Abstract
Tissue factor (TF)-expressing tumor-derived extracellular vesicles (EVs) can promote metastasis and pre-metastatic niche formation, but the mechanisms by which this occurs remain largely unknown. We hypothesized that generation of activated factor X (FXa) by TF expressed on tumor-derived EV could activate protease-activated receptors (PARs) on non-activated endothelial cells to induce a pro-adhesive and pro-inflammatory phenotype. We obtained EV from TF-expressing breast (MDA-MB-231) and pancreatic (BxPC3 and Capan-1) tumor cell lines. We measured expression of E-selectin and secretion of interleukin-8 (IL-8) in human umbilical vein endothelial cells after exposure to EV and various immunologic and chemical inhibitors of TF, FXa, PAR-1, and PAR-2. After 6 h of exposure to tumor-derived EV (pretreated with factor VIIa and FX) in vitro, endothelial cells upregulated E-selectin expression and secreted IL-8. These changes were decreased with an anti-TF antibody, FXa inhibitors (FPRCK and EGRCK), and PAR-1 antagonist (E5555), demonstrating that FXa generated by TF-expressing tumor-derived EV was signaling through endothelial PAR-1. Due to weak constitutive PAR-2 expression, these endothelial responses were not induced by a PAR-2 agonist peptide (SLIGKV) and were not inhibited by a PAR-2 antagonist (FSLLRY) after exposure to tumor-derived EV. In conclusion, we found that TF-expressing cancer-derived EVs activate quiescent endothelial cells, upregulating E-selectin and inducing IL-8 secretion through generation of FXa and cleavage of PAR-1. Conversion of resting endothelial cells to an activated phenotype by TF-expressing cancer-derived EV could promote cancer metastases.
Highlights
The majority of epithelial tumors upregulate tissue factor (TF) [1] and tumor-derived extracellular vesicles (EVs), including exosomes (30–150 nm) and membrane-derived microparticles (150–1,000 nm), expressing TF antigen and procoagulant activity have been isolated from plasma of cancer patients [2,3,4] and conditioned media from cell lines [5,6,7]
We demonstrate that TF-expressing EVs induce a pro-adhesive and pro-inflammatory phenotype in quiescent endothelial cells
Our results suggest that protease-activated receptors (PARs)-1 is the dominant receptor on unstimulated endothelial cells for the TF–factor VIIa (FVIIa)–FX complex on tumor-derived EV
Summary
The majority of epithelial tumors upregulate tissue factor (TF) [1] and tumor-derived extracellular vesicles (EVs), including exosomes (30–150 nm) and membrane-derived microparticles (150–1,000 nm), expressing TF antigen and procoagulant activity have been isolated from plasma of cancer patients [2,3,4] and conditioned media from cell lines [5,6,7]. TF-expressing tumor-derived EV promoted premetastatic niche formation and metastasis in mice by initiating coagulation and recruiting bone. TF-Expressing EVs Activate Endothelial Cells marrow-derived monocytic cells [8], which was prevented by inhibition of TF-triggered thrombin generation. Normal quiescent endothelial cells act as a barrier to cancer metastasis, but pro-inflammatory mediators can activate endothelial cells to induce expression of pro-inflammatory adhesion molecules and secretion of pro-inflammatory cytokines. Such endothelial responses can potentially promote cancer metastasis by facilitating recruitment of tumor cells or bone marrow-derived hematopoietic cells involved in pre-metastatic niche formation [8,9,10]. Activation of endothelial cells by EV has been observed previously, resulting in increased vascular permeability, angiogenesis, and cancer metastasis [10,11,12,13,14]; the mechanisms remain largely unknown
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