Melanin Phantoms

Phantoms were prepared as solid dilutions of eumelanin (Sepia officinalis, > 99%; Sigma-Aldrich, Steinheim, Germany; 3.5 × 1018 spins/gram of melanin) by trituration with increasing amounts of sucrose (> 99%, Fluka Biochemika, Steinheim, Germany). The powder was weighed on Scotch® Magic tape with melanin quantity ranging between 75 µg to 1 mg. The diameter of the circle occupied by the powder was chosen as less than 8 mm (diameter of the loop coil). The scotch tape used did not present any EPR signal. After weighing, the tape was covered with a second tape and the obtained film was used for measurement purposes.

Cell Culture

Two melanoma cell lines were used: B16F10luc pigmented melanoma cells and nonpigmented WM-266–4 melanoma cells were purchased from ATCC (Manassas, Virginia, USA) and Rockland Immunochemicals (Limerick, PA, USA) respectively. B16F10Luc and WM2664 melanoma cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM; Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco™, Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated at 37 °C with 5% CO2. Cells were used for inoculation in animals after 3 passages of cell culture.

Tumor Inoculation in Mice

All animal experiments were carried out in accordance with the local ethics committee for animal care of the Université catholique de Louvain (Protocol #2023/UCL/MD/06) and with the European Directive 2010/63/EU.

Intra Dermal Skin Injection

Mice were anesthetized with inhaled isoflurane mixed with air (3% for induction, 1.5% for maintenance). Five-week-old female NMRI nude mice (n = 8) (Elevage Janvier, Le Genest St. Isle, France) were inoculated via an intradermal injection of 200 k cells in 50 µL PBS of murine luciferase-encoding B16F10Luc pigmented melanoma cells or 200 k in 50 µL PBS cells of human WM2664 melanoma cells (used as non-pigmented control cells). Tumors were measured as 2 perpendicular diameters (length and width) of the tumor, to determine the surface area in mm2.

Subcutaneous Lymph Node Injection

For the proof-of-principle, we used an injection of cells into the lymph nodes to insure the exact localization of melanoma cells. Mice were anesthetized with inhaled isoflurane mixed with air (3% for induction, 1.5% for maintenance). Five-week-old female NMRI nude mice (n = 8) (Elevage Janvier, Le Genest St. Isle, France) were injected at the tail base with 20 µL of 0.5% Evans Blue dye (Alfa Aesar, Haverhill, MA, USA). After 24 h, a small incision was performed in the iliac region. The lymph nodes were identified as small blue dots because of the accumulation of the dye. 200 k cells in 20 µL PBS of murine luciferase-encoding B16F10Luc melanoma cells were injected directly in one of the right or the left iliac lymph node. After injection, the wound was closed with a sterile suture thread.

Lung Metastasis Model

Mice were anesthetized with inhaled isoflurane mixed with air (3% for induction, 1.5% for maintenance). Five-week-old female NMRI nude mice (n = 8) (Elevage Janvier, Le Genest St. Isle, France) were injected intravenously with 750 k cells in 50 µL PBS of Luciferase-encoding murine B16F10Luc melanoma cells in the tail vein. Melanoma cells are known to accumulate in the lungs after IV injection [28].

Measurement on Human Skin

Films containing melanin described previously were taped onto the surface of a human arm belonging to a healthy volunteer (Phototype III) who signed an informed consent form.

Multi-harmonic and CW EPR Measurements in vivo and in vitro

EPR measurements were performed using a clinical EPR spectrometer (Clin-EPR LLC, Lyme, NH) operating at 1.15 GHz equipped with the eSpect++ expansion module for multi-harmonic EPR analysis (Novilet, Poznan, Poland) (Fig. 1a). Measurements were carried out using the following parameters: center field, 41.6 mT; modulation amplitude, 0.3 mT or 0.6 mT; microwave power, 10 mW; modulation frequency, 20.4 kHz; time constant, 5 ms; scan time, 5 s; sweep width, 5 mT; number of acquisitions, 5 scans. Automatic tuning was performed by the system, followed by a manual adjustment for optimization. A sealed capillary containing 10 μL of a 2 mM solution of 15N-PDT (4-oxo-2,2,6,6-tetramethylpiperidine-d16-15N-1-oxyl; CDN Isotopes, Pointe-Claire, Canada) was fixed along the axis of the resonator with the tip of the capillary just at the surface of the loop coil. 15N-PDT was used for field positioning knowing that the EPR signal of melanin appears between the two lines of PDT (Fig. 1b) [11]. For in vitro calibrations, films containing melanin were placed on top of a solid surface and ten independent EPR measurements were performed by acquisitioning a series of five scans. For in vivo measurements, the area of interest (skin tumor or area identified thanks to the bioluminescence for the lymph node and metastatic models) was placed under a loop-gap surface coil resonator (Novilet, Poznan, Poland) with an inner diameter of 8 mm placed in the center of the magnet. A film (Parafilm® “M,” Neenah, WI, USA) 10 × 20 cm was folded three times resulting in eight layers, and placed between the coil and the area of interest. This method was previously optimized to allow rapid tuning of the coil on lossy biological samples [4, 13]. Mice were anesthetized with inhaled isoflurane mixed with air (3% for induction, 1.5% for maintenance). Measurements were recorded at days 5, 8, 11, 13, 15 after B16F10Luc tumor induction in the skin, 5, 8, 11, 14 days after WM2664 tumor induction in the skin, and 5, 7, 9, 11, 14 days after B16F10Luc tumor injection in the lymph nodes. The tumor diameter was measured with a digital caliper (Traceable® Products, 12,554 Galveston Road Suite B230 Webster, TX 77598 USA). Two acquisition modes were used: 1st harmonic mode (CW) and multi-harmonic mode, both possible on the same resonator. First, the 1st harmonic signal was measured with a modulation amplitude of 0.3 mT (corresponding to the experimental conditions used in the previous clinical trial carried out on patients with lesions suspects of being melanomas) [11]. As the clinical EPR system was upgraded with the capability to deliver larger modulation amplitude, another 1st harmonic analysis was applied at modulation amplitude of 0.6 mT. For multi-harmonic analysis, the analysis range for multi-harmonic data processing was set up to 100 harmonics, and the cut off frequency of the low-pass filter used in the post-acquisition was 700 kHz. Peak-to-peak signal amplitude of the recorded signal was measured and calculated as a ratio over the noise recorded.

Fig. 1

A: Clinical EPR spectrometer (Clin-EPR, on the left) equipped with a module for multi-harmonic analysis (Novilet, on the right). B: EPR spectrum recorded in vivo on skin melanoma in mice. The EPR spectrum contains signals coming from melanin (center, green arrow) and the doublet of 15N-PDT (low and high field signals, red arrows; external reference placed in a sealed capillary attached to the coil)

Bioluminescence Measurements

While superficial melanoma can be easily visualized, the invasion in deeper tissues such as in lymph nodes and lungs required to use bioluminescence to assess melanoma cells expansion. That is the reason why we selected a B16 melanoma model expressing luciferase (B16F10luc). Tumors developed in the lymph node and metastasis models were monitored by bioluminescence imaging, using IVIS Spectrum in vivo Imaging System (PerkinElmer, Waltham, MA, USA). The mice were anesthetized with inhaled isoflurane mixed with air (3% for induction, 1.5% for maintenance). D-luciferin (PerkinElmer, 150 mg/kg) was injected intra-peritoneally (IP). After 25 min, the mice were placed in the bioluminescence imaging setup, and the bioluminescence signal was measured as the total flux of photons detected from the tumor (p/s) (the tumor area is specified in a defined region of interest on the camera image). A preliminary kinetics experiment allowed us to the optimal time point for in vivo imaging.