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The capture of tumour-derived extracellular vesicles (TEVs) by cells in the tumour microenvironment (TME) contributes to metastasis and notably to the formation of the pre-metastatic niche (PMN). However, due to the challenges associated with modelling release of small EVs in vivo, the kinetics of PMN formation in response to endogenously released TEVs have not been examined. Here, we have studied the endogenous release of TEVs in mice orthotopically implanted with metastatic human melanoma (MEL) and neuroblastoma (NB) cells releasing GFP-tagged EVs (GFTEVs) and their capture by host cells to demonstrate the active contribution of TEVs to metastasis. Human GFTEVs captured by mouse macrophages in vitro resulted in transfer of GFP vesicles and the human exosomal miR-1246. Mice orthotopically implanted with MEL or NB cells showed the presence of TEVs in the blood between 5 and 28 days after implantation. Moreover, kinetic analysis of TEV capture by resident cells relative to the arrival and outgrowth of TEV-producing tumour cells in metastatic organs demonstrated that the capture of TEVs by lung and liver cells precedes the homing of metastatic tumour cells, consistent with the critical roles of TEVs in PMN formation. Importantly, TEV capture at future sites of metastasis was associated with the transfer of miR-1246 to lungmacrophages, liver macrophages, and stellate cells. This is the first demonstration that the capture of endogenously released TEVs is organotropic as demonstrated by the presence of TEV-capturing cells only in metastatic organs and their absence in non-metastatic organs. The capture of TEVs in the PMN induced dynamic changes in inflammatory gene expression which evolved to a pro-tumorigenic reaction as the niche progressed to the metastatic state. Thus, our work describes a novel approach to TEV tracking in vivo that provides additional insights into their role in the earliest stages of metastatic progression. [ABSTRACT FROM AUTHOR]

The potential role of osteoblasts in bone and bone marrow (BM) metastases in neuroblastoma (NBL) remains unclear. In this study, we examined the effect of NBL cells on the osteoblastic differentiation of BM-derived mesenchymal stromal cells (BMMSC). We show that the presence of NBL cells enhanced the osteoblastic differentiation of BMMSC driven by bone morphogenetic protein (BMP)-4, in the absence of any effect on NBL cell proliferation. Expression profiles of BMMSC driven toward osteoblastic differentiation revealed an increase in vascular endothelial growth factor A ( Vegfa) expression in the presence of NBL cells. We demonstrated that NBL cells increased BMMSC-derived VEGFA mRNA and protein and that this was enhanced by BMP-4. However, in similar conditions, neither the addition of an mVEGFA blocking antibody nor exogenous recombinant (r) mVEGFA affected osteoblastic differentiation. In contrast, siRNA- mediated knock-down of VEGFA in BMMSC prevented osteoblastic differentiation in BMP-4-treated cocultures, an effect that was not reversed in the presence of rmVEGFA. An analysis of murine bones injected with hNBL cells revealed an increase of mVEGFA producing cells near tumor cells concomitantly with an increase in Vegfa and Runx2 mRNA. This coincided with an increase in osteoclasts, in Rankl/Opg mRNA ratio and with the formation of osteolytic lesions. Thus NBL cells promote osteoblastogenesis in the BM by increasing VEGFA expression in BMMSC. Our study provides a new insight into the role of VEGFA in NBL metastases by pointing to the role of stroma-derived intracrine VEGFA in osteoblastogenesis. [ABSTRACT FROM AUTHOR]

The bone marrow (BM) niche is a microenvironment promoting survival, dormancy and therapeutic resistance in tumor cells. Central to this function are mesenchymal stromal cells (MSCs). Here, using neuroblastoma (NB) as a model, we demonstrate that NB cells release an extracellular vesicle (EVs) whose protein cargo is enriched in exosomal proteins but lacks cytokines and chemokines. Using three different purification methods, we then demonstrate that NB-derived exosomes were captured by MSCs and induced the production of pro-tumorigenic cytokines and chemokines, including interleukin-6 (IL-6), IL-8/CXCL8, vascular endothelial cell growth factor and monocyte-chemotactic protein-1, with exosomes prepared by size exclusion chromatography having the highest activity. We found no correlation between the IL-6 and IL-8/CXCL8 stimulatory activity of exosomes from eight NB cell lines and their origin, degree of MYCN amplification, drug resistance and disease status. We then demonstrate that the uptake of NB exosomes by MSCs was associated with a rapid increase in ERK1/2 and AKT activation, and that blocking ERK1/2 but not AKT activation inhibited the IL-6 and IL-8/CXCL8 production by MSCs without affecting exosome uptake. Thus, we describe a new mechanism by which NB cells induce in MSCs an inflammatory reaction that contributes to a favorable microenvironment in the BM. [ABSTRACT FROM PUBLISHER]