Combination therapy with BRAF and MEK inhibitors significantly improves survival in BRAF mutated melanoma patients but is unable to prevent disease recurrence due to the emergence of drug resistance. Cancer stem cells (CSCs) have been involved in these long-term treatment failures. We previously reported in lung cancer that CSCs maintenance is due to altered lipid metabolism and dependent upon Stearoyl-CoA-desaturase (SCD1)-mediated upregulation of YAP and TAZ. On this ground, we investigated the role of SCD1 in melanoma CSCs. SCD1 gene expression data of melanoma patients were downloaded from TCGA and correlated with disease progression by bioinformatics analysis and confirmed on patient’s tissues by qRT-PCR and IHC analyses. The effects of combination of BRAF/MEKi and the SCD1 inhibitor MF-438 were monitored by spheroid-forming and proliferation assays on a panel of BRAF-mutated melanoma cell lines grown in 3D and 2D conditions, respectively. SCD1, YAP/TAZ and stemness markers were evaluated in melanoma cells and tissues by qRT-PCR, WB and Immunofluorescence. We first observed that SCD1 expression increases during melanoma progression. BRAF-mutated melanoma 3D cultures enriched for CSCs overexpressed SCD1 and were more resistant than 2D differentiated cultures to BRAF and MEK inhibitors. We next showed that exposure of BRAF-mutated melanoma cells to MAPK pathway inhibitors enhanced stemness features by upregulating the expression of YAP/TAZ and downstream genes but surprisingly not SCD1. However, SCD1 pharmacological inhibition was able to downregulate YAP/TAZ and to revert at the same time CSC enrichment and resistance to MAPK inhibitors. Our data underscore the role of SCD1 as prognostic marker in melanoma and promote the use of SCD1 inhibitors in combination with MAPK inhibitors for the control of drug resistance.

In the past decade, adipose tissue has become a highly interesting source of adult stem cells for plastic surgery and regenerative medicine. The adipose source offers two options for the isolation of regenerative cells: the enzymatic digestion an expensive time-consuming procedure lacking a common standard operating protocol, or the non-enzymatic dissociation methods based on mechanical forces to break the processed adipose tissue. Here, we propose innovative inexpensive non-enzymatic protocols to collect and concentrate clinically useful regenerative cells from adipose tissue by centrifugation of the infranatant fraction of lipoaspirate as first step, usually discarded as a byproduct of the surgical procedure, and by fat shaking and wash as second enrichment step. The isolated cells were characterized according to the criteria proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) to define human mesenchymal stem cells, and the results were compared with matched lipoaspirate samples processed with collagenase. The results demonstrated the usability of these new procedures as an alternative to fat grafting for treating stem cell-depleted tissues and for specific application requiring minimal or null soft tissue augmentation, such as skin diseases including severe burn and post-oncological scaring, chronic non-healing wounds, and vitiligo.

Adipose tissue-derived stem cells are considered to be a promising source in the field of cell therapy and regenerative medicine. In addition to direct cell replacement using adipose tissue or purified stem cells, intercellular molecule exchange by the adipose tissue complex, a vast array of bioactive secretory factors, demonstrated beneficial effects by reducing tissue damage and stimulation of endogenous repair. However, for therapeutic purposes, the use of secretome derivatives, such as full conditioned media or purified exosomes generated in vitro, may present considerable disadvantages for cell manufacturing, storage, product safety, and their potential as a ready-to-go therapeutic product. In this study, the effect of a liquid fraction of lipoaspirates isolated intraoperatively from 28 healthy donors was evaluated for their protective effect against oxidative stress and senescence, proliferation, and migration in vitro on normal human melanocytes, keratinocytes, and fibroblasts. Immunoenzymatic quantification of several growth factors and important signal molecules was used to define the biological profile of physiological adipose tissue secretome. Adipose tissue extracellular fraction (AT-Ex), isolated from lipoaspirate, exhibited significant potential for skin repair. AT-Ex augmented dermal and epidermal cell proliferation in a dose-dependent manner without promoting cancer cell growth. Moreover, migration of dermal fibroblasts, an important phenomenon implicated in endogenous repair, was enhanced by AT-Ex treatment. AT-Ex has a positive impact on oxidative stress damage when cells are exposed to extrinsic hostile factors and prevent a fibroblast senescence phenotype including paracrine functions associated with skin aging. Collectively, our findings propose natural systems carrying the physiological balance of in-vivo produced secretome that could improve cutaneous wound healing and tissue repair. This approach, representing an innovative perspective and therapeutic strategy in regenerative medicine, could also be combined with autologous stem cell grafts to treat chronic nonhealing wounds, stable vitiligo, severe burns, and post-oncological scarring.

Solid tumors are complex systems characterized by dynamic interactions between neoplastic cells, non-tumoral cells, and extracellular components. Among all the stromal cells that populate tumor microenvironment, fibroblasts are the most abundant elements and are critically involved in disease progression. Cancer-associated fibroblasts (CAFs) have pleiotropic functions in tumor growth and extracellular matrix remodeling implicated in local invasion and distant metastasis. CAFs additionally participate in the inflammatory response of the tumor site by releasing a variety of chemokines and cytokines. It is becoming clear that understanding the dynamic, mutual melanoma–fibroblast relationship would enable treatment options to be amplified. To better characterize melanoma-associated fibroblasts, here we analyzed low-passage primary CAFs derived from advanced-stage primary skin melanomas, focusing on the immuno-phenotype. Furthermore, we assessed the expression of several CAF markers and the production of growth factors. To deepen the study of CAF–melanoma cell crosstalk, we employed CAF-derived supernatants and trans-well co-culture systems to evaluate the influences of CAFs on (i) the motogenic ability of melanoma cells, (ii) the chemotherapy-induced cytotoxicity, and (iii) the release of mediators active in modulating tumor growth and spread.

The role of Wnt/-catenin signaling pathway in melanoma epithelial-to-mesenchymal-like switching: evidences from patients-derived cell lines