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LPCC/Pfizer 2011. Tagus TANK award 2018 (grant no. 1/2018). SFRH/BD/100970/2014 SFRH/BPD/108686/2015 The formation of distant metastasis resulting from vascular dissemination is one of the leading causes of mortality in non-small cell lung cancer (NSCLC). This metastatic dissemination initiates with the adhesion of circulating cancer cells to the endothelium. The minimal requirement for the binding of leukocytes to endothelial E-selectins and subsequent transmigration is the epitope of the fucosylated glycan, sialyl Lewis x (sLex), attached to specific cell surface glycoproteins. sLex and its isomer sialyl Lewis a (sLea) have been described in NSCLC, but their functional role in cancer cell adhesion to endothelium is still poorly understood. In this study, it was hypothesised that, similarly to leukocytes, sLe glycans play a role in NSCLC cell adhesion to E-selectins. To assess this, paired tumour and normal lung tissue samples from 18 NSCLC patients were analyzed. Immunoblotting and immunohistochemistry assays demonstrated that tumour tissues exhibited significantly stronger reactivity with anti‑sLex/sLea antibody and E-selectin chimera than normal tissues (2.2- and 1.8-fold higher, respectively), as well as a higher immunoreactive score. High sLex/sLea expression was associated with bone metastasis. The overall α1,3-fucosyltransferase (FUT) activity was increased in tumour tissues, along with the mRNA levels of FUT3, FUT6 and FUT7, whereas FUT4 mRNA expression was decreased. The expression of E-selectin ligands exhibited a weak but significant correlation with the FUT3/FUT4 and FUT7/FUT4 ratios. Additionally, carcinoembryonic antigen (CEA) was identified in only 8 of the 18 tumour tissues; CEA-positive tissues exhibited significantly increased sLex/sLea expression. Tumour tissue areas expressing CEA also expressed sLex/sLea and showed reactivity to E-selectin. Blot rolling assays further demonstrated that CEA immunoprecipitates exhibited sustained adhesive interactions with E-selectin-expressing cells, suggesting CEA acts as a functional protein scaffold for E-selectin ligands in NSCLC. In conclusion, this work provides the first demonstration that sLex/sLea are increased in primary NSCLC due to increased α1,3-FUT activity. sLex/sLea is carried by CEA and confers the ability for NSCLC cells to bind E-selectins, and is potentially associated with bone metastasis. This study contributes to identifying potential future diagnostic/prognostic biomarkers and therapeutic targets for lung cancer. preprint published

SFRH/BD/100970/2014 This work was supported by the LPCC/Pfizer2011 and Portuguese Foundation for Science and Technology (FCT) - SFRH/BD/100970/2014 (MAC) and Premio Santander/ Totta - UNL, Bluepharma/UC (PAV), by the National Institutes of Health, in particular, the National Heart Lung Blood Institute (NHLBI) Program of Excellence in Glycosciences (PEG) grant PO1 HL107146 (RS) and by the GlycoCan Marie Curie Actions (grant agreement number 676421). All the funding bodies supported the acquisition for all the required material for experiments and analysis, besides a fellowship for MAC. Background: The E-selectin ligands expressed by cancer cells mediate adhesion of circulating cancer cells to endothelial cells, as well as within tissue microenvironments important for tumor progression and metastasis. The identification of E-selectin ligands within cancer tissue could yield new biomarkers for patient stratification and aid in identifying novel therapeutic targets. The determinants of selectin ligands consist of sialylated tetrasaccharides, the sialyl Lewis X and A (sLeX and sLeA), displayed on protein or lipid scaffolds. Standardized procedures for immunohistochemistry make use of the antibodies against sLeX and/or sLeA. However, antibody binding does not define E-selectin binding activity. Methods: In this study, we developed an immunohistochemical staining technique, using E-selectin-human Ig Fc chimera (E-Ig) to characterize the expression and localization of E-selectin binding sites on paraffin-embedded sections of different cancer tissue. Results: E-Ig successfully stained cancer cells with high specificity. The E-Ig staining show high reactivity scores in colon and lung adenocarcinoma and moderate reactivity in triple negative breast cancer. Compared with reactivity of antibody against sLeX/A, the E-Ig staining presented higher specificity to cancer tissue with better defined borders and less background. Conclusions: The E-Ig staining technique allows the qualitative and semi-quantitative analysis of E-selectin binding activity on cancer cells. The development of accurate techniques for detection of selectin ligands may contribute to better diagnostic and better understanding of the molecular basis of tumor progression and metastasis. publishersversion published