Description:
<jats:title>Abstract</jats:title><jats:sec>
<jats:title>Background</jats:title>
<jats:p>Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression.</jats:p>
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<jats:title>Methods</jats:title>
<jats:p>EVs were isolated by differential ultracentrifugation from cell culture supernatant as well as plasma samples from healthy individuals (<jats:italic>n</jats:italic> = 27) and breast cancer patients (<jats:italic>n</jats:italic> = 41). EVs were thoroughly characterized by electron microscopy, nanoparticle tracking analysis, immunoblot, and flow cytometry. ROR transfer to target cells was observed using microscopy-based assays and biodistribution experiments were conducted in syngeneic mice. EV impact on cancer cell migration and invasion was tested in functional assays.</jats:p>
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<jats:title>Results</jats:title>
<jats:p>We observed that the supernatant of ROR-overexpressing cells was sufficient for transferring the receptors to ROR-negative cells. Analyzing the secretome of the ROR-overexpressing cells, we detected a high enrichment of ROR1/2 on large and small EVs, but not on large oncosomes. Interestingly, the majority of ROR-positive EVs remained attached to the target cell surface after 24 h of stimulation and was quickly removed by treatment with trypsin. Nonetheless, ROR-positive EVs increased migration and invasion of breast cancer cells, even after chemically inhibiting EV uptake, in dependence of RhoA downstream signaling. In vivo, ROR-depleted EVs tended to distribute less into organs prone for the formation of breast cancer metastases. ROR-positive EVs were also significantly elevated in the plasma of breast cancer patients and allowed to separate them from healthy controls.</jats:p>
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<jats:title>Conclusions</jats:title>
<jats:p>The oncogenic Wnt receptors ROR1/2 are transferred via EVs to the surface of ROR-negative cancer cells, in which they induce an aggressive phenotype supporting tumor progression.</jats:p>
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