Epithelial-mesenchymal plasticity and circulating tumor cells: Travel companions to metastases.

Epithelial-mesenchymal transitions (EMTs) associated with metastatic progression may contribute to the generation of hybrid phenotypes capable of plasticity. This cellular plasticity would provide tumor cells with an increased potential to adapt to the different microenvironments encountered during metastatic spread. Understanding how EMT may functionally equip circulating tumor cells (CTCs) with an enhanced competence to survive in the bloodstream and niche in the colonized organs has thus become a major cancer research axis. We summarize here clinical data with CTC endpoints involving EMT. We then review the work functionally linking EMT programs to CTC biology and deciphering molecular EMT-driven mechanisms supporting their metastatic competence. Developmental Dynamics 247:432-450, 2018. © 2017 Wiley Periodicals, Inc.

Soluble factors regulated by epithelial-mesenchymal transition mediate tumour angiogenesis and myeloid cell recruitment.

Epithelial-mesenchymal transition (EMT) programmes provide cancer cells with invasive and survival capacities that might favour metastatic dissemination. Whilst signalling cascades triggering EMT have been extensively studied, the impact of EMT on the crosstalk between tumour cells and the tumour microenvironment remains elusive. We aimed to identify EMT-regulated soluble factors that facilitate the recruitment of host cells in the tumour. Our findings indicate that EMT phenotypes relate to the induction of a panel of secreted mediators, namely IL-8, IL-6, sICAM-1, PAI-1 and GM-CSF, and implicate the EMT-transcription factor Snail as a regulator of this process. We further show that EMT-derived soluble factors are pro-angiogenic in vivo (in the mouse ear sponge assay), ex vivo (in the rat aortic ring assay) and in vitro (in a chemotaxis assay). Additionally, conditioned medium from EMT-positive cells stimulates the recruitment of myeloid cells. In a bank of 40 triple-negative breast cancers, tumours presenting features of EMT were significantly more angiogenic and infiltrated by a higher quantity of myeloid cells compared to tumours with little or no EMT. Taken together, our results show that EMT programmes trigger the expression of soluble mediators in cancer cells that stimulate angiogenesis and recruit myeloid cells in vivo, which might in turn favour cancer spread.

Vimentin prevents a miR-dependent negative regulation of tissue factor mRNA during epithelial-mesenchymal transitions and facilitates early metastasis.

Epithelial-mesenchymal transitions (EMTs) are high-profile in the field of circulating tumor cells (CTCs). EMT-shifted CTCs are considered to encompass pre-metastatic subpopulations though underlying molecular mechanisms remain elusive. Our previous work identified tissue factor (TF) as an EMT-induced gene providing tumor cells with coagulant properties and supporting metastatic colonization by CTCs. We here report that vimentin, the type III intermediate filament considered a canonical EMT marker, contributes to TF regulation and positively supports coagulant properties and early metastasis. Different evidence further pointed to a new post-transcriptional regulatory mechanism of TF mRNA by vimentin: (1) vimentin silencing accelerated TF mRNA decay after actinomycin D treatment, reflecting TF mRNA stabilization, (2) RNA immunoprecipitation revealed enriched levels of TF mRNA in vimentin immunoprecipitate, (3) TF 3'-UTR-luciferase reporter vector assays implicated the 3'-UTR of TF mRNA in vimentin-dependent TF regulation, and (4) using different TF 3'UTR-luciferase reporter vectors mutated for potential miR binding sites and specific Target Site Blockers identified a key miR binding site in vimentin-dependent TF mRNA regulation. All together, these data support a novel mechanism by which vimentin interferes with a miR-dependent negative regulation of TF mRNA, thereby promoting coagulant activity and early metastasis of vimentin-expressing CTCs.

Flow Cytometry Assessment of Procoagulant Platelets Using a Dithiol-Reactive Probe.

Flow cytometry assessment of platelets using the combination of GSAO [4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], a dithiol-reactive probe, and P-selectin, a platelet activation marker, is a novel and powerful assay in the identification and quantification of the procoagulant subpopulation of platelets that has the capacity to support thrombin generation. In this chapter, we provide the flow cytometry protocols aimed at the study of procoagulant platelets under resting and agonist-stimulated conditions in whole blood and washed platelets of both human and murine (mouse) samples.

Tissue Factor Induced by Epithelial-Mesenchymal Transition Triggers a Procoagulant State That Drives Metastasis of Circulating Tumor Cells.

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.