Simple SummaryThe melanoma-conditioned microenvironment promotes immune escape and tumor progression, contributing to resistance to anti-melanoma immunotherapy in a large group of treated patients. While two-dimensional cultures lack tissue-context, animals poorly predict human immune responses. Thus, a clear need exists for more physiological human models. We previously described an in vitro human melanoma-in-skin (Mel-RhS) model with the SK-MEL-28 cell line mirroring features of invasive melanoma. Here, we further investigated the tumor-induced microenvironment of this model, generated five additional Mel-RhS models (A375, COLO829, G361, MeWo, and RPMI-7951), and performed in-depth analysis of invasion, immune modulation, angiogenesis, and their respective mediators. We present three-dimensional models allowing for the study of diverse tumor-intrinsic properties that could be used to investigate efficacy of therapeutic interventions. By mimicking different stages of melanoma and related features, each Mel-RhS can be further individually tailored to obtain a physiologically evermore relevant model to study specific mechanisms underlying melanomagenesis and progression.AbstractInvasion, immune modulation, and angiogenesis are crucial in melanoma progression. Studies based on animals or two-dimensional cultures poorly recapitulate the tumor-microenvironmental cross-talk found in humans. This highlights a need for more physiological human models to better study melanoma features. Here, six melanoma cell lines (A375, COLO829, G361, MeWo, RPMI-7951, and SK-MEL-28) were used to generate an in vitro three-dimensional human melanoma-in-skin (Mel-RhS) model and were compared in terms of dermal invasion and immune modulatory and pro-angiogenic capabilities. A375 displayed the most invasive phenotype by clearly expanding into the dermal compartment, whereas COLO829, G361, MeWo, and SK-MEL-28 recapitulated to different extent the initial stages of melanoma invasion. No nest formation was observed for RPMI-7951. Notably, the integration of A375 and SK-MEL-28 cells into the model resulted in an increased secretion of immune modulatory factors (e.g., M-CSF, IL-10, and TGFβ) and pro-angiogenic factors (e.g., Flt-1 and VEGF). Mel-RhS-derived supernatants induced endothelial cell sprouting in vitro. In addition, observed A375-RhS tissue contraction was correlated to increased TGFβ release and α-SMA expression, all indicative of differentiation of fibroblasts into cancer-associated fibroblast-like cells and reminiscent of epithelial-to-mesenchymal transition, consistent with A375′s most prominent invasive behavior. In conclusion, we successfully generated several Mel-RhS models mimicking different stages of melanoma progression, which can be further tailored for future studies to investigate individual aspects of the disease and serve as three-dimensional models to assess efficacy of therapeutic strategies.