Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. For this reason, besides clinical and preclinical studies, novel in vitro models for the assessment of cancer ...response to drugs and radiation are being developed. In such context, three-dimensional (3D)-engineered cellular microenvironments, compared to unrealistic two-dimensional (2D) monolayer cell culture, provide a model closer to the in vivo configuration. Concerning cancer treatment, while X-ray radiotherapy and chemotherapy remain the current standard, proton beam therapy is an appealing alternative as protons can be efficiently targeted to destroy cancer cells while sparing the surrounding healthy tissue. However, despite the treatment’s compelling biological and medical rationale, little is known about the effects of protons on GBM at the cellular level. In this work, we designed novel 3D-engineered scaffolds inspired by the geometry of brain blood vessels, which cover a vital role in the colonization mechanisms of GBM cells. The architectures were fabricated by two-photon polymerization (2PP), cultured with U-251 GBM cells and integrated for the first time in the context of proton radiation experiments to assess their response to treatment. We employed Gamma H2A.X as a fluorescent biomarker to identify the DNA damage induced in the cells by proton beams. The results show a higher DNA double-strand breakage in 2D cell monolayers as compared to cells cultured in 3D. The discrepancy in terms of proton radiation response could indicate a difference in the radioresistance of the GBM cells or in the rate of repair kinetics between 2D cell monolayers and 3D cell networks. Thus, these biomimetic-engineered 3D scaffolds pave the way for the realization of a benchmark tool that can be used to routinely assess the effects of proton therapy on 3D GBM cell networks and other types of cancer cells.
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IJS, KILJ, NUK, PNG, UL, UM
Cancer patients are at increased risk of developing thrombosis, comorbidity that has been associated with increased neutrophil counts and the formation of neutrophil extracellular traps (NETs). ...Interleukin-1β (IL-1β) modulates the expression of granulocyte colony-stimulating factor (G-CSF), a cytokine that promotes cancer-associated neutrophilia and NET generation. Herein, we combined a murine breast cancer model with a flow-restriction thrombosis model to evaluate whether the IL-1β blockade could interfere with cancer-associated thrombosis. Mice bearing metastatic 4T1 tumors exhibited high neutrophil counts as well as elevated expression of G-CSF and IL-1β in their tumors. On the other hand, mice bearing non-metastatic 67NR tumors showed no elevation in neutrophil counts and displayed low expression levels of G-CSF and IL-1β in their tumors. 4T1 tumor-bearing mice but not 67NR tumor-bearing mice exhibited a NET-dependent prothrombotic state. Pharmacological blockade of IL-1 receptor (IL-1R) decreased the primary growth of 4T1 tumors and reduced the systemic levels of myeloperoxidase, cell-free DNA (cfDNA) and G-CSF, without interfering with the neutrophil counts. Most remarkably, the blockade of IL-1R abolished the prothrombotic state observed in 4T1 tumor-bearing mice. Overall, our results demonstrate that IL-1β might be a feasible target to attenuate cancer-associated thrombosis, particularly in cancer types that rely on increased G-CSF production and involvement of NET formation.
Tissue Factor (TF) is the initiator of blood coagulation but also functions as a signal transduction receptor. TF expression in breast cancer is associated with higher tumor grade, metastasis and ...poor survival. The role of TF signaling on the early phases of metastasis has never been addressed. Here, we show an association between TF expression and metastasis as well as cancer stemness in 574 breast cancer patients. In preclinical models, blockade of TF signaling inhibited metastasis tenfold independent of primary tumor growth. TF blockade caused a reduction in epithelial-to-mesenchymal-transition, cancer stemness and expression of the pro-metastatic markers Slug and SOX9 in several breast cancer cell lines and in ex vivo cultured tumor cells. Mechanistically, TF forms a complex with β1-integrin leading to inactivation of β1-integrin. Inhibition of TF signaling induces a shift in TF-binding from α3β1-integrin to α6β4 and dictates FAK recruitment, leading to reduced epithelial-to-mesenchymal-transition and tumor cell differentiation. In conclusion, TF signaling inhibition leads to reduced pro-metastatic transcriptional programs, and a subsequent integrin β1 and β4-dependent reduction in metastasic dissemination.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Cancer-associated thrombosis is one of the major causes of worse prognosis among tumor-bearing patients. Extracellular vesicles derived from cancer cells, which can be divided mainly into ...microvesicles and exosomes, can participate in several tumor progression phenomena. Tumor-derived microvesicles positive for tissue factor (TF) have been associated with thrombotic risk in certain cancer types. Cancer cell-derived exosomes, however, have not. In this study we evaluated the capacity of extracellular vesicles (EVs, containing both microvesicles and exosomes) derived from breast-cancer cell lines in promoting platelet activation, aggregation and plasma coagulation, in experiments that access both TF-dependent and -independent activities.
EVs were isolated from the conditioned media of two human mammary carcinoma cell lines: MDA-MB-231 (highly invasive) and MCF-7 (less invasive). TF-independent EV/platelet interaction, platelet P-selectin exposure and aggregation were evaluated. Western blotting, plasma clotting and platelet aggregation in the presence of plasma were performed for the measurement of TF-dependent activity in EVs.
Interaction between MDA-MB-231 EVs and washed platelets led to increased platelet P-selectin exposure and platelet aggregation compared to MCF-7 EVs. MDA-MB-231 EVs had higher TF protein levels and TF-dependent procoagulant activity than MCF-7 EVs. Consequently, TF-dependent platelet aggregation was also induced by MDA-MB-231 EVs, but not by MCF-7 EVs.
Our results suggest that MDA-MB-231 EVs induce TF-independent platelet activation and aggregation, as well as TF-dependent plasma clotting and platelet aggregation by means of thrombin generation. In this context, aggressive breast cancer-derived EVs may contribute to cancer-associated thrombosis.
•Extracellular vesicles (EVs) derived from breast cancer cell lines interact with platelets.•MDA-MB-231 EVs induce TF-independent platelet activation and aggregation.•MDA-MB-231 EVs induce TF-dependent plasma clotting.•Thrombin generated by MDA-MB-231 EVs promotes platelet aggregation in plasma.•MCF-7 EVs were very much less efficient in eliciting pro-hemostatic responses.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Neutrophil extracellular traps (NETs) have been implicated in several hallmarks of cancer. Among the protumor effects, NETs promote epithelial-mesenchymal transition (EMT) in different cancer models. ...EMT has been linked to an enhanced expression of the clotting-initiating protein, tissue factor (TF), thus favoring the metastatic potential. TF may also exert protumor effects by facilitating the activation of protease-activated receptor 2 (PAR2). Herein, we evaluated whether NETs could induce TF expression in breast cancer cells and further promote procoagulant and intracellular signaling effects via the TF/PAR2 axis. T-47D and MCF7 cell lines were treated with isolated NETs, and samples were obtained for real-time PCR, flow cytometry, Western blotting, and plasma coagulation assays. In silico analyses were performed employing RNA-seq data from breast cancer patients deposited in The Cancer Genome Atlas (TCGA) database. A positive correlation was observed between neutrophil/NETs gene signatures and TF gene expression. Neutrophils/NETs gene signatures and PAR2 gene expression also showed a significant positive correlation in the bioinformatics model. In vitro analysis showed that treatment with NETs upregulated TF gene and protein expression in breast cancer cell lines. The inhibition of ERK/JNK reduced the TF gene expression induced by NETs. Remarkably, the pharmacological or genetic inhibition of the TF/PAR2 signaling axis attenuated the NETs-induced expression of several protumor genes. Also, treatment of NETs with a neutrophil elastase inhibitor reduced the expression of metastasis-related genes. Our results suggest that the TF/PAR2 signaling axis contributes to the pro-cancer effects of NETs in human breast cancer cells.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Extracellular vesicles, such as microvesicles (MVs), were identified as important players in tumor progression and acquisition of an aggressive phenotype. Tissue factor (TF) is a transmembrane ...protein that initiates the blood coagulation cascade. In tumor cells, TF has been associated with aggressiveness and cancer progression. Previous studies demonstrate that TF is incorporated into MVs secreted by tumor cells; however, it is unknown whether TF is actively involved in the release of MVs. Here, we investigated the influence of TF expression on the release of MVs. TF silencing was achieved through CRISPR/Cas9 approaches in the human breast cancer cell line, MDA-MB-231. TF knockout in MDA-MB-231 cells efficiently reduced TF-dependent signaling and procoagulant activity. Remarkably, silencing of TF caused a significant decrease in the number of MVs released by MDA-MB-231 cells. We also observed an increase in actin-positive membrane projections in TF knockout cells and a reduction in RhoA expression when compared to TF-expressing cells. Treatment of MDA-MB-231 cells with the RhoA-ROCK signaling pathway inhibitor, fasudil, significantly reduced the release of MVs. Taken together, our results suggest a novel and relevant role for TF in tumor biology by playing an active role in the MVs secretion.
•TF knockout in MDA-MB-231 cells reduced TF/FVIIa signaling and coagulation activity.•Silencing of TF in MDA-MB-231 cells decreased the release of MVs.•RhoA expression is decreased in TF-silenced MDA-MB-231 cells.•The ROCK inhibitor, fasudil, decreases MVs shedding by MDA-MB-231 cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The establishment of prothrombotic states during cancer progression is well reported but the precise mechanisms underlying this process remain elusive. A number of studies have implicated the ...presence of the clotting initiator protein, tissue factor (TF), in circulating tumor-derived extracellular vesicles (EVs) with thrombotic manifestations in certain cancer types. Tumor cells, as well as tumor-derived EVs, may activate and promote platelet aggregation by TF-dependent and independent pathways. Cancer cells and their secreted EVs may also facilitate the formation of neutrophil extracellular traps (NETs), which may contribute to thrombus development. Alternatively, the presence of polyphosphate (polyP) in tumor-derived EVs may promote thrombosis through a TF-independent route. We conclude that the contribution of EVs to cancer coagulopathy is quite complex, in which one or more mechanisms may take place in a certain cancer type. In this context, strategies that could attenuate the crosstalk between the proposed pro-hemostatic routes could potentially reduce cancer-associated thrombosis.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Glioblastoma (GBM) patients have one of the highest risks of venous thromboembolism (VTE), which is even further increased upon treatment with chemotherapy. Tissue factor (TF) is the initiator of the ...extrinsic coagulation pathway and expressed by GBM cells. In this study, we aimed to examine the effect of routinely used chemotherapeutic agents Temozolomide (TMZ) and Lomustine (LOM) on TF procoagulant activity and expression in GBM cells in vitro. Three human GBM cell lines (U-251, U-87, U-118) were exposed to 100 µM TMZ or 30 µM LOM for 72 h. TF procoagulant activity was assessed via an FXa generation assay and TF gene and protein expression through qPCR and Western blotting. The externalization of phosphatidylserine (PS) was studied using Annexin V flow cytometry. Treatment with TMZ and LOM resulted in increased procoagulant activity in all cell lines. Furthermore, both agents induced procoagulant activity in the supernatant and tumor-cell-secreted extracellular vesicles. In line, TF gene and protein expression were increased upon TMZ and LOM treatment. Additionally, PS externalization and induction of inflammatory-associated genes were observed. Overall, the chemotherapeutic modalities TMZ and LOM induced procoagulant activity and increased TF gene and protein expression in all GBM cell lines tested, which may contribute to the increased VTE risk observed in GBM patients undergoing chemotherapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor which is activated upon cleavage of its N-terminal region. PAR2 has been associated with many aspects regarding tumor progression, ...such as the production of pro-tumoral cytokines. Granulocyte colony-stimulating factor (G-CSF) is a cytokine essential to neutrophil production and maturation, and it is often overexpressed in tumors. In this study, we evaluated the ability of PAR2 to modulate G-CSF expression. PAR2 and G-CSF were significantly more expressed in metastatic (4T1 and MDA-MB-231) as compared to non-metastatic (67NR and MCF7) breast cancer cell lines. In addition, PAR2 stimulation by a synthetic agonist peptide significantly increased G-CSF gene expression in the metastatic cell lines. Knockdown of PAR2 in 4T1 cells decreased G-CSF expression and secretion. In addition, treatment of 4T1 with the commercial PAR2 antagonist, ENMD-1068, significantly decreased G-CSF expression. cBioPortal analyses of the TCGA database showed a significant co-occurrence of G-CSF and PAR2 gene overexpression in breast cancer samples. In conclusion, our data suggest that PAR2 contributes to G-CSF expression in breast cancer cells, possibly favoring tumor progression.
•PAR2 and G-CSF are upregulated in metastatic breast cancer cell lines.•Activation of PAR2 increases G-CSF expression in human and murine breast cancer cell lines.•Genetic or pharmacological inhibition of PAR2 reduces G-CSF expression in the 4T1 cell line.•There is a significant co-occurrence of G-CSF and PAR2 overexpression in human breast cancer samples.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
In this study, the role of de-palmitoylation of tissue factor (TF) in the decryption of its activity was explored. TF-tGFP constructs were prepared by mutagenesis-substitution at Cys245 to prevent or ...mimic palmitolyation. Additionally, to reduce TF de-palmitoylation, the expression of palmitoyl-protein thioesterases (PPT) was suppressed. Other TF mutants were prepared with altered flexibility, hydrophobicity or length of the transmembrane domain. The outcome of these alterations on fXa-generation, fVIIa binding, Ser253 phosphorylation and TF-microvesicle release were assessed in endothelial cells, and the influence on endothelial and MCF-7 cell proliferation and apoptosis was analysed. Preventing TF palmitoylation (TFSer245-tGFP), increasing the hydrophobicity (TFPhe241-tGFP) or lengthening (TFLongTM-tGFP) of the transmembrane domain enhanced fXa-generation in resting cells compared to cells expressing TFWt-tGFP, but fXa-generation was not further increased following PAR2 activation. Extending the available length of the transmembrane domain enhanced the TF-tGFP release within microvesicles and Ser253 phosphorylation and increased cell proliferation. Moreover, prevention of PKCα-mediated Ser253 phosphorylation with Gö6976 did not preclude fXa-generation. Conversely, reducing the hydrophobicity (TFSer242-tGFP), shortening (TFShortTM-tGFP) or reducing the flexibility (TFVal225-tGFP) of the transmembrane domain suppressed fXa-generation, fVIIa-HRP binding and Ser253 phosphorylation following PAR2 activation. PPT knock-down or mimicking palmitoylation (TFPhe245-tGFP) reduced fXa-generation without affecting fVIIa binding. This study has for the first time shown that TF procoagulant activity is regulated through de-palmitoylation, which alters the orientation of its transmembrane domain and is independent of TF phosphorylation. However, Ser253 phosphorylation is facilitated by changes in the orientation of the transmembrane domain and can induce TF-cellular signalling that influences cellular proliferation/apoptosis.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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