Signature human melanoma cell surface glycans license vascular adhesion through galectin-3 Exploring Glycomic Strategies to Enhance CAR-T Cell Efficacy Against Diffuse Large B Cell Lymphoma
Abstract
Introduction and Objective. Melanoma is a relatively rare yet highly lethal form of skin cancer, and despite recent therapeutic advances, only ~30% of patients with metastatic melanoma (MM) survive beyond five years. Understanding the molecular mechanisms that drive melanoma cells to systemic dissemination and extravasation is fundamental for predicting metastatic potential and developing novel anti-melanoma therapies. Previous studies revealed that human MM cell surfaces, unlike normal melanocytes, possess an abundance of linear poly-N-acetyllactosamine glycans (poly-LacNAcs) characteristically recognized by a family of β-galactoside-binding proteins known as galectins (Gal). We hypothesized that circulating galectins, namely Gal-3 and -8, which have been functionally associated with melanoma progression, bind to MM cell surface poly-LacNAcs, facilitating cell "bridging" to vascular endothelial cells (ECs) to initiate tumor cell adhesion and subsequent extravasation into distant tissues. Methods. Using flow cytometry, we analyzed Gal-3- and -8-binding activity on MM A375/A2058/UACC62/SKMEL5 cells and human vascular EC (HUVEC) surfaces. Using RT-qPCR, we analyzed the RNA expression levels of fucosyltransferases enzymes (FUT3-7), which are crucial for the formation of sialyl Lewis X and A (sLeX/A) that mediate E-selectin- dependent-adhesion. Additionally, we used parallel-plate chamber assays to simulate the physiological shear stress of circulating MM cells flowing over human ECs in the presence and absence of Gal-3 and -8. Data was analyzed and represented using relative mean fluorescence intensity (MFI), RQ, or fold change ± the standard error of the mean (SEM); statistical analysis was conducted using GraphPad Prism 8 software, employing the Holm-Sidak method to control for Type I errors with alpha value at 0.05 (p≤0.05). Experiments were performed at least in triplicate to meet statistical significance. Results. Flow cytometry data revealed the absence of E-selectin-binding glycans (sLeX/A) on MM cells, as well as robust Gal-3 and -8 binding activity. RT-qPCR data revealed a lack of FUT3-7 mRNA expression, which is necessary for sLeX/A formation, and parallel plate flow chamber assays showed that preincubated MM cells with Gal-3 supported MM cell adhesion to the HUVEC under physiologic shear flow. Conclusions-Implications. The data suggested that exogenous Gal-3 significantly enhanced MM cell adhesion to ECs compared to untreated or galectin-inhibited control groups. These findings support our premise that MM cells may use a galectin-dependent adhesion as a mechanism for metastatic spread, distinct from the established dependence on vascular E-selectin. In all, these results suggested that galectins and their ligands on MM cell surfaces can be targets for innovative therapeutics tailored to melanoma patients, who commonly possess higher levels of Gal-3 and -8.
Keywords
Melanoma, Cancer, Metastasis, Galectins, Glycobiology
Presentation Type
Oral Presentation
Signature human melanoma cell surface glycans license vascular adhesion through galectin-3 Exploring Glycomic Strategies to Enhance CAR-T Cell Efficacy Against Diffuse Large B Cell Lymphoma
Introduction and Objective. Melanoma is a relatively rare yet highly lethal form of skin cancer, and despite recent therapeutic advances, only ~30% of patients with metastatic melanoma (MM) survive beyond five years. Understanding the molecular mechanisms that drive melanoma cells to systemic dissemination and extravasation is fundamental for predicting metastatic potential and developing novel anti-melanoma therapies. Previous studies revealed that human MM cell surfaces, unlike normal melanocytes, possess an abundance of linear poly-N-acetyllactosamine glycans (poly-LacNAcs) characteristically recognized by a family of β-galactoside-binding proteins known as galectins (Gal). We hypothesized that circulating galectins, namely Gal-3 and -8, which have been functionally associated with melanoma progression, bind to MM cell surface poly-LacNAcs, facilitating cell "bridging" to vascular endothelial cells (ECs) to initiate tumor cell adhesion and subsequent extravasation into distant tissues. Methods. Using flow cytometry, we analyzed Gal-3- and -8-binding activity on MM A375/A2058/UACC62/SKMEL5 cells and human vascular EC (HUVEC) surfaces. Using RT-qPCR, we analyzed the RNA expression levels of fucosyltransferases enzymes (FUT3-7), which are crucial for the formation of sialyl Lewis X and A (sLeX/A) that mediate E-selectin- dependent-adhesion. Additionally, we used parallel-plate chamber assays to simulate the physiological shear stress of circulating MM cells flowing over human ECs in the presence and absence of Gal-3 and -8. Data was analyzed and represented using relative mean fluorescence intensity (MFI), RQ, or fold change ± the standard error of the mean (SEM); statistical analysis was conducted using GraphPad Prism 8 software, employing the Holm-Sidak method to control for Type I errors with alpha value at 0.05 (p≤0.05). Experiments were performed at least in triplicate to meet statistical significance. Results. Flow cytometry data revealed the absence of E-selectin-binding glycans (sLeX/A) on MM cells, as well as robust Gal-3 and -8 binding activity. RT-qPCR data revealed a lack of FUT3-7 mRNA expression, which is necessary for sLeX/A formation, and parallel plate flow chamber assays showed that preincubated MM cells with Gal-3 supported MM cell adhesion to the HUVEC under physiologic shear flow. Conclusions-Implications. The data suggested that exogenous Gal-3 significantly enhanced MM cell adhesion to ECs compared to untreated or galectin-inhibited control groups. These findings support our premise that MM cells may use a galectin-dependent adhesion as a mechanism for metastatic spread, distinct from the established dependence on vascular E-selectin. In all, these results suggested that galectins and their ligands on MM cell surfaces can be targets for innovative therapeutics tailored to melanoma patients, who commonly possess higher levels of Gal-3 and -8.