Cancer cells induced to express mesenchymal phenotype release exosome-like extracellular vesicles carrying tissue factor

Abstract

Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes has not been studied. EVs are heterogeneous membrane structures that originate either at the surface (microparticles), or within (exosomes) activated or transformed cells, and are involved in intercellular trafficking of bioactive molecules. Here, we show that epithelial cancer cells (A431, DLD-1) adopt mesenchymal features (epithelial-to-mesenchymal transition-like state) upon activation of epidermal growth factor receptor (EGFR) coupled with blockade of E-cadherin. This treatment leads to a coordinated loss of EGFR and tissue factor (TF) from the plasma membrane and coincides with a surge in emission of small, exosome-like EVs containing both receptors. TF (but not EGFR) is selectively up-regulated in EVs produced by mesenchymal-like cancer cells and can be transferred to cultured endothelial cells rendering them highly procoagulant. We postulate that epithelial-to-mesenchymal transition-like changes may alter cancer cell interactions with the vascular systems through altered vesiculation and TF shedding.

FIGURE 1.

Mesenchymal cell subpopulations within epithelial cancers. Staining for vimentin expression in sections of human head and neck and vulvar cancer (A and B), as well as A431 xenografts in immunodeficient (SCID) mice (C). A, examples of grade I (left) or grade II (right) head and neck tumors: notable presence of vimentin-positive cells in both stromal and tumor cell compartments. B, transitory phenotypes in epidermoid vulvar carcinoma with different degrees of vimentin-positivity among cells with epithelial morphology (arrows). C, cellular heterogeneity of subcutaneous A431 tumor xenografts in immunodeficient mice. Tumors, which originate from morphologically uniform cancer cells driven by amplification of EGFR, contain both epidermoid vimentin-negative and mesenchymal vimentin-positive (arrows) cellular subpopulations, the latter indicative of an ongoing EMT.

FIGURE 2.

Induction of the mesenchymal phenotype in A431 cancer cells. A, induction of the EMT-like phenotype in vitro by a combined blockade of E-cadherin (SHE78-7 antibody) and stimulation of EGFR (TGFα, 50 ng/ml). The consequences of either individual or combined treatments on the cobblestone morphology of A431 colonies (upper row; phase contrast microscopy), architecture of the actin cytoskeleton (middle panel, phalloidin and DAPI fluorescent staining), and on the cellular morphology (bottom panel, SEM) are shown as indicated. Of note is the dissociation of cell-cell contacts, residual cytoneme-like protrusions (white arrows), and elongated morphology in cells treated with both SHE78-7 and TGFα. Scale bars are given in the respective panels. B, induction of vimentin expression in A431 cells treated as indicated. SHE78-7 and TGFα induce vimentin expression individually, but their effects are most pronounced under co-treatment conditions inducing EMT-like morphology. C, EMT-like changes are not associated with major changes in cell number (cell count). NS, non-significant statistically (p > 0.05; n = 3). WB, Western blot.

FIGURE 3.

The impact of mesenchymal phenotype induction on the expression patterns of EGFR and TF in cancer cells. A, differential impact of EMT inducing treatments on the levels of EGFR and TF in A431 cells (Western blotting; WB). There is a notable and expected decrease in levels of EGFR upon treatment with TGFα alone or in combination with SHE78-7, whereas these treatments up-regulate TF expression. B, changes in subcellular localization of EGFR and TF upon induction of EMT-like state with both receptors depleted from the plasma membrane. The cells were treated as indicated and subjected to immunofluorescent staining for EGFR and TF. C, computation of TF and EGFR co-localization in confocal images of A431 cells under epithelial and mesenchymal growth conditions. There is a reduction in the overlap between the respective signals in SHE78-7/TGFα-treated cells (***, p < 0.0005) (n = 3).

FIGURE 4.

Accumulation of TF at cell junctions in A431 epithelial cells. A, staining for TF in A431 cells. At low density, even unstimulated A431 cells exhibit weaker presence of TF at the plasma membrane and this changes at the cell-cell interface once the cells make contact. B, transmission electron microscopy image following immunogold staining for TF shows a more pronounced accumulation of TF signal (fine black dots) around cell-to-cell junction. C and D, TF localization at the cell-cell junctions co-localizes with junction proteins such as β-catenin (C) and especially γ-catenin (D).

FIGURE 5.

Quantitative and qualitative changes in cancer cell vesiculation profile as a function of mesenchymal transition. A, heterogeneity of EVs produced by A431 cells in culture (transmission electron microscopy; TEM). Of note is the presence of EVs ranging in size between 50 and 200 nm. B, Surface characteristics of A431 cells in their epithelial (left) and mesenchymal state (right). Untreated A431 cells (left panel) exhibit relatively smooth apical surfaces, form tight intercellular contacts, and deploy multiple intercellular membrane bridges. In contrast, many of the A431 cells in SHE78-7/TGFα treated cultures (right panel) are isolated from one another, assume elongated morphology, and exhibit a very complex and rough surface architecture with deployment of numerous filopodia-like processes. C, nanoparticle tracking analysis (NTA) of control and SHE78-7/TGFα-treated A431 cells. Untreated cells emit a wide spectrum of EVs of different sizes (as in A), whereas cells treated with SHE/TGFα, a mesenchymal phenotype inducing mixture, produce a distinct peak within the size range of exosomes (20–50 nm). Only particles between 0 to 300 nm are shown to maintain the resolution. (**, p < 0.005; ***, p < 0.0005) (n = 3).

FIGURE 6.

The impact of epithelial and mesenchymal culture conditions on the EV-mediated emission of EGFR and TF by A431 cancer cells. A, differential content of EGFR and TF in EVs isolated from supernatants of control A431 cells and upon indicated treatments (Western blotting (WB); flotillin-1 present in EVs is used as a loading control, also compare Fig. 5A). B, quantification of TF in the conditioned medium of A431 cells induced to undergo mesenchymal transition (TF ELISA). The effects of individual treatments are markedly less pronounced than the combined exposure to SHE78-7/TGFα (n = 3). C, sustained emission of EGFR as cargo of EVs into the A431 conditioned media (n = 3). D, concentration of the TF signal as a function of the SHE78-7/TGFα treatment in the P4 fraction of the conditioned media corresponding to small (exosome-like) EVs, relative to larger EVs (P2 fraction; TF ELISA). The removal of small EVs (P4) by ultracentrifugation resulted in depletion of the TF signal from the cell culture supernatants, suggesting the absence of soluble (EV-unrelated) TF in this material. P4 and P2 fractions were defined as in the text by differential centrifugation protocols (n = 2) (*, p < 0,05). NS, not significant; ND, not detectable.

FIGURE 7.

Stimulation of EGFR and blockade of E-cadherin induce mesenchymal phenotype and exosomal TF emission in DLD-1 cells. A, stimulation of DLD-1 colorectal adenocarcinoma cells with SHE78-7 antibody and TGFα provokes cell scattering, elongation, and mesenchymal appearance. B, marked release of TF into conditioned medium of DLD-1 cells stimulated with the SHE/TGFα mixture (TF ELISA) (n = 3) (*, p < 0,05). C, increase in production of exosomal-like EVs by DLD-1 cells stimulated with SHE78-7 and TGFα (n = 2) (*, p < 0.05).

FIGURE 8.

Extracellular vesicle-mediated transfer of tumor-derived TF to endothelial cells. A, EV-mediated transfer of membrane fluorescence from PKH26-labeled A431 cells to cultured endothelium (HUVEC). Following a 24-h incubation with EVs generated by control (EVs Ctrl) or SHE78-7/TGFα-stimulated/mesenchymal tumor cells (EVs MES), HUVEC cells avidly incorporated EV-associated fluorescence (∼20% cells were gated as positive), regardless of treatment. B, differential acquisition of TF positivity by HUVEC cells exposed to EVs from control and SHE78-7/TGFα-treated A431 cells (Western blotting; WB). The incorporated TF signal is severalfold stronger in the case of HUVEC incubated with EVs released by stimulated A431 cells relative to control. C, TF-containing EVs isolated from A431 cells cultured under mesenchymal conditions transfer their procoagulant phenotype to HUVEC more robustly than their control counterparts (EVs from untreated A431 cells). TF procoagulant activity assay that measures activation of the coagulation factor X to Xa was conducted on HUVEC cells treated as in B (n = 2) (**, p < 0.005; ***, p < 0.0005). NS, not significant statistically.

FIGURE 9.

Circulating TF and TAT complexes in tumor bearing mice. A, heterogeneous pattern of TF staining (red) in A431 tumor xenografts resembles the heterogeneity of vimentin expression (compare Fig. 1C and (4)). TF-positive cells are found in the proximity of blood vessels (green, CD105 staining; blue, DAPI (nuclei)). B, circulating human (tumor-derived) TF in plasma of mice harboring A431 tumors measured by ELISA. C, TAT complexes indicative of the activated coagulation system parallel the presence of TF in plasma of tumor bearing mice and are absent in tumor-free mice (TAT ELISA). *, p < 0.05; **, p < 0.005.