There is increasing need of a more human cell model to recapitulate the in-vivo cell-cell interactions, presenting physiological relevance. Unraveling the cell molecular interactions of complex molecular machines in their cellular context is of prime importance for understanding fundamental physiological processes and bridge the gap between animal models and human clinical trials. In this context human 3D in-vitro models have a potential for pre-clinical research. Magnetic resonance spectroscopy (MRS) of intact living cells gives the possibility to revile information about cell-cell and cell-extracellular matrix (ECM) interactions. [1,2]. The carcinogenic process, i.e. transform into tumor cells leads to intracellular changes, such as immortalization, independence of growth signals and metabolic changes. These significantly modify the intracellular and the tumor microenvironment [3]. Physiological processes rely on the concerted action of molecular entities in and across different cellular compartments. MRS has the potential to diagnose many tumors and to characterize their metastatic potential. Therefore the meaning of intensity changes of MRS signals associated to cells metabolism is of fundamental importance. With this respect, studies in intact cultured cells that maintain cells under controlled conditions are a unique tool for high-lighting aspects of metabolism. In this study we present the metabolic profile of highly metastatic human melanoma cells line WM793.

For in-vitro MRS, the studied melanoma cells WM793 were grown in 75cm² TC flasks (Sarstedt) at 37^oC in 95% humidified air containing 5% CO₂ incubator. The applied culture medium was RPMI1640 (pH 7.4, Sigma), supplemented with 10% of Fetal Bovine Serum (Sigma) and 1% of antibiotics (50.5 units/ml penicillin, 50.5μg/ml streptomycin and 101μg/ml neomycin, Sigma). Cells were trypsinized to detach as a thin tissue layer from flask, transferred into 15mL tube and centrifuged at 2000 RPM for 2min. The trypsin supernatant was removed and cells quasi pellet was transferred into the designed setup. The tube with cells was filled within the RPMI (Fig 1).

All experiments were performed on a 9.4T/26cm horizontal magnet with a 12cm gradient coil insert (400mT/m,120µs)(Varian/Agilent). A home-built single loop surface coil of 15mm-diameter was used as transceiver. Localization of the living cell was performed with a fast spin-echo image and used to place the 1H-MRS acquisition voxel (Fig 2). Spectroscopy was acquired with the SPECIAL sequence [4] in a 2.5x4x5mm³ voxel with the following parameters (TE/TR=2.8/4000ms, 400 averages). Vapor water suppression as outer volume suppression module were applied. Field inhomogeneity was corrected using the FASTMAP protocol. The acquisition time was of 25min. Spectrum was quantified using LCModel using water signal as reference.

The proposed procedure with an acquisition time of 25min allowed to acquire high quality spectra with S/N=26 and line width of 6Hz measured on the water resonance (Fig. 3). The relatively short acquisition time is believed to minimize the eventual exposure of cells to high hypoxia environment. Peaks of metabolites characteristic for dividing cells populations, like choline and amino acids such as alanine, glutamate, taurine, and lactate were detected in addition to the metabolism of glucose, acetate, ascorbate and macromolecules (Table 1). A few resonances were not clearly identified in this preliminary study, with peaks at 3.45, 3.82 and 3.87, which may correspond to glycogen. Further investigations are required to establish the true nature of these signals. Metabolites detected in this study were also reported by Bacchi et al. in their experiment on the lyophilized extracts from Tm1 murine melanoma cells [5]. These primary results encourage us to continue investigation of in-vitro living cells MRS study and to go further for primary cells line from eradicated tumors of clinical patients. Furthermore this method allows us to perform investigation on experimental treatments with for example anticancer drug by monitoring the change of metabolites level.We demonstrate the feasibility of characterizing in-vitro living melanoma cells by 1H-MRS. A total number of 9 metabolites were quantified in the study, covering energy markers (Glc, Lac, Ace), amino acids (Glu, Ala) and anti-oxidant (Tau, Asc) and cell membrane precursor (Cho). In-vitro living cells MRS is believed to provide new insight on the characterization of cancer and also to help in conception of treatments.