Abstract
Rationale: Gastric cancer (GC) is a leading cause of cancer-related mortality, particularly in advanced stages where prognosis and targeted treatment remain challenging. The glycocalyx, a dense network of glycans and glycoproteins, is critical for tumor progression and immune evasion, yet its molecular signatures are poorly understood. This study investigates glycan-based biomarkers of aggressiveness, focusing on paucimannosidic N-glycans, a previously underexplored glycosylation pattern in cancer. Methods: High-throughput N-glycome analysis was performed on gastric tumors of varying aggressiveness, followed by Galanthus Nivalis Lectin (GNL) immunostaining to assess paucimannosidic glycans across tumor stages. Comparative analysis was performed against clinically relevant GC biomarkers (E-cadherin, p53, MSI, sTn, sLeA). TCGA analysis correlated key paucimannose-associated glycosyltransferases with clinical outcomes. Glycoproteomics identified glycoproteins carrying paucimannoses, later validated using immunoassays in tumor tissues for clinical relevance. Additionally, serum samples were analyzed to evaluate the non-invasive potential of GNL reactivity and associated glycoproteins. Results: Aggressive gastric tumors were significantly enriched in paucimannosidic N-glycans, a feature not previously reported in this malignancy. Lectin immunoblotting confirmed their disease specificity, with expression increasing with tumor progression. GNL staining outperformed established biomarkers in prognostic accuracy. TCGA analysis of more than 400 cases showed a strong correlation between high paucimannose-associated glycosyltransferase expression and poor prognosis. Glycoproteomics unexpectedly revealed paucimannose N-glycans primarily on intracellular ribosomal proteins, though key membrane proteins like MMP9 displayed aberrant paucimannosylation. MMP9 expression increased with tumor stage and grade, with tumors co-expressing MMP9 and paucimannosidic glycans exhibiting the worst prognosis. In serum, only MMP9 demonstrated diagnostic potential as a circulating biomarker, whereas GNL did not show a significant association. Conclusions: This study provides the first comprehensive characterization of the GC glycome, linking paucimannosidic N-glycans to tumor aggressiveness and poor clinical outcome. These glycans demonstrated superior prognostic performance compared to established clinical biomarkers. Their association with MMP9 further suggests a key role in disease progression. Together, these findings suggest that alterations in N-glycosylation, including paucimannosylated glycoproteins, hold promise for future prognostic and therapeutic applications in gastric cancer.