Staining of E-selectin ligands on paraffin-embedded sections of tumor tissue

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.

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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.