Nucleolin-SLe <sup>A</sup> Glycoforms as E-Selectin Ligands and Potentially Targetable Biomarkers at the Cell Surface of Gastric Cancer Cells

Abstract

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide, with half of patients developing metastases within 5 years after treatment, urging the need for novel biomarkers for diagnosis and more efficient therapeutic targeting. The sialyl-Lewis A (SLeA) antigen, a terminal glycoepitope of glycoproteins and glycolipids, offers tremendous potential towards this objective. This glycan is rarely expressed by healthy tissues and blood cells but is often present in cancer cells of high metastatic potential, as well as in the metastases. The SLeA antigen is also directly involved in E-selectin mediated metastasis, making it an ideal target to control disease dissemination. To improve further on cancer specificity, we have zoomed in on the SLeA-glycoproteome of six distinct GC cell models. Emphasis was set on glycoproteins showing affinity for E-selectin. A bioinformatics-assisted algorithm, used for the first time in this study, identified nucleolin (NCL), a nuclear protein frequently observed at the cell-surface in cancer cells, as a potential targetable biomarker potentially involved in metastasis development. Several immunoassays, including western blot and in situ proximity ligation reinforced the existence of NCL-SLeA glycoforms at the cancer cells surface and in GC. The NCL-SLeA glycophenotype was associated with decreased survival and was not reflected in relevant healthy tissues, supporting its tremendous potential for precise cancer targeting with limited off-target effects. To our knowledge, this is also the first report describing SLeA in a protein preferentially found in the nucleus, setting a new paradigm for cancer biomarkers discovery and targeted therapies. Funding Statement: Portuguese Foundation for Science and Technology (FCT) for the human resources grants: PhD grant BD/103571/2014 (EF), SFRH/BD/111242/2015 (AP), SFRH/BD/146500/2019 (MRS), SFRH/BD/142479/2018 (JS), SFRH/BD/105355/2014 (RA), SFRH/BD/127327/2016 (CG); and FCT assistant researcher grant CEECIND/03186/2017 (JAF). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The authors also acknowledge FCT the funding for CI-IPOP research unit (PEst-OE/SAU/UI0776/201), the Portuguese Oncology of Porto Research Centre (CI-IPOP-29-2014; CI-IPOP-58-2015; CI-IPOPProj.70-bolsa2019-GPTE) and PhD Programs in Biomedical and Pathology and Molecular Genetics of ICBAS-University of Porto. The authors EF, DF and MRS also acknowledge the financial support of the Research Group of Digestive Cancers (GICD) and the “Early stage cancer treatment, driven by context of molecular imaging (ESTIMA)” framework (NORTE-01-0145-FEDER-000027), IPO-Score (DSAIPA/DS/0042/2018) and “Towards a single therapy with a synergistic combination against triple negative breast cancer and neuroblastoma by nucleolin mediated multicellular targeting” (EU joint call ENMed/0006/2015) for financial support. This article is also a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundacao para a Ciencia e a Tecnologia/Ministerio da Ciencia, Tecnologia e Ensino Superior in the framework of the project Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274). Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: All procedures concerning the inclusion of patients were approved by the institutional Ethics Committee after patient’s informed consent.