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Melanoma tumors have been shown to comprise both invasive and proliferative cell subpopulations. These populations are highly plastic, thus hampering full characterization and therapeutic targeting of dormant and partially dedifferentiated invasive cells. We have reported, previously, that melanoma cells grown in a serum-free neural crest medium, in which they propagate as spheroids, show higher invasiveness and increased immune escape. In addition, in spheroids, we showed the increased expression of several genes which are involved in pluripotency, differentiation, and invasion. We therefore proposed that these culture conditions favor the polarization of proliferative melanoma cells toward an invasive state. As plasticity may suggest a reversible polarization, the aim of this report is to assess the transient phenotype of invasive cells generated through this procedure. We provide evidence that spheroid cells mimic dormant populations, and that this phenotype is fully reversible when cells are reintroduced into culture media that contain serum in which they grow as a monolayer. We also show that most transcriptional deregulations can be reversed. To further explain this plasticity in melanoma cells, we explored the epigenetic status of four gene promoters, assuming changes in acetylation or dimethylation on histone 3. We show reversible modifications on lysine 9 and lysine 4. We propose that spheroids allow the transient polarization of melanoma cells toward enhanced dormancy, loss of differentiation, and invasiveness, thereby reproducing the properties and plasticity of invasive subpopulations in melanoma tumors. This in-vitro model will allow further characterization and targeting of melanoma invasive cell populations.

Background: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells., Methodology/principal Findings: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity., Conclusion/significance: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.