The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90–120 nm; small exosome vesicles, Exo-S, 60–80 nm) and discovered an abundant population of non-membranous nanoparticles termed ‘exomeres’ (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.

Glycoconjugates constitute a major class of biomolecules which include glycoproteins, glycosphingolipids and proteoglycans. Glycans are involved in several physiological and pathological conditions, such as host–pathogen interactions, cell differentiation, migration, tumour invasion and metastisation, cell trafficking and signalling. Cancer is associated with glycosylation alterations in glycoproteins and glycolipids. This review describes various aspects of protein glycosylation with the focus on alterations associated with human cancer. The application of these glycosylation modifications as biomarkers for cancer detection in tumour tissues and serological assays is summarised.

Objectives Since liver transplant (LT) was introduced to treat patients with familial amyloid polyneuropathy carrying the V30M mutation (ATTR-V30M), ocular and cardiac complications have developed. Long-term central nervous system (CNS) involvement was not investigated. Our goals were to: (1) identify and characterise focal neurological episodes (FNEs) due to CNS dysfunction in ATTR-V30M patients; (2) characterise neuropathological features and temporal profile of CNS transthyretin amyloidosis. Methods We monitored the presence and type of FNEs in 87 consecutive ATTR-V30M and 35 non-ATTR LT patients. FNEs were investigated with CT scan, EEG and extensive neurovascular workup. MRI studies were not performed because all patients had cardiac pacemakers as part of the LT protocol. We characterised transthyretin amyloid deposition in the brains of seven ATTR-V30M patients, dead 3–13 years after polyneuropathy onset. Results FNEs occurred in 31% (27/87) of ATTR-V30M and in 5.7% (2/35) of the non-ATTR transplanted patients (OR=7.0, 95% CI 1.5 to 33.5). FNEs occurred on average 14.6 years after disease onset (95% CI 13.3 to 16.0) in ATTR-V30M patients, which is beyond the life expectancy of non-transplanted ATTR-V30M patients (10.9, 95% CI 10.5 to 11.3). ATTR-V30M patients with FNEs had longer disease duration (OR=1.24; 95% CI 1.07 to 1.43), renal dysfunction (OR=4.65; 95% CI 1.20 to 18.05) and were men (OR=3.57; 95% CI 1.02 to 12.30). CNS transthyretin amyloidosis was already present 3 years after polyneuropathy onset and progressed from the meninges and its vessels towards meningocortical vessels and the superficial brain parenchyma, as disease duration increased. Conclusions Our findings indicate that CNS clinical involvement occurs in ATTR-V30M patients regardless of LT. Longer disease duration after LT can provide the necessary time for transthyretin amyloidosis to progress until it becomes clinically relevant. Highly sensitive imaging methods are needed to identify and monitor brain ATTR. Disease modifying therapies should consider brain TTR as a target.

Sialyl-Lewis X (SLe^x) is a sialylated glycan antigen expressed on the cell surface during malignant cell transformation and is associated with cancer progression and poor prognosis. The increased expression of sialylated glycans is associated with alterations in the expression of sialyltransferases (STs). In this study we determined the capacity of ST3GAL3 and ST3GAL4 sialyltransferases to synthesize the SLe^x antigen in MKN45 gastric carcinoma cells and evaluated the effect of SLe^x overexpression in cancer cell behavior both in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. The activation of tyrosine kinase receptors and their downstream molecular targets was also addressed. Our results showed that the expression of ST3GAL4 in MKN45 gastric cancer cells leads to the synthesis of SLe^x antigens and to an increased invasive phenotype both in vitro and in the in vivo CAM model. Analysis of phosphorylation of tyrosine kinase receptors showed a specific increase in c-Met activation. The characterization of downstream molecular targets of c-Met activation, involved in the invasive phenotype, revealed increased phosphorylation of FAK and Src proteins and activation of Cdc42, Rac1 and RhoA GTPases. Inhibition of c-Met and Src activation abolished the observed increased cell invasive phenotype. In conclusion, the expression of ST3GAL4 leads to SLe^x antigen expression in gastric cancer cells which in turn induces an increased invasive phenotype through the activation of c-Met, in association with Src, FAK and Cdc42, Rac1 and RhoA GTPases activation.

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.