3D co-culture models underline metastatic melanoma cell sensitivity to vemurafenib.

Melanoma cell sensitivity to targeted therapy molecules is dependent on the tumor microenvironment (cell-cell and cell-extracellular matrix interactions). Three dimensional (3D) in vitro cell culture systems better reflect the native structural architecture of tissues and are attractive to investigate cellular interactions. We have developed and compared several metastatic melanoma (MM) models: melanoma cells (SK-MEL-28 and SK-MEL-3, BRAF V600E mutant and SK-MEL-2 BRAF wt) cultured as a monolayer (2D) and cocultured on 3D dermal equivalents with fibroblasts to better unravel factors modulating cell sensitivity to a BRAF inhibitor (BRAFi, vemurafenib). Cell sensitivity to treatments was evaluated under various aspects: cell proliferation (cell counting, EdU incorporation, MTS assay), MAPK and PKB/Akt signaling pathway analysis (Western blotting), apoptosis (TUNEL), cytokine and growth factor release (ELISA), and histology (3D models). SK-MEL-28 and SK-MEL-3 cell proliferation inhibition upon BRAFi treatment was observed in both models, without apoptosis induction. SK-MEL-2 cell line was clearly resistant to BRAFi when cultured as a monolayer but not when cocultured with 3D fibroblasts embedded in a type I collagen matrix. Conditioned-media provided by 3D fibroblasts (dermal equivalents) underlined 2D SK-MEL-2 cell sensitivity to BRAFi. Cell culture supernatant analysis revealed that dermal equivalents released some soluble factors (interleukin [IL]-6, IL-8, hepatocyte growth factor, transforming growth factor); these secretions were modified during vemurafenib treatment. The heterotypic 3D melanoma model we have established summarizes paracrine signalization by stromal cells and type I collagen matrix, mimicking the natural microenvironment of cutaneous MM. This model could be a powerful tool for predicting drug efficiency. Impact Statement Three dimensional in vitro cell culture systems better reflect the native structural architecture of tissues and are attractive to investigate cancer cell sensitivity to drugs. We have developed and compared several metastatic melanoma (MM) models cultured as a monolayer (2D) and cocultured on three dimensional (3D) dermal equivalents with fibroblasts to better unravel factors modulating cell sensitivity to vemurafenib, a BRAF inhibitor. The heterotypic 3D melanoma model we have established summarizes paracrine signalization by stromal cells and type I collagen matrix, mimicking the natural microenvironment of cutaneous MM, and allows for the identification of potent sensitive melanoma cells to the drug. This model could be a powerful tool for predicting drug efficiency.