Abstract
The metastatic cascade describes the escape of primary tumour cells to distant secondary sites. Cells at the leading tumour edge are thought to undergo epithelial-mesenchymal transition (EMT), to enhance their motility and invasion for spreading. Whether EMT cells directly promote tumour angiogenesis, and the role of exosomes (30-150 nm extracellular vesicles) remains largely unknown. We examined the functional effects of exosomes from MDCK cells, MDCK cells stably expressing YBX1 (MDCKYBX1, intermediate EMT), and Ras-transformed MDCK cells (21D1 cells, complete EMT). 2F-2B cell motility and tube formation (length and branching) was significantly increased following supplementation with MDCKYBX1 or 21D1 exosomes, but not MDCK exosomes. Next, Matrigel™ plugs containing exosome-supplemented 2F-2B cells were subcutaneously injected into mice. Systemic perfusion was only observed for plugs supplemented with MDCKYBX1 or 21D1 exosomes. Comparative proteomics revealed that 21D1 exosomes contained VEGF-associated proteins, while MDCKYBX1 exosomes were enriched with activated Rac1 and PAK2. To validate, 2F-2B cells and HUVECs were pre-treated with PAK inhibitors prior to exosome supplementation. PAK inhibition nullified the effects of MDCKYBX1 exosomes by reducing the tube length and branching to baseline levels. By contrast, the effects of 21D1 exosomes were not significantly decreased. Our results demonstrate for the first time that oncogenic cells undergoing EMT can communicate with endothelial cells via exosomes, and establish exosomal Rac1/PAK2 as angiogenic promoters that may function from early stages of the metastatic cascade.
Highlights
Interactions occurring between tumour cells at the invasive front and cells in the tumour microenvironment (TM) promote cancer invasion and metastasis [1,2,3,4]
We have previously observed that over-expression of Y-box binding protein 1 (YBX1) in MadinDarby canine kidney (MDCK) cells induces p-epithelial-mesenchymal transition (EMT), and causes elevated release of soluble secreted proteins (TGF-β, Colony stimulating factor 1 (CSF-1), NGF, VGF, ADAM9 and ADAM17) associated with promoting angiogenesis [23]
We focussed on the functional contribution exosomes derived from increasingly oncogenic EMT cells (MDCK < MDCKYBX1 < Ras-transformed MDCK cells (21D1)) may have on inducing angiogenesis in recipient endothelial cells
Summary
Interactions occurring between tumour cells at the invasive front and cells in the tumour microenvironment (TM) promote cancer invasion and metastasis [1,2,3,4]. Cells at the leading tumour edge are thought to undergo epithelial-mesenchymal transition (EMT) in response to a variety of growth factors, signalling molecules and transcription factors [5]. EMT is involved in the process of cancer cell intravasation into blood and/or lymph vessels [6, 7]. The contribution of EMT cells to the recruitment of endothelial cells and the formation of blood vessels remains not well understood. Tumour angiogenesis and vessel formation can be controlled by signals in the TM that permit vascular remodelling via a series of controlled events [8, 9]. Growth factors (VEGF, PDGF, FGF), signalling molecules (TGF-β, Notch, Wnt, ANG and TIE), integrins (αvβ and αvβ5) and proteases (membrane type 1-MMP and MMP9) are known to regulate endothelial cell functions and neovascularisation [11]. Given that extracellular vesicles (EV) have recently been reported as modulators of the TM [12, 13], we speculate that they may promote angiogenesis
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