extracellular vesicles (eVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Human serum is a rich source of readily accessible eVs; however, the separation of eVs from serum proteins and non-eV lipid particles represents a considerable challenge. in this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 µl of human serum and their separation from non-eV protein and lipid particles present in serum. the size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of eVs from soluble protein was determined by calculating the ratio of eV particle count to protein concentration. finally, lipoprotein particles co-isolated with eVs was determined by Western blot analysis of lipoprotein markers ApoB and Apoe. overall, this study reveals that the choice of eV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants. Extracellular vesicles (EVs) were originally identified in reticulocytes as a means of disposing of obsolete membrane proteins such as α4β1 and transferrin receptor during reticulocyte maturation 1-3 , and have since been shown to participate in cell-cell signalling via transfer of proteins, nucleic acids and metabolites 4-6. EVs have been identified in a diverse range of human biofluids including serum, plasma, urine, saliva, breast milk, amniotic fluid, ascites fluid, cerebrospinal fluid and bile 7,8. These EVs are classified into three groups; exosomes, microvesicles and apoptotic bodies depending on their size, biogenesis and method of cellular release. Microvesicles and apoptotic bodies generally range from 100 to 1000 nm and 1-4 µm respectively, and are formed by budding from the plasma membrane 4,9. In contrast, exosomes have a diameter of 30-150 nm and are formed by inward budding of the late endosome lumen to form a multivesicular body (MVB) that is secreted by fusion with the plasma membrane 10. The overlap in exosome and microvesicle size (100-150 nm) and density (1.08-1.19 g/ml) makes it difficult to distinguish the two groups and as a result exosomes are often defined by their content of endosome-associated proteins including tetraspanins CD9, CD63, and CD81. However, since microvesicles from haematopoietic cells are also enriched for endosomal proteins such as CD63 and CD81 11 exosomes and microvesicles <150 nm are collectively referred to as small extracellular vesicles (sEVs) 12. EV secretion has been shown to be elevated in response to inflammation 13 , hypoxia 14,15 and an acidic microenvironment 16,17 and is associated with human diseases such as cancer, where secretion levels have b...

IntroductionThe adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells.MethodsMCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation.ResultsJAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients.ConclusionsOur findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and β1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.

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Breast cancer is a complex and heterogeneous disease that arises from epithelial cells lining the breast ducts and lobules. Correct adhesion between adjacent epithelial cells is important in determining the normal structure and function of epithelial tissues, and there is accumulating evidence that dysregulated cell-cell adhesion is associated with many cancers. This review will focus on one cell-cell adhesion complex, the tight junction (TJ), and summarize recent evidence that TJs may participate in breast cancer development or progression. We will first outline the protein composition of TJs and discuss the functions of the TJ complex. Secondly we will examine how alterations in these functions might facilitate breast cancer initiation or progression; by focussing on the regulatory influence of TJs on cell polarity, cell fate and cell migration. Finally we will outline how pharmacological targeting of TJ proteins may be useful in limiting breast cancer progression. Overall we hope to illustrate that the relationship between TJ alterations and breast cancer is a complex one; but that this area offers promise in uncovering fundamental mechanisms linked to breast cancer progression.