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.
B-RAF is the most frequently mutated protein kinase in human cancers.1 The finding that oncogenic mutations in BRAF are common in melanoma2 followed by the demonstration that these tumors are dependent on the RAF/MEK/ERK pathway3 offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients.,Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity.,Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts.4 Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032.5 In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment.,This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumor regressions.,At higher drug exposures afforded by a new amorphous drug formulation,4,5 greater than 80% inhibition of ERK phosphorylation in the tumors of patients correlated with clinical response.,Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily.5 These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.