Preclinical assessment of novel therapies to fight cancer requires models that reflect the human physiology and immune response.,Here, we established an in vitro three-dimensional (3D) reconstructed organotypic human melanoma-in-skin (Mel-RhS) model to investigate cellular and molecular features of tumor formation over a period of 6 weeks.,Tumor nests developed over time at the epidermal-dermal junction and spread towards the dermis, in places disrupting the basement membrane.,This coincided with secretion of matrix metalloproteinase 9 (MMP-9) by melanoma cells.,These features resemble the initial stages of invasive melanoma.,Interestingly, while the SK-MEL-28 cell line did not secrete detectable levels of interleukin-10 (IL-10) in traditional two-dimensional monolayers, it did express IL-10 in the 3D Mel-RhS, as did the surrounding keratinocytes and fibroblasts.,This cellular cross-talk-induced secretion of IL-10 in the Mel-RhS indicated the generation of an immune suppressive microenvironment.,Culture supernatants from Mel-RhS interfered with monocyte-to-dendritic-cell differentiation, leading to the development of M2-like macrophages, which was in part prevented by antibody-mediated IL-10 blockade.,Indeed, high-dimensional single-cell analysis revealed a shift within the monocyte population away from a CD163+PD-L1+ M2-like phenotype upon IL-10 blockade.,Thus, the 3D configuration of the Mel-RhS model revealed a role for IL-10 in immune escape through misdirected myeloid differentiation, which would have been missed in classical monolayer cultures.,The online version of this article (10.1007/s00262-020-02626-4) contains supplementary material, which is available to authorized users.
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.,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.,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.