The incidence of malignant melanoma, the most dangerous form of skin cancer, is rising each year. However, some aspects of the tumor initiation and development are still unclear, and the current method of diagnosis, based on the visual aspect of the tumor, shows limitations. For these reasons, developments of new techniques are ongoing to improve basic knowledge on the disease and diagnosis of tumors in individual patients. This paper shows how electron paramagnetic resonance (EPR), a method able to detect free radicals trapped in melanin pigments, has recently brought its unique value to this specific field. The general principles of the method and the convenience of melanin as an endogenous substrate for EPR measurements are explained. Then, the way by which EPR has recently helped to assess the contribution of ultraviolet rays (UVA and UVB) to the initiation of melanoma is described. Finally, we describe the improvements of EPR spectrometry and imaging in the detection and mapping of melanin pigments inside ex vivo and in vivo melanomas. We discuss how these advances might improve the diagnosis of this skin cancer and point out the present capabilities and limitations of the method.

MRI cell tracking is a promising technique for tracking various cell types in living animals. Usually, cells are incubated with iron oxides so that the particles are taken up before the cells are injected in vivo. In the present study, we aimed to monitor migration of luciferase-expressing mouse renal cancer cells (RENCA-luc) after intrarenal or intrasplenic injection. These cells were labelled using Molday Ion Rhodamine B (MIRB) fluorescent superparamagnetic iron oxide particles. Their fate after injection was first assessed using ex vivo X-band electron paramagnetic resonance (EPR) spectroscopy. This biodistribution study showed that RENCA-luc cells quickly colonized the lungs and the liver after intrarenal and intrasplenic injection, respectively. Bioluminescence imaging (BLI) studies confirmed that this cell line preferentially metastasized to these organs. Early tracking of labelled RENCA-luc cells in the liver using high-field MRI (11.7 T) was not feasible because of a lack of sensitivity. MRI of MIRB-labelled RENCA-luc cells after injection in the left kidney was then performed. T 2 -and T 2 *-weighted images showed that the labelled cells induced hypointense signals at the injection site. Nevertheless, the hypointense regions tended to disappear after several days, mainly owing to dilution of the MIRB iron oxides with cell proliferation. In conclusion, EPR is well adapted to ex vivo analysis of tissues after cell tracking experiments and allows short-term monitoring of metastasizing cells. MRI is a suitable tool for checking labelled cells at their injection site, but dilution of the iron oxides owing to cell division remains a major limitation. BLI remains the most suitable technique for long-term monitoring of metastatic cells.

Malignant melanoma is a tumor characterized by the uncontrolled proliferation of melanocytes, mainly in skin, but also in eyes. Its incidence is rising each year. To improve the diagnosis and treatment of the tumor, it is essential to develop new effective methods to early detect and characterize melanoma. Previously, we demonstrated in a single-shot study that it was possible to map free radicals of melanin pigments using an electron paramagnetic resonance (EPR)-based method. Furthermore, we demonstrated that X-Band (9 GHz) EPR spectrometry was an accurate tool to assess the growth stage of a pigmented tumor. The aim of the present study was to investigate the ability of EPR imaging to detect and localize melanin pigments inside melanin phantoms, B16 melanoma tumor models and resected human melanomas. We show that EPR can provide an accurate image of synthetic samples, both in terms of shape and size, with errors always lower than 10% compared to the real size. Regarding melanoma studies, the ability of EPR imaging to map accurately the melanoma was depending on the concentration of melanin in the sample, which is proportional to the growth stage of the tumor and the consequent signal-to-noise ratio (SNR) provided by the EPR signal intensity. This led us to define an operational concept, considering SNR and interferences with other EPR signals, to determine when EPR imaging was feasible.

The clinical outcome of melanoma depends on the local and distant spread of the disease at the time of diagnosis, as the estimated 5-year survival rate is about 100% for superficial melanoma diagnosed early, but less than 10% for melanoma that has disseminated to major organs such as lungs. There is a crucial need for new effective methods for the detection and the characterization of melanomas. In the pre-clinical setting, this will help to understand the factors that contribute to the malignancy while the transfer into the clinic will contribute to an early effective treatment of patients. Melanoma lesions can be detected by electron paramagnetic resonance (EPR) using paramagnetic properties of melanin pigments. As part of the development of EPR imaging to characterize melanomas, we evaluated in the present study the usefulness of EPR to report on the extension of lung metastases by comparing the method with bioluminescence imaging using B16 melanoma cells expressing luciferase. B16 melanoma cells were injected subcutaneously or intravenously in C57/BL6 mice. The primary tumors or the lung colonization by melanoma cells was measured after several delay periods to obtain several degrees of invasiveness. The animals were measured in-vivo with bioluminescence after i.v. injection of luciferin. The primary tumors or lungs were then excised. After freeze-drying, the content of melanin in lungs was measured and imaged by EPR at 9 GHz. We observed a direct relationship between the EPR intensity and the bioluminescence intensity. Another tumor model (KHT sarcoma), non-pigmented but expressing luciferase, was used to confirm that the EPR signal was directly linked to the melanin pigment present in the tumors.

It has been known for a long time that the melanin pigments present in normal skin, hair, and most of malignant melanomas can be detected by electron paramagnetic resonance (EPR) spectrometry. In this study, we used EPR imaging as a tool to map the concentration of melanin inside ex vivo human pigmented and nonpigmented melanomas and correlated this cartography with anatomopathology. We obtained accurate mappings of the melanin inside pigmented human melanoma samples. The signal intensity observed on the EPR images correlated with the concentration of melanin within the tumors, visible on the histologic sections. In contrast, no EPR signal coming from melanin was observed from nonpigmented melanomas, therefore demonstrating the absence of EPR-detectable pigments inside these particular cases of skin cancer and the importance of pigmentation for further EPR imaging studies on melanoma.