detect endogenous free radical melanin in melanocytic skin lesions and could potentially discriminate between benign atypical nevi and malignant melanoma lesions. We recently succeeded in demonstrating the ability of clinical EPR to noninvasively detect the endogenous melanin free radical in skin lesions of patients. However, the signal-to-noise ratio (SNR) was extremely low warranting further research to boost the sensitivity of detection. In the present study, we assessed the performance of a clinical EPR system with the capability to perform multi-harmonic (MH) analysis for the detection of melanin., Procedures: The sensitivity of MH-EPR was compared with a classical continuous wave (CW)-EPR (1st harmonic) detection in vitro in melanin phantoms, in vivo in melanoma models with cells implanted in the skin, in lymph nodes and having colonized the lungs, and finally on phantoms placed at the surface of human skin., Results: In vitro, we observed an increase in SNR by a factor of 10 in flat melanin phantoms when using MH analysis compared to CW combined with an increase in modulation amplitude. In B16 melanomas having grown in the skin of hairless mice, we observed a boost in sensitivity in vivo similar to that observed in vitro with the capability to detect melanoma cells at an earlier stage of development. MH-EPR was also able to detect non-invasively the melanin signal coming from melanoma cells present in lymph nodes as well as in lungs. We also observed a boost of sensitivity using phantoms of melanin placed at the surface of human skin., Conclusions: Overall, our results are paving the way for new clinical trials that will use MH clinical EPR for the characterization of pigmented skin lesions., (© 2024. The Author(s).)

This 30-color full spectrum flow cytometry panel was developed and optimized for in-depth analysis T cells immunophenotype in tumor microenvironment and peripheral lymphoid organs. The panel presented here first identify the main cell subsets including myeloid cells, B cells, NKT cells, γδ T cells, CD4 + T cells and CD8 + T cells. For CD4 + T cells or CD8 + T cells, the panel includes markers for further characterization by including a selection of activation status(CD44, CD62L, CD69, Ki67, CD127, KLRG1 and CXCR3), costimulatory/co-inhibitory molecules (ICOS, OX-40, PD-1, LAG3, TIM-3, CTLA-4 and TIGIT), pro-inflammatory/anti-inflammatory cytokines (IFN-γ, TNF-α and IL-10) and cytotoxic molecules (Perforin, Granzymes B and CD107a). The panel has been tested on the tumor infiltrating T cells and corresponding spleen T cells in B16-F10 murine melanoma models., Competing Interests: Declaration of Competing Interest All the authors declare no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Melanin exists in the most of melanoma lesions. Melanin plays an important role in melanoma progression, metastasis, therapy response, and the overall survival of patients. Therefore, melanin is a critical target for melanoma diagnosis and therapy. Many melanin targeting probes, such as radioisotope-labeled benzamide analogs, have been developed for melanoma diagnosis using positron emission tomography (PET). The N-(2-(diethylamino)-ethyl)- 18 F-5-fluoropicolinamide ( 18 F-P3BZA) probe is one of the benzamide analogs and has been preliminarily tested for clinical diagnosis of melanoma in our recent studies. It has shown high specificity and favorable in vivo performance for PET of melanoma. Herein, we describe the detailed synthesis protocol of 18 F-P3BZA and PET/CT imaging procedure for animal models and patients.

In this study, the melanoma targeting property of 67 Ga-NODAGA-GGNle-CycMSH hex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-D-Phe-Arg-Trp-Lys]-CONH 2 } was determined on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NODAGA as a radiometal chelator for facile room temperature radiolabeling of NODAGA-GGNle-CycMSH hex . The IC 50 value of NODAGA-GGNle-CycMSH hex was 0.87 ± 0.12 nM on B16/F10 melanoma cells. 67 Ga-NODAGA-GGNle-CycMSH hex was readily prepared at room temperature with greater than 98% radiolabeling yield and displayed MC1R-specific binding on B16/F10 melanoma cells. The B16/F10 melanoma uptake of 67 Ga-NODAGA-GGNle-CycMSH hex was 10.31 ± 0.78, 14.96 ± 1.34, 13.7 ± 3.33 and 10.4 ± 2.2% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Approximately 85% of the injected dose was cleared out the body via urinary system at 2 h post-injection. 67 Ga-NODAGA-GGNle-CycMSH hex showed high tumor/blood, tumor/muscle and tumor/skin uptake ratios after 2 h post-injection. Overall, 67 Ga-NODAGA-GGNle-CycMSH hex could be easily prepared at room temperature and exhibited favorable melanoma targeting property, suggesting the potential use of NODAGA as a radiometal chelator for facile room temperature radiolabeling of α-MSH peptides., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A mitochondrial pH sensing fluorescent probe namely 2-(2-(6-hydroxynaphthalen-2-yl)vinyl)-3-(6-(triphenyl-phosphonio)hexyl)benzothiazol-3-ium bromide (HTBT2) was designed and facilely synthesized via the Knoevenagel condensation reaction. HTBT2 displayed a linear fluorescence enhancement at 612 nm in response to pH changes between 8.70 and 7.20. The pK a value was determined to be 8.04 ± 0.02, which might be ideal for mitochondrial pH (pH mito ∼8.0) detection. HTBT2 also exhibited a remarkable large Stokes shift of 176 nm, which could diminish the interference of excitation light. The results of live cell imaging studies suggested that HTBT2 showed excellent targeting ability for mitochondria. Importantly, it was successfully applied to visualize mitochondrial pH changes in live cells and differentiate the pH mito difference between cancer cell lines and normal cell lines. Our results consistently supported that HTBT2 held practical promise for the investigation of physiological processes related to pH mito changes and clinical potential for cancer cell differentiation.

The rise of programmed death-1 (PD-1)/PD-L1 immune checkpoint inhibitor therapy has been one of the most promising developments in melanoma research. However, not all the melanoma patients respond to such immune checkpoint blockade. There is a great need of biomarkers for appropriate melanoma patient selection and therapeutic efficacy monitoring. The objective of this study is to develop a novel radiolabeled anti-PD-L1 antibody fragment, as an imaging biomarker, for evaluating the in vivo PD-L1 levels in melanoma. The Df-conjugated F(ab') 2 fragment of the anti-mouse PD-L1 antibody was successfully synthesized and radiolabeled with 89 Zr. Both Df-F(ab') 2 and 89 Zr-Df-F(ab') 2 maintained the nano-molar murine PD-L1 targeting specificity and affinity. 89 Zr-Df-F(ab') 2 showed less uptake in normal liver tissue in mice compared with its full antibody counterpart 89 Zr-Df-anti-PD-L1. Positron emission tomography (PET)/computed tomography images clearly showed that 89 Zr-Df-F(ab') 2 possessed superior pharmacokinetics and imaging contrast over the radiolabeled full antibody, with much earlier and higher tumor uptake (5.5 times more at 2 h post injection) and much lower liver background (51% reduction at 2 h post injection). The specific and high murine PD-L1-targeting uptake at tumor foci coupled with fast clearance of 89 Zr-Df-F(ab') 2 highlighted its potential for in vivo PET imaging of murine PD-L1 levels and future development of radiolabeled anti-human PD-L1 fragment for potential application in melanoma patients.

Transmission mode terahertz time-domain spectroscopy system was employed to image BALB/c mouse skin tissue slices containing melanoma. The melanoma was unambiguously identified in the frequency region of 0.6-1.8 THz because melanoma has a higher refractive index as well as a higher absorption coefficient than the normal region of the skin tissue. Based on the results of hematoxylin-eosin staining and mass weighing, it was further suggested that the higher density of nucleic acids, higher water content, and lower fat content in the melanoma compared to the normal region are major factors responsible for melanoma's higher refractive index and absorption coefficient than normal tissue. The present work validates that terahertz time-domain spectroscopy imaging technique is possible to be used for the diagnosis of melanoma., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

A noninvasive electrochemical melanoma detection approach based on using adhesive tapes for collecting and fixing cells from a suspicious skin area and transferring the cells into a scanning electrochemical microscope (SECM) is presented. The adhesive layer collects the cells reproducibly and keeps them well adhered on the tape during experiments in an electrolyte solution. A melanoma biomarker, here the intracellular enzyme tyrosinase (TYR), was imaged on the tape-collected cells without further cell lysing using antibodies that were labeled with horseradish peroxidase (HRP). The HRP labels catalyzed the oxidation of a dissolved redox-active species, which was detected at a soft microelectrode, gently brushed in contact mode over the tape. The melanoma biomarker was first detected on tape-stripped samples with murine melanoma cells of different concentrations. Thereafter, increasing levels of TYR were recorded in cells that were collected from the skin of melanoma mouse models representing three different stages of tumor growth. Additionally, SECM results of tape-stripped different human melanoma cell lines were confirmed by previous studies based on traditionally fixed and permeabilized cells.

Objective: Early detection plays a role in the prognosis of melanoma, the most aggressive skin cancer. 64 Cu- and 68 Ga-labeled alpha-melanocyte-stimulating hormone (α-MSH) analogs targeting the melanocortin-1 receptor are promising positron emission tomography (PET) tracers for detecting melanoma, and the use of 18 F-labeling will further contribute to the detectability and availability. However, the high radiochemistry demand related to the conventional 18 F-labeling methods has restricted the development of 18 F-labeled α-MSH analogs. A recently developed radiofluorination method using aluminum-fluoride (Al 18 F) offers a simple, efficient, and time-saving labeling procedure compared to the conventional 18 F-labeling methods. Herein, we sought to establish a simple preparation method for an 18 F-labeled α-MSH analog using Al 18 F, and we examined its potential for the early detection of melanoma., Methods: A 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA)-conjugated α-MSH analog (NOTA-GGNle-CycMSH hex ) was prepared by the Fmoc solid-phase strategy. NOTA-GGNle-CycMSH hex was labeled with Al 18 F by heating at 105 °C using a microwave synthesizer for 15 min. Biodistribution study was conducted on B16/F10-luc melanoma-bearing mice at 30 min, 1 h and 3 h after injection of Al 18 F-NOTA-GGNle-CycMSH hex . PET imaging was conducted on melanoma-bearing mice at 1 h post-injection. One day prior to the PET imaging, bioluminescence imaging was also performed., Results: Al 18 F-NOTA-GGNle-CycMSH hex was readily prepared with a high radiochemical yield (94.0 ± 2.8%). The biodistribution study showed a high accumulation of Al 18 F-NOTA-GGNle-CycMSH hex in the tumor at 30 min and 1 h post-injection (6.69 ± 1.49 and 7.70 ± 1.71%ID/g, respectively). The tumor-to-blood ratio increased with time: 3.46 ± 0.89, 12.67 ± 1.29, and 35.27 ± 9.12 at 30 min, 1 h, and 3 h post-injection, respectively. In the PET imaging, Al 18 F-NOTA-GGNle-CycMSH hex clearly visualized the tumors and depicted very small tumors (< 3 mm)., Conclusions: We successfully prepared Al 18 F-NOTA-GGNle-CycMSH hex in a simple and efficient manner. Al 18 F-NOTA-GGNle-CycMSH hex showed high tumor accumulation and clearly visualized very small tumors in melanoma-bearing mice. These findings suggest that Al 18 F-NOTA-GGNle-CycMSH hex will be a promising PET tracer for melanoma imaging at an earlier stage.

A novel hydrosoluble near-infrared (NIR) fluorescent probe that could specifically identify tyrosinase has been successfully constructed and applied for imaging of tyrosinase in living cells and zebrafish. Notably, the probe has been successfully applied to the diagnosis of melanoma in a xenogeneic mouse model.

Magnetic hyperthermia is an oncological therapy where magnetic nanostructures, under a radiofrequency field, act as heat transducers increasing tumour temperature and killing cancerous cells. Nanostructure heating efficiency depends both on the field conditions and on the nanostructure properties and mobility inside the tumour. Such nanostructures are often incorrectly bench-marketed in the colloidal state and using field settings far off from the recommended therapeutic values. Here, we prepared nanoclusters composed of iron oxide magnetite nanoparticles crystallographically aligned and their specific absorption rate (SAR) values were calorimetrically determined in physiological fluids, agarose-gel-phantoms and ex vivo tumours extracted from mice challenged with B16-F0 melanoma cells. A portable, multipurpose applicator using medical field settings; 100 kHz and 9.3 kA m -1 , was developed and the results were fully analysed in terms of nanoclusters' structural and magnetic properties. A careful evaluation of the nanoclusters' heating capacity in the three milieus clearly indicates that the SAR values of fluid suspensions or agarose-gel-phantoms are not adequate to predict the real tissue temperature increase or the dosage needed to heat a tumour. Our results show that besides nanostructure mobility, perfusion and local thermoregulation, the nanostructure distribution inside the tumour plays a key role in effective heating. A suppression of the magnetic material effective heating efficiency appears in tumour tissue. In fact, dosage had to be increased considerably, from the SAR values predicted from fluid or agarose, to achieve the desired temperature increase. These results represent an important contribution towards the design of more efficient nanostructures and towards the clinical translation of hyperthermia.

Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software "Cancer Cell Finder" that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.

This study presents, attenuated total reflection Fourier transforms infrared spectroscopy of dried serum samples in an effort to assess biochemical changes induced by non-Hodgkin's lymphoma and subcutaneous melanoma. An EL4 mouse model of non-Hodgkin lymphoma and a B16 mouse model of subcutaneous melanoma are used to extract a snapshot of tumor-associated alteration in the serum. The study of both cancer-bearing mouse models in wild types and their corresponding control types, emphasizes the diagnostic potential of this approach as a screening technique for non-Hodgkin lymphoma and melanoma skin cancer. Infrared absorbance values of the different spectral bands, hierarchical clustering and integral values of the component bands by curve fitting, show statistically significant differences (student's t-test, two-tailed unequal variance p-value < 0.05) between spectra representing healthy and tumorous mouse. This technique may thus be useful for having individualized route maps for rapid evaluation of lymphoma and melanoma status and associated therapeutic modalities.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

The quantification of circulating tumor cells (CTCs) has been considered a potentially powerful tool in cancer diagnosis and prognosis, as CTCs have been shown to appear very early in cancer development. Great efforts have been made to develop methods that were less invasive and more sensitive to detect CTCs earlier. There is growing evidence that CTC clusters have greater metastatic potential than single CTCs. Therefore, the detection of CTC clusters is also important. This chapter is aimed to introduce a noninvasive technique for CTCs detection named in vivo flow cytometry (IVFC), which has been demonstrated to be capable of monitoring CTCs dynamics continuously. Furthermore, IVFC could be helpful for CTC cluster enumeration.

Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of (111)In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies.

Thromboembolic events are one of the world's leading causes of death among patients. Embolus or clot formations have several etiologies including paraneoplastic, post-surgery, cauterization, transplantation, or extracorporeal circuits. Despite its medical significance, little progress has been made in early embolus detection, screening and control. The aim of our study is to test the utility of the in vivo photoacoustic (PA) flow cytometry (PAFC) technique for non-invasive embolus detection in real-time. Using in vivo PAFC, emboli were non-invasively monitored in the bloodstream of two different mouse models. The tumor-free mouse model consisted of two groups, one in which the limbs were clamped to produce vessel stasis (7 procedures), and one where the mice underwent surgery (7 procedures). The melanoma-bearing mouse model also consisted of two groups, one in which the implanted tumor underwent compression (8 procedures), and one where a surgical excision of the implanted tumor was performed (8 procedures). We demonstrated that the PAFC can detect a single embolus, and has the ability to distinguish between erythrocyte-rich (red) and leukocyte/platelet-rich (white) emboli in small vessels. We show that, in tumor-bearing mice, the level of circulating emboli was increased compared to tumor-free mice (p = 0.0013). The number of circulating emboli temporarily increased in the tumor-free control mice during vessel stasis (p = 0.033) and after surgical excisions (signed-rank p = 0.031). Similar observations were noted during tumor compression (p = 0.013) and after tumor excisions (p = 0.012). For the first time, it was possible to detect unlabeled emboli in vivo non-invasively, and to confirm the presence of pigmented tumor cells within circulating emboli. The insight on embolus dynamics during cancer progression and medical procedures highlight the clinical potential of PAFC for early detection of cancer and surgery-induced emboli to prevent the fatal thromboembolic complications by well-timed therapy.

In recent years the authors have reported on (99m)Tc(CO)3-labeled peptides that serve as carriers for biomolecules or radiopharmaceuticals to the tumors. In continuation of that work they report the synthesis of a pentapeptide (Met-Phe-Phe-Gly-His; pep-1), a hexapeptide (Met-Phe-Phe-Asp-Gly-His; pep-2), and a tetrapeptide (Asp-Gly-Arg-His; pep-3) and the attachment of 3-amino-1,2,4-triazole to the β carboxylic function of the aspartic acid unit of pep-2 and pep-3. The pharmacophores were radiolabeled in high yields with [(99m)Tc(CO)3(H2O)3](+) metal aqua ion, characterized for their stability in serum and saline, as well as in His solution, and found to be substantially stable. B16F10 cell line binding studies showed favorable uptake and internalization. In vivo behavior of the radiolabeled triazolyl peptides was assessed in mice bearing induced tumor. The (99m)Tc(CO)3-triazolyl pep-3 demonstrated rapid urinary clearance and comparatively better tumor uptake. Imaging studies showed visualization of the tumor using (99m)Tc(CO)3-triazolyl pep-3, but due to high abdominal background, low delineation occurred. Based on the results further experiments will be carried out for targeting tumor with triazolyl peptides.

Regional lymph node status is an important prognostic indicator of tumor aggressiveness. However, early diagnosis of metastasis using intranodal pressure, at a stage when lymph node size has not changed significantly, has not been investigated. Here, we use an MXH10/Mo-lpr/lpr mouse model of lymph node metastasis to show that intranodal pressure increases in both the subiliac lymph node and proper axillary lymph node, which are connected by lymphatic vessels, when tumor cells are injected into the subiliac lymph node to induce metastasis to the proper axillary lymph node. We found that intranodal pressure in the subiliac lymph node increased at the stage when metastasis was detected by in vivo bioluminescence, but when proper axillary lymph node volume (measured by high-frequency ultrasound imaging) had not increased significantly. Intravenously injected liposomes, encapsulating indocyanine green, were detected in solid tumors by in vivo bioluminescence, but not in the proper axillary lymph node. Basic blood vessel and lymphatic channel structures were maintained in the proper axillary lymph node, although sinus histiocytosis was detected. These results show that intranodal pressure in the proper axillary lymph node increases at early stages when metastatic tumor cells have not fully proliferated. Intranodal pressure may be a useful parameter for facilitating early diagnosis of lymph node metastasis., (© 2015 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Recently, much effort has been made to develop effective dermatological depigmenting compounds. In this study, we investigated the novel candidate compound, AP736 (an adamantyl benzylbenzamide derivative), and its effects on melanogenesis in B16F10 melanoma cells, as well as the mechanisms involved. AP736 has been reported to exert anti-melanogenic effects in melanocytes in vitro and in artificial skin equivalents through the inhibition of key melanogenic enzymes and the suppression of the cAMP-protein kinase A (PKA)-cAMP response element‑binding protein (CREB) signaling pathway. Thus, we examined another pathway of melanogenesis involving the effects of AP736 on the glycogen synthesis kinase 3β (GSK3β) pathway. Melanin content and tyrosinase activity were measured using a spectrophotometer after the cells were treated with AP736. The AP736-induced activation of signaling pathways was examined by western blot analysis. We confirmed that AP736 decreased melanin production in a dose-dependent manner; however, it did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme. The expression of microphthalmia-associated transcription factor, tyrosinase, and related signal transduction pathways was also investigated. The Wnt signaling pathway is deeply involved in melanogenesis; therefore, phosphorylation by GSK3β was assessed following treatment with AP736. AP736 induced GSK3β phosphorylation (inactivation), but it did not alter the level of β-catenin. Furthermore, the expression of α-melanocyte-stimulating hormone-induced tyrosinase was downregulated by AP736. Our data suggest that AP736 exerts hypopigmentary effects through the downregulation of tyrosinase via GSK3β phosphorylation.

Contrast-enhanced MRI lymphography shows potential to identify alterations in lymph drainage through lymph nodes (LNs) in cancer and other diseases. MRI studies have typically used low molecular weight gadolinium contrast agents, however larger gadolinium-loaded nanoparticles possess characteristics that could improve the specificity and sensitivity of lymphography. The performance of three gadolinium contrast agents with different sizes and properties was compared by 3T MRI after subcutaneous injection. Mice bearing B16-F10 melanoma footpad tumors were imaged to assess tumor-induced alterations in lymph drainage through tumor-draining popliteal and inguinal LNs versus contralateral uninvolved drainage. Gadolinium lipid nanoparticles were able to identify tumor-induced alterations in contrast agent drainage into the popliteal LN, while lower molecular weight or albumin-binding gadolinium agents were less effective. All of the contrast agents distributed in foci around the cortex and medulla of tumor-draining popliteal LNs, while they were restricted to the cortex of non-draining LNs. Surprisingly, second-tier tumor-draining inguinal LNs exhibited reduced uptake, indicating that tumors can also divert LN drainage. These characteristics of tumor-induced lymph drainage could be useful for diagnosis of LN pathology in cancer and other diseases. The preferential uptake of nanoparticle contrasts into tumor-draining LNs could also allow selective targeting of therapies to tumor-draining LNs.

GLUT's (facilitative glucose transporters) over-expression in tumor cells has allowed the detection of several cancer types, using a glucose analogue ((18)F-FDG) with PET images, worldwide. New glucose analogs radiolabeled with (99m)Tc could be a less-expensive and more accessible alternative for diagnosis using SPECT imaging. d-Glucose ((99m)Tc-IDAG) and 2-d-deoxyglucose ((99m)Tc-AADG) organometallic complexes were proposed and studied as potential (18)F-FDG surrogates. The glucose complexes were prepared and evaluated as potential cancer imaging agents, in a melanoma tumor model. Iminodiacetic acid (IDA) and aminoacetate (AA) moieties were chosen as chelating system for radiolabeling with (99m)Tc. Tumor uptake of the formed complexes was evaluated in B16 murine cell line in vitro and in vivo in melanoma bearing C57BL/6 mice. In vitro and in vivo studies were conducted with (18)F-FDG in order to compare the uptake of (99m)Tc-glucose complexes in the tumor model. IDAG and AADG compounds were synthesized and radiolabeled with (99m)TcO4(-) to obtain the (99m)Tc-IDAG and (99m)Tc-AADG complexes in high yield and stability. In vitro cell studies showed maximum uptake at 60 min for complexes, (99m)Tc-IDAG and (99m)Tc-AADG, with 6% and 2%, respectively. Biodistribution studies showed high tumor uptake one hour post-injection, reaching tumor-to-muscle ratios of 12.1 ± 3.73 and 2.88 ± 1.40 for (99m)Tc-IDAG and (99m)Tc-AADG, respectively. SPECT and micro-SPECT-CT images acquired after the injection of (99m)Tc-IDAG showed accumulation in tumor sites, suggesting that this glucose complex would be a promising candidate for cancer imaging., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

With available MRI techniques, primary and metastatic liver cancers that are associated with high mortality rates and poor treatment responses are only diagnosed at late stages, due to the lack of highly sensitive contrast agents without Gd(3+) toxicity. We have developed a protein contrast agent (ProCA32) that exhibits high stability for Gd(3+) and a 10(11)-fold greater selectivity for Gd(3+) over Zn(2+) compared with existing contrast agents. ProCA32, modified from parvalbumin, possesses high relaxivities (r1/r2: 66.8 mmol(-1)⋅s(-1)/89.2 mmol(-1)⋅s(-1) per particle). Using T1- and T2-weighted, as well as T2/T1 ratio imaging, we have achieved, for the first time (to our knowledge), robust MRI detection of early liver metastases as small as ∼0.24 mm in diameter, much smaller than the current detection limit of 10-20 mm. Furthermore, ProCA32 exhibits appropriate in vivo preference for liver sinusoidal spaces and pharmacokinetics for high-quality imaging. ProCA32 will be invaluable for noninvasive early detection of primary and metastatic liver cancers as well as for monitoring treatment and guiding therapeutic interventions, including drug delivery.

Introduction: The aim of this study was to examine whether the substitution of the Lys linker with the aminooctanoic acid (Aoc) and polyethylene glycol (PEG) linker could substantially decrease the non-specific renal uptake of (99m)Tc-labeled Arg-Gly-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptides., Methods: The RGD motif {Arg-Gly-Asp-DTyr-Asp} was coupled to [Cys(3,4,10), D-Phe(7), Arg(11)]α-MSH₃₋₁₃ via the Aoc or PEG₂ linker to generate RGD-Aoc-(Arg(11))CCMSH and RGD-PEG-(Arg(11))CCMSH. The biodistribution results of (99m)Tc-RGD-Aoc-(Arg(11))CCMSH and (99m)Tc-RGD-PEG₂-(Arg(11))CCMSH were examined in M21 human melanoma-xenografted nude mice., Results: The substitution of Lys linker with Aoc and PEG₂ linker significantly reduced the renal uptake of (99m)Tc-RGD-Aoc-(Arg(11))CCMSH and (99m)Tc-RGD-PEG₂-(Arg(11))CCMSH by 58% and 63% at 2h post-injection. The renal uptake of (99m)Tc-RGD-Aoc-(Arg(11))CCMSH and (99m)Tc-RGD-PEG₂-(Arg(11))CCMSH was 27.93 ± 3.98 and 22.01 ± 9.89% ID/g at 2 h post-injection. (99m)Tc-RGD-Aoc-(Arg(11))CCMSH displayed higher tumor uptake than (99m)Tc-RGD-PEG₂-(Arg(11))CCMSH (2.35 ± 0.12 vs. 1.71 ± 0.25% ID/g at 2 h post-injection). The M21 human melanoma lesions could be clearly visualized by SPECT/CT using (99m)Tc-RGD-Aoc-(Arg(11))CCMSH as an imaging probe., Conclusions: The favorable effect of Aoc and PEG₂ linker in reducing the renal uptake provided a new insight into the design of novel dual receptor-targeting radiolabeled peptides., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Melanin pigment represents an attractive target to address specific treatment to melanoma cells, such as cytotoxic radionuclides. However, less than half of the patients have pigmented metastases. Hence, specific marker is required to stratify this patient population before proceeding with melanin-targeted radionuclide therapy. In such a context, we developed fluorinated analogues of a previously studied melanin-targeting ligand, N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide (ICF01012). These latter can be labeled either with (18)F or (131)I/(125)I for positron emission tomography imaging (melanin-positive patient selection) and targeted radionuclide therapy purposes. Here we describe the syntheses, radiosyntheses and preclinical evaluations on melanoma-bearing mice model of several iodo- and fluoro(hetero)aromatic derivatives of the ICF01012 scaffold. After preliminary planar gamma scintigraphic and positron emission tomography imaging evaluations, [(125)I]- and [(18)F]-N-[2-(diethylamino)ethyl]-4-fluoro-3-iodobenzamides ([(125)I]4, [(18)F]4) were found to be chemically and biologically stable with quite similar tumor uptakes at 1 h p.i. (9.7 ± 2.6% ID/g and 6.8 ± 1.9% ID/g, respectively)., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

This unit describes protocols for developing tumors in mice, including subcutaneous growth, pulmonary metastases of B16 melanoma, and spontaneous melanoma in B-Raf V600E/PTEN deletion transgenic mouse models. Two immunization methods to prevent B16 tumor growth are described using B16.GM-CSF and recombinant vaccinia virus. A therapeutic approach is also included that uses adoptive transfer of tumor antigen-specific T cells. Methods including CTL induction, isolation, testing, and genetic modification of mouse T cells for adoptive transfer by using retrovirus-expressing genes of interest are provided. Additional sections, including growing B16 melanoma, enumerating pulmonary metastases, tumor imaging technique, and use of recombinant viruses for vaccination, are discussed together with safety concerns., (Copyright © 2015 John Wiley & Sons, Inc.)

Several radiolabeled alpha-melanocyte stimulating hormone (α-MSH) analogs have been studied for their abilities to target melanoma tumor cells through specific recognition and binding to the melanocortin receptor 1 (MCR1). In this work, a lactam bridgecyclized α-MSH analog was labeled with (99m) via the hydrazinonicotinamide (HYNIC) chelator and characterized for its melanoma tumor targeting properties. The bifunctional chelating agent HYNIC-Boc was attached to the N-terminus of the MSH peptide followed by the lactam cyclization, resulting in the HYNIC-cyc-MSH analog. The lactam cyclized peptide displayed high affinity and specificity for MC1-receptors present on B16/F1 melanoma tumor cells, exhibiting an IC50 of 6.48 nM. HYNIC-cyc-MSH was radiolabeled with (99m)Tc using two common co-ligands, tricine and EDDA. In vitro, the radiochemical stability, cell binding and efflux properties were similar between the peptides radiolabeled with tricine and EDDA as co-ligands. In vivo, biodistribution studies (n=4) demonstrated that (99m)Tc- HYNIC-cyc-MSH/tricine had superior tumor to muscle and tumor to blood ratios than (99m)Tc-HYNIC-cyc-MSH/EDDA at early time points. Planar gamma imaging of melanoma bearing mice showed that 99mTc-HYNIC-cyc-MSH/tricine was able to clearly visualize tumors, underscoring the potential utility of (99m)Tc labeled lactam cyclized MSH molecules as melanoma imaging agents.

Nutrient demand is a fundamental characteristic of rapidly proliferating cells. Vitamin B12 is vital for cell proliferation; thus neoplastic cells have an increased demand for this essential nutrient. In this study we exploited the vitamin B12 uptake pathway to probe the nutritional demand of proliferating cells with a radiolabeled B12 derivative in various preclinical tumor models. We describe the synthesis and biological evaluations of copper-64-labeled B12 -ethylenediamine-benzyl-1,4,7-triazacyclononane-N,N',N''-triacetic acid (B12 -en-Bn-NOTA-(64) Cu), the first example of a B12 derivative for positron emission tomography (PET) imaging. Small-animal imaging and pharmacological evaluation show high tumor uptake ranging from 2.20 to 4.84% ID g(-1) at 6 h post-administration. Competition studies with excess native B12 resulted in a 95% decrease in tumor accumulation, indicating the specificity of this radiopharmaceutical for B12 endocytotic transport proteins. These results show that a vitamin B12 PET radiopharmaceutical has potential utility for non-invasive imaging of enhanced nutrient demand in proliferating cells., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oligometastasis can be defined as a state of limited metastases that is potentially amenable to ablative local therapy; the success of such therapy depends on whether or not additional occult metastases exist. A model is presented here to predict occult metastases given detectable oligometastases. Predictions were based on Bayes' theorem, in conjunction with descriptions of the statistical distributions for the sizes and numbers of hematogenous metastases. The background probability for occult metastases in individuals with oligometastases increased markedly with relatively minor increases in metastatic potential. With each additional metastasis detected the chance of further occult metastases increased. These latter increases were incremental and proportionately smaller with the more metastatic tumors. Long disease free intervals had a major effect to decrease in the probability of further occult disease. Demonstration of oligometastases depends heavily upon the sensitivity of radiological imaging techniques, where the proportion of detectable metastases relates to the position of the distribution of metastasis growth times with respect to the detection threshold. Given the limitations of radiological methods, and the possibility that the oligometastases detected may be the only disease, an aggressive approach appears indicated., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction was used to incorporate alkyne-functionalized dipicolylamine (DPA) ligands (1 and 3) for fac-[M(I)(CO)3](+) (M = Re/(99m)Tc) complexation into an α-melanocyte stimulating hormone (α-MSH) peptide analogue. A novel DPA ligand with carboxylate substitutions on the pyridyl rings (3) was designed to increase the hydrophilicity and to decrease in vivo hepatobiliary retention of fac-[(99m)Tc(I)(CO)3](+) complexes used in single photon emission computed tomography (SPECT) imaging studies with targeting biomolecules. The fac-[Re(I)(CO)3(3)] complex (4) was used for chemical characterization and X-ray crystal analysis prior to radiolabeling studies between 3 and fac-[(99m)Tc(I)(OH2)3(CO)3](+). The corresponding (99m)Tc complex (4a) was obtained in high radiochemical yields, was stable in vitro for 24 h during amino acid challenge and serum stability assays, and showed increased hydrophilicity by log P analysis compared to an analogous complex with nonfunctionalized pyridine rings (2a). An α-MSH peptide functionalized with an azide was labeled with fac-[M(I)(CO)3](+) using both click, then chelate (CuAAC reaction with 1 or 3 followed by metal complexation) and chelate, then click (metal complexation of 1 and 3 followed by CuAAC with the peptide) strategies to assess the effects of CuAAC conditions on fac-[M(I)(CO)3](+) complexation within a peptide framework. The peptides from the click, then chelate strategy had different HPLC tR's and in vitro stabilities compared to those from the chelate, then click strategy, suggesting nonspecific coordination of fac-[M(I)(CO)3](+) using this synthetic route. The fac-[M(I)(CO)3](+)-complexed peptides from the chelate, then click strategy showed >90% stability during in vitro challenge conditions for 6 h, demonstrated high affinity and specificity for the melanocortin 1 receptor (MC1R) in IC50 analyses, and led to moderately high uptake in B16F10 melanoma cells. Log P analysis of the (99m)Tc-labeled peptides confirmed the enhanced hydrophilicity of the peptide bearing the novel, carboxylate-functionalized DPA chelate (10a') compared to the peptide with the unmodified DPA chelate (9a'). In vivo biodistribution analysis of 9a' and 10a' showed moderate tumor uptake in a B16F10 melanoma xenograft mouse model with enhanced renal uptake and surprising intestinal uptake for 10a' compared to predominantly hepatic accumulation for 9a'. These results, coupled with the versatility of CuAAC, suggests this novel, hydrophilic chelate can be incorporated into numerous biomolecules containing azides for generating targeted fac-[M(I)(CO)3](+) complexes in future studies.

Melanoma is the most dangerous form of skin cancer and its incidence is rising each year. Because the current methods of diagnosis based on the visual aspect of the tumor show limitations, several new techniques are emerging to help in this diagnosis, amongst which are magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR). The origin of the typical contrast pattern observable in melanoma in T1 - and T2 -weighted images remains to be elucidated and is a source of controversy. In addition, melanin could create sufficient magnetic inhomogeneities to allow its visualization on T2 *-weighted images using high-field MRI. In order to elucidate the possible role of melanin in the MRI contrast of melanoma, the present study was designed to correlate the paramagnetic content in melanin pigment to the contrast on T1 -, T2 - and T2 *-weighted images. MR images were obtained in vivo at 11.7 T using four types of experimental tumors with different pigmentations (B16, HBL, LND1 melanomas and KHT sarcomas). The paramagnetic content in melanin pigment was measured by EPR. No significant correlation was observed between the content in melanin and the relaxation times T1 , T2 and T2 *, emphasizing that the presence of pigment alone has negligible effect on the MRI contrast., (Copyright © 2014 John Wiley & Sons, Ltd.)

Background: Effective chemotherapy rapidly reduces the spin-lattice relaxation of water protons (T1) in solid tumours and this change (ΔT1) often precedes and strongly correlates with the eventual change in tumour volume (TVol). To understand the biological nature of ΔT1, we have performed studies in vivo and ex vivo with the allosteric mTOR inhibitor, everolimus., Methods: Mice bearing RIF-1 tumours were studied by magnetic resonance imaging (MRI) to determine TVol and T1, and MR spectroscopy (MRS) to determine levels of the proliferation marker choline and levels of lipid apoptosis markers, prior to and 5 days (endpoint) after daily treatment with vehicle or everolimus (10 mg/kg). At the endpoint, tumours were ablated and an entire section analysed for cellular and necrotic quantification and staining for the proliferation antigen Ki67 and cleaved-caspase-3 as a measure of apoptosis. The number of blood-vessels (BV) was evaluated by CD31 staining. Mice bearing B16/BL6 melanoma tumours were studied by MRI to determine T1 under similar everolimus treatment. At the endpoint, cell bioluminescence of the tumours was measured ex vivo., Results: Everolimus blocked RIF-1 tumour growth and significantly reduced tumour T1 and total choline (Cho) levels, and increased polyunsaturated fatty-acids which are markers of apoptosis. Immunohistochemistry showed that everolimus reduced the %Ki67+ cells but did not affect caspase-3 apoptosis, necrosis, BV-number or cell density. The change in T1 (ΔT1) correlated strongly with the changes in TVol and Cho and %Ki67+. In B16/BL6 tumours, everolimus also decreased T1 and this correlated with cell bioluminescence; another marker of cell viability. Receiver-operating-characteristic curves (ROC) for everolimus on RIF-1 tumours showed that ΔT1 had very high levels of sensitivity and specificity (ROCAUC = 0.84) and this was confirmed for the cytotoxic patupilone in the same tumour model (ROCAUC = 0.97)., Conclusion: These studies suggest that ΔT1 is not a measure of cell density but reflects the decreased number of remaining viable and proliferating tumour cells due to perhaps cell and tissue destruction releasing proteins and/or metals that cause T1 relaxation. ΔT1 is a highly sensitive and specific predictor of response. This MRI method provides the opportunity to stratify a patient population during tumour therapy in the clinic.

Multimodal imaging offers the potential to improve diagnosis and enhance the specificity of photothermal cancer therapy. Toward this goal, gadolinium-conjugated gold nanoshells are engineered and it is demonstrated that they enhance contrast for magnetic resonance imaging, X-ray, optical coherence tomography, reflectance confocal microscopy, and two-photon luminescence. Additionally, these particles effectively convert near-infrared light to heat, which can be used to ablate cancer cells. Ultimately, these studies demonstrate the potential of gadolinium-nanoshells for image-guided photothermal ablation., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Benzamide can specifically bind to melanoma cells. A 18F-labeled benzamide derivative, [18F]N-(2-diethylaminoethyl)-4-[2-(2-(2-fluoroethoxy) ethoxy)ethoxy]benzamide ([18F]FPBZA), was developed as a promising PET probe for primary and metastatic melanoma., Methods: [18F]FPBZA was synthesized via a one-step radiofluorination in this study. The specific uptake of [18F]FPBZA was studied in B16F0 melanoma cells, A375 amelanotic melanoma cells, and NB-DNJ-pretreated B16F0 melanoma cells. The biological characterization of [18F]FPBZA was performed on mice bearing B16F0 melanoma, A375 amelanotic melanoma, or inflammation lesion., Results: [18F]FPBZA can be prepared efficiently with a yield of 40-50%. The uptake of [18F]FPBZA by B16F0 melanoma cells was significantly higher than those by A375 tumor cells and NB-DNJ-pretreated B16F0 melanoma cells. B16F0 melanoma displayed prominent uptake of [18F]FPBZA at 2 h (7.81±0.82%ID/g), compared with A375 tumor and inflammation lesion (3.00±0.71 and 1.67±0.56%ID/g, resp.). [18F]FPBZA microPET scan clearly delineated B16F0 melanoma but not A375 tumor and inflammation lesion. In mice bearing pulmonary metastases, the lung radioactivity reached 4.77±0.36%ID/g at 2 h (versus 1.16±0.23%ID/g in normal mice)., Conclusions: Our results suggested that [18F]FPBZA PET would provide a promising and specific approach for the detection of primary and metastatic melanoma lesions.

If parameters for lateral diffusion of lipids within membranes are macroscopic metaphors of the angular magnetic moment of the Bohr magneton then the energy emission should be within the visible wavelength for applied ~1 µT magnetic fields. Single or paired digital photomultiplier tubes (PMTs) were placed near dishes of ~1 million B16 mouse melanoma cells that had been removed from incubation. In very dark conditions (10(-11) W/m(2)) different averaged (RMS) intensities between 5 nT and 3.5 µT were applied randomly in 4 min increments. Numbers of photons were recorded directly over or beside the cell dishes by PMTs placed in pairs within various planes. Spectral analyses were completed for photon power density. The peak photon emissions occurred around 1 µT as predicted by the equation. Spectra analyses showed reliable discrete peaks between 0.9 and 1.8 µT but not for lesser or greater intensities; these peak frequencies corresponded to the energy difference of the orbital-spin magnetic moment of the electron within the applied range of magnetic field intensities and the standard solution for Rydberg atoms. Numbers of photons from cooling cells can be modified by applying specific intensities of temporally patterned magnetic fields. There may be a type of "cellular" magnetic moment that, when stimulated by intensity-tuned magnetic fields, results in photon emissions whose peak frequencies reflect predicted energies for fundamental orbital/spin properties of the electron and atomic aggregates with large principal quantum numbers.

Purpose: Combined therapeutic and diagnostic agents, "theranostics" are emerging valuable tools for noninvasive imaging and drug delivery. Here, we report on a solid biodegradable multifunctional nanoparticle that combines both features., Methods: Poly(lactide-co-glycolide) nanoparticles were engineered to confine superparamagnetic iron oxide contrast for magnetic resonance imaging while enabling controlled drug delivery and targeting to specific cells. To achieve this dual modality, fatty acids were used as anchors for surface ligands and for encapsulated iron oxide in the polymer matrix., Results: We demonstrate that fatty acid modified iron oxide prolonged retention of the contrast agent in the polymer matrix during degradative release of drug. Antibody-fatty acid surface modification facilitated cellular targeting and subsequent internalization in cells while inducing clustering of encapsulated fatty-acid modified superparamagnetic iron oxide during particle formulation. This induced clustered confinement led to an aggregation within the nanoparticle and, hence, higher transverse relaxivity, r2 , (294 mM(-1) s(-1) ) compared with nanoparticles without fatty-acid ligands (160 mM(-1) s(-1) ) and higher than commercially available superparamagnetic iron oxide nanoparticles (89 mM(-1) s(-1) )., Conclusion: Clustering of superparamagnetic iron oxide in poly(lactide-co-glycolide) did not affect the controlled release of encapsulated drugs such as methotrexate or clodronate and their subsequent pharmacological activity, thus highlighting the full theranostic capability of our system., (Copyright © 2013 Wiley Periodicals, Inc.)

Tumor recurrence represents a major clinical challenge. Our data show that emergent recurrent tumors acquire a phenotype radically different from that of their originating primary tumors. This phenotype allows them to evade a host-derived innate immune response elicited by the progression from minimal residual disease (MRD) to actively growing recurrence. Screening for this innate response predicted accurately in which mice recurrence would occur. Premature induction of recurrence resensitized MRD to the primary therapy, suggesting a possible paradigm shift for clinical treatment of dormant disease in which the current expectant approach is replaced with active attempts to uncover MRD before evolution of the escape phenotype is complete. By combining screening with second-line treatments targeting innate insensitivity, up to 100% of mice that would have otherwise relapsed were cured. These data may open new avenues for early detection and appropriately timed, highly targeted treatment of tumor recurrence irrespective of tumor type or frontline treatment.

A simple and facile synthetic strategy is developed to prepare a new class of multifunctional hybrid nanoparticles (NPs) that can integrate a magnetic core with silver nanocrystals embedded in porous carbon shell. The method involves a one-step solvothermal synthesis of Fe3O4@C template NPs with Fe3O4nanocrystals in the core protected by a porous carbon shell, followed by loading and in situ reduction of silver ions in the carbon shell in water at room temperature. The core-satellite and dumbbell-like nanostructures of the resulted Fe3O4@C-Ag hybrid NPs can be readily controlled by loading amount of silver ions. The hybrid NPs can efficiently catalyze the reduction reaction of organic dyes in water. The easy magnetic separation and high stability of the catalytically active silver nanocrystals embedded in the carbon shell enable the hybrid NPs to be recycled for reuse as catalysts. The hybrid NPs can also overcome cellular barriers to enter the intracellular region and light up the mouse melanoma B16F10 cells in multicolor modal, with no cytotoxicity. Such porous carbon protected Fe3O4@C-Ag hybrid NPs with controllable nanostructures and a combination of magnetic and noble metallic components have great potential for a broad range of applications in the catalytic industry and biomedical field.

In small-animal studies, calipers are the standard method used for measurement of external tumor size. However, as tumors are not usually prolate spheroids, this may lead to inaccuracies in the data. Also, correlations vary according to the kind and size of tumors. Tumors were generated by transplanting B16 mouse melanoma cells into the back of Balb/c nude mice. True volumes were measured by calipers, an in-vivo stereo imaging system, and as a standard uptake value (SUV) by (18)F-fluorodeoxyglucose ([(18)F]FDG)-PET. Correlations between measurements were analyzed. Correlation with the true volume was higher for measurements using the in-vivo stereo imaging system (r = 0.876) than with calipers (r = 0.744). Measurement of melanoma volume has a larger measuring error when performed using a caliper compared with measurements performed by stereo imaging when the volume of the melanoma is small. Correlation of the volume and PET-SUV by a caliper is low as the size of the melanoma increases. This same relationship exists with the comparison of stereo imaging and PET-SUV. The correlation between the SUV of [(18)F]FDG-PET and tumor volume with the melanoma is expected to be important in related future studies.

Multicolor carbon nanoparticles (CNPs) were prepared, characterized and developed as fluorescent probes for cell imaging. The fluorescent CNPs were prepared with a facile hydrothermal oxidation route by using linear polysaccharide cellulose and cyclic oligosaccharide cyclodextrin as carbon sources. The characterizations by transmission electron microscopy show that the prepared cellulose-CNPs and cyclodextrin-CNPs are spherical, well-dispersed in water with average diameters of 100 nm and 76 nm, respectively. Under the excitation of UV light, the CNPs are strongly luminescent with an excitation-dependent emission behavior and bathochromic emission properties. The fluorometric methods show that the cellulose-CNPs and cyclodextrin-CNPs are strongly fluorescent with fluorescence quantum yield of 7.47% and 4.49%, respectively. The multicolor CNPs have excellent photostability toward photobleaching. Strong near-infrared fluorescence of the carbon nanoparticles was observed with a 632.8 nm excitation wavelength laser. The oxidative metal ions like Hg(II), Cu(II) and Fe(III) show an quench effect on the fluorescence intensity of the CNPs. The multicolor CNPs were successfully used as fluorescent probes for mouse melanoma cells imaging. The results indicate that the multicolor CNPs derived from cellulose and cyclodextrin may have a great potential for the applications in bioimaging., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Tumor angiogenesis is important in the growth and metastasis of malignant tumors. In our previous study, we demonstrated that an arginine-arginine-leucine (RRL) peptide is a tumor endothelial cell-specific binding sequence that may be used as a molecular probe for the imaging of malignant tumors in vivo. The aim of the present study was to further explore the characteristics of 131I‑RRL by biodistribution tests, and to estimate the radiation dosimetry of 131I‑RRL for humans using mice data. The RRL peptide was radiolabeled with 131I by a chloramine-T (CH-T) method. The radiolabeling efficiency and radiochemical purity were then characterized in vitro. 131I‑RRL was injected intravenously into B16 xenograft-bearing Kunming mice. Biodistribution analysis and in vivo imaging were performed periodically. The radiation dosimetry in humans was calculated according to the organ distribution and the standard medical internal radiation dose (MIRD) method in mice. All data were analyzed by statistical and MIRDOSE 3.1 software. The labeling efficiency of 131I‑RRL reached 70.0±2.91% (n=5), and the radiochemical purity exceeded 95% following purification. In mice bearing B16 xenografts, 131I‑RRL rapidly cleared from the blood and predominantly accumulated in the kidneys, the stomach and the tumor tissue. The specific uptake of 131I‑RRL in the tumor increased over time and was significantly higher than that of the other organs, 24-72 h following injection (P<0.05). The ratio of tumor-to-skeletal muscle (T/SM) tissue exceeded 4.75, and the ratio of the tumor-to-blood (T/B) tissue peaked at 3.36. In the single-photon emission computed tomography (SPECT) imaging of Kunming mice bearing B16 xenografts, the tumors were clearly identifiable at 6 h, and significant uptake was evident 24-72 h following administration of 131I‑RRL. The effective dose for the adult male dosimetric model was estimated to be 0.0293 mSv/MBq. Higher absorbed doses were estimated for the stomach (0.102 mGy/MBq), the small intestines (0.0699 mGy/MBq), the kidneys (0.0611 mGy/MBq) and the liver (0.055 mGy/MBq). These results highlight the potential of 131I‑RRL as a ligand for the SPECT imaging of tumors. Administration of 131I‑RRL led to a reasonable radiation dose burden and was safe for human use.

Aiming at the design of specific melanocortin-1 receptor (MC1R) targeted imaging probes, we report on the effect of different azolyl-ring substitution patterns (carboxylate at the 4-position and/or methyl groups at the 3,5 positions) of pyrazolyl-diamine bifunctional chelators (Pz(2)-Pz(4)) on the pharmacokinetic profile of the (99m)Tc(CO)3-labeled lactam bridge-cyclized α-melanocyte stimulating hormone derivative, βAlaNleCycMSH(hex). Three pyrazolyl-diamine-containing chelators were conjugated to βAlaNleCycMSHhex, with the resulting peptide conjugates displaying subnanomolar MC1R binding affinity. Biodistribution studies in B16F1 melanoma-bearing mice show that all radiopeptides present a good melanoma uptake. The introduction of a carboxylate group in the azolyl-ring leads to a remarkable reduction of the kidney (>89%) and liver (>91%) accumulation for (99m)Tc(CO)3-Pz(3)-βAlaNleCycMSH(hex) and (99m)Tc(CO)3-Pz(4)-βAlaNleCycMSH(hex) when compared to the radiopeptide (99m)Tc(CO)3-Pz(1)-βAlaNleCycMSH(hex), where that group is absent. The good tumor uptake and favorable tumor-to-nontarget-organs ratios of (99m)Tc(CO)3-Pz(3)-βAlaNleCycMSH(hex) and (99m)Tc(CO)3-Pz(4)-βAlaNleCycMSH(hex) highlights the potential of both compounds as melanoma imaging agents.

In this study, the Huisgen reaction has been used to functionalise a carborane cage with a lipophilic moiety and a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand to obtain a new Gd boron neutron-capture therapy (BNCT)/magnetic resonance imaging (MRI) agent. The introduction of the triazole units has been accomplished under both heterogeneous conditions, by the use of a Cu-supported ionic-liquid catalyst, and homogeneous conditions. The ability of the Gd complex of the synthesised ligand to form stable adducts with low-density lipoproteins (LDLs) has been evaluated and then MRI has been performed on tumour melanoma cells incubated in the presence of a Gd-complex/LDL imaging probe. It has been concluded that the high amount of intracellular boron necessary to perform BNCT can be reached even in the presence of a relatively low-boron-containing LDL concentration., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The incidence of malignant melanoma is rising more rapidly than that of any other cancer in the United States. The melanocortin 1 receptor (MC1R) is overexpressed in most human melanoma metastases, thus making it a promising target for imaging and therapy of melanomas. We have previously reported the development of a peptidomimetic ligand with high specificity and affinity for MC1R. Here, we have conjugated near-infrared fluorescent dyes to the C-terminus of this ligand via lysine-mercaptopropionic acid linkers to generate MC1R specific optical probes (MC1RL-800, 0.4 nM K(i); and MC1RL-Cy5, 0.3 nM K(i)). Internalization of the imaging probe was studied in vitro by fluorescence microscopy using engineered A375/MC1R cells and B16F10 cells with endogenous MC1R expression. The in vivo tumor targeting of MC1RL-800 was evaluated by intravenous injection of probe into nude mice bearing bilateral subcutaneous A375 xenograft tumors with low MC1R expression and engineered A375/MC1R tumors with high receptor expression. Melanotic B16F10 xenografts were also studied. Fluorescence imaging showed that the agent has higher uptake values in tumors with high expression compared to low (p < 0.05), demonstrating the effect of expression levels on image contrast-to-noise. In addition, tumor uptake was significantly blocked by coinjection of excess NDP-α-MSH peptide (p < 0.05). In conclusion, the MC1R-specific imaging probe developed in this study displays excellent potential for the intraoperative detection of regional node involvement and for margin detection during melanoma metastasis resection.

The 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphinic acid) chelators TRAP and NOPO are complexing copper-64 with similar efficiency as 1,4,7-triazacyclononane-triacetic acid (NOTA). The kinetic stability of Cu-64-labelled TRAP-peptides is sufficient for PET imaging at early time points (1-2 h post injection). For labelling of TRAP conjugates, Cu-64 can be recommended as an alternative to Ga-68 to achieve higher resolution of PET images.

Engineering peptide-based targeting agents with residues for site-specific and stable complexation of radionuclides is a highly desirable strategy for producing diagnostic and therapeutic agents for cancer and other diseases. In this report, a model N-S-N(Py) ligand (3) and a cysteine-derived α-melanocyte stimulating hormone (α-MSH) peptide (6) were used as novel demonstrations of a widely applicable chelation strategy for incorporation of the [M(I)(CO)(3)](+) (M = Re, (99m)Tc) core into peptide-based molecules for radiopharmaceutical applications. The structural details of the core ligand-metal complexes as model systems were demonstrated by full chemical characterization of fac-[Re(I)(CO)(3)(N,S,N(Py)-3)](+) (4) and comparative high-performance liquid chromatography (HPLC) analysis between 4 and [(99m)Tc(I)(CO)(3)(N,S,N(Py)-3)](+) (4a). The α-MSH analogue bearing the N-S-N(Py) chelate on a modified cysteine residue (6) was generated and complexed with [M(I)(CO)(3)](+) to confirm the chelation strategy's utility when applied in a peptide-based targeting agent. Characterization of the Re(I)(CO)(3)-6 peptide conjugate (7) confirmed the efficient incorporation of the metal center, and the (99m)Tc(I)(CO)(3)-6 analogue (7a) was explored as a potential single photon emission computed tomography (SPECT) compound for imaging the melanocortin 1 receptor (MC1R) in melanoma. Peptide 7a showed excellent radiolabeling yields and in vitro stability during amino acid challenge and serum stability assays. In vitro B16F10 melanoma cell uptake of 7a reached a modest value of 2.3 ± 0.08% of applied activity at 2 h at 37 °C, while this uptake was significantly reduced by coincubation with a nonlabeled α-MSH analogue, NAPamide (3.2 μM) (P < 0.05). In vivo SPECT/X-ray computed tomography (SPECT/CT) imaging and biodistribution of 7a were evaluated in a B16F10 melanoma xenografted mouse model. SPECT/CT imaging clearly visualized the tumor at 1 h post injection (p.i.) with high tumor-to-background contrast. Blocking studies with coinjected NAPamide (10 mg per kg of mouse body weight) confirmed the in vivo specificity of 7a for MC1R-positive tumors. Biodistribution results with 7a yielded a moderate tumor uptake of 1.20 ± 0.09 percentage of the injected radioactive dose per gram of tissue (% ID/g) at 1 h p.i. Relatively high uptake of 7a was also seen in the kidneys and liver at 1 h p.i. (6.55 ± 0.36% ID/g and 4.44 ± 0.17% ID/g, respectively), although reduced kidney uptake was seen at 4 h p.i. (3.20 ± 0.48% ID/g). These results demonstrate the utility of the novel [M(I)(CO)(3)](+) chelation strategy when applied in a targeting peptide.