Figure 1. Tumorigenicity and Metastatic Capabilities Were Acquired by melan-a Melanocytes after Detachment/Re-adhe- sion Cycles. (A) Tumorigenicity assay in vivo. Mice were subcutane- ously injected with melan-a melanocyte lineage and its derived cell lines 4C, 4C112 and 4C11+ (2.5x105 cells per mouse). Tumor volume was measured during 100 days (n = 5 animals for each group, P,0.01 by One-Way ANOVA test for significance). (B) Metastasis capacity in vivo. Lungs of mice were analyzed after tail vein injection of 4C112 and 4C11+ melanoma cell lines (2.5x105 cells per mouse). Melan-a and 4C non-tumorigenic melanocyte lineages were used as negative controls. Tumor multiplicity is indicated by the number of lung tumor nodules formed by 4C11+ melanoma cell line (n = 5 animals for each group, P,0.0001 by One-Way ANOVA test for significance). (C) Survival curves of mice injected intravenously with 4C112 and 4C11+ melanoma cell lines (2.5x105 cells per mouse) (n = 5 animals for each group, P,0.0001 by Log-rank test for significance). Poor clinical outcomes were observed for all animals injected with 4C11+ metastatic melanoma cell line. doi:10.1371/journal.pone.0044800.g001 Novel Candidate Biomarkers in Melanomas array approach was used as a platform screening (GeneChip Mouse Genome 430_2.0, format 49; Affymetrix Inc., Santa Clara, CA). In the chosen arrays, there are 45,101 probe sets designed to investigate approximately 34,000 mouse transcripts (the whole genome from Mus musculus). The experiments were carried out in three biological replicates. Class-comparison experiments were performed, and transcripts were selected as statistically significant by the unpairwise two-class SAM analysis (FDR and Q- values,0.05) by using 700 random permutations as follows: I) gene expression profile of the parental melan-a melanocyte lineage versus its 4C pre-malignant melanocyte counterpart: 528 up- and 573 down-regulated genes in 4C pre-malignant melanocytes when compared with melan-a melanocyte lineage (stage I of melan-a malignant transformation); II) gene expression profile of 4C pre- malignant melanocyte lineage versus its 4C112 non-metastatic melanoma cell counterpart: 112 up- and 51 down-regulated genes in 4C112 non-metastatic melanoma cell line when compared with 4C pre-malignant melanocyte lineage (stage II of melan-a malignant transformation); III) gene expression profile of 4C112 non-metastatic melanoma cell line versus its 4C11+ metastatic melanoma cell counterpart: 1294 up- and 1387 down-regulated genes in 4C11+ metastatic melanoma cell line when compared with 4C112 non-metastatic melanoma cell (stage III of melan-a malignant transformation) (Figure 2A). PANTHER software was used to determine the pathway profiles deregulated in the course of melan-a malignant transformation. Therefore, we investigated whether the proteins encoded by the genes identified as differentially expressed during the establishment of a melanoma- like phenotype were connected in known protein interaction signaling pathways. Following detailed manual annotation, the resulting pathways were ranked according to the number of genes involved in each pathway. Within the group corresponding to the transition from melan-a to 4C, representative genes from 22 and 32 pathways had undergone up- and down-regulation, respective- ly. Besides that, within the group corresponding to 4C to 4C112 transition, we observed that representative genes from 3 and 5 pathways had undergone up- and down-regulation, respectively. In addition, representative genes from 39 and 37 pathways had undergone up- and down-regulation, respectively, within the group corresponding to 4C112 to 4C11+ transition (Figure 2B). Additionally, since we have observed no intersection between up- and down-regulated genes within each group (Figure 2A) the presence of a switch of genes was found for pathways within the groups ma versus 4C, and 4C112 versus 4C11+ as follows: I) ma versus 4C: Angiotensin II-stimulated, Inflammation mediated by chemokine and cytokine, Dopamine receptor, Endogenous cannabinoid, Endothelin, GABA-B receptor, Heterotrimeric G-protein, Integrin, PI3 kinase, VEGF, and Wnt. II) 4C112 versus 4C11+: 5HT receptor, Angiogenesis, Inflammation mediated by chemokine and cytokine, EGFR, Endothelin, FGF, Histamine receptor, Integrin, Interleukin, Oxidative stress, Oxytocin receptor, PDGF, Ras, Thyrotropin-releasing hormone, Toll receptor, and VEGF. (Figure 2B). Moreover, genes that significantly contributed to stage-specific pathways were also identified. Thus, deregulation of pathways Aminobutyrate degradation and Phenylethylamine degradation characterized the transition from melan-a melanocytes to 4C pre- malignant melanocyte lineage. Besides that, the pathways Blood coagulation and General transcription by RNA polymerase I marked the transition from 4C pre-malignant melanocytes to 4C112 non- metastatic melanoma cell line. Furthermore, sixteen signaling pathways marked the acquisition of a metastatic phenotype as follows: 2 Arachidonoylglycerol biosynthesis, Adenine/hypoxanthine salvage, Apoptosis, B cell activation, DNA replication, Fas, FGF, Fructose galactose metabolism, N-acetylglucosamine metabolism, P53, P53 feedback loops 2, Purine metabolism, Ras, Synaptic vesicle trafficking, T cell activation, and PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44800