Abstract 1085: Prostate cancer secreted microRNAs influence early steps in bone metastasis via osteoclast precursors
Authors: Erozenci L. Ayse , Wang Ning , Geldof Albert A. , Mulder Jorn , Jimenez Connie R. , Moorselaar R. Jeroen van , Gartland Allison , Vries Teun J. de , Bijnsdorp Irene V. 
Published: August 2018

Abstract
BACKGROUND: Bone metastasis is a complex process involving reciprocal interplay between cancer and bone cells. Metastatic prostate cancer (PCA) cells secrete extracellular vesicles (EVs), that contain microRNAs (miRNAs), and these can be found in blood. We hypothesize that precursor osteoclasts scavenge PCA EVs, leading to a manipulation of their behavior thereby driving metastasis.
METHODS: To test how EV-miRNAs affect metastasis, we stably overexpressed two metastasis-related miRNAs in PCA cells. Bone metastases were induced by inoculating miRNA overexpressing PCA cells into nude mice via intracardiac route with or without the presence of subcutaneous tumors. Tumor progression was monitored by F-luciferase signal (IVIS) while bones were analyzed by microCT ex vivo. miRNAs expression was analyzed by qRT-PCR on precursor osteoclasts isolated from mice blood. Osteoclast differentiation assays were performed using human peripheral mononucleated blood cells on bone chips together with RANKL and M-CSF to drive differentiation.
RESULTS: In vivo, intracardiac injection of PC3 cells that overexpress selected miRNAs accelerated the formation of bone metastases (0-85%) compared to control PC3 cells (43%). To determine whether EVs secreted by PCA cells affect osteoclasts, human monocytes were allowed to differentiate to osteoclasts while exposing them to PCA EVs. EVs secreted by control PCA cell lines increased osteoclast differentiation by 22.5% (p&lt;0.01), compared to EVs secreted by RWPE1 noncancerous prostate cells. Exposure to EVs secreted by the miRNA overexpressing PCA cells further enhanced osteoclast formation by an additional 10% (p&lt;0.05). To test whether pro-metastatic miRNAs enhance metastases when mouse osteoclast precursors are pre-stimulated (via EVs or soluble factors), subcutaneous tumours of pro-metastatic cells were grown for 2 weeks. In circulating osteoclast precursors of mice that were pre-stimulated with PCA factors that were secreted by subcutaneous tumors (2 weeks), the miRNA levels were increased by 1.5 - 4 fold (p&lt;0.05), demonstrating that these cells scavenge the secreted tumor miRNAs. Subsequently, mice received an intracardiac injection with matching cells to induce metastases. Trabecular bone volume of &gt;15% was used as indicator for metastasis induced bone loss. Mice that grew both a subcutaneous tumor and also received an intracardiac injection to drive metastasis formation led to an ~30% increased number of mice with &gt;15% bone loss (p&lt;0.01). This indicates that osteoclasts may be manipulated by the cancer cell-secreted factors to form a pro-metastatic niche.
CONCLUSION: PCA miRNAs secreted via EVs stimulate osteoclast formation, potentially increasing the formation of overt metastases. Further investigation is needed to unravel novel pathways that can be targeted to prevent bone metastasis.
Citation Format: L. Ayse Erozenci, Ning Wang, Albert A. Geldof, Jorn Mulder, Connie R. Jimenez, R. Jeroen van Moorselaar, Allison Gartland, Teun J. de Vries, Irene V. Bijnsdorp. Prostate cancer secreted microRNAs influence early steps in bone metastasis via osteoclast precursors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1085.