Tumour metastasis is the main cause of cancer related deaths. Metastasis is an intricate multi-step process that requires the acquisition of several cancer cell features, including the modulation of tumour cell migration, adhesion, invasion, and immune evasion. Changes in the cellular glycosylation are associated with malignant transformation of cancer cells, tumour progression and ultimately, metastasis formation. Glycans have major impact on cellular signalling and on the regulation of tumour cell-cell adhesion and cell-matrix interaction. Glycans drive the interplay between the cancer cells and the tumour microenvironment. In this review, we summarize the roles of glycan alterations in tumour progression, such as acquisition of oncogenic features due to modulation of receptor tyrosine kinases, proteoglycans, cadherins and integrins. We also highlight the importance of key glycan binding proteins such as selectins, siglecs and galectins, which are pivotal in the modulation of immune response. An overview on glycans as cancer biomarkers is also presented.

Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state‐of‐the‐art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrep^TM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour‐associated glycan sialyl‐Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer‐related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates.

Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that SDC4 knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression.

In an era when cancer glycobiology research is exponentially growing, we are witnessing a progressive translation of the major scientific findings to the clinical practice with the overarching aim of improving cancer patients’ management. Many mechanistic cell biology studies have demonstrated that heparan sulfate (HS) glycosaminoglycans are key molecules responsible for several molecular and biochemical processes, impacting extracellular matrix properties and cellular functions. HS can interact with a myriad of different ligands, and therefore, hold a pleiotropic role in regulating the activity of important cellular receptors and downstream signalling pathways. The aberrant expression of HS glycan chains in tumours determines main malignant features, such as cancer cell proliferation, angiogenesis, invasion and metastasis. In this review, we devote particular attention to HS biological activities, its expression profile and modulation in cancer. Moreover, we highlight HS clinical potential to improve both diagnosis and prognosis of cancer, either as HS-based biomarkers or as therapeutic targets.

Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells’ glycocalyx and extracellular matrix. By acting as scaffolds for protein–protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell–extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood. In this study, we established glycoengineered gastric cancer cell models lacking either exostosin-like glycosyltransferase 2 (EXTL2) or EXTL3 and revealed their regulatory roles in both HS and chondroitin sulfate (CS) biosynthesis and structural features. We showed that EXTL3 is key for initiating the synthesis of HS chains in detriment of CS biosynthesis, intervening in the fine-tuned balance of the HS/CS ratio in cells, while EXTL2 functions as a negative regulator of HS biosynthesis, with impact over the glycoproteome of gastric cancer cells. We demonstrated that KO of EXTL2 enhanced HS levels along with concomitant upregulation of Syndecan-4, which is a major cell surface carrier of HS. This aberrant HS expression profile promoted a more aggressive phenotype, characterized by higher cellular motility and invasion, and impaired activation of Ephrin type-A 4 cell surface receptor tyrosine kinase. Our findings uncover the biosynthetic roles of EXTL2 and EXTL3 in the regulation of cancer cell GAGosylation and proteoglycans expression and unravel the functional consequences of aberrant HS/CS balance in cellular malignant features.