Prostate cancer cell-derived exosomes impair osteoclastic differentiation of bone marrow-derived primary osteoclast progenitor cells.

<p>Primary osteoclast progenitor cells were isolated from mouse bone marrow and subsequently expanded <i>in vitro</i> for 6 days. Osteoclast precursor cells were then cultured in complete α-MEM culture media supplemented with M-CSF (M; 30 ng/mL) in the absence or presence of TRAMP-C1 prostate cancer cell-derived exosomes alone or in combination with the osteoclastogenic factor RANKL (R; 2 ng/mL) before further analysis. Exosomes were added at two different concentrations, either at a lower concentration of 10 ng/10³ seeded cells (Exo1) or a higher concentration, 50 ng/10³ seeded cells (Exo2). (A) Proliferation (MTT) analysis of osteoclast precursor cells after incubation in the absence (M-CSF-treatment only; M) or presence of exosomes from TRAMP-C1 prostate cancer tumor cells at two different concentrations (M+Exo1 and M+Exo2, respectively). Data are obtained from 8 wells per treatment group and presented as means ± SEM. (B) Osteoclast formation analysis for bone marrow-derived osteoclast progenitor cells cultured in complete α-MEM culture media supplemented with M-CSF (M) and treated with RANKL (MR) alone or in combination with exosomes from TRAMP-C1 tumor cells at two different concentrations (MR+Exo1 and MR+Exo2, respectively). After 96 h of culture, the cells were stained for tartrate-resistant acid phosphatase (TRAP) using a Leukocyte Acid Phosphatase kit, and cells positive for TRAP with ≥ 3 nuclei were considered osteoclasts and counted. Data are obtained from 4 wells per treatment group and presented as means ± SEM, ** <i>p</i> ≤ 0.01, *** <i>p</i> ≤ 0.001.