Abstract
BackgroundExtracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells.MethodsTo probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments.ResultsWe find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content.ConclusionIn summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs.6Ak9CS6CfK5Ku3JS4e63coVideo Graphical abstract
Highlights
Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication
We have demonstrated that DisBa-01 affinity for αvβ3 integrin (Kd 4.63 × 10− 7 M) is one hundred times higher than its affinity for α5β1 integrin (Kd 7.62 × 10− 5 M), which makes this protein an excellent tool to study the roles of αvβ3 integrin in the adhesion and migration processes [15, 16, 27]
Integrin inhibition affects uptake of tumor SEVs by breast epithelial cells far, our experiments show that adhesion and uptake of purified SEVs by MCF10A cells are reduced by DisBa-01 using a transwell co-culture system
Summary
Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Cancer cells and neighboring host cells interact with each other and with extracellular matrix (ECM) proteins including collagen, laminin, vitronectin, and fibronectin [1]. Altei et al Cell Communication and Signaling (2020) 18:158 They include larger EVs (LEVs) such as shed microvesicles (100–1000 nm) from the cell membrane, [7] and SEVs, which include exosomes (50–150 nm) secreted from endocytic pathways [10, 11]. Integrins are critical adhesion receptors for ECM proteins that support cell adhesion and drive cell migration. Amongst the particular sequences recognized by integrins, the RGD (Arg-GlyAsp) motif is found in many ECM proteins including vitronectin, fibronectin and laminin [12]. RGD is usually recognized by both α5β1 and αvβ integrins, these two integrins play divergent roles in cell adhesion and migration. In addition to the RGD motif, integrin and ECM conformations are crucial to their interaction, indicating a complex mechanism [16, 17]
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