Viktória Koroknai

Background: Pulmonary endothelial cells are increasingly recognized as active regulators of organ-specific metastasis; however, the mechanisms by which they influence melanoma invasion remain incompletely understood. Methods: Six primary melanoma-derived cell lines were first assessed for their invasive responses to HPMEC-conditioned medium. In a separate HPMEC co-culture invasion model, invasive and non-invasive melanoma subpopulations were isolated and analyzed for the expression of 88 chemokine and cytokine receptor genes. Candidate receptors were subsequently validated in primary melanoma tissues (n = 15), while their corresponding ligands were analyzed in patient plasma samples (n = 30). Results: HPMEC-conditioned medium enhanced invasion in half of the melanoma cell lines. Exploratory receptor-expression analyses identified candidate genes, including CCR2, IL11RA, IL15RA, TNFRSF11B, and TNFRSF12A, with expression patterns potentially associated with the observed invasive responses, while CCR4 expression was consistently elevated in all invasive subpopulations. In an exploratory analysis of a small primary melanoma tissue cohort, CCR2 and TNFRSF11B expression was significantly higher in tumors from patients with lung metastases, while IL11RA expression was upregulated compared with that in non-metastatic tumors. Melanoma cells consistently induced endothelial IL-8 expression, whereas plasma concentrations of the corresponding ligands did not differ significantly between patient groups. Conclusions: The elevated expression of CCR2, IL11RA, and TNFRSF11B observed in primary melanomas associated with lung metastases suggests that these factors may contribute to the development or progression of pulmonary metastases. These findings highlight the potential role of bidirectional communication between endothelial cells and melanoma cells in metastatic progression.

Background: The metastatic dissemination of melanoma involves adhesion of circulating tumor cells within organ-specific vascular beds; however, the relative contribution of the endothelial environment versus that of the melanoma-intrinsic molecular state remains unclear. Materials and Methods: We quantified the in vitro adhesion of primary (n = 5) and metastatic (n = 3) melanoma cell lines to human hepatic, brain, and pulmonary endothelial cells under co-culture conditions, and we profiled the expression of 86 adhesion- and extracellular-matrix-related genes in melanoma and endothelial cells. Results: Adhesion was highest for the hepatic endothelium, intermediate for the pulmonary endothelium, and lowest for the brain endothelium. This endothelial preference was conserved in both primary and metastatic melanoma cells, though metastatic cells exhibited higher absolute adhesion. The linear mixed-effect models revealed that the effects of adhesion state on melanoma gene expression were modest and varied by endothelial type, whereas melanoma origin had more widespread and larger effects (mean absolute standardized coefficients of 0.32–0.47 versus 0.60–0.87, respectively). The expression of three genes (SPP1, ITGA11, and MMP2) was associated with melanoma origin in all endothelial types. Spearman’s co-expression analysis revealed endothelial-type-specific gene networks, and within-sample permutation confirmed the non-random coordination in all three endothelial types. Conclusions: Our findings support a model in which endothelial organ specificity contributes to melanoma cell adhesion behavior and associated transcriptional patterns, highlighting the importance of the vascular interface as a biologically active mediator of early metastatic cell–endothelium interactions.

Metastatic spread remains the primary cause of mortality in melanoma. Our aim was to investigate the role of dermal endothelial cells in modulating melanoma cell invasiveness and cytokine/chemokine pattern. Primary melanoma cell lines were co-cultured with human dermal endothelial cells and assessed using Matrigel invasion assays. Invasive and non-invasive subpopulations were separated for gene expression analyses, and candidate molecules were further evaluated in patient tissue and plasma samples. Co-culture of melanoma and dermal endothelial cells revealed altered expression of several cytokine receptor genes (CCR5, CXCR7, IL1RAPL2, IL4R, IL6ST, IL18R1, IL22RA2, TNFRSF10A, TNFRSF11B, and TNFRSF21). Analysis of clinical melanoma samples showed significant downregulation of IL1RAPL2 and TNFRSF10A in cutaneous metastases, whereas IL6ST expression correlated with Breslow thickness of the primary tumor rather than metastatic site. Proteome profiling of dermal endothelial cells revealed alterations in Midkine, GROα, MIP-3α, IL-8, and SDF-1 following co-culture with melanoma cells. Plasma measurements in melanoma patients confirmed elevated Midkine levels in skin metastases and decreased MIP-3α in metastatic disease. These results highlight potential cytokine and chemokine-mediated pathways involved in melanoma dermal invasion and cutaneous metastasis. While some findings did not reach statistical significance, concordant trends between in vitro and patient-derived data suggest their relevance and warrant further investigation in larger cohorts.

Recent evidence has highlighted the critical role of lipids in tumor biology. In this study, we analyzed the plasma lipid profiles of 151 melanoma patients (University of Debrecen, Department of Dermatology, Hungary) to examine the associations between specific lipid species and commonly used LDL-C lipid parameters, as well as the Breslow thickness and ulceration of primary tumors. Our analysis included patients who underwent primary tumor resection, comprising 83 individuals without metastases and 68 with metastases at the time of blood sampling. Lipid profiling was performed using the Lipidyzer™ platform, which targets over 1100 lipid species. Following quality control filtering, 802 lipids were included in the subsequent analyses. Ten lipids were found to have decreased plasma levels, while one lipid exhibited elevated plasma levels, both associated with an increased risk of higher Breslow thickness. Additionally, patients with thicker tumors (≥2 mm) demonstrated significantly higher LDL-C levels after adjusting for age, sex, therapy, and tumor presence (p = 0.032). Using forward stepwise regression, we identified a combination of four lipids—(CE(20:5), LCER(24:1), PE(P18:1/18:1), and LPE(18:2))—that demonstrated the strongest correlation with Breslow depth (AUC = 0.779, as determined by ROC analysis). Additionally, we identified 11 lipids significantly associated with tumor ulceration. Stepwise regression analysis further revealed two lipids (FFA(16:0) and PC(15:0/18:1)) capable of predicting tumor ulceration with an AUC score of 0.740. These findings suggest that individual lipid metabolism may influence tumor thickness and ulceration during the development and progression of primary melanoma.

The aim of this study was to apply a state-of-the-art quantitative lipidomic profiling platform to uncover lipid alterations predictive of melanoma progression. Our study included 151 melanoma patients; of these, 83 were without metastasis and 68 with metastases. Plasma samples were analyzed using a targeted Lipidyzer™ platform, covering 13 lipid classes and over 1100 lipid species. Following quality control filters, 802 lipid species were included in the subsequent analyses. Total plasma lipid contents were significantly reduced in patients with metastasis. Specifically, levels of two out of the thirteen lipid classes (free fatty acids (FFAs) and lactosylceramides (LCERs)) were significantly decreased in patients with metastasis. Three lipids (CE(12:0), FFA(24:1), and TAG47:2-FA16:1) were identified as more effective predictors of melanoma metastasis than the well-known markers LDH and S100B. Furthermore, the predictive value substantially improved upon combining the lipid markers. We observed an increase in the cumulative levels of five lysophosphatidylcholines (LPC(16:0); LPC(18:0); LPC(18:1); LPC(18:2); LPC(20:4)), each individually associated with an elevated risk of lymph node metastasis but not cutaneous or distant metastasis. Additionally, seventeen lipid molecules were linked to patient survival, four of which (CE(12:0), CE(14:0), CE(15:0), SM(14:0)) overlapped with the lipid panel predicting metastasis. This study represents the first comprehensive investigation of the plasma lipidome of melanoma patients to date. Our findings suggest that plasma lipid profiles may serve as important biomarkers for predicting clinical outcomes of melanoma patients, including the presence of metastasis, and may also serve as indicators of patient survival.

The early detection of melanoma relapse can improve patient survival; thus, there is a great need for easily accessible biomarkers that facilitate the diagnosis of metastatic disease. We investigated the diagnostic effect of blood biomarkers such as lactate dehydrogenase (LDH), S100B, and osteopontin in the detection of metastases. Clinical data and peripheral blood samples of 206 melanoma patients were collected (no metastasis, N = 120; metastasis, N = 86). The discriminative power of blood biomarkers, patient demographics, and clinicopathological parameters of primary melanomas were evaluated using univariate and multivariate logistic regression models and receiver operating characteristic (ROC) analysis to determine the area under the curve (AUC). Plasma osteopontin levels showed a significant and independent effect on the probability of metastasis, similar to serum S100B levels. In addition, the location of the primary tumor on the lower extremities and the American Joint Committee on Cancer (AJCC) categories pT2b-3a, pT3b-4a, and pT4b were associated with the diagnosis of metastasis. Importantly, the combination of the three blood biomarkers and primary tumor localization and AJCC pT category yielded excellent discrimination (AUC: training set: 0.803; validation set: 0.822). In conclusion, plasma osteopontin can be classified as a melanoma biomarker; moreover, by combining clinicopathological prognostic variables, the diagnostic effect of blood biomarkers in the detection of metastatic melanoma can be improved.

Osteopontin (OPN) is a secreted integrin‐binding protein that plays a role in inflammation, cellular viability, cell adhesion and migration, cancer development, and diabetes through different mechanisms. The splice variants of OPN can play essential roles in cancer development, progression, and metastasis formation; however, limited data are available about the role of OPN isoforms in human malignant melanoma. Our goal was to define the gene expression patterns of five OPN variants (OPN4, OPN5, OPNa, OPNb, and OPNc), integrin, and CD44 receptor genes in primary and metastatic melanoma‐originated cell lines (n = 19), and to explore the association of the expression patterns with clinicopathological parameters. We evaluated the invasive property of the cell lines and investigated the potential association between the invasion and gene expression of OPN isoforms. We found a significant rise in the expression of OPNc in the invasive cell lines compared to the noninvasive cells and detected significantly higher expression of the OPN splice variants in melanoma cell lines originating from more advanced stages tumors than cell lines originating from early‐stage melanomas. The correlation analysis revealed that all five OPN variants positively correlated with ITGB3 and ITGA9, whereas OPN5 positively correlated with ITGB1, ITGAV, ITGA6, and CD44. OPN can activate extracellular signal‐regulated kinase signaling through binding to α9β1 integrin, promoting melanoma tumor cell migration. It is possible that such associations between OPN splice variants and integrin receptors may play a role in melanoma progression. In conclusion, our findings suggest that high expression of OPNc correlates with the invasive behavior of melanoma cells.

Cytokines and chemokines (chemotactic cytokines) are soluble extracellular proteins that bind to specific receptors and play an integral role in the cell-to-cell signaling network. In addition, they can promote the homing of cancer cells into different organs. We investigated the potential relationship between human hepatic sinusoidal endothelial cells (HHSECs) and several melanoma cell lines for the expression of chemokine and cytokine ligands and receptor expression during the invasion of melanoma cells. In order to identify differences in gene expression related to invasion, we selected invasive and non-invasive subpopulations of cells after co-culturing with HHSECs and identified the gene expression patterns of 88 chemokine/cytokine receptors in all cell lines. Cell lines with stable invasiveness and cell lines with increased invasiveness displayed distinct profiles of receptor genes. Cell lines with increased invasive capacity after culturing with conditioned medium showed a set of receptor genes (CXCR1, IL1RL1, IL1RN, IL3RA, IL8RA, IL11RA, IL15RA, IL17RC, and IL17RD) with significantly different expressions. It is very important to emphasize that we detected significantly higher IL11RA gene expression in primary melanoma tissues with liver metastasis as well, compared to those without metastasis. In addition, we assessed protein expression in endothelial cells before and after co-culturing them with melanoma cell lines by applying chemokine and cytokine proteome arrays. This analysis revealed 15 differentially expressed proteins (including CD31, VCAM-1, ANGPT2, CXCL8, and CCL20) in the hepatic endothelial cells after co-culture with melanoma cells. Our results clearly indicate the interaction between liver endothelial and melanoma cells. Furthermore, we assume that overexpression of the IL11RA gene may play a key role in organ-specific metastasis of primary melanoma cells to the liver.

Cytokines and chemokines (chemotactic cytokines) are soluble extracellular proteins that bind to specific receptors and play an integral role in the cell-to-cell signaling network, in addition can promote the homing of cancer cells into different organs. We investigated the potential relationship between human hepatic sinusoidal endothelial cells (HHSECs) and several melanoma cell lines for the expression of chemokine and cytokine ligand and receptor expression during invasion of melanoma cells. In order to define the invasion related gene expression differences, we selected invasive and non-invasive subpopulations of cells after co-culturing with HHSECs. In addition, we determined protein expression of the endothelial cells before and after co-culturing with melanoma cell lines originated from primary tumors. Cell lines with increased invasive capacity after culturing with conditioned medium showed a set of receptor genes (CXCR1, IL1RL1, IL1RN, IL3RA, IL8RA, IL11RA, IL15RA, IL17RC, and IL17RD) with significantly different expression. It is very important note, that the IL11RA gene expression level was also significantly increased in primary melanoma tissues with liver metastasis as well. Proteome arrays revealed 15 differentially expressed proteins (including CD31, VCAM-1, ANGPT2, CXCL8, and CCL20) in the hepatic endothelial cells after co-cultured with melanoma cells. Our findings clearly indicate the interaction between liver endothelial- and melanoma cells. Based on our data, we assume that overexpression of the IL11RA gene might has key role in the formation of organ specific metastasis to the liver by primary melanoma cells.