Extracellular Vesicles from Osteotropic Triple-Negative Breast Cancer Cells Transfer miRNAs to Bone Cells Reducing Collagen Expression and Bone Matrix Mineralisation

<b>Background/Objectives</b>: Bone metastases are a common complication of breast cancer. In our previous study, we reported that extracellular vesicles released by osteotropic human (MDA-MB-231) and murine (4T1) breast cancer cells disrupt bone homeostasis by enhancing osteoclast differentiation and impairing osteoblast function. Based on these findings, we investigated whether microRNAs contained within tumour-derived EVs could mediate these bone-altering effects. <b>Methods</b>: MDA-MB-231- and 4T1-EVs were tagged with the RNA-specific fluorophore SYTORNA and employed to treat mouse primary bone marrow macrophages (BMMs) and osteoblasts (OBs). We also performed RNAseq on MDA-MB-231- and 4T1-EVs to assess their miRNAs content. Finally, we evaluated the effect of selected miRNA-mimics on OBs, BMMs and HUVEC cells. <b>Results</b>: Fluorescence microscopy demonstrated EV-RNAs shuttling to recipient cells, while RNA sequencing on MDA-MB-231- and 4T1-EVs revealed that, of the top 20 expressed miRNAs, 10 were common. Among them, we first focused on the following four: miR-26a-5p, miR-24-3p, miR-29a-3p, and miR-29b-3p, which were linked to bone biology. We confirmed their presence in MDA-MB-231-/4T1-EVs by qPCR. Then, we evaluated their EV-mediated shuttling to BMMs and OBs using affinity tags. Among all the conditions tested, miR-29a and miR-29b were the best-shuttled miRNAs, with efficiency between 50-100% in both OBs and BMMs, both for MDA-MB-231- and 4T1-EVs. Finally, to test whether miR-29a and miR-29b could have a functional role in bone cells, OBs were transfected with miR-29a and 29b-mimics, discovering that this treatment reduced collagen1α1 and 1α2 mRNA as well as the OBs' mineralisation ability, while the same miRNA mimics were found to have no effect on osteoclastogenesis or on in vitro angiogenesis. <b>Conclusions</b>: MDA-MB-231- and 4T1-EVs shuttle miRNAs to bone cells, which likely contributes to OBs' activity impairment.