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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

Figure S1. Example of application of staining of E-selectin ligands by Immunofluorescence. Staining of E-selectin ligands in CF1_T cells. The breast cancer cell line CF1_T has a high content of E-selectin ligands and it was obtained and cultured as described by Carrascal et al. (2017). Cells were cultured on glass coverslips overnight and then fixed with 3.7% paraformaldehyde. After blocking with 1% bovine serum albumin, cells were stained with E-Ig chimera in the presence of 2 mM CaCl2. The final step included anti-human Ig antibody conjugated with fluorescein (FITC, green), in the presence of PBS containing 2 mM CaCl2 (A). Control experiments were processed in the absence of CaCl2 (B). After permeabilization with 0.1% TritonX-100, F-actin was stained with Alexa Fluor 568 phalloidin (Molecular Probes, Leiden, Netherlands). Images were acquired with a Leica TCS SP2 AOBS confocal microscope. A representative cross-section confocal images were selected after Z-stacking. (PPTX 230 kb)

Figure S1. Example of application of staining of E-selectin ligands by Immunofluorescence. Staining of E-selectin ligands in CF1_T cells. The breast cancer cell line CF1_T has a high content of E-selectin ligands and it was obtained and cultured as described by Carrascal et al. (2017). Cells were cultured on glass coverslips overnight and then fixed with 3.7% paraformaldehyde. After blocking with 1% bovine serum albumin, cells were stained with E-Ig chimera in the presence of 2 mM CaCl2. The final step included anti-human Ig antibody conjugated with fluorescein (FITC, green), in the presence of PBS containing 2 mM CaCl2 (A). Control experiments were processed in the absence of CaCl2 (B). After permeabilization with 0.1% TritonX-100, F-actin was stained with Alexa Fluor 568 phalloidin (Molecular Probes, Leiden, Netherlands). Images were acquired with a Leica TCS SP2 AOBS confocal microscope. A representative cross-section confocal images were selected after Z-stacking. (PPTX 230 kb)