candidate biomarkers commonly perturbed in metastases. Fur- thermore, it provides a platform for testing new agents in combination therapies. Finally, the molecular markers deregulated earlier in melanoma development should be validated in the future in a large cohort of human specimens as a prognostic signature. Materials and Methods Model of Study This work was done using an in vitro mouse melanoma model in which the deregulation of cellular adhesion configured the carcinogenic stimulus to melan-a (ma) melanocyte lineage. This experimental model developed by our research group was previously described [14,15]. It does not use physical, chemical or environmental carcinogens or genetic manipulations, and can easily be reproduced to study mechanisms of melanoma genesis in a spectrum of distinct stages of tumor progression [15]. Melan-a, that is the first known established lineage of non- tumorigenic mouse melanocytes, is a spontaneous immortal line of pigmented melanocytes derived from normal epidermal melano- blasts from embryos of inbred C57BL mice. Melan-a cells grow in adherent condition and require 12-O-tetradecanoyl phorbol 13- acetate (PMA) to grow [14]. Recently, our group has showed no structural chromosomal abnormalities in melan-a cell line based on karyotype analysis [17]. Thus, melan-a melanocytes provide an excellent parallel non-tumorigenic lineage for studies of the molecular basis of melanoma pathophysiology. Shortly, four sequential anchorage blockade cycles of melan-a melanocytes for 96 hours culminate with the establishment of the melan-a derived subline corresponding to a non-tumorigenic, but pre-malignant melanocyte lineage, which was called 4C. Apart from this intermediate phenotype associated with melanomas, distinct melanoma cell lines (4C112 and 4C11+, among others) were established by limiting dilution of spheroids found after a new anchorage blockade cycle of the 4C pre-malignant melanocyte lineage. To perform repetitive deadhesion cycles, melan-a melanocytes were plated at 1% agarose-coated plates, as previously described [15]. We have characterized the 4C as a pre-malignant melanocyte lineage since it presents a higher anoikis resistance [49] and an aberrant differentiation program (Camila Ferreira de Souza, manuscript in preparation) relative to its parental melan-a cell line. Therefore, we are using a time- dependent model of melanoma progression that was developed at five different time-points to carefully analyze the molecular basis of the tumorigenic conversion of melan-a melanocytes subjected to a sustained stress condition, which was promoted by consecutive detachment/re-adhesion cycles. Cell Culture Mouse melan-a melanocyte lineage was established by Prof. Dorothy C. Bennett (Division of Biomedical Sciences, St. George’s, University of London, UK) [14] and kindly provided by Prof. Michel Rabinovitch (Departamento de Parasitologia, Universidade Federal de Sa˜o Paulo, Brazil). Melan-a melanocytes were grown in RPMI 1640 pH 6.9 medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY), 200 nM PMA (Sigma-Aldrich, Saint Louis, MO) and antibiotics (Gibco) at 37uC with 5% CO2 in air. Cell sublines derivatives from melan-a melanocytes after sequential detachment/re-adhesion cycles (4C, 4C112 and 4C11+) were grown under the same condition as the parental melan-a melanocyte lineage, except for PMA, since they lost the requirement of this factor to grow. Novel Candidate Biomarkers in Melanomas Human primary melanocytes from neonatal foreskin were isolated after informed consent signed by patients according to the Ethics Committee from Hospital Universita´rio (HU-USP, CEP 943/09) and kindly provided by Prof. Silvya S. Maria-Engler (Departamento de Ana´lises Cl´ınicas e Toxicolo´ gicas, Universidade de Sa˜o Paulo, Brazil). These cells were maintained in basal medium for melanocytes 254CF supplemented with Human Melanocyte Growth Supplement (HMGS) and 0.2 mM calcium chloride (Gibco). Five short-term cultures of human melanoma cell lines representing lymph node and systemic metastases (Mel-2, Mel-3, Mel-11, Mel-14 and Mel-33) were patient-derived and maintained in RPMI 1640 pH 7.2 medium (Gibco) supplemented with 15% FBS (Gibco BRL), 2 mM L-glutamine, 1 mM sodium pyruvate and antibiotics (Gibco). These cell lines, freshly derived from surgery after informed consent signed by patients according to human subjects’ research protocol approved by the Ethics Committee of the Fundac¸a˜o Antoˆnio Prudente, Hospital A.C. Camargo (CEP 096/98, 31/03/98), were a kindly gift from Dr. De´bora Castanheira Pereira da Silva (Hospital A.C. Camargo). Mice Studies All in vivo studies were carried out in accordance with the protocol approved by the Institutional Animal Care and Ethics Committee of the Universidade Federal de Sa˜o Paulo (CEP 1509/ 07 and 0389/09 from P.X. and C.F.S., respectively). Female syngeneic C57Bl/6 mice were obtained at 6-8 weeks of age from Biote´rio Central (Universidade Federal de Sa˜o Paulo, Brazil). Animals were maintained on a 12 hours light/dark cycle and were given free access to food in accordance to the International Guiding Principles for Biomedical Research Involving Animals (CIOMS; Genebra, 1985). Melan-a, 4C, 4C112 and 4C11+ cell lines were harvested after trypsin treatment of subconfluent monolayers, counted and suspended in phosphate-buffered saline (PBS). To perform the in vivo tumorigenesis assay, 2.5x105 4C112 and 4C11+ melanoma cells per mouse were inoculated subcutaneously. Animals were checked daily to monitor the presence of palpable tumors during 100 days. The growth tumor curves were determined by measuring the tumor volume (mm3) as follows: d26D/2, in which d corresponds to the smallest tumor diameter and D to the largest one. To perform the in vivo experimental metastasis assay, 2.5x105 4C112 and 4C11+ melanoma cells per mouse were inoculated intravenously into lateral tail vein. Mice were sacrificed, lungs were surgically excised, and tumor multiplicities were evaluated by measuring the presence and the number of metastatic pulmonary foci. In both experiments, non-tumorigenic melan-a and 4C melanocyte lineages were used as negative controls. Each experimental group consisted of five animals. Statistical signifi- cance (P,0.05) was determined using One-way ANOVA test. Mice survival curves after intravenously inoculation of 4C112 and 4C11+ melanoma cells were calculated by the Kaplan-Meier method, with the statistical significance (P,0.05) evaluated by the Log-rank test. All statistics were carried out using GraphPad Prism software version 5 for Windows (GraphPad, San Diego, CA). Spectral Karyotyping Analysis (SKY) In order to investigate structural chromosomal abnormalities and rearrangements as indicators of karyotype evolution in melanomas, SKY was performed using the ASI (Applied Spectral Imaging, Vista, CA) kit for mice. Chromosome preparations were hybridized and stained in accordance to manufacturer’s instruc- tions. Imaging of the signals was carried out using the Spectra Cube (ASI) system mounted on an Axioplan 2 microscope (Carl Zeiss) with a 63x/1.4 oil objective and the Case Data Manager 4.0 PLOS ONE | www.plosone.org 15 September 2012 | Volume 7 | Issue 9 | e44800