Breast cancer cells release a large quantity of biocargo-bearing extracellular vesicles (EVs), which mediate intercellular communication within the tumour microenvironment and promote
metastasis. To identify EV-bound
proteins related to
metastasis, we used mass spectrometry to profile EVs from highly and poorly metastatic
breast cancer lines of human and mouse origins. Comparative mass spectrometry indicated that
integrins, including αv and β1 subunits, are preferentially enriched in EVs of highly metastatic origin over those of poorly metastatic origin. These results are consistent with our histopathological findings, which show that
integrin αv is associated with
disease progression in
breast cancer patients.
Integrin αv colocalizes with the multivesicular-body marker CD63 at a higher frequency in the tumour and is enriched in circulating EVs of
breast cancer patients at late stages when compared with circulating EVs from early-stage patients. With a magnetic bead-based flow cytometry assay, we confirmed that
integrins αv and β1 are enriched in the CD63+ subsets of EVs from both human and mouse highly metastatic cells. By analysing the level of
integrin αv on circulating EVs, this assay could predict the metastatic potential of a xenografted mouse model. To explore the export mechanism of
integrins into EVs, we performed immunoprecipitation mass spectrometry and identified members of the
galectin family as potential shuttlers of
integrin αvβ1 into EVs. In particular, knockdown of
galectin-3, but not
galectin-1, causes a reduction in the levels of cell surface
integrins β1 and αv, and decreases the colocalization of these
integrins with CD63. Importantly, knockdown of
galectin-3 leads to a decrease of
integrin αvβ1 export into the EVs concomitant with a decrease in the metastatic potential of
breast cancer cells. Moreover, inhibition of the
integrin αvβ1 complex leads to a reduction in the binding of EVs to
fibronectin, suggesting that
integrin αvβ1 is important for EV retention in the extracellular matrix. EVs retained in the extracellular matrix are taken up by fibroblasts, which differentiate into cancer associated fibroblasts. In summary, our data indicate an important link between EV-bound
integrin αvβ1 with
breast cancer metastasis and provide additional insights into the export of
integrin αvβ1 into EVs in the context of
metastasis.