Extracellular vesicles derived from prostate cancer cells impair osteoblastic functions

Introduction Prostate cancer (PCa) is the second leading cause of cancer-related death in men and is characterized by a predominant metastatic-tropism to bone. Tumor cell-derived extracellular vesicles (EVs) are key regulators of cancer initiation and progression. However, the role of tumor-derived EVs in the establishment and maintenance of the tumor microenvironment in bone remains unclear. Here, we evaluated the effects of PCa-derived EVs on osteoblasts in vitro and in vivo.

Methods EVs were isolated from the murine osteotropic PCa cell line RM1-BM by serial ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analyzer and Western blot analyses. Murine primary osteoblasts (OB) were differentiated from bone marrow stromal cells and maintained in osteogenic media for 7 days. Internalization of PKH26-labeled EVs was detected using fluorescence microscopy and flow cytometry. In addition, OB were cultured with different concentrations (20, 50, 100 µg/ml) of PCa-EVs to assess dose-dependent effects on OB metabolic activity (Cell Titer Blue assay), vitality (crystal violet), and mineralization capacity (alizarin red staining). Also, Next Generation Sequencing was performed to identify molecular alterations in the gene expression of osteoblasts after EV treatment. Finally, the effect of PCa-EVs on osteoblasts in vivo was evaluated in an ectopic bone mouse model, after implantation of alginate beads containing PCa-EVs and OB or untreated OB.

Results PCa-derived EVs displayed the characteristic cup-shape and had the expected size of 50 to 100 nm. Moreover, Western blot analysis showed the expression of two commonly-reported EV markers, CD63 and CD9. Fluorescence microscopy revealed the uptake of PCa-EVs into osteoblasts as early as after 1 h. After 24 h, OB were densely packed with tumor EVs, indicating an efficient cellular uptake by OB. Treatment of 7 days differentiated OB with PCa-EVs showed a dose-dependent increase of cell metabolic activity [+24 %; +40 %; +58 %; p < 0.001] and viability [+32 %; +52 %; +65 %; p < 0.001] compared to untreated cells. In contrast, mineral deposition was significantly reduced by increasing the concentration of EVs [-4 % to -17 %; p < 0.001]. Gene set enrichment analysis of OB treated with PCa-EVs versus normal OB shows a significant downregulation of osteoblastic markers [p < 0.02] and an upregulation of the inflammatory factors [p < 0.001], adipogenesis [p < 0.008], TNFα and IL-6 signaling pathway [p < 0.001]. In line with the in vitro studies, analysis of ectopic ossification by µCT revealed significantly decreased bone formation in beads containing PCa-EVs and OB compared to untreated OB (p < 0.05).

Discussion The results of this study highlight the importance of EVs in cell to cell communication in vitro and in vivo. The alteration of the OB activity suggests that PCa-EVs could facilitate the initial communication between the primary tumor and site of metastasis.

Korrespondenzadresse Giulia Furesi, Technische Universität Dresden, Department of Medicine III, Fetscherstr. 74, 01307 Dresden, Germany,

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