DISSECTING MELANOMA-TUMOUR MICROENVIRONMENT INTERACTIONS DURING TUMOUR PROGRESSION

Despite the latest advances in melanoma management, patients frequently experience relapse and die of metastases. The development of new approaches to better mimic the metastatic process and test novel combination therapies are therefore urgently required. It was recently shown that not transformed cells in the tumour ecosystem could act as key players in driving cancer development, progression, spreading and metastatic niche formation. To deepen the current knowledge of how melanoma reacts to a changing microenvironment and how the cross-talk between melanoma and the surrounding cells impacts on therapy response, we established an innovative, flexible and highly reproducible three-dimensional (3D) model. Making use of our platform of PDXs and primary patient cultures, we proved that patient-derived cells, and not human cell lines, allow us to closer recapitulate the cellular organization of the melanoma tissue. Besides, we determine that the addition of microenvironmental components in the culture, such as fibroblasts and endothelial cells, fosters melanoma invasion of collagen matrix in vitro. Furthermore, we show that 3D cultures are not only tumorigenic in immunodeficient mice (NSG), but that we could also score differences in the metastatic potential of melanomas exposed to fibroblasts and endothelium in hetero-cultures, compared to homo-culture controls, further supporting the in vitro data. In order to dissect the mechanism behind melanoma phenotypical alterations induced by fibroblasts and/or endothelial cells, we inspected the transcriptional and phenotypic profiles of our 3D cultures. We, therefore, assess the upregulation of pathways related to invasion, EMT and ECM remodelling during organotypic culture growth. Notably, L1 cell adhesion molecule (L1CAM/CD171) expression results highly increased in melanoma cells' interaction with either fibroblasts and/or endothelial cells. L1CAM overexpression on melanoma cells proved to be necessary, but not sufficient, to mimic environmental interactions in in vitro invasion assays, still highly prompting melanoma aggressiveness in in vivo experimental models of metastasis. These data imply a fundamental role of TME in driving L1CAM activation. Accordingly, its depletion reduces melanoma cells invasion capability in vitro, while strikingly abolishing the melanoma metastatic properties in NSG mice, even when transplanted with hetero-organotypic cultures. Indeed, all the mice transplanted with L1CAM silenced cells displayed lower metastatic burden and prolonged overall survival. Transcriptional analyses also evidence melanoma transcriptional reprogramming upon L1CAM variation, particularly involving epithelial-to-mesenchymal transition. Plasminogen Activator Inhibitor 1 (PAI1) or SERPINE1, one of the prominent members of the serpins superfamily, is among the secreted factors mostly altered in the hetero-cultures and its production is strictly dictated by melanoma/stroma cross-talk. Interestingly, the increase in L1CAM levels is able to sensitize melanoma to anti-PAI-1 agent cytotoxic effects, while it is only partially effective on L1CAM low expressing cells. Even if further studies are required to determine the reciprocal role of the two proteins in the context of the organotypic culture to then translate it to the tumour niche, our results suggest the existence of an L1CAM/SERPINE1 axis involved in melanoma TME-driven acquisition of invasive and metastatic phenotype, possibly exploitable as a novel vulnerability of highly aggressive cancer cell. Taken together our data indicate that fibroblasts and endothelial cells are able to reprogram melanoma cells' transcriptome and phenotype in our organotypic cultures. Thus, our model is a powerful tool to unveil the mechanisms behind melanoma and surrounding ecosystem relationships during tumour progression, as well as to identify new targetable vulnerabilities during tumour evolution, finally allowing to test of innovative combination treatments in a complex and relevant system.