Prostate cancer (PCa) is the most common cancer and the second cause of cancer-related death in males. When patients have (bone) metastases, no curative therapy is available. MicroRNAs (miRNAs) have been reported for their contribution to PCa metastases, however there is still limited understanding about their actual biological involvement. Cancer cells secrete small vesicles (exosomes) that are enriched for miRNAs. miRNAs within these exosomes potentially have a function in cell-signalling. Secreted exosomes can, when taken up at distant locations, potentially change the microenvironment creating a pre-metastatic niche. The aim of the present study was to investigate the influence of secreted PCa-exosomes on osteoclasts. Exosomes from in vitro PCa cell cultures (PC-3, MatLyLu and DU145) and urine were isolated by ultracentrifugation. By small RNA sequencing the miRNAs profile was determined in urine-exosomes from PCa patients. Uptake of PKH67-fluorescently-labelled-exosomes was determined by FACS measurement. Peripheral blood mononuclear cells were isolated by ficol paque separation. Osteoclastogenesis of mouse bone marrow was performed in the presence of M-CSF and RANKL, and osteoclast formation was determined by the TRAP-activity assay. Bone resorption as a measure of osteoclast activity was assessed with Coommassie Brilliant Blue staining. We identified two miRNAs that were differentially expressed in urine-exosomes from metastatic prostate cancer patients compared with patients without metastasis. These were highly present in the blood-exosomes of metastatic patients and also in exosomes retrieved form in vitro prostate cancer cell lines. Exosomes were almost exclusively (90%) taken up by human monocytes, which are potential precursors for osteoclasts. After PCa exosome stimulation, osteoclasts differentiation was stimulated, and large multinucleated cells with >10 nuclei/osteoclast were formed, compared with controls with 5-10 nuclei (P<0.005). Moreover, we found that exosome-stimulated-osteoclasts were slightly more active in bone resorption, compared with normal osteoclasts. In conclusion, we show that PCa exosomes stimulate osteoclast formation and activity.
Disclosure: The authors declared no competing interests. Stichting VUmc CCA is kindly acknowledged for financial support.