Extracellular vesiscles derived from prostate cancer cells influence osteoblast activity

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 EVs on osteoblasts in vitro and in vivo.

Methods EVs were isolated from the murine PCa cell line RM1-BM by serial ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analyzer (NTA) and western blot (WB) 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 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 (NGS) was performed to identify molecular alterations in the gene expression of osteoblasts after EVs treatment. Finally, osteogenic capacity of PCa-EVs in vivo was evaluated in an ectopic mouse model, after implantation of beads containing tumor EVs-OB or normal OB.

Results Analysis by TEM revealed the characteristic cup-shape of the isolated EVs. NTA confirmed the expected size (50-100 nm). Moreover, WB analysis showed the expression of two commonly-reported EV markers, CD63 and CD9. Fluorescence microscopy displayed cells with EVs as early as after 1 h. After 24 h, OB were densely packed with EVs, indicating an efficient cellular uptake of EVs by OB. Treatment of 7 days differentiated OB with 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]. Additionally, analysis of ossification by µCT revealed significantly decreased bone formation in beads containing tumor EVs-OB compared to untreated OB (P < 0.05).

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

Keywords Prostate cancer, Bone metastasis, Extracellular vesicles, Osteoblasts

Korrespondenzadresse Giulia Furesi, Medical Center Department of Medicine III and Center for Healthy Aging, Division of Endocrinology, Diabetes, and Bone Diseases Dresden, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany

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Publication History

Article published online:
05 March 2021