Background and Objective: We recently reported that rapidly migratory, “amoeboid” prostate cancer (PCa) cells shed large (1-10µm diameter), bioactive extracellular vesicles (EV), termed large oncosomes (LO), whose abundance correlates with tumor aggressiveness (Di Vizio et al., Cancer Res. 2009; Di Vizio et al. Am J Pathol. 2012). Increasing evidence supports an important role for EVs in mechanisms of communication between cancer cells and the surrounding microenvironment. EVs can activate signal transduction as well as transfer biomolecules to recipient cells, processes that may promote oncogenesis and/or enhance tumor progression. The aim of this study was to investigate the molecular mechanisms of LO internalization into recipient cells and LO-mediated intercellular communication. Results: LO stimulated the migration of tumor and endothelial cells and contained active MMP2 and MMP9, key proteases involved in tumor cell invasion. Quantitative LC-MS/MS SILAC analysis of LO and exosomes demonstrated enrichment of specific proteins in LO in comparison with exosomes. Fluorescently labeled-LO were internalized into recipient cells and maintained their stability as discrete microvesicles that were localized in the perinuclear space at early time points and into the nucleus at later times. Because of this transfer of LO to the nuclear compartment, we investigated whether exposure to LO altered the activity of transcription factors in recipient cells, hypothesizing a possible novel mechanism of LO-mediated intracellular communication. Interestingly, the degree of internalization, as quantitatively assessed by flow cytometry, varied among different recipient cells, including benign and cancer prostate epithelial cells, immortalized myofibroblasts, cancer associated fibroblasts and endothelial cells. These results suggest cell-specific affinities for target cells and, perhaps, finely regulated mechanisms underlying LO-internalization. Moreover selective inhibition of pathways important in EV internalization indicated that the LO enter cells through an active endocytic process, rather than passively fusing with the plasma membrane of recipient cells. Internalization of LO resulted in altered expression of transcription factors. Conclusions: Our results show for the first time that internalization of intact LO might be necessary for their biological activity in recipient cells. These findings also suggest that the functional role of LO in the tumor microenvironment might be mediated by modulation of transcription factors.

Citation Format: Valentina R. Minciacchi, Matteo Morello, Sungyong You, Wei Yang, Mariana Sobreiro, Cristiana Spinelli, Mandana Zandian, Mirja Rotinen, Samantha Morley, Rosalyn Adam, Michael R. Freeman, Dolores Di Vizio. Large oncosomes are internalized and functionally modulate transcription factors in recipient cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4868. doi:10.1158/1538-7445.AM2014-4868