Overexpression Of Tissue Factor Research Articles

Activation of \u03b21 integrins and caveolin-1 by TF/FVIIa promotes IGF-1R signaling and cell survival

The tissue factor/coagulation factor VIIa (TF/FVIIa) complex induces transactivation of the IGF-1 receptor (IGF-1R) in a number of different cell types. The mechanism is largely unknown. The transactivation leads to protection from apoptosis and nuclear translocation of the IGF-1R. The aim of this study was to clarify the signaling pathway between TF and IGF-1R after FVIIa treatment with PC3 and DU145 prostate or MDA-MB-231 breast cancer cells as model systems. Protein interactions, levels, and phosphorylations were assessed by proximity ligation assay or flow cytometry in intact cells and by western blot on cell lysates. The transactivation of the IGF-1R was found dependent on TF/FVIIa-induced activation of β1-integrins. A series of experiments led to the conclusion that the caveolae protein caveolin-1 prevented IGF-1R activation in resting cells via its scaffolding domain. TF/FVIIa/β1-integrins terminated this inhibition by activation of Src family kinases and subsequent phosphorylation of caveolin-1 on tyrosine 14. This phosphorylation was not seen after treatment with PAR1 or PAR2 agonists. Consequently, the protective effect of FVIIa against apoptosis induced by the death receptor agonist TRAIL and the de novo synthesis of cyclin D1 induced by nuclear IGF-1R accumulation were both significantly reduced by down-regulation of β1-integrins or overexpression of the caveolin-1 scaffolding domain. In conclusion, we present a plausible mechanism for the interplay between TF and IGF-1R involving FVIIa, β1-integrins, Src family proteins, and caveolin-1. Our results increase the knowledge of diseases associated with TF and IGF-1R overexpression in general but specifically of TF-mediated signaling with focus on cell survival.

Iron-Deficiency and Estrogen Are Associated With Ischemic Stroke by Up-Regulating Transferrin to Induce Hypercoagulability.

Epidemiological studies have identified an associate between iron deficiency (ID) and the use of oral contraceptives (CC) and ischemic stroke (IS). To date, however, the underlying mechanism remains poorly understood. Both ID and CC have been demonstrated to upregulate the level and iron-binding ability of Tf (transferrin), with our recent study showing that this upregulation can induce hypercoagulability by potentiating FXIIa/thrombin and blocking antithrombin-coagulation proteases interactions. To investigate whether Tf mediates IS associated with ID or CC and the underlying mechanisms. Tf levels were assayed in the plasma of IS patients with a history of ID anemia, ID anemia patients, venous thromboembolism patients using CC, and ID mice, and in the cerebrospinal fluid of some IS patients. Effects of ID and estrogen administration on Tf expression and coagulability and the underlying mechanisms were studied in vivo and in vitro. High levels of Tf and Tf-thrombin/FXIIa complexes were found in patients and ID mice. Both ID and estrogen upregulated Tf through hypoxia and estrogen response elements located in the Tf gene enhancer and promoter regions, respectively. In addition, ID, administration of exogenous Tf or estrogen, and Tf overexpression promoted platelet-based thrombin generation and hypercoagulability and thus aggravated IS. In contrast, anti-Tf antibodies, Tf knockdown, and peptide inhibitors of Tf-thrombin/FXIIa interaction exerted anti-IS effects in vivo. Our findings revealed that certain factors (ie, ID and CC) upregulating Tf are risk factors of thromboembolic diseases decipher a previously unrecognized mechanistic association among ID, CC, and IS and provide a novel strategy for the development of anti-IS medicine by interfering with Tf-thrombin/FXIIa interactions.

86/90Y-Labeled Monoclonal Antibody Targeting Tissue Factor for Pancreatic Cancer Theranostics.

Pancreatic cancer is highly aggressive, with a median survival time of less than 6 months and a 5-year overall survival rate of around 7%. The poor prognosis of PaCa is largely due to its advanced stage at diagnosis and the lack of efficient therapeutic options. Thus, the development of an efficient, multifunctional PaCa theranostic system is urgently needed. Overexpression of tissue factor (TF) has been associated with increased tumor growth, angiogenesis, and metastasis in many malignancies, including pancreatic cancer. Herein, we propose the use of a TF-targeted monoclonal antibody (ALT836) conjugated with the pair 86/90Y as a theranostic agent against pancreatic cancer. For methods, serial PET imaging with 86Y-DTPA-ALT836 was conducted to map the biodistribution the tracer in BXPC-3 tumor-bearing mice. 90Y-DTPA-ALT836 was employed as a therapeutic agent that also allowed tumor burden monitoring through Cherenkov luminescence imaging. The results were that the uptake of 86Y-DTPA-ALT836 in BXPC-3 xenograft tumors was high and increased over time up to 48 h postinjection (p.i.), corroborated through ex vivo biodistribution studies and further confirmed by Cherenkov luminescence Imaging. In therapeutic studies, 90Y-DTPA-ALT836 was found to slow tumor growth relative to the control groups and had significantly smaller (p < 0.05) tumor volumes 1 day p.i. Histological analysis of ex vivo tissues revealed significant damage to the treated tumors. The conclusion is that the use of the 86/90Y theranostic pair allows PET imaging with excellent tumor-to-background contrast and treatment of TF-expressing pancreatic tumors with promising therapeutic outcomes.

3308The critical roles of coagulation factors in inducing brown adipose tissue dysfunction and systemic metabolic disorder in obesity

Abstract Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model. Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. BAT specific PAR1 KO mice improved glucose intolerance and thermogenic response under a metabolically stressed condition. In differentiated brown adipocytes, FXa markedly increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes.

BSCI-23. TISSUE FACTOR SIGNALING ENHANCES METASTATIC BRAIN CANCER MALIGNANCY

Brain metastases are on the rise and remain one of the most refractory malignancies worldwide. Currently, the standard approach for therapy involves surgery and radiation. However, this approach usually produces only a modest increase in survival. We recently discovered that Tissue Factor (TF) strongly enhances the malignancy of gliomas via protease-activated receptor 2 (PAR2) signaling, though its role in brain metastases is not as well understood. In this study, we further explored the significance of TF in lung cancer brain metastases, showing that genetic and pharmacological targeting of TF-PAR2 signaling may decrease malignancy and increase the efficacy of radiotherapy. Studies were performed using patient-derived brain metastases coming from lung carcinoma. Markers of malignancy were measured by BrdU incorporation for cell proliferation, Matrigel-coated transwell migration, soft agar colony formation for anchorage-independent growth, limiting dilution assay for tumor initiation capacity, and clonogenic cell survival assay to measure radiation sensitivity. Low transcription of the TF gene is a favorable prognostic marker for overall survival in TCGA lung cancer patients (54.7 vs 41.9 months, P=0.0053), with 74% longer progression-free survival (102.7 vs 59.1 months, P=0.0012). TF knockdown significantly reduced tumor malignancy as determined by cell proliferation, invasion, colony formation, and in vivo growth. Conversely, TF overexpression increased tumor malignancy and promoted cancer stem-like behavior, as indicated by CD44 and CD133 expression, extreme limiting dilution assay, and anchorage-independent growth. A PAR2 antagonist, I-191, inhibited TF-mediated signaling and reduced cell proliferation by 51.3% (P< 0.001). TF knockdown and I-191 increased radiation sensitivity. Exogenous treatment of lung cancer cells with recombinant TF suppressed radiation-induced apoptosis, and this effect was blocked with I-191. These data show that TF-PAR2 signaling may represent a novel therapeutic strategy to reduce the malignancy of brain metastasis and increase the efficacy of radiation.

Abstract 852: The Pathological Role of Coagulation Factors in Promoting Brown Adipose Tissue Dysfunction and Systemic Metabolic Disorder in Obesity

Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model. Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. BAT specific PAR1 KO mice improved glucose intolerance and thermogenic response under a metabolically stressed condition. In differentiated brown adipocytes, FXa markedly increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes

Hydroxychloroquine reverses the prothrombotic state in a mouse model of antiphospholipid syndrome: Role of reduced inflammation and endothelial dysfunction.

Antiphospholipid antibodies (aPL) promote endothelial dysfunction, inflammation and procoagulant state. We investigated the effect of hydroxychloroquine (HCQ) on prothrombotic state and endothelial function in mice and in human aortic endothelial cells (HAEC). Human aPL were injected to C57BL/6 mice treated or not with HCQ. Vascular endothelial function and eNOS were assessed in isolated mesenteric arteries. Thrombosis was assessed both in vitro by measuring thrombin generation time (TGT) and tissue factor (TF) expression and in vivo by the measurement of the time to occlusion in carotid and the total thrombosis area in mesenteric arteries. TGT, TF, and VCAM1 expression were evaluated in HAEC. aPL increased VCAM-1 expression and reduced endothelium dependent relaxation to acetylcholine. In parallel, aPL shortened the time to occlusion and extended thrombus area in mice. This was associated with an overexpression of TF and an increased TGT in mice and in HAEC. HCQ reduced clot formation as well as TGT, and improved endothelial-dependent relaxations. Finally, HCQ increased the p-eNOS/eNOS ratio. This study provides new evidence that HCQ improves procoagulant status and vascular function in APS by modulating eNOS, leading to an improvement in the production of NO.

A Phase 2 Study of PCI-27483, a Factor VIIa Inhibitor in Combination with Gemcitabine for Advanced Pancreatic Cancer

Objectives: Tissue factor overexpression is associated with tumor progression, venous thromboembolism, and worsened survival in patients with cancer. Tissue factor and activated factor VII (FVIIa) complex may contribute to tumor invasiveness by promoting cell migration and angiogenesis. The study objective was to evaluate safety, pharmacokinetics, and efficacy of PCI-27483, a selective FVIIa inhibitor. Methods: This was an open-label, multicenter phase 2 trial of patients with advanced pancreatic cancer. Part A of the study was an intrapatient dose escalation lead-in portion in patients concurrently receiving gemcitabine, and in part B, patients were randomized 1: 1 to the recommended phase 2 dose combination PCI-27483–gemcitabine versus gemcitabine alone. Results: Target international normalized ratio (between 2.0–3.0) was achieved following PCI-27483 treatment. Overall safety of PCI-27483–gemcitabine (n = 26) was similar to gemcitabine alone (n = 16), with a higher incidence of mostly low-grade bleeding events (65% vs. 19%). Progression-free survival (PFS) and overall survival (OS) were not significantly different between patients treated with PCI-27483–gemcitabine (PFS: 3.7 months, OS: 5.7 months) and those treated with gemcitabine alone (PFS: 1.9 months, OS: 5.6 months). Conclusions: Targeted inhibition of the coagulation cascade was achieved by administering PCI-27483. PCI-27483–gemcitabine was well tolerated, but superiority to single agent gemcitabine was not demonstrated.

Tissue factor over-expression in platelets of patients with anti-phospholipid syndrome: induction role of anti-β2-GPI antibodies.

Anti-phospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis and pregnancy morbidity. It is well known that in these patients thrombosis may be the result of a hypercoagulable state related to anti-β2-glycoprotein I (β2-GPI) antibodies. Moreover, platelets may play a role in thrombotic manifestations by binding of anti-β2-GPI antibodies. Platelets express tissue factor (TF), the major initiator of the clotting cascade, after activation. We primarily analyzed whether anti-β2-GPI antibodies may trigger a signal transduction pathway leading to TF expression in human platelets. Platelets from healthy donors were incubated with affinity purified anti-β2-GPI antibodies for different times. Platelet lysates were analyzed for phospho-interleukin-1 receptor-associated kinase 1 (IRAK), phospho-p65 nuclear factor kappaB (NF-κB) and TF by Western blot. IRAK phosphorylation was observed as early as 10min of anti-β2-GPI treatment, with consequent NF-κB activation, whereas TF expression, detectable at 45min, was significantly increased after 4h of anti-β2-GPI treatment. Virtually no activation was observed following treatment with control immunoglobulin IgG. We then analyzed TF expression in platelets from 20 APS patients and 20 healthy donors. We observed a significant increase of TF in APS patients versus control subjects (P<0·0001). This work demonstrates that anti-β2-GPI antibodies may trigger in vitro a signal transduction pathway in human platelets, which involves IRAK phosphorylation and NF-κB activation, followed by TF expression. Furthermore, ex vivo, platelets of APS patients showed a significantly increased expression of TF. These findings support the view that platelets may play a role in the pathogenesis of APS, with consequent release of different procoagulant mediators, including TF.

Overexpression of tissue factor induced atherothrombosis in apolipoprotein E−/− mice via both enhanced plaque thrombogenicity and plaque instability

The mechanisms leading to atherothrombosis from “vulnerable plaque” are more complex than initially proposed. We aimed to clarify whether plaque thrombogenicity is critical in atherothrombosis in mice. In a murine model of plaque destabilization, we enhanced plaque thrombogenicity by systemically overexpressing murine tissue factor (TF) by adenovirus-mediated gene transfer. The potential effects and mechanisms of TF on plaque destabilization were examined in cultured human aortic smooth muscle cells (HASMCs), RAW264.7 cells and human umbilical vein endothelial cells (HUVECs). To elucidate the TF noncoagulant effects on plaque destabilization, TF-overexpressed mice were treated with the protease-activated receptor 2 (PAR-2) antagonist ENMD-1068. In TF-overexpressing apolipoprotein (E)–deficient (ApoE−/−) mice, 67% (8 of 12) of carotid plaques exhibited plaque disruption and atherothrombosis. Moreover, 58% (7 of 12) showed plaque hemorrhage, including 1 due to plaque disruption, 4 neovascularization and 2 both. In contrast, only 17% (2 of 12) of control mice showed atherothrombosis, both with plaque hemorrhage but no neovascularization. On PCR, TF overexpression increased the expression of inflammatory factors. In cultured cells, the TF–FVIIa complex enhanced the expression of inflammatory factors and a vicious cycle of inflammation. Also, TF–FVIIa complex induced intra-plaque angiogenesis via PAR-2. ENMD-1068 treatment significantly inhibited the expression of inflammatory factors and neovascularization, and the incidence of intra-plaque hemorrhage decreased in TF-overexpressing mice. In conclusions, TF overexpression enhanced plaque thrombogenicity, which played a pivotal role in atherothrombosis in ApoE−/− mice. In addition, TF promoted plaque instability by activating inflammatory and proangiogenic effects via TF–FVIIa/PAR-2 signaling.

Occurrence of Venous Thromboembolism in Patients with Lung Cancer and Its Anticoagulant Therapy

目前，肺癌的发病率和死亡率在全世界范围内居首位。静脉血栓栓塞症（venous thromboembolism, VTE）是一种公认的肺癌并发症，也是肺癌患者的主要死因之一。癌症自身因素、患者自身因素以及治疗相关因素都是导致肺癌患者发生VTE的主要原因。肿瘤细胞可产生组织因子（tissue factor, TF）、癌性促凝物质（cancer procoagulant, CP）、炎症因子和细胞因子，从而直接激活凝血；其中TF过度表达是肿瘤患者血栓形成的主要机制之一。2016年美国胸外科医师协会（American College of Chest Physicians, ACCP）发布的第10版肿瘤患者VTE防治指南（AT-10）指出，抗凝治疗是肺癌患者合并VTE的基本治疗措施；其中低分子肝素（low molecular-weight-heparin, LWMH）被认为是首选的抗凝药物，但要注意出血风险。

Effect of Tissue Factor on Colorectal Cancer Stem Cells.

Tissue factor (TF) expression increases cancer stem cell (CSC) activity in breast and lung cancer. There are ongoing studies focused on targeting CSCs via anti-TF treatment, for breast and lung cancer therapy. Herein, the aim was to determine whether targeting TF could have an anti-CSC therapeutic role in colorectal cancer (CRC). Evaluation of colonosphere-forming efficiency (CFE) and aldehyde dehydrogenase (ALDH) expression level was used to quantify CSC activity in two CRC cell lines, after TF knockdown (TFKD) or TF over-expression (TFOE). TFKD resulted in increased levels of ALDH in SW620 (1.31±0.04-fold, p<0.001) and DLD-1 (1.63±0.14-fold, p=0.04) cells. CFE was increased in SW620 (1.21±0.23% vs. 2.03±0.29%, p=0.01) and DLD-1 (0.41±0.12% vs. 0.68±0.9%, p=0.01) cells. Conversely, TFOE decreased ALDH expression (0.72±0.04-fold, p=0.001) and CFE (0.33±0.05% vs. 0.66±0.14%, p=0.006) in DLD-1, but had no impact on SW620 cells. In the examined CRC cell lines, TF expression was inversely related to CSC activity suggesting that anti-TF therapies may not have a role in CRC treatment.

Pathophysiology of Trousseau’s syndrome

SummaryClinically relevant clotting abnormalities in cancer patients are referred to as Trousseau’s syndrome. While thrombotic complications such as venous thromboembolism are most frequent in every day’s practice, cancer patients may also experience severe bleeding symptoms due to complex systemic coagulopathies, including disseminated intravascular coagulation, haemolytic thrombotic microangiopathy, and hyperfibrinolysis. The pathophysiology of Trousseau’s syndrome involves all aspects of Virchow’s triad, but previous basic research has mainly focused on the cellular and molecular mechanisms underlying blood hypercoagulability in solid cancers and haematological malignancies. In this regard, over-expression of tissue factor (TF), the principal initiator of the extrinsic coagulation pathway, by primary tumour cells and increased shedding of TF-bearing plasma microparticles are critical to both thrombus formation and cancer progression. However, novel findings on intrinsic contact activation in vivo, such as the release of polyphosphates or DNA by activated platelets and neutrophils, respectively, have pointed to additional pathways in the complex pathophysiology of Trousseau’s syndrome.

ImmunoPET imaging of tissue factor expression in pancreatic cancer with 89Zr-Df-ALT-836

Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies, including pancreatic cancer. Additionally, high TF expression was shown to strongly correlate with poor prognoses and decreased survival in pancreatic cancer patients. Herein, we exploited the potential targeting of TF for positron emission tomography (PET) imaging of pancreatic cancer. The TF-targeted tracer was developed through radiolabeling of the anti-human TF monoclonal antibody (ALT-836) with 89Zr. The tracer was characterized by fluorescence microscopy and flow cytometry assays in BXPC-3 and PANC-1 cells, two pancreatic cancer cell lines with high and low TF expression levels, respectively. Non-invasive PET scans were acquired in tumor-bearing mice injected with 89Zr-Df-ALT-836. Additionally, ex vivo biodistribution, blocking, and histological studies were performed to establish the affinity and specificity of 89Zr-Df-ALT-836 for TF in vivo. 89Zr-labeling of Df-ALT-836 was achieved in high yield and good specific activity. Flow cytometry and microscopy studies revealed no detectable difference in TF-binding affinity between ALT-836 and Df-ALT-836 in vitro. Longitudinal PET scans unveiled a lasting and prominent 89Zr-Df-ALT-836 uptake in BXPC-3 tumors (peak at 31.5±6.0%ID/g at 48h post-injection; n=3), which was significantly abrogated (2.3±0.5%ID/g at 48h post-injection; n=3) when mice were pre-injected with a blocking dose (50mg/kg) of unlabeled ALT-836. Ex vivo biodistribution data confirmed the accuracy of the PET results, and histological analysis correlated high tumor uptake with in situ TF expression. Taken together, these results attest to the excellent affinity and TF-specificity of 89Zr-Df-ALT-836. With elevated, persistent, and specific accumulation in TF-positive BXPC-3 tumors, PET imaging using 89Zr-Df-ALT-836 promises to open new avenues for improving future diagnosis, stratification, and treatment response assessment in pancreatic cancer patients.

Impact of haemostatic mechanisms on pathophysiology of preeclampsia

Preeclampsia (PE) is disorder of new onset hypertension and proteinuria during the second half of pregnancy. There is increasing evidence to implicate placental over-expression of tissue factor and PAR-1 in the pathophysiology of PE. Excessive activation of platelets, neutrophils and the complement system may also contribute to the placental pathology and maternal endothelial responsible for the symptoms of PE. Increased knowledge in this field may identify new therapeutic strategies for the treatment of PE.

In Vivo Generation of Engraftable Murine Hematopoietic Stem Cells from Induced Pluripotent Stem Cells through Hemogenic Endothelium

IntroductionGeneration of engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has long been thought an ultimate goal in the field of hematology. Numerous in vitro differentiation protocols, including trans-differentiation and forward programming approaches, have been reported but have so far failed to generate fully functional HSCs. We have previously demonstrated proof-of-concept for the in vivo generation of fully functional HSCs from induced PSCs (iPSCs) through teratoma formation (Suzuki et al., 2013). However, this method is time-consuming (taking over two months), HSCs are generated at low frequencies, and additionally require co-injection on OP9 stromal cells and SCF/TPO cytokines. Here, we present optimization of in vivo HSC generation via teratoma formation for faster, higher-efficiency HSC generation and without co-injection of stromal cells or cytokines.ResultsFirst, we screened reported in vitro trans-differentiation and forward programming strategies for their ability to generate HSCs in vivo within the teratoma assay. We tested iPSCs transduced with the following dox-inducible TF overexpression vectors: (1) Gfi1b, cFOS and Gata2 (GFG), which induce hemogenic endothelial-like cells from fibroblast (Pereira et al.,2013); (2) Erg, HoxA9 and Rora (EAR), which induce short-term hematopoietic stem/progenitor cell (HSPC) formation during embryoid body differentiation (Doulatov et,al., 2013); and (3) Foxc1, which is highly expressed the CAR cells, a critical cell type for HSC maintenance (Oomatsu et al.,2014). We injected iPSCs into recipient mice, without co-injection of stromal cells or cytokines, and induced TF expression after teratoma formation by dox administration. After four weeks, GFG-derived teratomas contained large numbers of endothelial-like and epithelial-like cells, and importantly GFG-derived hematopoietic cells could also be detected. EAR-teratomas also generated hematopoietic cells, although at lower frequencies. By contrast, hematopoietic cells were not detected in control teratomas or Foxc1-teratomas. Through use of iPSCs generated from Runx1-EGFP mice (Ng et al. 2010), and CUBIC 3D imaging technology (Susaki et al. 2014), we were further able to demonstrate that GFG-derived hematopoietic cells were generated through a haemogenic endothelium precursor. Next, we assessed whether HSPC-deficient recipient mice would allow greater expansion of teratoma-derived HSCs. This was achieved by inducing c-kit deletion within the hematopoietic compartment of recipient mice (Kimura et al., 2011) and resulted in a ten-fold increase in the peripheral blood frequency of iPSC-derived hematopoietic cells. We further confirmed similar increases in iPSC-derived bone marrow cells, and in vivo HSC expansion, through bone marrow transplantation assays. Finally, we have been able to shorten the HSC generation time in this assay by five weeks through use of transplantable teratomas, rather than iPSCs.ConclusionsWe have demonstrated that GFG-iPSCs induce HSC generation within teratomas, via a hemogenic endothelium precursor, and that use of HSPC-deficient recipient mice further promotes expansion of teratoma-derived HSCs. These modifications now allow us to generate engraftable HSCs without co-injection of stromal cells or cytokines. Additionally, use of transplantable teratomas reduced HSC generation times as compared with the conventional assay. These findings suggest that our in vivo system provides a promising strategy to generate engraftable HSCs from iPSCs. DisclosuresNo relevant conflicts of interest to declare.

Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer.

BackgroundTissue Factor (TF) forms a proteolytically active complex together with coagulation factor VIIa (FVIIa) and functions as the trigger of blood coagulation or alternatively activates cell signaling. We recently described that EphA2 of the Eph tyrosine kinase receptor family is cleaved directly by the TF/FVIIa complex. The aim of the present study was to further characterize the cross-talk between TF/FVIIa and EphA2 using in vitro model systems and human cancer specimens.MethodsCleavage and phosphorylation of EphA2 was studied by Western blot. Subcellular localization of TF and EphA2 was investigated by a proximity ligation assay and confocal microscopy. Phalloidin staining of the actin cytoskeleton was used to study cell rounding and retraction fiber formation. Expression of TF and EphA2 in human colorectal cancer specimens was examined by immunohistochemistry.ResultsTF and EphA2 co-localized constitutively in MDA-MB-231 cells, and addition of FVIIa resulted in cleavage of EphA2 by a PAR2-independent mechanism. Overexpression of TF in U251 glioblastoma cells lead to co-localization with EphA2 at the leading edge and FVIIa-dependent cleavage of EphA2. FVIIa potentiated ephrin-A1-induced cell rounding and retraction fiber formation in MDA-MB-231 cells through a RhoA/ROCK-dependent pathway that did not require PAR2-activation. TF and EphA2 were expressed in colorectal cancer specimens, and were significantly correlated.ConclusionsThese results suggest that TF/FVIIa-EphA2 cross-talk might potentiate ligand-dependent EphA2 signaling in human cancers, and provide initial evidence that it is possible for this interaction to occur in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2375-1) contains supplementary material, which is available to authorized users.

PO-50 - The effect of tissue factor expression on colorectal cancer cell proliferation

Tissue factor (TF) is abnormally expressed in many cancers including colorectal and is associated with a poor cancer prognosis. Colorectal cancer cell lines expressing TF produce faster growing tumours. In lung cancer, TF inhibition has been shown to reduce proliferation We aimed to determine if TF expression and activity increases cellular proliferation in colorectal cancer cell lines. DLD-1 and SW620 colorectal cell lines were transduced with cDNA to over express TF (TF+ve). Proliferation was determined by Alamar blue assay where level of absorption indicates the number of living cells, expressed as an arbitrary unit of absorption (u). Factor VIIa (TF ligand) at increasing concentrations was used to determine the effect of TF activity on proliferation. Downstream marker of TF activity (MAPK phosphorylation) was assessed by Western blot and correlated with proliferation. There was a significant increase in proliferation in both DLD-1 TF+ve and SW620 TF+ve compared to their negative controls at 42 hours (DLD1 TF+ve: 5,455u (SD 2,485u) vs 2,246u (SD 1,107u) p<0.001); SW620 TF+ve: 414u (SD 96u) vs 286u (SD 114u) p<0.05). When factor VIIa (FVIIa) was added in concentrations from 0nM to the supra-physiological concentration of 25nM there was a dose-dependent increase in proliferation up to physiological levels (0.1nM) which was further increased in the TF+ve cell lines. Fold change from baseline 0 vs 0.1nM FVIIa (DLD-1: 3.22u (SD 0.61u) vs 6.17u (SD 2.21u) p<0.05; SW620 2.33u (SD 2.21u) vs 4.69u (SD 0.61u) p<0.05). The increase in proliferation was reflected in the phosphorylation of MAPK which was increased by TF overexpression alone and further increased by FVIIa in a dose-dependent fashion. Increased TF expression and activation is associated with increased cellular proliferation. This effect appears to be exerted via MAPK pathways. Tissue factor may provide a therapeutic target in colorectal cancer.

PO-11 - Thrombin and cancer stem-like cells: in vitro support for breast cancer anticoagulation

Cancer stem-like cells (CSCs) are tumour initiating, resistant to chemotherapy and play a role in metastasis. We have previously shown thrombin pathway signalling regulates CSC activity: thrombin pathway activation with FVIIa or through TF overexpression increases CSC activity whilst TF siRNA knockdown decreases it. We hypothesise that NOACs, by targeting specific factors in the thrombin pathway, may inhibit CSC activity. In breast cancer cell lines, to determine the effect of i) thrombin and ii) dabigatran (Boehringer Ingelheim), a direct thrombin inhibitor on cancer stem cell activity. MDA-MB-231, MCF-7, SKBR3 and MDA-MB-157 cell lines representing the spectrum of breast cancer subtypes were cultured with: i) 0.1 NIH Units/ml human thrombin (Sigma) ii) 0.5μM Dabigatran (clinically relevant plasma concentration). Mammosphere culture is an established in vitro assay to measure CSC activity. Mammosphere forming efficiency (MFE) was calculated from the proportion of plated cells forming mammospheres in non-adherent culture, (each experiment, n=≥4, two-tailed independent t-test). Thrombin increased MFE in the high PAR-1 expressing MDA-MB-231 and MDA-MB-157 cell lines, but not the low PAR-1 expressing MCF-7/SKBR3 cell lines as compared to untreated controls (MDA-MB-231 mean (range): thrombin treated: 0.91 (0.70-1.11) vs control: 0.73 (0.51-0.93)%, p=<0.04; MDA-MB-157: thrombin treated: 0.58 (0.45-0.69) vs control: 0.37 (0.31-0.47)%, p=<0.001). Dabigatran abrogated the stimulatory effect of thrombin on MFE in thrombin-treated MDA-MB-231 and MDA-MB-157 cells (MDA-MB-231 mean (range) thrombin+Dabigatran: 1.10 (0.83-1.41) vs thrombin only: 1.45 (1.32-1.66)%, p=<0.01); MDA-MB-157 thrombin+Dabigatran: 0.48 (0.39-0.56) vs thrombin only: 0.58 (0.45-0.69)%, p=<0.05). The stimulation of mammosphere formation by thrombin and reduction by thrombin inhibitor treatment indicates a functional relationship between cancer stem-like cells and coagulation. This suggests possible clinical utility of novel oral anticoagulants in targeting this critical cancer cell subpopulation.

Utility of epirubicin-incorporating micelles tagged with anti-tissue factor antibody clone with no anticoagulant effect.

Tissue factor (TF), an initiator of the extrinsic blood coagulation cascade, is overexpressed in different types of cancer. Tissue factor overexpression is also known as a poor prognostic factor in pancreatic cancer. We recently developed anti‐TF antibody (clone1849)‐conjugated epirubicin‐incorporating micelles (NC‐6300), and reported that this anti‐TF1849‐NC‐6300 showed enhanced antitumor activity against TF‐high expressed human pancreatic cancer cells, when compared with NC‐6300 alone. However, clone 1849 antibody inhibited TF‐associated blood coagulation activity. We studied another anti‐TF antibody, clone 1859, which had no effect on blood coagulation and prepared anti‐TF1859‐NC‐6300. In addition, to determine the optimum size of the antibody fragment to conjugate with NC‐6300, three forms of the 1859 antibody (whole IgG, F[ab’]2, and Fab’) were conjugated to NC‐6300. The antitumor effect of each anti‐TF1859‐NC‐6300 was studied in vitro and in vivo, using two human pancreatic cancer cell lines, BxPC3 with high‐expressed TF, and SUIT2 with low levels of TF. In vitro, all forms of anti‐TF1859‐NC‐6300 showed higher cytocidal effects than NC‐6300 in BxPC3, whereas this enhanced effect was not observed in SUIT2. Likewise, all forms of anti‐TF1859‐NC‐6300 significantly suppressed tumor growth when compared to NC‐6300 in the BxPC3, but not in the SUIT2, xenograft model. Among the three forms of conjugates, anti‐TF1859‐IgG‐NC‐6300 had a higher antitumor tendency in TF‐high expressed cells. Thus, we have confirmed an enhanced antitumor effect of anti‐TF1859‐NC‐6300 in a TF‐high expressing tumor; anti‐TF1859‐IgG‐NC‐6300 could be used to simplify the manufacturing process of the antibody–micelle conjugation for future clinical studies.