Physicochemical aspects of the tumour microenvironment as drivers of vasculogenic mimicry.

Tumour vascularisation is vital for cancer sustainment representing not only the main source of nutrients and oxygen supply but also an escape route for single or clustered cancer cells that, once detached from the primary mass, enter the blood circulation and disseminate to distant organs. Among the mechanisms identified to contribute to tumour vascularisation, vasculogenic mimicry (VM) is gaining increasing interest in the scientific community representing an intriguing target for cancer treatment. VM indeed associates with highly aggressive tumour phenotypes and strongly impairs patient outcomes. Differently from vessels of healthy tissues, tumour vasculature is extremely heterogeneous and tortuous, impeding efficient chemotherapy delivery, and at the meantime hyperpermeable and thus extremely accessible to metastasising cancer cells. Moreover, tumour vessel disorganisation creates a self-reinforcing vicious circle fuelling cancer malignancy and progression. Because of the inefficient oxygen delivery and metabolic waste removal from tumour vessels, many cells within the tumour mass indeed experience hypoxia and acidosis, now considered hallmarks of cancer. Being strong inducers of vascularisation, therapy resistance, inflammation and metastasis, hypoxia and acidosis create a permissive microenvironment for cancer progression and dissemination. Along with these considerations, we decided to focus our attention on the relationship between hypoxia/acidosis and VM. Indeed, besides tumour angiogenesis, VM is strongly influenced by both hypoxia and acidosis, which could potentiate each other and fuel this vicious circle. Thus, targeting hypoxia and acidosis may represent a potential target to treat VM to impair tumour perfusion and cancer cell sustainment.

Nintedanib-αVß6 Integrin Ligand Conjugates Reduce TGFß-Induced EMT in Human Non-Small Cell Lung Cancer.

Growth factors and cytokines released in the lung cancer microenvironment promote an epithelial-to-mesenchymal transition (EMT) that sustains the progression of neoplastic diseases. TGFß is one of the most powerful inducers of this transition, as it induces overexpression of the fibronectin receptor, αvß6 integrin, in cancer cells which, in turn, is strongly associated with EMT. Thus, αvß6 integrin receptors may be exploited as a target for the selective delivery of anti-tumor agents. We introduce three novel synthesized conjugates, in which a selective αvß6 receptor ligand is linked to nintedanib, a potent kinase inhibitor used to treat advanced adenocarcinoma lung cancer in clinics. The αvß6 integrin ligand directs nintedanib activity to the target cells of the tumor microenvironment, avoiding the onset of negative side effects in normal cells. We found that the three conjugates inhibit the adhesion of cancer cells to fibronectin in a concentration-dependent manner and that αvß6-expressing cells internalized the conjugated compounds, thus permitting nintedanib to inhibit 2D and 3D cancer cell growth and suppress the clonogenic ability of the EMT phenotype as well as intervening in other aspects associated with the EMT transition. These results highlight αvß6 receptors as privileged access points for dual-targeting molecular conjugates engaged in an efficient and precise strategy against non-small cell lung cancer.

Extracellular Acidosis Differentially Regulates Estrogen Receptor ß-Dependent EMT Reprogramming in Female and Male Melanoma Cells.

Clinical outcomes of melanoma patients pointed out a gender disparity that supports a correlation between sex hormone activity on estrogen receptors (ER) and melanoma development and progression. Here, we found that the epithelial-to-mesenchymal transition (EMT) of melanoma cells induced by extracellular acidosis, which is a crucial hallmark of solid cancers, correlates with the expression of ERß, the most representative ER on melanoma cells. Extracellular acidosis induces an enhanced expression of ERß in female cells and EMT markers remain unchanged, while extracellular acidosis did not induce the expression of ERß in male cells and EMT was strongly promoted. An inverse relationship between ERß expression and EMT markers in melanoma cells of different sex exposed to extracellular acidosis was revealed by two different technical approaches: florescence-activated cell sorting of high ERß expressing cell subpopulations and ERß receptor silencing. Finally, we found that ERß regulates EMT through NF-κB activation. These results demonstrate that extracellular acidosis drives a differential ERß regulation in male and female melanoma cells and that this gender disparity might open new perspectives for personalized therapeutic approaches.

Glycolysis-derived acidic microenvironment as a driver of endothelial dysfunction in systemic sclerosis.

OBJECTIVES: SSc is an autoimmune disease characterized by peripheral vasculopathy and skin and internal organ fibrosis. Accumulating evidence underlines a close association between a metabolic reprogramming of activated fibroblasts and fibrosis. This prompted us to determine the metabolism of SSc dermal fibroblasts and the effect on the vasculopathy characterizing the disease. METHODS: A Seahorse XF96 Extracellular Flux Analyzer was used to evaluate SSc fibroblast metabolism. In vitro invasion and capillary morphogenesis assays were used to determine the angiogenic ability of endothelial cells (ECs). Immunofluorescence, flow cytometry and real-time PCR techniques provided evidence of the molecular mechanism behind the impaired vascularization that characterizes SSc patients. RESULTS: SSc fibroblasts, compared with controls, showed a boosted glycolytic metabolism with increased lactic acid release and subsequent extracellular acidification that in turn was found to impair EC invasion and organization in capillary-like networks without altering cell viability. A molecular link between extracellular acidosis and endothelial dysfunction was identified as acidic ECs upregulated MMP-12, which cleaves and inactivates urokinase-type plasminogen activator receptor, impairing angiogenesis in SSc. Moreover, the acidic environment was found to induce the loss of endothelial markers and the acquisition of mesenchymal-like features in ECs, thus promoting the endothelial-to-mesenchymal transition process that contributes to both capillary rarefaction and tissue fibrosis in SSc. CONCLUSION: This study showed the relationship of the metabolic reprogramming of SSc dermal fibroblasts, extracellular acidosis and endothelial dysfunction that may contribute to the impairment and loss of peripheral capillary networks in SSc disease.

The Carbonic Anhydrase IX inhibitor SLC-0111 as emerging agent against the mesenchymal stem cell-derived pro-survival effects on melanoma cells.

Mesenchymal stem cells (MSC) take part to solid tumour-associated stroma and critically influence progression of malignancy. Our study represents a striking example of melanoma progression to a more malignant and resistant phenotype promoted by MSC and the possibility to contrast this diabolic liaison using CAIX inhibitors. In particular, we demonstrated that melanoma cells exposed to a MSC-conditioned medium switch to a more malignant phenotype, characterised by resistance to programmed cell death and endowed with an epithelial-to-mesenchymal transition and stem cell characteristics. These effects were reversed abrogating MSC CAIX activity using SLC-0111, a CAIX inhibitor. Moreover, the acquisition by melanoma cells of a Vemurafenib-resistant phenotype upon MSC-conditioned medium exposure was removed when MSC were treated with SLC-0111. Therefore, MSC may profoundly reprogramme melanoma cells towards a wide resistant phenotype through CAIX involvement, as the use of SLC-0111 is able to contrast the development of this highly risky adaptation for disease progression.

A potentiated cooperation of carbonic anhydrase IX and histone deacetylase inhibitors against cancer.

The emergence of tumour recurrence and resistance limits the survival rate for most tumour-bearing patients. Only, combination therapies targeting pathways involved in the induction and in the maintenance of cancer growth and progression might potentially result in an enhanced therapeutic efficacy. Herein, we provided a prospective combination treatment that includes suberoylanilide hydroxamic acid (SAHA), a well-known inhibitor of histone deacetylases (HDACs), and SLC-0111, a novel inhibitor of carbonic anhydrase (CA) IX. We proved that HDAC inhibition with SAHA in combination with SLC-0111 affects cell viability and colony forming capability to greater extent than either treatment alone of breast, colorectal and melanoma cancer cells. At the molecular level, this therapeutic regimen resulted in a synergistically increase of histone H4 and p53 acetylation in all tested cell lines. Overall, our findings showed that SAHA and SLC-0111 can be regarded as very attractive combination providing a potential therapeutic strategy against different cancer models.

The carbonic anhydrase IX inhibitor SLC-0111 sensitises cancer cells to conventional chemotherapy.

Drug combination represents one of the most accredited strategies of cancer therapy able to improve drug efficacy and possibly overcome drug resistance. Among the agents used to complement conventional chemotherapy, carbonic anhydrase IX (CAIX) inhibitors appear as one of the most suitable, as markers of hypoxic and acidic cancer cells which do not respond to chemo- and radiotherapy. We performed preclinical in vitro assays to evaluate whether the SLC-0111 CAIX inhibitor co-operates and potentiates the cytotoxic effects of conventional chemotherapeutic drugs in A375-M6 melanoma cells, MCF7 breast cancer cells, and HCT116 colorectal cancer cells. Here, we demonstrate that the SLC-0111 CAIX inhibitor potentiates cytotoxicity of Dacarbazine and Temozolomide currently used for advanced melanoma treatment. SLC-0111 also increases breast cancer cell response to Doxorubicin and enhances 5-Fluorouracil cytostatic activity on colon cancer cells. These findings disclose the possibility to extend the use of CAIX inhibitors in the combination therapy of various cancer histotypes.

SOX2 as a novel contributor of oxidative metabolism in melanoma cells.

BACKGROUND: Deregulated metabolism is a hallmark of cancer and recent evidence underlines that targeting tumor energetics may improve therapy response and patient outcome. Despite the general attitude of cancer cells to exploit the glycolytic pathway even in the presence of oxygen (aerobic glycolysis or "Warburg effect"), tumor metabolism is extremely plastic, and such ability to switch from glycolysis to oxidative phosphorylation (OxPhos) allows cancer cells to survive under hostile microenvironments. Recently, OxPhos has been related with malignant progression, chemo-resistance and metastasis. OxPhos is induced under extracellular acidosis, a well-known characteristic of most solid tumors, included melanoma. METHODS: To evaluate whether SOX2 modulation is correlated with metabolic changes under standard or acidic conditions, SOX2 was silenced and overexpressed in several melanoma cell lines. To demonstrate that SOX2 directly represses HIF1A expression we used chromatin immunoprecipitation (ChIP) and luciferase assay. RESULTS: In A375-M6 melanoma cells, extracellular acidosis increases SOX2 expression, that sustains the oxidative cancer metabolism exploited under acidic conditions. By studying non-acidic SSM2c and 501-Mel melanoma cells (high- and very low-SOX2 expressing cells, respectively), we confirmed the metabolic role of SOX2, attributing SOX2-driven OxPhos reprogramming to HIF1α pathway disruption. CONCLUSIONS: SOX2 contributes to the acquisition of an aggressive oxidative tumor phenotype, endowed with enhanced drug resistance and metastatic ability.

The acidic microenvironment as a possible niche of dormant tumor cells.

Although surgical excision, chemo-, and radio-therapy are clearly advanced, tumors may relapse due to cells of the so-called "minimal residual disease". Indeed, small clusters of tumor cells persist in host tissues after treatment of the primary tumor elaborating strategies to survive and escape from immunological attacks before their relapse: this variable period of remission is known as "cancer dormancy". Therefore, it is crucial to understand and consider the major concepts addressing dormancy, to identify new targets and disclose potential clinical strategies. Here, we have particularly focused the relationships between tumor microenvironment and cancer dormancy, looking at a re-appreciated aspect of this compartment that is the low extracellular pH. Accumulating evidences indicate that acidity of tumor microenvironment is associated with a poor prognosis of tumor-bearing patients, stimulates a chemo- and radio-therapy resistant phenotype, and suppresses the tumoricidal activity of cytotoxic lymphocytes and natural killer cells, and all these aspects are useful for dormancy. Therefore, this review discusses the possibility that acidity of tumor microenvironment may provide a new, not previously suggested, adequate milieu for "dormancy" of tumor cells.

Changes in Faecal Microbiota Profile and Plasma Biomarkers following the Administration of an Antioxidant Oleuropein-Rich Leaf Extract in a Rat Model Mimicking Colorectal Cancer.

Oleuropein (OLE), a phenolic compound particularly abundant in the olive leaves, has been reported to have beneficial activities against colorectal cancer (CRC). In vitro studies suggested that these latter could be due to a modulation of the intestinal microbiota. Aiming to evaluate if OLE could affect the intestinal microbiota and the plasma metabolome, an antioxidant oleuropein-rich leaf extract (ORLE) was administered for one week to PIRC rats (F344/NTac-Apcam1137), a genetic model mimicking CRC. ORLE treatment significantly modulated the gut microbiota composition. Plasma metabolomic profiles revealed a significant predictive ability for amino acids, medium-chain fatty acids, and aldehydes. Pathway analysis revealed a significant decrease in phosphatidylcholine accumulation (LogFC = -1.67) in PIRC rats. These results suggest a significant effect of ORLE administration on faecal microbiota profiles and plasma metabolomes, thereby offering new omics-based insights into its protective role in CRC progression.

Divergent regulation of long non-coding RNAs H19 and PURPL affects cell senescence in human dermal fibroblasts.

Cellular senescence is a permanent cell growth arrest that occurs in response to various intrinsic and extrinsic stimuli and is associated with cellular and molecular changes. Long non-coding RNAs (lncRNAs) are key regulators of cellular senescence by affecting the expression of many important genes involved in senescence-associated pathways and processes. Here, we evaluated a panel of lncRNAs associated with senescence for their differential expression between young and senescent human dermal fibroblasts (NHDFs) and studied the effect of a known senomorphic compound, resveratrol, on the expression of lncRNAs in senescent NHDFs. As markers of senescence, we evaluated cell growth, senescence-associated (SA)-ß-Gal staining, and the expression of p21, Lamin B1 and γH2AX. We found that H19 and PURPL were the most altered lncRNAs in replicative, in doxorubicin (DOXO) and ionising radiation (IR)-induced senescence models. We then investigated the function of H19 and PURPL in cell senescence by siRNA-mediated silencing in young and senescent fibroblasts, respectively. Our results showed that H19 knockdown reduced cell viability and induced cell senescence and autophagy of NHDFs through the regulation of the PI3K/AKT/mTOR pathway; conversely, PURPL silencing reversed senescence by reducing (SA)-ß-Gal staining, recovering cell proliferation with an increase of S-phase cells, and reducing the p53-dependent DNA damage response. Overall, our data highlighted the role of H19 and PURPL in the senescent phenotype and suggested that these lncRNAs may have important implications in senescence-related diseases.

Oxidative Stress and Natural Antioxidants in Osteoporosis: Novel Preventive and Therapeutic Approaches.

This review reports in detail the cellular and molecular mechanisms which regulate the bone remodeling process in relation to oxidative stress (OS), inflammatory factors, and estrogen deficiency. OS is considered an important pathogenic factor of osteoporosis, inducing osteocyte apoptosis and varying levels of specific factors, such as receptor activator κB ligand (RANKL), sclerostin, and, according to recent evidence, fibroblast growth factor 23, with consequent impairment of bone remodeling and high bone resorption. Bone loss increases the risk of fragility fractures, and the most commonly used treatments are antiresorptive drugs, followed by anabolic drugs or those with a double effect. In addition, recent data show that natural antioxidants contained in the diet are efficient in preventing and reducing the negative effects of OS on bone remodeling and osteocytes through the involvement of sirtuin type 1 enzyme. Indeed, osteocytes and some of their molecular factors are considered potential biological targets on which antioxidants can act to prevent and reduce bone loss, as well as to promote bone anabolic and regenerative processes by restoring physiological bone remodeling. Several data suggest including antioxidants in novel therapeutic approaches to develop better management strategies for the prevention and treatment of osteoporosis and OS-related bone diseases. In particular, anthocyanins, as well as resveratrol, lycopene, oleuropein, some vitamins, and thiol antioxidants, could have protective and therapeutic anti-osteoporotic effects.

The CAIX inhibitor SLC-0111 exerts anti-cancer activity on gastric cancer cell lines and resensitizes resistant cells to 5-Fluorouracil, taxane-derived, and platinum-based drugs.

Gastric cancer (GC) is the fifth most frequent malignancy and the fourth leading cause of worldwide cancer-related death. Despite the usage of multimodal perioperative chemotherapy (pCT), GC progressively gains chemoresistance, thereby, the identification of suitable targets to overcome drug resistance is fundamental. Amongst the potential biomarkers, carbonic anhydrase IX (CAIX) - associated with a poor prognosis of several solid cancers - has gained the most attention. In a cohort of GC patients who received perioperative FLOT (i.e., Leucovorin, 5-Fluouracil, Docetaxel, and Oxaliplatin) or FOLFOX (i.e., Leucovorin, 5-Fluouracil, and Oxaliplatin), non-responder patients showed an increased expression of tumor CAIX compared to responder group. Moreover, GC cell lines induced to be resistant to 5-Fluouracil, Paclitaxel, Cisplatin, or the combination of 5-Fluorouracil, Oxaliplatin, and Docetaxel, overexpressed CAIX compared to the control. Accordingly, CAIX-high-expressing GC cells showed increased therapy resistance compared to low-expressing cells. Notably, SLC0111 significantly improved the therapy response of both wild-type and resistant GC cells. Overall, these data suggest a correlation between CAIX and GC drug resistance highlighting the potential of SLC-0111 in re-sensitizing GC cells to pCT.

Lactate Maintains BCR/Abl Expression and Signaling in Chronic Myeloid Leukemia Cells Under Nutrient Restriction.

This study was directed to deepen the effects of nutrient shortage on BCR/Ablprotein expression and signaling in chronic myeloid leukemia (CML) cells. The backbone of the study was cell culture in medium lacking glucose, the consumption of which we had previously shown to drive BCR/Ablprotein suppression, and glutamine, the other main nutrient besides glucose. In this context, we focused on the role of lactate, the main by-product of glucose metabolism under conditions of rapid cell growth, in particular as a modulator of the maintenance of CML stem/progenitor cell potential, a crucial determinant of disease course and relapse of disease. The results obtained indicated that lactate is a powerful surrogate of glucose to prevent the suppression of BCR/Abl signaling and is therefore capable to maintain BCR/Abl-dependent CML stem/progenitor cell potential. A number of metabolism-related functional and phenotypical features of CML cells were also determined. Among these, we focused on the effect of lactate on oxygen consumption rate, the dependence of this effect on the cell surface lactate carrier MCT-1, and the relationship of the lactate effect to pyruvate and to the activity of mitochondrial pyruvate carrier.

Nintedanib-Containing Dual Conjugates Targeting αVß6 Integrin and Tyrosine Kinase Receptors as Potential Antifibrotic Agents.

αVß6 Integrin plays a fundamental role in the activation of transforming growth factor-ß (TGF-ß), the major profibrotic mediator; for this reason, αVß6 ligands have recently been forwarded to clinical phases for the therapy of fibrotic diseases. Herein, we report the synthesis and in vitro biological evaluation as antifibrotic agents of three new covalent conjugates, constituted by c(AmpLRGDL), an αVß6 integrin-recognizing small cyclopeptide, and nintedanib, a tyrosine kinase inhibitor approved for idiopathic pulmonary fibrosis (IPF) treatment. One of these conjugates recapitulates optimal in vitro antifibrotic properties of the two active units. The integrin ligand portion within the conjugate plays a role in inhibiting profibrotic stimuli, potentiating the nintedanib effect and favoring the selective uptake of the conjugate in cells overexpressing αVß6 integrin. These results may open a new perspective on the development of dual conjugates in the targeted therapy of IPF.

Oleuropein from olive leaf extracts and extra-virgin olive oil provides distinctive phenolic profiles and modulation of microbiota in the large intestine.

The interest in the modulation of gut microbiota by polyphenols from olives and derived products is increasing. In this work, phenolic leaf extracts (PLE) were in vitro faecal fermented to evaluate the changes in phenolic profiles and the impact on microbiota, using a commercial extra-virgin olive oil (EVOO) as reference. The in vitro fermentation decreased oleuropein content in PLE, determining an increase of hydroxytyrosol and other phenolic metabolites. An increase (p < 0.05) of hydroxytyrosol (LogFC = 6.02; VIP score = 1.05) was also observed in fermented EVOO. Besides, PLE significantly (p < 0.05) changed amino acids (LogFC = 6.1) and fatty acids (LogFC = 5.9) profile of the faeces. Metagenomic sequencing revealed that Coriobacteriaceae at the family level, and Collinsella at the genus level, were the most affected by PLE fermentation. These findings support the modulation of the gut microbiota exerted by phenolics from PLE and EVOO.

miR-214-Enriched Extracellular Vesicles Released by Acid-Adapted Melanoma Cells Promote Inflammatory Macrophage-Dependent Tumor Trans-Endothelial Migration.

The understanding of the molecular mechanisms leading to melanoma dissemination is urgently needed in view of the identification of new targets and the development of innovative strategies to improve patients' outcomes. Within the complexity of tumor intercellular communications leading to metastatic dissemination, extracellular vesicles (EV) released by tumor cells are central players. Indeed, the ability to travel through the circulatory system conveying oncogenic bioactive molecules even at distant sites makes EV capable of modulating recipient cells to facilitate metastatic dissemination. The dynamic remodeling of the tumor microenvironment might influence, along with a number of other events, tumoral EV release. We observed that, in melanoma, extracellular acidosis increases the release of EV enriched in miR-214, an onco-miRNA involved in melanoma metastasis. Then, miR-214-enriched EV were found to induce a state of macrophage activation, leading to an overproduction of proinflammatory cytokines and nitric oxide. Such an inflammatory microenvironment was able to alter the endothelial cell permeability, thereby facilitating the trans-endothelial migration of melanoma cells, a crucial step in the metastatic cascade. The use of synthetic miR-214 inhibitors and miR-214 overexpression allowed us to demonstrate the key role of miR-214 in the EV-dependent induction of macrophage activation. Overall, our in vitro study reveals that the release of tumor miR-214-enriched EV, potentiated by adapting tumor cells to extracellular acidosis, drives a macrophage-dependent trans-endothelial migration of melanoma cells. This finding points to miR-214 as a potential new therapeutic target to prevent melanoma intravasation.

An Oleocanthal-Enriched EVO Oil Extract Induces the ROS Production in Gastric Cancer Cells and Potentiates the Effect of Chemotherapy.

Oleocanthal, a minor polar compound in extra-virgin olive (EVO) oil, contains anticancer properties, which should be encouraged in its use in oncology. Gastric Cancer (GC), a very aggressive human cancer, is often diagnosed at advanced stages, when surgery is substituted or supported by chemotherapy (CT). However, CT frequently fails due to the patient's resistance to the treatment. Thus, the aim of this study is to verify whether an OC-enriched EVO oil extract fraction (OCF) may be useful in order to overcome a resistance to GC. We evaluated the OCF effects on an AGS gastric adenocarcinoma cell line wild type (AGS wt) and on its subpopulations resistant to 5-fluorouracil (5FUr), Paclitaxel (TAXr) or cisplatin (CISr). We found that a 60 µM dose of the OCF acts on the AGS wt, 5FUr and TAXr, leading to the cell cycle inhibition and to a ROS production, but not on CISr cells. Resistance of CISr to the OCF seems to be due to higher levels of antioxidant-enzymes that can counteract the OCF-induced ROS production. Moreover, using the OCF plus 5-fluorouracil, Paclitaxel or cisplatin, we found a potentiating effect compared with a mono-treatment in all resistant GC cells, including CISr. In conclusion, the use of the OCF in the management of GC has shown very interesting advantages, opening-up the possibility to evaluate the efficacy of the OCF in vivo, as a valid adjuvant in the treatment of resistant GC.

Extra Virgin Olive Oil and Cardiovascular Protection in Chronic Kidney Disease.

The high mortality related to chronic kidney disease (CKD) is not only due to the disease itself; in fact, CKD also represents an important risk factor for cardiovascular (CV) morbidity and mortality. Among the functional foods that seems to have cardioprotective action, extra virgin olive oil (EVOO) plays a pivotal health-promoting role. The aim of this study was to evaluate the possible cardioprotective effects of an EVOO containing a very high content (>900 ppm) of minor phenolic compounds (MPCs). The selected EVOO was analyzed by HPLC-DAD-MS to establish the MPC content. The Olea extract obtained from the selected EVOO was tested against the RAW 264.7 cell line in order to investigate its anti-inflammatory activity. We enrolled 40 CKD patients under conservative therapy for in vivo clinical testing. All CKD patients consumed 40 mL/day of raw EVOO for 9 weeks (T1). At baseline (T0) and at T1, we monitored the patients' blood and urinary parameters. The patients' body composition was assessed using bioelectrical impedance analysis and the carotid intima-media thickness (CIMT) using ultrasound imaging. At T1, we observed a decrease in inflammatory parameters, CIMT, and oxidative stress biomarkers. We also noticed improvements in lipid and purine metabolism, atherogenic indices, and body composition. Thus, this study highlighted the cardioprotective action of EVOO in nephropathic patients.

uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells.

Malignant melanoma is a highly aggressive skin cancer characterized by an elevated grade of tumor cell plasticity. Such plasticity allows adaptation of melanoma cells to different hostile conditions and guarantees tumor survival and disease progression, including aggressive features such as drug resistance. Indeed, almost 50% of melanoma rapidly develop resistance to the BRAFV600E inhibitor vemurafenib, with fast tumor dissemination, a devastating consequence for patients outcomes. Vasculogenic mimicry (VM), the ability of cancer cells to organize themselves in perfused vascular-like channels, might sustain tumor spread by providing vemurafenib-resistant cancer cells with supplementary ways to enter into circulation and disseminate. Thus, this research aims to determine if vemurafenib resistance goes with the acquisition of VM ability by aggressive melanoma cells, and identify a driving molecule for both vemurafenib resistance and VM. We used two independent experimental models of drug-resistant melanoma cells, the first one represented by a chronic adaptation of melanoma cells to extracellular acidosis, known to drive a particularly aggressive and vemurafenib-resistant phenotype, the second one generated with chronic vemurafenib exposure. By performing in vitro tube formation assay and evaluating the expression levels of the VM markers EphA2 and VE-cadherin by Western blotting and flow cytometer analyses, we demonstrated that vemurafenib-resistant cells obtained by both models are characterized by an increased ability to perform VM. Moreover, by exploiting the CRISPR-Cas9 technique and using the urokinase plasminogen activator receptor (uPAR) inhibitor M25, we identified uPAR as a driver of VM expressed by vemurafenib-resistant melanoma cells. Thus, uPAR targeting may be successfully leveraged as a new complementary therapy to inhibit VM in drug-resistant melanoma patients, to counteract the rapid progression and dissemination of the disease.