Metastatic Melanoma Cell Lines Research Articles

Inhibition of glycolysis and Src/Akt signaling reduces Caveolin-1-enhanced metastasis

Metastasis is the leading cause of cancer-related deaths, making the development of novel, more effective therapies imperative to alleviate patient suffering. Metabolic switching is a hallmark of cancer cells that facilitates metastasis. Cancer cells obtain most of their energy and intermediate metabolites, which are required to proliferate and metastasize, through aerobic glycolysis. Previous work from our laboratory has shown that Caveolin-1 (CAV1) expression in cancer cells promotes glycolysis and metastasis. Here, we sought to determine if limiting glycolysis reduced CAV1-enhanced metastasis and to identify the mechanism(s) involved. We evaluated the effects of the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) in metastatic melanoma and breast cancer cell lines expressing or not CAV1. Non-cytotoxic concentrations of 2-DG (1 mM) inhibited the migration of B16-F10 melanoma and MDA-MB-231 breast cancer cells. CAV1-mediated activation of Src/Akt signaling was required for CAV1-enhanced migration and was blocked in the presence of 2-DG. Moreover, inhibition of Akt reduced CAV1-enhanced lung metastasis of B16-F10 cells. Collectively, these findings highlight the importance of CAV1-induced metabolic reprogramming for metastasis and point towards possible therapeutic approaches to prevent metastatic disease by inhibiting glycolysis and Src/Akt signaling.

Antiproliferative potential of essential oil from the leaves of Croton floribundus(Euphorbiaceae) against human metastatic melanoma cell lines.

Melanoma is a cancer type with high lethality, metastatic capacity, and limited therapeutic options. Different essential oils have been reported with antitumoral potential. Thus, the essential oil (EO) of the leaves of C. floribundus was obtained by hydrodistillation and analyzed by GC-MS. The majority of substances annotated were β-selinene, E-Caryophyllene, and Premnaspirodiene. The cytotoxic activity of EO was evaluated on three melanoma cell lines SKMEL-147, WM-1366, and CHL-1, which are representative of metastatic melanoma with different mutation profiles. The IC50 values found for EO were lower than temozolomide (reference drug) in all melanoma cell lines. In addition, the selectivity of EO was upward when compared to the reference drug. Interestingly, the WM-1366 cell line was the most responsive, and these findings are very promising considering that it has shown high resistance to the plethora of compounds. Thus, the C. floribundus EO is a promising source to drive further studies for the development of new treatments for metastatic melanoma, which is urgently relevant given the resistance of this pathology to current treatments.

RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition.

The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

Highly sensitive ratiometric fluorescent fiber matrices for oxygen sensing with micrometer spatial resolution

AbstractOxygen (O2)-sensing matrices are promising tools for the live monitoring of extracellular O2 consumption levels in long-term cell cultures. In this study, ratiometric O2-sensing membranes were prepared by electrospinning, an easy, low-cost, scalable, and robust method for fabricating nanofibers. Poly(ε-caprolactone) and poly(dimethyl)siloxane polymers were blended with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride, which was used as the O2-sensing probe, and rhodamine B isothiocyanate, which was used as the reference dye. The functionalized scaffolds were morphologically characterized by scanning electron microscopy, and their physicochemical profiles were obtained by Fourier transform infrared spectroscopy, thermogravimetric analysis, and water contact angle measurement. The sensing capabilities were investigated by confocal laser scanning microscopy, performing photobleaching, reversibility, and calibration curve studies toward different dissolved O2 (DO) concentrations. Electrospun sensing nanofibers showed a high response to changes in DO concentrations in the physiological-pathological range from 0.5% to 20% and good stability under ratiometric imaging. In addition, the sensing systems were highly biocompatible for cell growth promoting adhesiveness and growth of three cancer cell lines, namely metastatic melanoma cell line SK-MEL2, breast cancer cell line MCF-7, and pancreatic ductal adenocarcinoma cell line Panc-1, thus recreating a suitable biological environment in vitro. These O2-sensing biomaterials can potentially measure alterations in cell metabolism caused by changes in ambient O2 content during drug testing/validation and tissue regeneration processes. Graphic abstract

Abstract 6745: Crafting hyaluronic acid-based nanoparticles for enhanced LN targeting as a potent priming tool in immunotherapy

Abstract Background: Cholesterol-conjugated hyaluronic acid, synthesized by grafting cholesterol moieties onto hyaluronic acid, spontaneously formed nano-sized hydrogel of approximately 30-100nm in water. This hyaluronic acid derivative (HA nanogel) has demonstrated the ability to encapsulate various modalities, including low- and medium-sized molecules, peptides, and proteins. Previous reports have highlighted its efficacy in solubilizing poorly water-soluble drugs and serving as a sustained-release matrix. In this study, we explore the potential of HA nanogels encapsulating antigen peptides with adjuvant as a cancer vaccine and a priming tool for combinational immunotherapy. Method: We prepared HA nanogels as a novel peptide carrier for cancer vaccines and attempted to encapsulate various peptides, including neoantigen (mERK2) peptides expressed in immune checkpoint inhibitor (ICI) therapy-resistant fibrosarcoma cell line CMS5a, gp-100 peptides expressed in metastatic melanoma cell line B16F10, and MAGEA4. Our experiments covered APC uptake, antigen-specific CTL induction, and anti-tumor studies to demonstrate the utility of HA nanogels in these applications. Results: HA nanogel efficiently formed stable complexes with mERK2, gp-100 and MAGEA4 long peptides, each with diameters of 30-100nm. The facile and simple vaccine formulation processes with HA nanogel achieved high drug content and high encapsulation efficiency of 90% or higher. Fluorescently labeled mERK2 long peptide encapsulated in HA nanogel vaccines were subcutaneously administered to BALB/c mice, and uptake analysis of mERK2 long peptides in draining lymph nodes (dLN) was performed using flow cytometry. In comparison to the group receiving mERK2 long peptide without nanogel encapsulation, the HA nanogel group exhibited higher uptake by macrophages and dendritic cells in the dLN. Administering HA nanogel vaccines containing mERK2 long peptides with CpG oligoDNA resulted in higher antigen-specific CD8+ T cells compared to the group receiving mERK2 long peptides without encapsulation. Strong anti-tumor effects were demonstrated in mice models with subcutaneously transplanted CMS5a and B16F10 cell lines. The enhanced efficacy through co-administration with ICI was also confirmed. Conclusion: HA nanogel vaccines efficiently deliver antigen peptides to macrophages and dendritic cells, inducing antigen-specific CTL responses, thereby demonstrating anti-tumor effects against cold tumors such as CMS5a and B16F10. HA nanogels have the potential not only as carriers for cancer vaccines but also as priming tools for combinational immunotherapy. Citation Format: Takashi Nakai, Fumiyasu Momose, Kohei Yabuuchi, Makiko Yamane, Tae Hayashi, Linan Wang, Yoshiyuki Nakagawa, Shogo Aso, Toru Katsumata, Tsuyoshi Shimoboji, Yoshihiro Miyahara. Crafting hyaluronic acid-based nanoparticles for enhanced LN targeting as a potent priming tool in immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6745.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617μM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24h of treatment at 70°C, after 1h treatments with different pH values, and β-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Pholiotic acid promotes apoptosis in human metastatic melanoma cells

Mushrooms produce a great variety of secondary metabolites that can be successful in both prevention and treatment of various cancers. In particular, higher Basidiomycete mushrooms contain various types of biologically active low-molecular compounds in fruiting bodies with suggested anticarcinogenic effects. The polyamine analogue {(2R)-2-[(S)-3-hydroxy-3-methylglutaryloxy] putrescine dicinnamamide} indicated with the name pholiotic acid, isolated for the first time by us from the fruiting bodies of the Basidiomycete Pholiota spumosa (Fr.) Sing. (Strophariaceae), inhibited the viability of human prostate cancer cells, such as other polyamine synthetic analogues that have shown antitumor activity in several types of cancer, including melanoma. Melanoma is an aggressive skin cancer that can metastasize to other organs and presents a high resistance to conventional therapies. In light of these considerations, the present study was therefore designed to assess whether this putrescine derivative could inhibit the growth of human metastatic melanoma cell lines, M14 and A2058. The results obtained demonstrate that this natural compound, at 12.5–50 μM concentration, was able to reduce cell viability of both cancer cells inducing cell death by intrinsic apoptotic pathway that probably involves PTEN activity, inhibition of Hsp70 expression and reactive oxygen species production. On the other hand, the increased expression of enzymes involved in polyamine catabolism trigger apoptotic cell death leading to polyamine depletion and generation of reactive oxygen species as by-products. In conclusion, these findings, starting point for further investigation, implement available our data to support pholiotic acid as an attractive potential chemopreventive agent, and provide a basis for further research into the use of this polyamine derivative as potential anticancer agent for melanoma in combination with existing therapies to improve treatment efficacy and overcome the obstacle of drug resistance.

Human Metastatic Melanoma Cell Lines Panel for In Vitro and In Vivo Investigations

The melanoma origin of cell lines obtained from the axillary lymph node (mel Kas, mel Pet, and mel Lap from patients with a verified diagnosis) was confirmed by the detection of the Melan A melanocyte marker expression. A hyperdiploid (2n+) for the mel Kas line; near-diploid (2n), and in some cells near-tertaploid (4n), and even hypo-octaploid (8n) set (172–179 chromosomes) in the mel Pet cell line; and a hypotetraploid (4n−) for the mel Lap line were detected by karyotypic analysis. All three cell lines are tumorigenic; however, mel Pet demonstrates tumor growth in Balb/c nude mice only in the presence of matrigel. All three lines showed a high expression of TUBB3 and PD-L1 markers, while ERa was low (minimum for mel Pet). Significant differences in the expression level were shown for the Cyt molecular marker. In the transplantation of cells to Balb/c nude mice, a stable expression level is observed only for TUBB3. For the rest of the markers, a decrease in their expression level of varying degrees was noted when the cells were growing in solid tumors in vivo. Mutations were detected in oncogenes (BRAF, EZH2, KIT, KRAS, NRAS, ROS1) and tumor suppressor genes (CDKN2A, FAT4, KMT2C, LRP1B, PTEN, PTPRB, TP53). The detailed characterization of the cell lines makes them valuable for various scientific and regulatory experiments, particularly those involving preclinical data on antiproliferative drugs for malignant melanoma or investigations into melanoma cell properties and progression.

Hellebrigenin induces apoptosis by triggering cellular inhibitor of apoptosis 1 and Jun N-terminal kinase pathway in melanoma cells

Abstract Background: Melanoma, the malignancy of melanocytes, is the most fatal form of skin cancer. Although various interventions are currently available, advanced stages of melanoma are still associated with a risk of metastatic recurrence. Objectives: The present study examined the anticancer activity of hellebrigenin, a natural product extracted from toad skin. Methods: Two types of metastatic melanoma cell lines, A2058 and HMY-1, were selected. Results: The findings revealed that hellebrigenin had cytotoxic effects on the two melanoma cell lines by triggering apoptosis: hellebrigenin activated cell arrest during the G2/M phase and downregulated regulators’ expression (e.g., cyclin-dependent kinase [CDK] 1, CDK 4, and cyclins E1, E2, A2, and D3). In intrinsic and extrinsic pathways of apoptosis, hellebrigenin activated the expression of the proapoptotic proteins t-BID, Bak, poly-ADP-ribose polymerase, Fas-associated death domain protein, death receptors 5, and cleaved caspases 3, 8, and 9; it also reduced the expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. Mitochondrial membrane depolarization and cell nucleus condensation were also observed. In addition, human apoptosis arrays revealed that hellebrigenin inhibited the expression of cellular inhibitor of apoptosis 1, which is crucial for blocking cell apoptosis. Regarding upstream signaling activity, hellebrigenin suppressed the phosphorylation of the Jun N-terminal kinase 1/2 pathway in both melanoma cell lines. Conclusion: Overall, hellebrigenin can be used as a potential anticancer agent in clinical settings for treating the advanced stages of melanoma.

Quaternary nanorods: promising versatile agents for cancer therapy, antimicrobial strategies and free radical neutralization

Cu2XSnS4 (CXTS; X: Zn, Mn or Co) nanorods (NRs) were prepared by a simple hot-injection method and their cytotoxic activities on human breast adenocarcinoma (MCF-7), metastatic melanoma (451Lu) and embryonic kidney (HEK-293) cell lines were investigated for the first time. The antibacterial activities of these nanoparticles on both gram-positive (S. aureus) and gram-negative (E. coli) pathogenic bacteria as well as their DPPH (2,2-diphenyl-1- picrylhydrazyl) scavenging activities were evaluated. The structural characterizations show that the obtained nanoparticles have the same crystal structure and are shaped as nanorods with an average 10–40 nm edge length. The data obtained in cytotoxicity studies revealed that the synthesized CXTS NRs have a significant inhibition effect, especially on cancer cell lines (MCF-7 and 451Lu), in a dose-dependent manner over a period of 24 h, demonstrating their potential for use as anticancer agents. In addition, the findings indicate that the synthesized nanorods possess strong antibacterial properties against the pathogenic microorganisms tested and display a marked antioxidant effect through efficient elimination of DPPH activity. This comprehensive study sheds light on the creation and development of CXTS NRs as promising versatile agents in various biomedical and environmental applications such as cancer therapy, antimicrobial strategies and the neutralization of free radicals.

Microfluidic development and biological evaluation of targeted therapy-loaded biomimetic nano system to improve the metastatic melanoma treatment

Optimizing current therapies is among next steps in metastatic melanoma (MM) treatment landscape. The innovation of this study is the design of production process by microfluidics of cell membrane (CM)-modified nanoparticles (NPs), as an emerging biomimetic platform that allows for reduced immune clearance, long blood circulation time and improved specific tumor targeting. To achieve melanoma selectivity, direct membrane fusion between synthetic liposomes and CMs extracted from MM cell line was performed by microfluidic sonication approach, then the hybrid liposomes were loaded with cobimetinib (Cob) or lenvatinib (Lenva) targeting agents and challenged against MM cell lines and liver cancer cell line to evaluate homotypic targeting and antitumor efficacy. Characterization studies demonstrated the effective fusion of CM with liposome and the high encapsulation efficiency of both drugs, showing the proficiency of microfluidic-based production. By studying the targeting of melanoma cells by hybrid liposomes versus liposomes, we found that both NPs entered cells through endocytosis, whereas the former showed higher selectivity for MM cells from which CM was extracted, with 8-fold higher cellular uptake than liposomes. Hybrid liposome formulation of Cob and Lenva reduced melanoma cells viability to a greater extent than liposomes and free drug and, notably, showed negligible toxicity as demonstrated by bona fide haemolysis test. The CM-modified NPs presented here have the potential to broaden the choice of therapeutic options in MM treatment.

AKT2 Loss Impairs BRAF-Mutant Melanoma Metastasis.

Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer due to its highly metastatic nature. Melanomas harboring oncogenic BRAFV600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain about whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and we investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT isoform-specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo, whereas AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby the inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that the elucidation of AKT2-specific functions in metastasis might inform therapeutic strategies to improve treatment options for melanoma patients.

Hypoxia-Induced Changes in L-Cysteine Metabolism and Antioxidative Processes in Melanoma Cells.

This study was performed on human primary (WM115) and metastatic (WM266-4) melanoma cell lines developed from the same individual. The expression of proteins involved in L-cysteine metabolism (sulfurtransferases, and cystathionine β-synthase) and antioxidative processes (thioredoxin, thioredoxin reductase-1, glutathione peroxidase, superoxide dismutase 1) as well as the level of sufane sulfur, and cell proliferation under hypoxic conditions were investigated. Hypoxia in WM115 and WM266-4 cells was confirmed by induced expression of carbonic anhydrase IX and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 by the RT-PCR and Western blot methods. It was shown that, under hypoxic conditions the inhibition of WM115 and WM266-4 melanoma cell proliferation was associated with decreased expression of thioredoxin reductase-1 and cystathionine β-synthase. These two enzymes may be important therapeutic targets in the treatment of melanoma. Interestingly, it was also found that in normoxia the expression and activity of 3-mercaptopyruvate sulfurtransferase in metastatic WM266-4 melanoma cells was significantly higher than in primary melanoma WM115 cells.

Establishment of SLC7A11-knockout mouse and its preliminary investigation in melanoma.

Solute carrier family 7 member 11 (SLC7A11)/xCT is an amino acid transporter that mediates the cystine uptake and glutamate export, participates in several malignant tumors' progression. However, the role of SLC7A11 on the occurrence and development of melanoma still remains unclear. Here, the transcribed mRNA encoding for Cas9 and sgRNA targeting SLC7A11 in vitro were microinjected into zygotes, to establish the SLC7A11 knockout (KO) mice (SLC7A11-/-). Further, we conducted melanoma-bearing mice using the metastatic melanoma cell line (B16-F10) to observe the melanoma development. There was no off-target in KO mice detected by T7E1 cleavage assay. The results showed that the tumor volume of KO mice was significantly lower than that of SLC7A11+/+ (WT) mice at 8d, 10d, 12d, 14d, and 16d (P < 0.05). The tumors of WT appeared to more disorganized morphology, more unbalanced nuclear-cytoplasmic ratio, less defined boundary, and increased tumor necrosis. And after SLC7A11 deletion, the expression of CXCL9 and TLR6 were significantly up-regulated, and that of NOS2 and CCL8 were significantly down-regulated (P < 0.01). Additionally, Ki67 immunostaining revealed lower proliferating cells in the tumors of SLC7A11 KO mice compared to WT mice. In summary, the deletion of SLC7A11 significantly inhibited the development of melanoma. Our results provide direct evidence to identify SLC7A11 as a novel target for molecular therapy and prognosis judgment of melanoma.

Biomineralized Polydopamine Nanoparticle-Based Sodium Alginate Hydrogels for Delivery of Anti-serine/Threonine Protein Kinase B-Rapidly Accelerated Fibrosarcoma siRNA for Metastatic Melanoma Therapy.

Malignant melanoma, as a highly aggressive skin cancer, is strongly associated with mutations in serine/threonine protein kinase B-RAF (BRAF, where RAF stands for rapidly accelerated fibrosarcoma). Targeted therapy with anti-BRAF small interfering RNA (siBRAF) represents a crucial aspect of metastatic melanoma treatment. In this study, an injectable hydrogel platform based on sodium alginate (SA), with multifunctions of photothermal and Ca2+-overload cell apoptosis, was explored as a siBRAF carrier for metastatic melanoma therapy. We employed polydopamine nanoparticles (PDAs) as a photothermal core and constructed a calcium phosphate (CaP) shell via biomineralization (PDA@CaP) to load siBRAF (PDA@siBRAF/CaP). The pH-sensitive CaP shell facilitated the release of Ca2+ under the weakly acidic tumor microenvironment, triggering the gelation of PDA@siBRAF/CaP-SA to localized release siBRAF at tumor sites with the interruption of the RAS-RAF-MEK-ERK (MAPK) pathway. Besides, the continuous release of Ca2+ could also lead to Ca2+-overload cell apoptosis. Moreover, the photothermal effect of PDA regulated the release kinetics, resulting in coordinated therapeutic abilities of individual components in the PDA@siBRAF/CaP-SA hydrogels. Consequently, the effective inhibition of tumor growth and metastasis was achieved in vitro and in vivo using a highly metastatic melanoma cell line B16F10 as the model, by combining photothermal ablation, Ca2+ overload, and BRAF silencing. Our work provides a proof-of-concept for an injectable hydrogel system that simultaneously targets multiple mechanisms involved in melanoma progression and has the potential to be translated into clinical use for the metastatic melanoma therapy.

Single-cell RNA sequencing of a poorly metastatic melanoma cell line and its subclones with high lung and brain metastasis potential reveals gene expression signature of metastasis with prognostic implication.

The molecular mechanisms underlying melanoma metastasis remain poorly understood. In this study, we aimed to delineate the mechanisms underlying gene expression alterations during metastatic potential acquisition and characterize the metastatic subclones within primary cell lines. We performed single-cell RNA sequencing of a poorly metastatic melanoma cell line (WM239A) and its subclones with high metastatic potential to the lung (113/6-4L) and the brain (131/4-5B1 and 131/4-5B2). Unsupervised clustering of 8173 melanoma cells identified three distinct clusters according to cell type ('Primary', 'Lung' and 'Brain' clusters) with differential expression of MITF and AXL pathways and putative cancer and cell cycle drivers, with the lung cluster expressing intermediate but distinct gene profiles between primary and brain clusters. Principal component (PC) analysis revealed that PC2 (the second PC), which was positively associated with MITF expression and negatively with AXL pathways, primarily segregated cell types, in addition to PC1 of the cell cycle pathway. Pseudotime trajectory and RNA velocity analyses suggested the existence of cellular subsets with metastatic potential in the Primary cluster and an association between PC2 signature alteration and metastasis potential acquisition. Analysis of The Cancer Genome Atlas melanoma samples by clustering into PC2-high and -low clusters by quartiles of PC2 signature expression revealed that the PC2-high cluster was an independent significant factor for poor prognosis (p-value = 0.003) with distinct genomic and transcriptomic characteristics, compared to the PC2-low cluster. In conclusion, we identified signatures of melanoma metastasis with prognostic significance and putative pro-metastatic subclones within a primary cell line.

The roles of different forms of IL-15 in human melanoma progression.

Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

Cinnamomum zeylanicum Blume Essential Oil Inhibits Metastatic Melanoma Cell Proliferation by Triggering an Incomplete Tumour Cell Stress Response.

Given the known pro-oxidant status of tumour cells, the development of anti-proliferative strategies focuses on products with both anti- and pro-oxidant properties that can enhance antitumour drug cytotoxicity. We used a C. zeylanicum essential oil (CINN-EO) and assessed its effect on a human metastatic melanoma cell line (M14). Human PBMCs and MDMs from healthy donors were used as normal control cells. CINN-EO induced cell growth inhibition, cell cycle perturbation, ROS and Fe(II) increases, and mitochondrial membrane depolarization. To assess whether CINN-EO could affect the stress response, we analysed iron metabolism and stress response gene expression. CINN-EO increased HMOX1, FTH1, SLC7A11, DGKK, and GSR expression but repressed OXR1, SOD3, Tf, and TfR1 expression. HMOX1, Fe(II), and ROS increases are associated with ferroptosis, which can be reversed by SnPPIX, an HMOX1 inhibitor. Indeed, our data demonstrated that SnPPIX significantly attenuated the inhibition of cell proliferation, suggesting that the inhibition of cell proliferation induced by CINN-EO could be related to ferroptosis. Concurrent treatment with CINN-EO enhanced the anti-melanoma effect of two conventional antineoplastic drugs: the mitochondria-targeting tamoxifen and the anti-BRAF dabrafenib. We demonstrate that CINN-EO-mediated induction of an incomplete stress response specifically in cancer cells affects the proliferation of melanoma cells and can enhance drug cytotoxicity.

Investigating the impact and the action mechanism of KCa3.1 blockers on a metastatic melanoma cell line.

Previous studies showed that the Ca2+ gated K+ channel KCa3.1 can play an important role in the progression of different cancer types like the Pancreatic Duct Adenocarcinoma (PDAC) and that the KCa3.1 blocker Clotrimazole decreases the proliferation of the PDAC line MiaPaCa-2 but not Panc-1, in which the channel is poorly expressed (Jäger, 2003, 10.1124/mol.65.3.6309). However, Clotrimazole also inhibits cytochrome P450; thus, proliferation might be influenced by this side effect. Nevertheless, studies in which KCa3.1 was silenced suggested that the channel is important for MiaPaCa-2 cell proliferation and migration (Bonito, 2016, 10.1007/s00424-016-1891-9). We are currently investigating the role of KCa3.1 in melanoma cell lines, checking if channel blockage alone can impact cell growth also in this type of cancer. To this purpose, we use two different channel blockers: the already mentioned Clotrimazole and Senicapoc, a more KCa3.1-selective blocker (Staal, 2017, 10.1007/s11064-017-2223-y). In order to better discriminate between unspecific toxicity and effects strictly correlated to channel blockage, we performed patch-clamp experiments and MTT viability assays on the melanoma line WM266 and on Panc-1, used as negative control. We found that Panc-1 cells, as expected, do not exhibit Ca2+-activated KCa3.1 currents, while such currents were present in WM266, where they were blocked by both Clotrimazole and Senicapoc. To test how channel blockage can impact cell proliferation, we performed MTT viability assays, which showed that Senicapoc affects the growth not only of WM266 but also, at a similar extent, of Panc-1, which lack KCa3.1 currents. This raises the question of whether Senicapoc can impact melanoma cells growth only by blocking the channel. To answer this question, we will perform further experiments (including migration assays) and we will present our newest data. Supported by AIRC and PRIN.

A strategy for uncovering germline variants altering anti-tumor CD8 T cell response

Among many factors known to alter the outcomes of T cell receptor (TCR)-induced proximal signaling, the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained challenging to address. Here, we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations (pTyr-SNVs) that directly impact TCR-induced proximal phosphotyrosine motif-based signaling pathways. We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin (OVA) antigens and retrovirally engineered syngeneic major histocompatibility complex (MHC) Class I restricted OVA TCR-transgenic CD8 T cells (OT-I). Using the synthetic version of pTyr-SNV rs1178800678-G/T-encoding integrin alpha 4 (ITGA4) p.S1027I variant as a prototype, we show that under identical TCR stimulation conditions, genetically determined membrane-proximal immunoreceptor tyrosin activation motif (ITAM) results in increased tyrosine phosphorylation of 70 kDa zeta-chain-associated protein (ZAP70) and the levels of cytotoxic effector molecule granzyme B (GZMB), which in turn result in enhanced cytotoxic activity against metastatic melanoma cell line. This strategy paves the way for rapid molecular and functional characterization of anti-tumor immune response-linked germline pTyr-SNVs so as to improve our understanding of the genetic basis of individual-to-individual differences in anti-tumor CD8 T cell response.