Cross-modulation between murine mast cells and the melanoma tumor microenvironment in a three-dimensional heterotypic culture model

Cutaneous melanoma is a highly aggressive malignant tumor, responsible for 60-80% of deaths among skin cancers. During its progression, transformed cells recruit adjacent cells (including fibroblasts and immune cells) to form the tumor microenvironment (TME) which is modulated by various signaling molecules. The role of infiltrating immune cells, such as mast cells, is controversial, presenting either protective effects or contributing to pathogenesis. Studies indicate that mast cells may have a pro-tumoral role in melanoma, but few evaluate the behavior of mast cell precursors in this context. Our aim is to assess how mast cells at different stages of differentiation affect and/or are affected by the melanoma TME.

Thus, we established a 3D culture model with murine melanoma cells and fibroblasts, to which mast cells at distinct stages of differentiation from C57BL/6 mice (CEUA 027/21) were added. The cells were co-cultured for 3 days, and the resulting spheroids were subjected to assays to investigate the growth and migratory capacity of the tumor mass and mast cell differentiation.

Our preliminary data suggest that mast cells do not significantly impact the growth and migratory capacity of the cells in the spheroids. However, we observed that mast cell "precursors" undergo differentiation in response to stimuli received in the spheroid within just 3 days, evidenced by a marked reduction in α4β7 integrins, which take much longer (2-3 weeks) when only mast cells were maintained in culture. We also found that fibroblasts cultured independently of anchorage are major producers of IL-6, which may be involved in the differentiation process of these "precursors." Thus, we intend to further explore the role of fibroblasts in the potential differentiation of mast cells, as well as identify the differentiation profile based on which proteases are expressed by mast cells in contact with spheroids compared to those maintained separately in culture.