THE INFLUENCE OF MELANOMA STEM CELLS ON TUMOR MICROENVIRONMENT REMODELING: EXPERIMENTAL INSIGHTS FROM IN VITRO STUDIES

Cancer stem cells (SCs) constitute a small group of tumor cells with SC-like properties, such as selfrenewal and resistance to conventional therapies. In melanoma, it is now well established that cancer SCs play a pivotal role in driving tumor initiation, progression and therapeutic resistance, especially through the cross-talk with stromal and immune cells infiltrating the tumor microenvironment (TME). Notably, cancer SCs possess the ability to protect themselves from immune surveillance by releasing soluble factors, metabolites, and cytokines, thereby creating an immunosuppressed TME. Since the precise molecular mechanisms governing the complex interplay between melanoma SCs and TME remains poorly understood, the aim of the present PhD thesis was to investigate the impact of melanoma SCs on neutrophils, macrophages, keratinocytes and fibroblasts, reported to actively participate in melanoma progression. A series of in vitro study was performed focusing on the phenotypic alterations of such immune/stromal cells under the influence of melanoma SCs. The effects of melanoma SC conditioned medium (SC-CM) on neutrophils was firstly evaluated. Using a neutrophil-like model obtained from HL60 cell line, it was found that melanoma SCs activated these immune cells, as indicated by the up-regulation of the CD66b and CD11b markers and induce their recruitment. Secretome analysis revealed that melanoma SC produce TGF-β, IL-6, and IL-8 cytokines, which are involved in chemoattraction and polarization of neutrophils towards a pro-tumoral (N2) phenotype. Accordingly, SC-CM promoted the activation of ERK and STAT3 pathways and the overexpression of CXCR2, NF-kB and P38 in neutrophils, pathways associated to a N2 phenotype. Activated neutrophil were also characterized by an augmented ROS and H2O2 production and the release of extracellular traps (NETs) in a cell death-independent manner. The acquired N2 phenotype was confirmed by ELISA assays showing an increased secretion of MMP-9 and pro-inflammatory cytokines like IL-8 and IL-6. In turn, SC-conditioned neutrophils contributed to maintain and sustain the stemness features of melanoma cells by boosting their spheres capacity and ABCG-2 expression. The second part of the project focused on the investigation of macrophage phenotypic remodeling induced by melanoma SCs. It was set-up an in vitro model by utilizing the human THP-1 monocyte cell line differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA). This cellular model was extensively characterized by morphological evaluations, Real-time PCR and ELISA assays that confirm its reliability. It was found that melanoma SCs contribute to the re-education of macrophages by inducing a mixed phenotype in these innate immune cells, characterized by the upregulation of both anti-tumoral (M1) and pro-tumoral (M2) markers, including IL-6, IL-12B, STAT1, IL-10, VEGF, and MARCO, after exposure to melanoma SCs-CM. It was also observed that melanoma SCs-activated macrophages were characterized by an increased expression of Arginase1, a well-described M2 markers associated with cancer immunosuppression and progression. Accordingly, ELISA data showed a higher secretion of IL-6, IL-10 and MMP-2 in macrophages exposed to melanoma SCs-CM compared with the control counterpart, reinforcing the idea of the acquisition of an M1/M2 mixed phenotype. To define the functional phenotype of melanoma SCs activated macrophages, it was assessed their ability to modulate NK cell cytotoxicity in vitro. Surprisingly, SCs-treated macrophages failed to enhance NK cell cytotoxic activity, indicating that despite expressing pro-inflammatory mediators, they lack the capacity to mount an effective immune response. Finally, the impact of melanoma SCs on the primary resident cells within the melanoma niche, such as fibroblasts and keratinocytes, was also studied. It was established a 3D tri-culture model consisting of human melanoma SCs, fibroblasts, and keratinocytes, using two different methodological approaches involving either low attachment 96-well plates or agarose molds. Spheroids were characterized through cryosection, immunofluorescence and confocal microscopy followed by quantitative analyses and nuclei segmentation with deep-learning-based algorithms. By employing these techniques, an early-stage melanoma cellular models were successfully reproduced by incorporating melanoma SCs directly into the tri-culture spheroids. Notably, melanoma SCs had a profound influence on keratinocyte differentiation, leading to a complete reorganization of the keratinocyte compartment around the fibroblast core. This resulted in a striking alteration of their typical stratification, with basal cells in contact with the fibroblasts core and more differentiated cells in the outer layer. Fibroblasts were also impacted by the presence of melanoma SCs, as evidenced by their reduced expression of collagen IV, mirroring the progressive rearrangement of the basement membrane, crucial for facilitating the spread of melanoma SCs. Overall, the obtained results provide an overview of the potential role of melanoma SCs in the TME, opening novel research areas for investigating the mediators involved in cell communication, and offering opportunities to develop in vitro 3D models that more accurately replicate the organizational complexity of the TME.