Supplementary Figure S1: Optimization of the triple immunostaining on different cell lines with known status of cytokeratins, TF and vimentin expression. Supplementary Figure S2: TF expression and pro-coagulant activity of EMT inducible cell lines. Supplementary Figure S3: Impact of TF blocking antibody on coagulant activity. Supplementary Figure S4: Regulation of TF by EMT transcription factors. Supplementary Figure S5: Induction of TF after de novo expression of Snail in MDA-MB-468. Supplementary Figure S6: Validation of TF shRNA. Supplementary Figure S7: Presence of fibrin fibers around lung colonizing MDA-MB-231 cells as revealed by transmission electron microscopy.
Funding
Fonds De La Recherche Scientifique
Fondation Contre le Cancer
the “Partenariat Hubert Curien-Tournesol”, the “Fonds spéciaux de la Recherche” (University of Liège), the “Centre Anticancéreux près l'Université de Liège”, the “Fonds Léon Fredericq” (University of Liège), the “Direction Générale Opérationnelle de l'Economie”, de l'Emploi et de la Recherche from the Service Public de Wallonie (SPW, Belgium), the Interuniversity Attraction Poles Programme (Brussels, Belgium), the “Actions de Recherche Concertées”
Australian Government Department of Health
Movember Foundation
Prostate Cancer Foundation of Australia
National Breast Cancer Foundation
ARTICLE ABSTRACT
Epithelial–mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT–TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 4270–82. ©2016 AACR.
