thromboplastin and Pancreatic-Neoplasms

Tissue factor (TF) is a procoagulant protein released from activated host cells, such as monocytes, and tumor cells on extracellular vesicles (EVs). TF + EVs are observed in the circulation of patients with various types of diseases. In this review, we will summarize the association between TF + EVs and activation of coagulation and survival in different types of diseases, including cancer, sepsis, and infections with different viruses, such as human immunodeficiency virus (HIV), influenza A virus (IAV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We will also discuss the source of TF + EVs in various diseases. EVTF activity is associated with thrombosis in pancreatic cancer patients and coronavirus disease 2019 patients (COVID-19) and with disseminated intravascular coagulation in cancer patients. EVTF activity is also associated with worse survival in patients with cancer and COVID-19. Monocytes are the major sources of TF + EVs in sepsis, and viral infections, such as HIV, Ebola virus, and SARS-CoV-2. In contrast, alveolar epithelial cells are the major source of TF + EVs in bronchoalveolar lavage fluid in COVID-19 and influenza A patients. These studies indicate that EVTF activity could be used as a biomarker to identify patients that have an increased risk of coagulopathy and mortality.

Topics: Biomarkers; COVID-19; Extracellular Vesicles; Humans; Pancreatic Neoplasms; SARS-CoV-2; Sepsis; Thromboplastin; Thrombosis

It has long been recognised that pancreatic cancer induces a hypercoagulable state that may lead to clinically apparent thrombosis. Although the relationship between pancreatic cancer and hypercoagulability is well described, the underlying pathological mechanism(s) and the interplay between these pathways remain a matter of intensive study. This review summarises existing data on epidemiology and pathogenesis of thrombotic complications in pancreatic cancer with a particular emphasis on novel pathophysiological pathways. Pancreatic cancer is characterised by high tumoural expression of tissue factor, activation of leukocytes with the release of neutrophil extracellular traps, the dissemination of tumour-derived microvesicles that promote hypercoagulability and increased platelet activation. Furthermore, other coagulation pathways probably contribute to these processes, such as those that involve heparanase, podoplanin and hypofibrinolysis. In the era in which heparin and its derivatives-the currently recommended therapy for cancer-associated thrombosis-might be superseded by direct oral anticoagulants, novel data from mouse models of cancer-associated thrombosis suggest the possibility of future personalised therapeutic approaches. In this dynamic era for cancer-associated thrombosis, the discovery of novel prothrombotic and proinflammatory mechanisms will potentially uncover pharmacological targets to prevent and treat thrombosis without adversely affecting haemostasis.

Topics: Animals; Anticoagulants; Antithrombin III; Cell-Derived Microparticles; Disease Models, Animal; Extracellular Traps; Factor VIII; Factor Xa Inhibitors; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinolysis; Glucuronidase; Heparin; Humans; Leukocytes; Membrane Glycoproteins; Mice; Molecular Targeted Therapy; Pancreatic Neoplasms; Protein C; Thrombophilia; Thromboplastin; Thrombosis

Chemotherapy is an independent risk factor of thromboembolic events in cancer patients. Various pathogenetic mechanisms have been hypothesized in the past, but until now their individual contribution to the risk of thrombosis has been hardly investigated in clinical trials. In recent years, studies increasingly suggested an association between the prothrombotic state in cancer patients and circulating tissue factor-exposing microparticles. In this review, we discuss the roles of tissue factor and microparticles with regard to chemotherapy-induced hypercoagulability.

Topics: Antineoplastic Agents; Body Mass Index; Breast Neoplasms; Cell Membrane Structures; Hemostasis; Humans; Leukocyte Count; Pancreatic Neoplasms; Platelet Count; Risk Factors; Thromboembolism; Thromboplastin

The paper presents aspects of pancreatic cancer associated thrombosis. Prospects of anticoagulation as an adjutant modality in pancreatic cancer treatment are also discussed.

Topics: Blood Coagulation; Fibrin; Humans; Intracranial Embolism and Thrombosis; Pancreatic Neoplasms; Thromboplastin; Whole Blood Coagulation Time

Thrombophlebitis has been associated with virtually all cancers, especially gastrointestinal, urogenital, and lung neoplasms. Although occurring infrequently in cancer patients, thrombophlebitis may appear before the cancer has become symptomatic and may lead to an earlier diagnosis of cancer. The phlebitic syndrome associated with cancer, although not unique, is distinctive. It is often recurrent and migratory, often involves unusual locations, and is often resistant to anticoagulation therapy. Pulmonary emboli are frequent complications. The pathogenesis of phlebitis in cancer patients is not well understood. Evidence suggests that many cancer patients are hypercoagulable, with abnormalities in platelets, coagulation factors, and the fibrinolytic system. These changes may results from the elaboration of thromboplastin-like substances from the cancer tissue.

Topics: Blood Coagulation; Fibrinolysis; Humans; Neoplasms; Pancreatic Neoplasms; Prognosis; Thrombophlebitis; Thromboplastin

The aim of the present study was to assess the role of tissue factor and serum-induced cell invasion in patients with advanced pancreatic cancer (APC). A cohort of 39 patients with APC, without thrombosis, receiving chemotherapy, were entered in a randomized controlled trial (ISRCTN = 76464767) of thromboprevention with weight-adjusted dalteparin (WAD). A total of 19 patients received WAD, the remaining 20 acting as a control group. Serum from baseline and week 8 was analysed for circulating-tissue factor antigen using ELISA. Circulating-tissue factor antigen rose from 324 pg/ml, [interquartile range (IQR) 282-347 pg/ml] to 356 pg/ml, (IQR 319-431 pg/ml) in controls (C), and decreased in the dalteparin-treated group (D) from 336 pg/ml (IQR 281-346 pg/ml) to 303 pg/ml (IQR 274-339 pg/ml). The difference in median percentage change between D and C was statistically significant [-4.0 (D) vs. 4.7 (C); P = 0.005, n = 39]. Serum-induced cellular invasion of MIA-Paca-2 cells in response to patient serum was studied using a Boyden chamber assay in 30 patients (14 WAD and 16 C). The median percentage change between C and D was significant [+54.9 (C) vs. -21.9 (D) P = 0.025, n = 30]. There was a weak correlation between BB-tissue factor reduction and cellular invasion reduction (Spearman) [0.384 (P = 0.037, n = 30)]. APC patients treated with WAD have lower tissue factor antigen levels and attenuated induction of cellular invasion in their blood. These assays may provide useful markers to guide appropriate dalteparin (and other low-molecular weight heparin) dosing schedules to optimize anticancer effects of dalteparin in APC.

Topics: Aged; Dalteparin; Disease Progression; Female; Humans; Male; Middle Aged; Molecular Weight; Neoplasm Invasiveness; Pancreatic Neoplasms; Thromboplastin; Venous Thromboembolism

Thrombosis is a common complication of advanced cancer, yet the cellular mechanisms linking malignancy to thrombosis are poorly understood. The unfolded protein response (UPR) is an ER stress response associated with advanced cancers. A proteomic evaluation of plasma from patients with gastric and non-small cell lung cancer who were monitored prospectively for venous thromboembolism demonstrated increased levels of UPR-related markers in plasma of patients who developed clots compared with those who did not. Release of procoagulant activity into supernatants of gastric, lung, and pancreatic cancer cells was enhanced by UPR induction and blocked by antagonists of the UPR receptors inositol-requiring enzyme 1α (IRE1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK). Release of extracellular vesicles bearing tissue factor (EVTFs) from pancreatic cancer cells was inhibited by siRNA-mediated knockdown of IRE1α/XBP1 or PERK pathways. Induction of UPR did not increase tissue factor (TF) synthesis, but rather stimulated localization of TF to the cell surface. UPR-induced TF delivery to EVTFs was inhibited by ADP-ribosylation factor 1 knockdown or GBF1 antagonism, verifying the role of vesicular trafficking. Our findings show that UPR activation resulted in increased vesicular trafficking leading to release of prothrombotic EVTFs, thus providing a mechanistic link between ER stress and cancer-associated thrombosis.

Topics: Carcinoma, Non-Small-Cell Lung; Endoribonucleases; Guanine Nucleotide Exchange Factors; Humans; Lung Neoplasms; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Proteomics; Thromboplastin; Unfolded Protein Response

The Quantikine

Topics: Extracellular Vesicles; Humans; Pancreatic Neoplasms; Thromboembolism; Thromboplastin

Tissue factor-procoagulant activity (TF-PCA) on cells is modified by multiple molecular mechanisms of encryption and decryption. The risk of thrombosis is higher for patients with a high tissue factor antigen level at registration as this enables patient's blood more PCA-high status before the onset of cancer-associated thromboembolism (CAT). ELISA, including the Quantikine assay with validation as performed in our study, can contribute to more precise prediction of CAT.

Topics: Humans; Pancreatic Neoplasms; Thromboembolism; Thromboplastin

Pancreatic cancer activates coagulation and increases risk of venous thromboembolism (VTE). We aimed at characterizing the association of hemostatic biomarkers and VTE with mortality and chemotherapy response.. Pancreatic cancer patients (N=145) were included in a prospective, observational cohort study (CATS [Vienna Cancer and Thrombosis Study]). Hemostatic biomarkers (D-dimer, extracellular vesicle-tissue factor activity, prothrombin fragment 1+2, fibrinogen, factor VIII, PAI-1 [plasminogen activator inhibitor 1], sP-selectin [soluble P-selectin], thrombin generation assay) were measured at inclusion. The impact of VTE on overall survival/progression-free survival (OS/PFS) was evaluated by multistate modeling. The association of biomarkers with OS was analyzed by Cox-regression and with PFS and disease control rate in patients initiating palliative chemotherapy (n=95) by Cox-regression and logistic regression. Multivariable analysis included stage, grade, sex, age, performance status, VTE (time-dependent), vascular infiltration/compression, and tumor marker levels (carbohydrate-antigen 19-9, carcinoembryonic antigen). VTE occurrence was associated with shorter OS (transition hazard ratio, 3.40 [95% CI, 2.05-5.64]) and shorter PFS (transition hazard ratio, 2.10 [1.16-3.79]). Median post-VTE OS/PFS in months was 5.5 [2.2-6.5] and 3.0 [1.5-3.9], compared with 13.4 [9.7-16.6] and 7.5 [5.9-9.8] in patients without VTE (both P<0.001). D-dimer, extracellular vesicle-tissue factor activity, PAI-1, and sP-selectin were associated with increased mortality (hazard ratio per doubling, 1.27 [1.00-1.61]; 1.63 [1.14-2.36]; 1.25 [1.06-1.47]; 1.52 [1.05-2.20]). In patients initiating palliative chemotherapy, higher D-dimer predicted shorter PFS (hazard ratio per doubling, 1.27 [1.01-1.60]) and lower disease control rate (odds ratio per doubling, 0.59 [0.36-0.98]).. VTE diagnosis is associated with shorter OS and PFS. Higher baseline levels of D-dimer, extracellular vesicle-tissue factor activity, PAI-1, and sP-selectin were independently prognostic for increased mortality, and D-dimer predicted response to palliative chemotherapy.

Topics: Aged; Anticoagulants; Antineoplastic Agents; Biomarkers; Disease Progression; Extracellular Vesicles; Female; Fibrin Fibrinogen Degradation Products; Hemostasis; Humans; Incidence; Male; Middle Aged; P-Selectin; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; Progression-Free Survival; Prospective Studies; Risk Assessment; Risk Factors; Thromboplastin; Time Factors; Treatment Outcome; Venous Thromboembolism

Tissue factor (TF) is an attractive target for cancer therapy due to its overexpression in multiple types of malignancies. In addition, TF has been reported to play functional roles in both cancer development and metastasis. Several groups have already developed antibody‑drug conjugates (ADCs) against TF for use as cancer treatments, and have demonstrated their efficacies in conventional subcutaneous xenograft models and patient‑derived xenograft models. However, no previous studies have investigated the effectiveness of anti‑TF ADC in an advanced‑stage cancer model. The present study developed an original humanized anti‑TF monoclonal antibody conjugated with monomethyl auristatin E, and evaluated its in vivo efficacy in a pancreatic cancer xenograft model with peritoneal dissemination. In vitro assays demonstrated that the anti‑TF ADC had potent binding affinity and cytotoxic activity against human pancreatic cancer cells that strongly expressed TF antigens. The anti‑TF ADC also exhibited greater antitumor effect than that of a control ADC in conventional subcutaneous xenograft models, with efficacy depending on the TF expression in the tumor tissues. Furthermore, the anti‑TF ADC significantly inhibited tumor growth in an orthotopic xenograft model, and extended the survival period in a murine peritoneal dissemination model. These results indicated that anti‑TF ADC has the potential to be an effective treatment not only for primary tumors, but also for those that are widely disseminated. Therefore, it can be concluded that ADC targeting TF may be a promising agent for advanced pancreatic cancer therapy.

Topics: Animals; Antibodies, Monoclonal, Humanized; Cell Line, Tumor; Female; Immunoconjugates; Mice; Mice, Inbred BALB C; Pancreatic Neoplasms; Peritoneum; Thromboplastin; Xenograft Model Antitumor Assays

Topics: Animals; Blood Coagulation; Cell Line, Tumor; Disease Models, Animal; Factor XI; Humans; Mice; Pancreatic Neoplasms; Thromboplastin; Thrombosis

Pancreatic cancer patients have an elevated risk of suffering from venous thrombosis. Among several risk factors that contribute to hypercoagulability of this malignancy, platelets possess a key role in the initiation of clot formation. Although single mechanisms of platelet activation are well-known in principle, combinations thereof and their potential synergy to mediate platelet activation is, in the case of pancreatic cancer, far from being clear. Applying an inhibitor screening approach using light transmission aggregometry, dense granule release, and thrombin formation assays, we provide evidence that a combination of tissue factor-induced thrombin formation by cancer cells and their platelet P-selectin binding is responsible for AsPC-1 and Capan-2 pancreatic cancer cell-mediated platelet activation. While the blockade of one of these pathways leads to a pronounced inhibition of platelet aggregation and dense granule release, the simultaneous blockade of both pathways is inevitable to prevent platelet aggregation completely and minimize ATP release. In contrast, MIA PaCa-2 pancreatic cancer cells express reduced levels of tissue factor and P-selectin ligands and thus turn out to be poor platelet activators. Consequently, a simultaneous blockade of thrombin and P-selectin binding seems to be a powerful approach, as mediated by heparin to crucially reduce the hypercoagulable state of pancreatic cancer patients.

Topics: Blood Platelets; Cell Line, Tumor; Humans; Ligands; P-Selectin; Pancreatic Neoplasms; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Risk Factors; Thrombin; Thrombophilia; Thromboplastin; Venous Thrombosis

Pancreatic cancer frequently involves cancer-associated thromboembolism, which is strongly associated with poor prognosis. Tissue factor, a blood coagulation factor largely produced in cancer patients as a component of extracellular vesicles, plays a key role in the incidence of cancer-associated thromboembolism in patients with pancreatic cancer. However, no prospective studies have been published on the relationship between tissue factor and cancer-associated thromboembolism or patient clinical characteristics, including recent chemotherapy regimens. Thus, we aimed to address this in a Japanese cohort of 197 patients and 41 healthy volunteers. Plasma tissue factor levels were measured by ELISAs preevaluated by tissue factor specificity. Multivariable analysis was used to identify independent predictors of cancer-associated thromboembolism. We found that the cancer-associated thromboembolism rate in the patient cohort was 6.6% (4.6%, venous thromboembolism; 2.0%, arterial thromboembolism). Tissue factor levels of 100 pg/mL or higher at patient registration were predictive of cancer-associated thromboembolism, with positive and negative predictive values of 23.1% and 94.6%, respectively. Multivariable analysis showed that plasma tissue factor levels were an independent predictive factor for cancer-associated thromboembolism, with a risk ratio of 5.54 (95% confidence interval, 1.02-30.09). Unlike in healthy volunteers and patients without cancer-associated thromboembolism, tissue factor levels were highly correlated with extracellular vesicles' procoagulant activity in patients developing cancer-associated thromboembolism. Taken together, our data show that the tissue factor levels at patient registration were a predictive factor for cancer-associated thromboembolism in this cohort of patients with pancreatic cancer.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Cohort Studies; Confidence Intervals; Enzyme-Linked Immunosorbent Assay; Extracellular Vesicles; Female; Humans; Japan; Male; Middle Aged; Multivariate Analysis; Pancreatic Neoplasms; Predictive Value of Tests; Risk; Thromboembolism; Thromboplastin; Venous Thromboembolism

The expression of active tissue factor (TF) on the surface of microvesicles (MVs) is essential for the activation of the coagulation system and transduction of the signaling pathways in cancer cells. In its use as a biomarker for cancer-associated venous thromboembolism (VTE), TF has shown high expression variability. As a contribution to this discussion, we present a study investigating plasma samples from patients with various progressive tumors at high risk for VTE.. Based on our previous study uncovering microvesicles (MVs), the larger ectosome-like extracellular vesicles (EV), as the major source of TF activity in EV preparations, we now determined TF activity on enriched MVs isolated from plasma of cancer patients and compared it with that on MVs from healthy individuals.. We found considerably higher amounts of MVs as well as higher levels of MV-bound TF activities in the plasma of cancer patients. We also show that preparations from plasma of cancer patients have the potency to induce ERK phosphorylation in a human tumor cell line through proteinase-activated receptor two (PAR2) activation.. We suggest that MVs instead of whole EV preparations, and TF activity rather than its antigenic quantification should be used in clinical studies for identifying patients with progressive tumors at high risk for VTE.

Topics: Aged; Case-Control Studies; Extracellular Vesicles; Female; Humans; Male; MAP Kinase Signaling System; Pancreatic Neoplasms; Phosphorylation; Thromboplastin; Venous Thromboembolism

The endothelium could be a potential target of cancer cell derived extracellular vesicles (CaCe-dEV). We investigated in vitro the effect of CaCe-dEV on the hemostatic balance of endothelial cells. Extracellular vesicles released from pancreas adenocarcinoma cells (BXPC3) or human breast cancer cells (MCF7) were isolated by differential centrifugation. Human umbilical vein endothelial cells (HUVEC) were cultured for 72 h in the presence or absence of CaCe-dEV. Subsequently, they were washed and re-cultivated over three cycles to get daughter cell generations (DG) which were not exposed to CaCe-dEV. Thrombin generation of normal platelet poor plasma (PPP) added in wells carrying HUVEC was assessed by the Calibrated Automated Thrombogram®. Tissue factor activity (TFa) and procoagulant phospholipid clotting time were assessed. Some traces of TFa were displayed by non-exposed HUVEC (0.18 ± 0.03 pM) and their EVs (1.2 ± 1.0 pM). Non-exposed HUVEC did not induce any detectable thrombin generation. BXPC3-dEV displayed significantly higher TFa as compared to MCF7-dEV (45 ± 5 pM versus 4.6 ± 2.3pM respectively; p < 0.05). HUVEC exposed to CaCe-dEV enhanced thrombin generation. BXPC3-dEV induced significantly higher thrombin generation as compared to those exposed to MCF7-dEV. The procoagulant properties of HUVEC, acquired upon exposure to CaCe-dEV were transferred to DG. In conclusion, CaCe-dEV lead to a procoagulant shift of endothelial cells which, upon exposure, display TFa and enhance thrombin generation which is transferred to DG of HUVEC. The potency of CaCe-dEV to induce procoagulant shift of HUVEC depends on the histological type of the cancer cells. The procoagulant shift of endothelial cells which is transferable to DG could be an additional mechanism - together with cancer-induced blood hypercoagulability - in the pathogenesis of cancer associated thrombosis.

Topics: Breast Neoplasms; Extracellular Vesicles; Female; Humans; Pancreas; Pancreatic Neoplasms; Thrombin; Thromboplastin

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

Topics: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Female; Mice; Pancreatic Neoplasms; Positron-Emission Tomography; Thromboplastin; Tissue Distribution; Yttrium Radioisotopes

Pancreatic cancer is a highly aggressive digestive system tumour with poor prognosis. Venous thromboembolism (VTE) is a well-known complication of pancreatic cancer, and tissue factor (TF) contributes to the generation of a hypercoagulable state and thrombotic disease in pancreatic cancer. Several studies showed that an elevated TF level was related to the development of VTE and influenced the survival of patients with pancreatic cancer. Thus, we wish to conduct a systematic review of literature to clarify the prognostic significance of TF in pancreatic cancer.. Studies comparing the circulating microparticle-associated TF (MP TF) level between patients who had pancreatic cancer with and without VTE will be included to evaluate the roles of TF in VTE development. Studies comparing the survival data between patients with high TF expression and low TF expression will also be included to explore the association of TF expression with patient survival. The outcomes are plasma MP TF level and survival endpoints (overall and progression-free survival), respectively. Primary studies of any type published in English will be included. Two reviewers will search Medline, EMBASE and Cochrane databases from inception to June 2020, retrieve relevant studies, and independently select the literatures and extract data from the included studies. The quality of each included study will be assessed by the Newcastle-Ottawa Scale score. The HR and 95% CI of each study will be pooled for survival outcome, and the standardised mean difference (SMD) with 95% CIs will be used for continuous outcomes. If meta-analysis is inappropriate, the result will only be reported qualitatively. Subgroup and sensitivity analyses will be considered to identify sources of heterogeneity. The Grades of Recommendation, Assessment, Development and Evaluation method will be applied to assess the level of evidence of this systematic review.. There are no concerning ethical issues. The results will be published.. CRD42019133665.

Topics: Gastrointestinal Neoplasms; Humans; Meta-Analysis as Topic; Pancreatic Neoplasms; Prognosis; Systematic Reviews as Topic; Thromboplastin; Venous Thromboembolism

Topics: Adenocarcinoma; Blood Coagulation Disorders; Humans; Pancreatic Neoplasms; Poland; Thromboplastin

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice, Nude; Neuroendocrine Tumors; Pancreatic Neoplasms; Promoter Regions, Genetic; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Signal Transduction; Thromboplastin; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Tissue factor (TF) has emerged as a critical factor in oncogenic events, leading to the development of TF‑targeted diagnostic and therapeutic approaches. A non‑invasive imaging method to evaluate target molecule expression with high sensitivity and high quantitative ability is imperative for selecting the appropriate patients for TF‑targeted therapy. To elucidate the potential of 111In‑labeled anti‑TF antibody 1849 (111In‑1849) as an immuno‑single photon emission computed tomography (SPECT) probe targeting TF, we evaluated TF‑dependent in vitro binding as well as in vivo biodistribution and tumor accumulation of 111In‑1849 in pancreatic cancer cells/models with varying TF expression levels. TF expression levels in five human pancreatic cancer cell lines, BxPC‑3, BxPC‑3‑TF‑knockout (BxPC‑3‑TFKO), Capan‑1, PSN‑1 and SUIT‑2, were examined by immunofluorescence. Binding of 111In‑1849 to each cell line was assessed. Biodistribution and imaging studies were also conducted in tumor‑bearing mice. Furthermore, the relationship of TF expression with cell binding and tumor uptake was analyzed. In the immunofluorescence studies, BxPC‑3 exhibited the highest TF expression, followed by Capan‑1, PSN‑1, SUIT‑2 and BxPC‑3‑TFKO. Cell binding assays revealed that BxPC‑3 cells had the highest 111In‑1849 binding, followed by PSN‑1, Capan‑1 and SUIT‑2; no binding was detected in BxPC‑3‑TFKO cells. The BxPC‑3 xenograft was clearly visualized on 111In‑1849 SPECT/CT, and the highest uptake was detected on day 4. The biodistribution of 111In‑1849 on day 4 revealed that tumor uptake ranged from 8.68 to 50.58% of the injected dose per gram of tissue; BxPC‑3 had the highest uptake and SUIT‑2 had the lowest. TF expression was significantly associated with cell binding (R2=0.79, P<0.05) and tumor uptake (R2=0.92, P<0.01). The association of 111In‑1849 uptake with TF expression suggests the potential application of non‑invasive imaging with radiolabelled 1849 for selecting the appropriate patients who would likely respond to TF‑targeted therapies in clinical practice.

Topics: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Humans; Immunoconjugates; Indium Radioisotopes; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Imaging; Pancreatic Neoplasms; Thromboplastin; Tissue Distribution; Tomography, Emission-Computed, Single-Photon; Xenograft Model Antitumor Assays

Topics: Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Immune Evasion; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Pancreatic Neoplasms; Receptor, PAR-1; Signal Transduction; Thrombin; Thromboplastin; Tumor Cells, Cultured; Tumor Microenvironment

The activation of protease-activated receptor (PAR)-2 by factor Xa (fXa) promotes the release of tissue factor-positive microvesicles (TF

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell-Derived Microparticles; Factor VIIa; Factor Xa Inhibitors; Female; Humans; Pancreatic Neoplasms; Pyrazoles; Pyridones; Receptor, PAR-2; Rivaroxaban; Signal Transduction; Thromboplastin

Tissue factor (TF) is known to be overexpressed in various cancers including pancreatic cancer. The upregulation of TF expression has been observed not only in tumor cells, but also in tumor stromal cells. Because of the potential of TF as a delivery target, several studies investigated the effectiveness of Ab-drug conjugates (ADCs) against TF for cancer therapy. However, it is still unclear whether anti-TF ADC can exert toxicity against both tumor cells and tumor stromal cells. Here, we prepared ADC using a rat anti-mouse TF mAb (clone.1157) and 2 types of in vivo murine pancreatic cancer models, one s.c. and other orthotopic with an abundant tumor stroma. We also compared the feasibility of bis-alkylating conjugation (bisAlk) with that of conventional maleimide-based conjugation (MC). In the s.c. models, anti-TF ADC showed greater antitumor effects than control ADC. The results also indicated that the bisAlk linker might be more suitable than the MC linker for cancer treatments. In the orthotopic model, anti-TF ADC showed greater in vivo efficacy and more extended survival time control ADC. Treatment with anti-TF ADC (20 mg/kg, three times a week) did not affect mouse body weight changes in any in vivo experiment. Furthermore, immunofluorescence staining indicated that anti-TF ADC delivered agents not only to TF-positive tumor cells, but also to TF-positive tumor vascular endothelial cells and other tumor stromal cells. We conclude that anti-TF ADC should be a selective and potent drug for pancreatic cancer therapy.

Topics: Alkylating Agents; Animals; Antineoplastic Agents, Immunological; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Administration Schedule; Female; Humans; Immunoconjugates; Maleimides; Mice; Mice, Transgenic; Pancreatic Neoplasms; Rats; Stromal Cells; Thromboplastin; Xenograft Model Antitumor Assays

Cancer patients are at high risk of developing deep venous thrombosis (DVT) and venous thromboembolism, a leading cause of mortality in this population. However, it is largely unclear how malignant tumors drive the prothrombotic cascade culminating in DVT.. Here, we addressed the pathophysiology of malignant DVT compared with nonmalignant DVT and focused on the role of tumor microvesicles as potential targets to prevent cancer-associated DVT. We show that microvesicles released by pancreatic adenocarcinoma cells (pancreatic tumor-derived microvesicles [pcMV]) boost thrombus formation in a model of flow restriction of the mouse vena cava. This depends on the synergistic activation of coagulation by pcMV and host tissue factor. Unlike nonmalignant DVT, which is initiated and propagated by innate immune cells, thrombosis triggered by pcMV was largely independent of myeloid leukocytes or platelets. Instead, we identified externalization of the phospholipid phosphatidylethanolamine as a major mechanism controlling the prothrombotic activity of pcMV. Disrupting phosphatidylethanolamine-dependent activation of factor X suppressed pcMV-induced DVT without causing changes in hemostasis.. Together, we show here that the pathophysiology of pcMV-associated experimental DVT differs markedly from innate immune cell-promoted nonmalignant DVT and is therefore amenable to distinct antithrombotic strategies. Targeting phosphatidylethanolamine on tumor microvesicles could be a new strategy for prevention of cancer-associated DVT without causing bleeding complications.

Topics: Adenocarcinoma; Animals; Bacteriocins; Blood Coagulation; Cell Line, Tumor; Cell-Derived Microparticles; Disease Models, Animal; Drug Design; Factor Xa; Fibrinolytic Agents; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Targeted Therapy; Pancreatic Neoplasms; Peptides; Phosphatidylethanolamines; Signal Transduction; Thromboplastin; Vena Cava, Inferior; Venous Thrombosis

Antibody fragment (Fab')-installed polyion complex (PIC) micelles were constructed to improve targetability of small interfering RNA (siRNA) delivery to pancreatic cancer cells. To this end, we synthesized a block copolymer of azide-functionalized poly(ethylene glycol) and poly(l-lysine) and prepared PIC micelles with siRNA. Then, a dibenzylcyclooctyne (DBCO)-modified antihuman tissue factor (TF) Fab' was conjugated to azido groups on the micellar surface. A fluorescence correlation spectroscopic analysis revealed that 1, 2, or 3 molecule(s) of Fab'(s) were installed onto one micellar nanoparticle according to the feeding ratio of Fab' (or DBCO) to micelle (or azide). The resulting micelles exhibited ∼40 nm in hydrodynamic diameter, similar to that of the parent micelles before Fab' conjugation. Flow cytometric analysis showed that three molecules of Fab'-installed PIC micelles (3(Fab')-micelles) had the highest binding affinity to cultured pancreatic cancer BxPC3 cells, which are known to overexpress TF on their surface. The 3(Fab')-micelles also exhibited the most efficient gene silencing activity against polo-like kinase 1 mRNA in the cultured cancer cells. Furthermore, the 3(Fab')-micelles exhibited high penetrability and the highest cellular internalization amounts in BxPC3 spheroids compared with one or two molecule(s) of Fab'-installed PIC micelles. These results demonstrate the potential of anti-TF Fab'-installed PIC micelles for active targeting of stroma-rich pancreatic tumors.

Topics: Antibodies, Neoplasm; Cell Cycle Proteins; Cell Line, Tumor; Drug Delivery Systems; Gene Silencing; Humans; Immunoglobulin Fab Fragments; Micelles; Pancreatic Neoplasms; Polo-Like Kinase 1; Polyethylene Glycols; Polylysine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; RNA, Small Interfering; Thromboplastin

Antibody-drug conjugates (ADCs) are currently considered to be promising agents for cancer therapy. However, especially in solid tumors, the uneven distribution of ADCs would decrease their efficacy in clinical studies. We suggest that in addition to optimizing ADC components, such as the linker structure and anticancer agent, it is necessary to consider the distribution of the ADC within tumor tissue. In this study, we established three kinds of anti-tissue factor (TF) ADCs: 1849ADC with a low k

Topics: Animals; Antineoplastic Agents, Immunological; Cell Line, Tumor; Female; Humans; Immunoconjugates; Mice, Inbred BALB C; Mice, Nude; Pancreatic Neoplasms; Thromboplastin

The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis.. Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated.. The addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE.. NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer.. This study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).

Topics: Adenocarcinoma; Animals; Chloroquine; DNA; Extracellular Traps; Female; Humans; Hydrolases; Hydroxychloroquine; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Platelet Aggregation; Protein-Arginine Deiminase Type 4; Receptor for Advanced Glycation End Products; Thrombelastography; Thrombophilia; Thromboplastin; Venous Thromboembolism

Essentials Tumor-bearing mice have larger venous clots than controls. Human tissue factor is present in clots in tumor-bearing mice. Inhibition of human tissue factor reduces clot size in tumor-bearing mice. This new mouse model may be useful to study mechanisms of cancer-associated thrombosis.. Background Pancreatic cancer patients have a high rate of venous thromboembolism. Human pancreatic tumors and cell lines express high levels of tissue factor (TF), and release TF-positive microvesicles (TF

Topics: Animals; Antibodies, Monoclonal; Blood Coagulation; Blood Platelets; Cell Line, Tumor; Cell-Derived Microparticles; Disease Models, Animal; Fibrin Fibrinogen Degradation Products; Fibrinolytic Agents; Heterografts; Humans; Male; Mice, Nude; Neoplasm Transplantation; Neutrophils; Pancreatic Neoplasms; Thromboplastin; Venous Thrombosis

Circulating microvesicles (MVs) are suggested to be important contributors to cancer-associated thrombosis due to the presence of surface-bound procoagulant molecules like tissue factor (TF) and phosphatidylserine (PS). Pancreatic cancer is considered to be one of the most prothrombotic malignancies. The aim of this study was to describe the impact of analytical variables on MV-associated thrombin generation in patients with pancreatic cancer and in healthy controls. MVs were isolated from citrated plasma and added to pooled normal plasma (PNP). Thrombin generation was measured by the calibrated automated thrombogram. The impact of corn trypsin inhibitor (CTI), anti-tissue factor pathway inhibitor (TFPI) antibodies and phospholipids was described. Antibodies against TF were used to assess TF-dependency, and MV-bound PS activity was measured with the Zymuphen MP-activity kit. MVs from the pancreatic cancer patients displayed higher thrombin generation and higher PS-activity than MVs from the healthy control group, while TF-dependency was observed in only 1 out of 13 patient samples. Adequate thrombin generation-curves were only achieved when CTI was omitted and anti-TFPI antibodies were added to PNP prepared in low contact-activating tubes. Addition of phospholipids reduced the significant differences between the two groups, and should be omitted. This modified thrombin generation assay could be useful for measurement of procoagulant circulating MVs, allowing the contribution from MVs affecting both the intrinsic and the extrinsic pathway to be measured.

Topics: Aged; Antibodies; Cell-Derived Microparticles; Female; Healthy Volunteers; Humans; Lipoproteins; Male; Middle Aged; Pancreatic Neoplasms; Phosphatidylserines; Phospholipids; Plant Proteins; Thrombin; Thromboplastin

Most cancer cells trigger thrombin generation (TG) to various extent. In the present study, we dissected the mechanisms responsible for the procoagulant activity of pancreatic adenocarcinoma cells (BXPC3), a highly thrombogenic cancer type, and breast cancer cells (MCF7), a less thombogenic tumor type. TG of normal plasma was assessed by the Thrombinoscope (CAT®) in the presence or absence of cancer cells. TG was also assessed in plasma depleted of clotting factors, in plasma spiked with tissue factor (TF) and/or procoagulant phospholipids, in plasma spiked with an anti-TF monoclonal antibody or with corn trypsin inhibitor (CTI). The presence of alternatively spliced TF (asTF), TF activity (TFa) and cancer procoagulant (CP) levels were determined. TFa and asTF were highly expressed by BXPC3 cells, compared to MCF7 cells, while CP levels were higher in MCF7 cells. BXPC3 cells had a stronger effect on TG than MCF7 cells. Accordingly, anti-TF had more inhibitory activity on TG triggered by BXPC3 cells while CTI had more pronounced inhibitory effect on TG triggered by MCF7 cells. TG enhancement by both BXPC3 and MCF7 cells was mediated by FVII and intrinsic tenase while FXII and FXI were also important for MCF7 cells. The induction of TG by BXPC3 cells was mainly driven by the TF pathway while TG generation triggered by MCF7 cells was also driven by FXII activation. Therefore, hypercoagulability results from a combination of the inherent procoagulant properties of cancer cell-associated TF as well as of procoagulant phospholipids in the plasma microenvironment.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Cell Line, Tumor; Colonic Neoplasms; Cysteine Endopeptidases; Factor XII; Female; HCT116 Cells; HT29 Cells; Human Umbilical Vein Endothelial Cells; Humans; MCF-7 Cells; Neoplasm Proteins; Ovarian Neoplasms; Pancreatic Neoplasms; Platelet-Rich Plasma; Thrombin; Thromboplastin

Increased levels of tissue factor-positive extracellular vesicles (TF+EVs) have been detected in the plasma of patients with various diseases, including cancer and endotoxemia. Levels of TF+EVs in plasma samples can be measured by antigen and activity assays. The aim of the present study was to visualize TF+EVs by laser scanning confocal microscopy (LSCM).. EVs were isolated from the supernatant of two cultured human pancreatic cancer cell lines (Panc-1 and BxPc-3), from untreated or lipopolysaccharide (LPS) treated whole blood, and from plasma of pancreatic cancer patients. EV-TF activity was determined using an in-house assay. The EVs were labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester, which is converted to the impermeant green fluorescent molecule carboxyfluorescein inside the EVs. EVs were either captured using annexin V and detected using a fluorescent-labeled anti-TF antibody, or captured using an anti-TF antibody and detected using fluorescent-labeled annexin V. EVs were visualized by LSCM.. TF+EVs were easily detected from high TF-expressing BxPc-3 cells using annexin V capture, whereas the addition of tyramide amplification was required to detect TF+EVs from low TF-expressing Panc-1 cells. Visualization of TF+EVs in plasma from LPS treated whole human blood and in plasma from pancreatic cancer patients required either capture with annexin V and detection with a fluorescent-labeled anti-TF antibody with tyramide signal amplification, or capture with an anti-TF antibody and detection with a fluorescent-labeled annexin V.. LSCM enables visualization of TF+EVs in the supernatant from cultured cells and in clinical samples.

Topics: Annexin A5; Cell Line; Cell Line, Tumor; Extracellular Vesicles; Humans; Lipopolysaccharide Receptors; Microscopy, Confocal; Pancreas; Pancreatic Neoplasms; Thromboplastin

ESSENTIALS: Cancer patients have a high rate of venous thrombosis (VT) but the underlying mechanisms are unknown. Tumor-derived, tissue factor-positive microvesicles in platelet activation in vitro and in vivo were studied. Tumor-derived, tissue factor-positive microvesicles enhanced VT in mice. Platelets may contribute to VT in some cancer patients, and this could be prevented with antiplatelet drugs.. Cancer patients have an approximately 4-fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor-derived, tissue factor-positive (TF(+) ) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF(+) tumor-derived MVs (TMVs) activate platelets in vitro and in mice.. We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc-3) and low (L3.6pl) levels of TF as models to study the effect of TF(+) TMVs on platelets and thrombosis.. We found that both types of TF(+) TMVs activated human platelets and induced aggregation in vitro in a TF and thrombin-dependent manner. Further, injection of BxPc-3 TF(+) TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF(+) TMV-enhanced thrombosis was reduced in Par4-deficient mice and in wild-type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF(+) TMV-induced platelet activation contributes to thrombosis in cancer patients.

Topics: Adenocarcinoma; Animals; Blood Platelets; Cell Line, Tumor; Cell-Derived Microparticles; Clopidogrel; Female; Flow Cytometry; Humans; Mice; Mice, Inbred C57BL; Neoplasms; Pancreatic Neoplasms; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Pulmonary Embolism; Thrombin; Thromboplastin; Thrombosis; Ticlopidine

Alternatively spliced Tissue Factor (asTF) is a secreted form of Tissue Factor (TF), the trigger of blood coagulation whose expression levels are heightened in several forms of solid cancer, including pancreatic ductal adenocarcinoma (PDAC). asTF binds to β1 integrins on PDAC cells, whereby it promotes tumor growth, metastatic spread, and monocyte recruitment to the stroma. In this study, we determined if targeting asTF in PDAC would significantly impact tumor progression. We here report that a novel inhibitory anti-asTF monoclonal antibody curtails experimental PDAC progression. Moreover, we show that tumor-derived asTF is able to promote PDAC primary growth and spread during early as well as later stages of the disease. This raises the likelihood that asTF may comprise a viable target in early- and late-stage PDAC. In addition, we show that TF expressed by host cells plays a significant role in PDAC spread. Together, our data demonstrate that targeting asTF in PDAC is a novel strategy to stem PDAC progression and spread.

Topics: Alternative Splicing; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; Thromboplastin

Tissue factor (TF) is the main initiator of the extrinsic coagulation cascade. However, TF also plays an important role in cancer. TF expression has been reported in 53%-89% of all pancreatic adenocarcinomas, and the expression level of TF has in clinical studies correlated with advanced stage, increased microvessel density, metastasis, and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker and as a companion diagnostic for TF-directed therapies currently under clinical development. Factor VII (FVII) is the natural ligand to TF. The purpose of this study was to investigate the possibility of using active site-inhibited FVII (FVIIai) labeled with (64)Cu for PET imaging of TF expression.. FVIIai was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu ((64)Cu-NOTA-FVIIai). Longitudinal in vivo PET imaging was performed at 1, 4, 15, and 36 h after injection of (64)Cu-NOTA-FVIIai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with (64)Cu-NOTA-FVIIai was investigated in subcutaneous pancreatic tumor models with different levels of TF expression and in a competition experiment. In addition, imaging of orthotopic pancreatic tumors was performed using (64)Cu-NOTA-FVIIai and PET/MRI. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry, and immunohistochemistry.. Longitudinal PET imaging with (64)Cu-NOTA-FVIIai showed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3, and 2.4 ± 0.3 percentage injected dose per gram at 1, 4, 15, and 36 h after injection, respectively. An increase in tumor-to-normal-tissue contrast was observed over the imaging time course. Competition with unlabeled FVIIai significantly (P < 0.001) reduced the tumor uptake. The tumor uptake observed in models with different TF expression levels was significantly different from each other (P < 0.001) and was in agreement with the TF level evaluated by TF immunohistochemistry staining. Orthotopic tumors were clearly visible on the PET/MR images, and the uptake of (64)Cu-NOTA-FVIIai was colocalized with viable tumor tissue.. (64)Cu-NOTA-FVIIai is well suited for PET imaging of tumor TF expression, and imaging is capable of distinguishing the TF expression level of various pancreatic tumor models.

Topics: Animals; Cell Line, Tumor; Copper Radioisotopes; Factor VII; Humans; Isotope Labeling; Magnetic Resonance Imaging; Mice; Multimodal Imaging; Neoplasm Transplantation; Pancreatic Neoplasms; Positron-Emission Tomography; Radiometry; Radiopharmaceuticals; Thromboplastin; Tissue Distribution

Pancreatic adenocarcinoma is a highly aggressive cancer, currently treated with limited success and dismal outcomes. New diagnostic and treatment strategies offer the potential to reduce cancer mortality. Developing highly specific noninvasive imaging probes for pancreatic cancer is essential to improving diagnostic accuracy and monitoring therapeutic intervention.. A bispecific heterodimer was synthesized by conjugating an anti-tissue factor (TF) Fab with an anti-CD105 Fab, via the bio-orthogonal "click" reaction between tetrazine (Tz) and trans-cyclooctene (TCO). The heterodimer was labeled with (64)Cu for PET imaging of nude mice bearing BXPC-3 xenograft and orthotopic pancreatic tumors.. PET imaging of BXPC-3 (TF/CD105(+/+)) xenograft tumors with (64)Cu-labeled heterodimer displayed significantly enhanced tumor uptake (28.8 ± 3.2 %ID/g; n = 4; SD) at 30 hours postinjection, as compared with each of their monospecific Fab tracers (12.5 ± 1.4 and 7.1 ± 2.6 %ID/g; n = 3; SD). In addition, the activity-concentration ratio allowed for effective tumor visualization (tumor/muscle ratio 75.2 ± 9.4 at 30 hours postinjection.; n = 4; SD). Furthermore, (64)Cu-NOTA-heterodimer enabled sensitive detection of orthotopic pancreatic tumor lesions with an uptake of 17.1 ± 4.9 %ID/g at 30 hours postinjection and tumor/muscle ratio of 72.3 ± 46.7.. This study demonstrates that dual targeting of TF and CD105 provided synergistic improvements in binding affinity and tumor localization of the heterodimer. Dual-targeted imaging agents of pancreatic and other cancers may assist in diagnosing pancreatic malignancies as well as reliable monitoring of therapeutic response. Clin Cancer Res; 22(15); 3821-30. ©2016 AACR.

Topics: Animals; Biomarkers; Cell Line, Tumor; Disease Models, Animal; Female; Flow Cytometry; Humans; Immunoglobulin Fab Fragments; Mice; Neprilysin; Pancreatic Neoplasms; Positron-Emission Tomography; Protein Multimerization; Radiopharmaceuticals; Thromboplastin; Tissue Distribution

Cancer-related venous thromboembolism (VTE) heralds a poor prognosis, especially in pancreatic adenocarcinoma (PAC). Tissue factor (TF) is implicated as one of the main culprits in PAC-associated VTE and disease progression.. In a prospective cohort study of 79 PAC patients, we measured plasma CA19-9 and microparticle-associated TF activity (MP-TF activity). In addition, we enumerated TF(+)MPs and MUC1(+)MPs in plasma (n=55), and studied the expression of TF, MUC1, CD31 and CD68 in tumour tissue (n=44).. Plasma MP-TF activity was markedly elevated in PAC patients with VTE compared with those without (median: 1925 vs 113 fM Xa min(-1); P<0.001) and correlated with the extent of thromboembolic events, metastatic disease and short survival. Similar results were found for CA19-9. Patients with massively progressing thrombosis and cerebral embolisms despite anticoagulant therapy (n=3) had the highest MP-TF activities (12 118-40 188 fM Xa min(-1)) and CA19-9 (40 730-197 000 kU l(-1)). All tumours expressed MUC1 and TF. MP-TF activity did not correlate with intensity of TF expression in adenocarcinoma cells, but corresponded with numbers of TF(+) macrophages in the surrounding stroma.. Circulating TF(+)MPs and mucins may concertedly aggravate coagulopathy in PAC. Understanding of underlying mechanisms may result in new treatment strategies for VTE prevention and improvement of survival.

Topics: Aged; Aged, 80 and over; CA-19-9 Antigen; Cohort Studies; Female; Humans; Male; Middle Aged; Pancreatic Neoplasms; Thromboplastin; Thrombosis

Venous thromboembolism constitutes one of the main causes of death during the progression of a cancer. We previously demonstrated that tissue factor (TF)-bearing cancer cell-derived microparticles accumulate at the site of injury in mice developing a pancreatic cancer. The presence of these microparticles at the site of thrombosis correlates with the size of the platelet-rich thrombus. The objective of this study was to determine the involvement of TF expressed by cancer cell-derived microparticles on thrombosis associated with cancer. We observed that pancreatic cancer cell derived microparticles expressed TF, its inhibitor tissue factor pathway inhibitor (TFPI) as well as the integrins αvβ1 and αvβ3. In mice bearing a tumor under-expressing TF, a significant decrease in circulating TF activity associated with an increase bleeding time and a 100-fold diminished fibrin generation and platelet accumulation at the site of injury were observed. This was mainly due to the interaction of circulating cancer cell-derived microparticles expressing TFPI with activated platelets and fibrinogen. In an ectopic model of cancer, treatment of mice with Clopidogrel, an anti-platelet drug, decreased the size of the tumors and restored hemostasis by preventing the accumulation of cancer cell-derived microparticles at the site of thrombosis. In a syngeneic orthotopic model of pancreatic cancer Clopidogrel also significantly inhibited the development of metastases. Together, these results indicate that an anti-platelet strategy may efficiently treat thrombosis associated with cancer and reduce the progression of pancreatic cancer in mice.

Topics: Animals; Blood Coagulation; Blotting, Western; Cell-Derived Microparticles; Clopidogrel; Disease Models, Animal; Flow Cytometry; Fluorescent Antibody Technique; Integrins; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; P-Selectin; Pancreatic Neoplasms; Platelet Activation; Platelet Aggregation Inhibitors; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Thrombosis; Ticlopidine; Tumor Cells, Cultured

Tissue factor (TF) triggers the extrinsic blood coagulation cascade and is highly expressed in various types of cancer. In this study, we investigated the antitumor effect of an antibody-drug conjugate (ADC) consisting of an anti-TF monoclonal antibody and monomethyl auristatin E (MMAE). MMAE was conjugated to an anti-human TF or anti-mouse TF antibody using a valine-citrulline linker that could be potentially hydrolyzed by cathepsin B in the acidic environment of the lysosome. The cytotoxic and antitumor effects of the ADCs against four pancreatic cancer cell lines were analyzed. Both the ADC with the anti-human TF antibody and that with the anti-mouse TF antibody were stable under physiological conditions. The anti-human ADC was internalized in TF-expressing human tumor cell lines, followed by effective MMAE release. The half maximal inhibitory concentration (IC50 ) of MMAE was approximately 1 nM for all of the cell lines used. Meanwhile, the IC50 of anti-human ADC was 1.15 nM in the cell lines showing high TF expression, while exceeding 100 nM in the cells showing low TF expression levels. Anti-human ADC with passive and active targeting ability exerted significant suppression of tumor growth as compared to that observed in the saline group (p < 0.01). Also significant tumor growth suppressions were seen at the anti-mouse ADC and control ADC groups compared to the saline group (p < 0.01) due to EPR effect. Because various clinical human cancers express highly amount of TF, this new anti-TF ADC may deserve a clinical evaluation.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Cell Line, Tumor; Female; Humans; Immunoconjugates; Mice; Mice, Inbred BALB C; Mice, Nude; Oligopeptides; Pancreatic Neoplasms; Thromboplastin; Xenograft Model Antitumor Assays

The risk of thrombotic complications such as deep vein thrombosis (DVT) during tumor development is well known. Tumors release into the circulation procoagulant microparticles (MPs) that can participate in thrombus formation following vessel injury. The importance of this MP tissue factor (TF) in the initiation of cancer-associated DVT remains uncertain.. To investigate how pancreatic cancer MPs promote DVT in vivo.. We combined a DVT mouse model in which thrombosis is induced by flow restriction in the inferior vena cava with one of subcutaneous pancreatic cancer in C57BL/6J mice. We infused high-TF and low-TF tumor MPs to determine the importance of TF in experimental cancer-associated DVT.. Both tumor-bearing mice and mice infused with tumor MPs subjected to 3 h of partial flow restriction developed an occlusive thrombus; fewer than one-third of the control mice did. We observed that MPs adhered to neutrophil extracellular traps (NETs), which are functionally important players during DVT, whereas neither P-selectin nor glycoprotein Ib were required for MP recruitment in DVT. The thrombotic phenotype induced by MP infusion was suppressed by hirudin, suggesting the importance of thrombin generation. TF carried by tumor MPs was essential to promote DVT, as mice infused with low-TF tumor MPs had less thrombosis than mice infused with high-TF tumor MPs.. TF expressed on tumor MPs contributes to the increased incidence of cancer-associated venous thrombosis in mice in vivo. These MPs may adhere to NETs formed at the site of thrombosis.

Topics: Animals; Antithrombins; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell-Derived Microparticles; Disease Models, Animal; Extracellular Traps; Hirudins; Ligation; Male; Mice, Inbred C57BL; Mice, Knockout; P-Selectin; Pancreatic Neoplasms; Platelet Glycoprotein GPIb-IX Complex; Regional Blood Flow; Thromboplastin; Vena Cava, Inferior; Venous Thrombosis

Circulating ('blood-borne') tissue factor (TF) is implicated in the pathogenesis of several chronic conditions, most notably cardiovascular disease, diabetes, and cancer. Full-length TF is an integral membrane protein, while alternatively spliced TF (asTF) can be secreted and, owing to its unique C-terminus, selectively detected in bio-specimens. The predictive and/or prognostic value of asTF in the circulation is unknown. In a retrospective study, we measured levels of circulating asTF in healthy subjects and individuals with acute coronary syndrome (ACS), diabetes mellitus (DM), ongoing ACS + DM, and pancreatic ductal adenocarcinoma (PDAC).. The prototype-tailored procedure (Diagnostica Stago) was used to measure asTF in plasma from 205 subjects.. There was no significant difference between the proportion of healthy subjects with asTF ≥200 pg/mL and those with ACS, DM, or ACS + DM. The proportion of pancreatic cancer patients (n = 43; PDAC: 42; pancreatic neuroendocrine tumor: 1) with asTF levels ≥200 pg/mL was significantly higher than in healthy subjects; asTF levels ≥200 pg/mL were detected more often in patients with unresectable disease irrespective of initial evaluation and/or preoperative carbohydrate antigen 19-9 (CA19-9) levels.. While asTF levels ≥200 pg/mL are not observed with increased frequency in patients with ACS and/or DM, they do occur more frequently in the plasma of patients with pancreatic cancer and are associated with lower likelihood of tumor resectability, irrespective of the preoperative diagnosis. asTF may thus have utility as a novel marker of aggressive pancreatic tumor phenotype.

Topics: Acute Coronary Syndrome; Alternative Splicing; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Case-Control Studies; Diabetes Mellitus; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; Prognosis; Retrospective Studies; Thromboplastin

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell-Derived Microparticles; Male; Pancreatic Neoplasms; Thromboplastin; Venous Thrombosis

Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Cell Line, Tumor; Electrophoresis, Polyacrylamide Gel; Female; Flow Cytometry; Heterografts; Humans; Immunoglobulin Fab Fragments; Immunoglobulin G; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Pancreatic Neoplasms; Surface Plasmon Resonance; Thromboplastin

Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF-overexpressing high-grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell-derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the procoagulant potential of PDAC cells and cell-derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

Topics: Alternative Splicing; Animals; Blood Coagulation; Blotting, Western; Carcinoma, Pancreatic Ductal; Flow Cytometry; Heterografts; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Pancreatic Neoplasms; Thromboplastin; Tissue Array Analysis

Highly elevated microparticle (MP)-associated tissue factor (TF) activity was found in patients with pancreatic cancer, one of the most prothrombotic malignancies. It remains to be elucidated whether MP-TF activity reflects the prothrombotic state in these patients. MP-TF activity levels and the TF-dependent and -independent effect of MPs on fibrin clot formation were determined in patients with metastatic pancreatic cancer (n = 27), in healthy individuals (n = 10) and in plasma samples from lipopolysaccharide (LPS)-stimulated blood (LPS-plasma), which is rich in monocyte-derived TF-bearing MPs. The median MP-TF activity was 1.06 pg/mL (range, from 0.19 to 10.34 pg/mL) in patients with pancreatic cancer, 0.61 pg/mL (range, from 0.36 to 0.79 pg/mL) in LPS-plasma, and 0.18 pg/mL (range, from 0.04 to 0.39 pg/mL) in healthy individuals. MPs derived from LPS-plasma had the strongest impact on fibrin clot formation time (median, 157.6 seconds; range, from 149.5 to 170.4 seconds). Fibrin clot formation occurred significantly later in MPs derived from patients with pancreatic cancer (median, 273.4 seconds; range, from 146.6 to 354.4 seconds; P < 0.001) and in healthy individuals (median, 299.0 seconds; range, from 261.1 to 417.9 seconds; P < 0.001). Only in MPs derived from LPS-plasma the fibrin clot formation time dependent strongly on TF (median prolongation after TF blockade: 68% in LPS-plasma, 10% in patients with pancreatic cancer, and 4% in healthy individuals). In conclusion, highly elevated MP-TF activity was found in patients with metastatic pancreatic cancer, but TF-bearing MPs had a small effect on fibrin clot formation. TF-bearing MPs might not be the main mediators of the prothrombotic state associated with pancreatic cancer. However, the small but significant increase in coagulation potential by TF-bearing MPs might contribute to the multifactorial pathogenesis of venous thromboembolism in pancreatic cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Case-Control Studies; Female; Fibrin; Humans; Male; Middle Aged; Neoplasm Metastasis; Pancreatic Neoplasms; Thromboplastin; Thrombosis

Topics: Adenocarcinoma; Female; Fibrin; Humans; Male; Neoplasm Metastasis; Pancreatic Neoplasms; Thromboplastin; Thrombosis

The metastasis-related molecules CD133, CD44v6 and human tissue factor (TF) have been shown to be associated with tumor invasion and metastasis. This study aimed to determine whether co-expression of these three molecules was associated with metastasis and overall prognosis in pancreatic carcinoma. We analyzed the expression profiles of these three molecules by immunohistochemistry and evaluated the relationship of their expression profiles with metastasis and prognosis in 109 pancreatic carcinomas. The results showed that the expression levels of CD133, CD44v6 and TF were increased in pancreatic carcinoma. Co-expression of CD133, CD44v6 and TF (tri-expression) was also detected in pancreatic carcinoma. Clinical analysis showed that individual expression of CD133, CD44v6 or TF was associated with vessel invasion, lymph node metastasis and liver metastasis, while tri-expression was associated with lymph node metastasis. Survival analysis showed that patients with co-expression of CD133 and TF or tri-expression had lower and the lowest overall survival rates, respectively. Univariate analysis showed that T-factor, lymph node metastasis, TNM stage, and individual levels or tri-expression of CD133, CD44v6 and TF were survival risk factors. Multivariate analysis showed that tri-expression of CD133, CD44v6 and TF was an independent predictor of survival. These results suggest that overexpression of CD133, CD44v6 and TF is associated with pancreatic carcinoma metastasis. Tri-expression of these three molecules may be a useful predictor for pancreatic carcinoma prognosis.

Topics: AC133 Antigen; Adult; Aged; Aged, 80 and over; Antigens, CD; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Hyaluronan Receptors; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Pancreatic Neoplasms; Peptides; Survival Analysis; Survival Rate; Thromboplastin

In this study, 52 patients were studied to elucidate the relative impact of resection of localized pancreaticobiliary adenocarcinoma (PBC) on circulating factors of tumour-associated angiogenesis e.g. tissue factor bearing microparticles (TFMP) and vascular endothelial growth factor (VEGF) and their clinicopathological significance to angiogenesis markers in cancer tissue from PBC patients. Angiogenesis array analysis on serum samples revealed that surgical resection of tumour lesion in PBC patients affects the levels of a panel of angiogenesis-related molecules, including VEGF that was verified by ELISA to significantly reduce (median & IQR: 1003(369-2000) vs. 457(159-834) pg/ml; p<0.05). Correspondingly, a significant decrease in the angiogenic activity (decreased capillary tube formation; p<0.05) of serum samples after the surgery was also found. Despite a decrease in number of circulating TFMP after surgery, this did not reach statistical significance; there was a significant reduction in pro-coagulant activity (prolonged prothrombin time, p<0.001) post-operatively. In addition, the activity of total microparticles (MP activity assay, p<0.05) was decreased significantly. Immunohistochemical staining of tumour tissue revealed a strong correlation between the microvessel density (MVD) and VEGF expression. Also, higher levels of circulating TFMP or TF related activity (prothrombin time) correlated significantly with TF expression and MVD on tumour tissues from PBC patients. These findings suggest that in pancreaticobiliary adenocarcinoma TF related angiogenesis drivers are equally significant to VEGF ones, raising the clinical question of whether the effectiveness of angiogenesis targeting studies could be improved through the 'dual' targeting of these pathways in PBC.

Topics: Adenocarcinoma; Aged; Cell-Derived Microparticles; Female; Humans; Male; Middle Aged; Neovascularization, Pathologic; Pancreas; Pancreatic Neoplasms; Thromboplastin; Thrombosis; Vascular Endothelial Growth Factor A

Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer. Overexpression of TF is considered to contribute to the high incidence of thrombotic complications and poor prognosis in patients with such cancers. Therefore, detection or targeting of TF may be a promising approach for the diagnosis and treatment of solid tumors that are known to overexpress the protein. Here, we used the recombinant DNA technology to develop an anti-TF single-chain Fv (scFv) of small size and high affinity for its target. The biochemical characteristics of the anti-TF scFv were evaluated using surface plasmon resonance (SPR) sensing and flow cytometry. The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10(-8) (KD), and that the protein showed significant binding to the cancer cells. Then, Alexa 647-labeled anti-TF scFv and anti-TF IgG were administered to mice bearing chemically induced spontaneous tumors. The maximum tumor to background ratios of anti-TF scFv and anti-TF IgG were obtained 3 and 24 h after the injections, respectively. This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.

Topics: Animals; Cell Line, Tumor; Female; Humans; Mice; Neoplasms, Experimental; Pancreatic Neoplasms; Single-Chain Antibodies; Skin Neoplasms; Surface Plasmon Resonance; Thromboplastin

Tissue factor (TF), the physiologic initiator of coagulation, is over-expressed in pancreatic cancer, and is associated with a pro-coagulant and pro-angiogenic state. We hypothesized that in patients with pancreaticobiliary cancers (PBC), elevated circulating microparticle-associated TF (MP-TF) activity would be associated with thrombosis and worsened survival.. Clinical data and plasma were obtained for consecutive patients with PBC seen at Roswell Park Cancer Institute from 2005-08. MP-TF activity levels were measured using a TF-dependent FXa generation assay.. The study population comprised 117 patients, including pancreatic (n=80), biliary (n=34) or unknown primary histologically consistent with PBC (n=3). Of these, 52 patients (44.5%) experienced thromboembolism, including pulmonary embolism (n=15), deep venous thrombosis (n=21) and other arterial or venous events (n=32). Mean TF was 2.15 (range 0.17- 31.01) pg/mL. Median survival was 98.5 days for MP-TF activity ≥ 2.5 pg/mL versus 231 days for MP-TF activity<2.5 pg/mL (p<0.0001). In multivariate analysis, elevated MP-TF activity was associated with both VTE (OR 1.4, 95% CI 1.1-1.6) and mortality (HR 2.5, 95% CI 1.4-4.5).. Elevated circulating MP-TF activity is associated with thrombosis and worsened survival in patients with PBC. MP-TF activity as a prognostic biomarker warrants further prospective evaluation.

Topics: Adult; Aged; Aged, 80 and over; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell-Derived Microparticles; Cohort Studies; Female; Humans; Male; Middle Aged; Pancreatic Neoplasms; Prognosis; Survival Analysis; Thromboembolism; Thromboplastin

Patients with pancreatic cancer have an unfavourable prognosis. A central role in pancreatic cancer progression has been suggested for tissue factor (TF), the main initiator of the blood coagulation cascade. We hypothesized that elevated levels of plasma microparticle (MP)-associated TF activity might indicate the presence of poorly differentiated pancreatic cancer, disease dissemination and infiltration of peripancreatic vessels.. MP-TF activity was measured in 73 pancreatic cancer patients and 22 healthy controls. Abdominal computerized tomography (CT) scans performed at study inclusion were investigated for probability of tumoural vascular invasion. In addition, intratumoural TF expression, D-dimer and CA 19-9 levels were determined.. MP-TF activity (pg/mL) was significantly higher in patients (median: 0·37 [range: 0·00-11·91]) than in controls (median: 0·05 [range: 0·00-0·76]; P < 0·001). When pancreatic cancer patients were compared with regard to stage and grade, significantly elevated levels of MP-TF activity were only present in those with poorly differentiated metastatic nonresectable tumours (n = 11, median: 2·95 [range: 0·25-11·91]). In three patients with poorly differentiated tumours, a high probability of vascular invasion was found (MP-TF activity in these cases: 2·95, 7·00 and 10·34). MP-TF activity correlated strongly with CA 19-9 (r = 0·60) and weakly with D-dimer (r = 0·33) levels. Immunohistochemical staining for TF was positive in 14 of 15 resected tumours. MP-TF activity was associated with an increased risk of mortality (HR: 1·8 per doubling in MP-TF activity, [95% CI: 1·4-2·4, P < 0·001]).. MP-TF activity might represent a biomarker for a poorly differentiated and invasive pancreatic cancer phenotype and poor survival.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; CA-19-9 Antigen; Cell-Derived Microparticles; Female; Fibrin Fibrinogen Degradation Products; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Staging; Pancreatic Neoplasms; Prognosis; Thromboplastin; Tomography, Spiral Computed; Vascular Neoplasms

Cancer histology influences the risk of venous thromboembolism and tissue factor (TF) is the key molecule in cancer-induced hypercoagulability. We investigated the relation between TF expression by pancreatic and breast cancer cells (BXPC3 and MCF7 respectively) and their capacity to trigger in vitro thrombin generation in normal human plasma. Flow cytometry and Western blot analysis for TF expression were performed using murine IgG1 monoclonal antibody against human TF. Real-time PCR for TFmRNA was also performed. Activity of TF expressed by cancer cells was measured with a specific chromogenic assay. Thrombin generation in PPP was assessed using calibrated automated thrombogram. Cancer cells were added to platelet poor plasma from healthy volunteers. In separate experiments cells were incubated with the anti-TF antibody at concentration that completely neutralized the activity of recombinant human TF on thrombin generation. BXPC3 cells expressed significantly higher amounts of functional TF as compared to MCF7 cells. Incubation of BXPC3 and MCF7 cells with PPP resulted in acceleration of the initiation phase of thrombin generation. BXPC3 cells manifested higher procoagulant potential than MCF7 cells. The incubation of BXPC3 or MCF7 cells with the anti-TF monoclonal antibody which resulted in reversal of their effect on thrombin generation. The present study establishes a link between the amount of TF expressed by cancer cells with their procoagulant activity. Both studied types of cancer cells trigger thrombin generation but they have different procoagulant potential. The procoagulant activity of BXPC3 and MCF7 cells is related to the amount of TF expressed. Kinetic parameters of thrombogram are the most relevant for the detection of the TF-dependent procoagulant activity of cancer cells. TF expression is one of the mechanisms by which cancer cells manifest their procoagulant potential but it is not the unique one. The present experimental model will allow the characterization the procoagulant fingerprint of cell lines from the same or different histological types of cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Flow Cytometry; Humans; MCF-7 Cells; Pancreatic Neoplasms; RNA, Messenger; Thrombin; Thromboplastin; Venous Thromboembolism

There is a clear need for better therapeutics and diagnostics for pancreatic cancer. We aimed to discover plasma membrane-associated proteins overexpressed in pancreatic cancer using quantitative proteomics and apply RNA interference (RNAi) to uncover proteins associated with cancer cell survival.. Cell surface glycoproteins from 5 pancreatic cancer cell lines were isolated, and differential analyses were performed using mass spectrometry and the "normoid" cell line Hs766T as the comparator. For validation, immunohistochemistry was performed on tissues from 10 independent patients and 2 normal donors. Correlation of protein and mRNA expression level was determined, and functional activity characterized using RNAi.. Integrin β6, CD46, tissue factor, and a novel protein, chromosome 14 open reading frame 1, were identified as overexpressed on pancreatic cancer cell lines. Immunohistochemistry demonstrated the 4 targets were overexpressed in 20% to 70% of primary pancreatic tumor specimens. Small interfering RNA knockdown resulted in a reduction of cellular proliferation by inhibiting DNA synthesis, blocking S-phase progression or induction of apoptosis.. By combining a mass spectrometry identification platform and an RNAi validation platform, we have identified a panel of cell surface glycoproteins that not only are overexpressed, but also play a functional role in pancreatic tumor cell survival.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Integrin beta Chains; Membrane Cofactor Protein; Membrane Glycoproteins; Membrane Proteins; Neoplasm Proteins; Pancreatic Neoplasms; Proteomics; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Thromboplastin

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a β3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via β3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.

Topics: Alternative Splicing; Animals; Cell Adhesion; Cell Line; Cell Movement; Cluster Analysis; Endothelial Cells; Gene Expression; Gene Expression Profiling; Integrins; Male; Mice; Mice, Inbred C57BL; Monocytes; Neovascularization, Physiologic; Pancreatic Neoplasms; Plaque, Atherosclerotic; Protein Binding; Protein Transport; Signal Transduction; Thromboplastin

Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti-human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Topics: Animals; Blood Coagulation; Cell Line, Tumor; Cell-Derived Microparticles; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Hemostasis; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; RNA, Messenger; Thromboplastin; Venous Thrombosis

Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types. The goal of this study was to develop a PET tracer for imaging of TF expression in pancreatic cancer.. ALT-836, a chimeric antihuman TF monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu. To compare the TF binding affinity of ALT-836 and NOTA-ALT-836, flow cytometry analysis was performed in 3 pancreatic cancer cell lines with different expression levels of TF (from low to high: PANC-1, ASPC-1, and BXPC-3). PET, biodistribution, blocking, and histology studies were performed on pancreatic tumor-bearing mice to evaluate the ability and specificity of (64)Cu-NOTA-ALT-836 to target TF in vivo.. There was no difference in TF binding affinity between ALT-836 and NOTA-ALT-836. (64)Cu-labeling was achieved with high yield and specific activity. Serial PET revealed that the uptake of (64)Cu-NOTA-ALT-836 in BXPC-3 tumors (high TF expression) was 5.7 ± 1.8, 10.4 ± 0.8, and 16.5 ± 2.6 percentage injected dose per gram at 4, 24, and 48 h after injection, respectively (n = 4), significantly higher than that in the PANC-1 and ASPC-1 tumors. Biodistribution data as measured by γ-counting were consistent with the PET findings. Blocking experiments and histology further confirmed the TF specificity of (64)Cu-NOTA-ALT-836.. Herein we report the first successful PET imaging of TF expression. Persistent and TF-specific uptake of (64)Cu-NOTA-ALT-836 was observed in pancreatic cancer models.

Topics: Animals; Cell Line, Tumor; Copper Radioisotopes; Female; Heterocyclic Compounds; Heterocyclic Compounds, 1-Ring; Humans; Immunoglobulin G; Mice; Pancreatic Neoplasms; Positron-Emission Tomography; Radiochemistry; Recombinant Proteins; Thromboplastin

Among cancers, pancreatic cancer is known to be associated with a higher incidence of venous thromboembolism (VTE). The aim of the study was to determine the implication of circulating tissue factor (TF) in VTE related to active pancreatic cancer. One hundred and sixty-four consecutive patients who participated to the Etude des Determinants et Interactions de la Thrombose veineuse (EDITH) study between January 2005 and August 2007 for symptomatic VTE related to active pancreatic cancer (n = 8), active cancer of other location (n = 42) or classified as unprovoked (n = 114) were included. TF activity (TFa) was measured in a one-stage kinetic chromogenic method. There were no differences of median TFa levels between patients with VTE related to cancer of other type than pancreas [2.01 pmol/l range (0.05-43.92)] and patients with unprovoked VTE [1.78 pmol/l (range 0.05-63.72), P = 0.21]. Median TFa levels were higher in patients with VTE related to pancreatic cancer [12.67 pmol/l (range 0.05-112.04)] than in patients with VTE related to cancer of other type [2.01 pmol/l (range 0.05-43.92), P = 0.02]. Higher levels of circulating TFa during the course of pancreatic cancer may explain the higher incidence of VTE associated with this type of cancer.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Female; Fibrin Fibrinogen Degradation Products; Humans; Incidence; Male; Middle Aged; Neoplasm Staging; Neoplasms; Pancreatic Neoplasms; Prospective Studies; Thromboplastin; Venous Thromboembolism

Tissue factor (TF), the initiating cell surface receptor for the blood coagulation cascade, plays an important role in malignant transformation of the pancreas, although the precise mechanism remains unresolved. Here, we report that the TF - factor VIIa complex in human pancreatic cancer cells produced a significant amount of MMP-9 and promoted invasion ability in vitro and invasion and metastasis in vivo. For treatment, we successfully developed an anti-human TF monoclonal antibody that inhibits both cellular signalling and blood coagulation cascade via TF. Invasive capability and MMP-9 expression were significantly reduced by the antibody. The antibody inhibited not only tumour invasion in the orthotopic model, but also haematogenous metastasis in the portal-injection liver metastasis model. In conclusion, the TF-VIIa complex plays an important role in invasion-metastasis by enhancing tumour cell infiltration ability and forming microthrombi. The newly established anti-human TF neutralisation antibody may be useful for the treatment of pancreatic and other invasive cancers.

Topics: Antibodies, Monoclonal; Blood Coagulation; Cell Transformation, Neoplastic; Factor VIIa; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Pancreatic Neoplasms; Second Messenger Systems; Signal Transduction; Thromboplastin; Tumor Cells, Cultured

Advanced pancreatic cancer is associated with a high risk of patients developing venous thromboembolism. This increased risk is thought to be tumour-driven and associated with tissue factor (TF) and microparticles. The aim of this study was to investigate the role of TF and phospholipid expression in the procoagulant properties of pancreatic cell lines and microparticles. Pancreatic cancer cell lines (MIA-PaCa-2, ASPC-1 and CFPAC-1) were assessed for expression of TF and microparticle release. Procoagulant potential was determined by a prothrombin time assay. Cell surface expression of TF was highest in CFPAC-1, with low expression on ASPC-1 and little/no expression on MIA-PaCa-2. Clotting time (CT) was cell number and TF-dependent (P < 0.001). Blocking of TF resulted in slower CT for CFPAC-1 and ASPC1 and prevented clotting in MIA-PaCa-2. Microparticles were shown to be procoagulant and the majority of procoagulant potential could be removed by passing cell-free media through a 0.1 μm filter. A dose-dependent CT was observed in both ASPC-1 and CFPAC-1 cell-free media. Furthermore, addition of duramycin prevented microparticle-supported coagulation. The data presented suggest a key role for cell and microparticle surface-expressed TF and phospholipids in coagulation and highlight duramycin-mediated disruption of clotting.

Topics: Adenocarcinoma; Adult; Aged; Bacteriocins; Blood Coagulation; Blood Coagulation Tests; Carcinoma; Cell Line, Tumor; Cell-Derived Microparticles; Culture Media, Conditioned; Female; Humans; Male; Middle Aged; Organ Specificity; Pancreas; Pancreatic Neoplasms; Peptides; Phosphatidylethanolamines; Phosphatidylserines; Prothrombin Time; Thrombin; Thromboplastin; Venous Thromboembolism

Cancer patients exhibit a high rate of thromboembolism (VTE). In this study, we analyzed levels of microparticle (MP) tissue factor (TF) activity in cancer patients with or without VTE. Blood was collected from cancer patients within 24 h of objectively diagnosed VTE (n=53) and from cancer patients without VTE (n=13). MPs were isolated from platelet poor plasma by centrifugation at 20,000g for 15 min. MP TF activity was measured using a two-stage chromogenic assay. Cancer patients with VTE had a significantly higher mean MP TF activity compared with cancer patients without VTE (1.7+/-3.8 pg/mL vs 0.6+/-0.4 pg/mL, p<0.05). Further prospective studies are required to determine if levels of MP TF activity may be a useful biomarker to identify patients at increased risk for VTE.

Topics: Biomarkers; Case-Control Studies; Cell-Derived Microparticles; Centrifugation; Chromogenic Compounds; Colonic Neoplasms; Humans; Lung Neoplasms; Neoplasms; Pancreatic Neoplasms; Thromboplastin; Venous Thromboembolism

Topics: ABO Blood-Group System; Factor VII; Humans; Pancreatic Neoplasms; Risk Assessment; Risk Factors; Thromboplastin; United States; Venous Thrombosis; von Willebrand Factor

Increased tissue factor (TF) expression is observed in many types of cancer, associated with more aggressive disease, and in thrombosis. The mechanism by which TF promotes tumor growth remains unclear. Anticoagulation has been shown to result in a trend toward improved survival; no direct antitumor effect has been shown in cancer patients. Alternatively spliced tissue factor (asTF) was recently described, in which exon 5 is deleted. Because of a frame-shift in exon 6, the transmembrane and cytoplasmic domains are replaced with a unique COOH-terminal domain, making asTF soluble. Both alternatively spliced human tissue factor (asHTF) and full-length tissue factor (flTF) are expressed in human pancreatic cancer lines and in pancreatic cancer specimens. We studied the role of asHTF and flTF in a mouse model of pancreatic cancer. Although lacking procoagulant activity, asTF promotes primary growth of human pancreatic cancer cells in mice and augments tumor-associated angiogenesis. This body of work suggests a new paradigm for the role of TF in pancreatic cancer: that asHTF contributes to cancer growth, independent of procoagulant activity.

Topics: Alternative Splicing; Animals; Anticoagulants; Cell Line, Tumor; Humans; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Pancreatic Neoplasms; Thromboplastin; Transfection

Topics: Humans; Pancreatic Neoplasms; Predictive Value of Tests; Thromboplastin; Venous Thromboembolism

Cancer, in particular mucinous adenocarcinoma, is associated with venous thromboembolism (VTE). Tissue factor (TF), initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF in tumors is associated with poor differentiation and poor prognosis.. We investigated the association between clinically manifest VTE and procoagulant properties of circulating microparticles (MP) isolated from blood of unselected pancreatic and breast adenocarcinoma patients' consecutive subjects, who presented with ultrasound or CT-scan confirmed VTE, and healthy subjects.. Patients with disseminated breast and pancreatic cancer had significantly increased levels of MP-associated TF activity compared with healthy controls, subjects with idiopathic acute VTE and non-metastatic cancer patients. Patients with both high MP-associated TF-activity and MP-associated epithelial mucin (MUC1) had a lower survival rate at 3-9 months follow-up than those with low TF-activity and no MUC1 expression: the likelihood of survival was 0.42 (95% CI: 0.19- 0.94) for an individual with these two predictor variables present, after adjustment for other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model.. Our results suggest an important role for MP-associated TF and MUC1 in the pathogenesis of thrombosis in disseminated mucinous adenocarcinoma patients. Future studies should reveal the mechanism underlying the observed associations.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Antigens, Neoplasm; Blood Coagulation; Breast Neoplasms; Case-Control Studies; Cell Differentiation; Cytoplasmic Vesicles; Female; Follow-Up Studies; Humans; Kaplan-Meier Estimate; Likelihood Functions; Male; Middle Aged; Mucin-1; Mucins; Neoplasm Invasiveness; Neoplasm Staging; Pancreatic Neoplasms; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Risk Assessment; Thromboembolism; Thromboplastin; Time Factors; Venous Thrombosis

Hemostatic activation is common in pancreatic cancer and may be linked to angiogenesis and venous thromboembolism. We investigated expression of tissue factor (TF), the prime initiator of coagulation, in noninvasive and invasive pancreatic neoplasia. We correlated TF expression with vascular endothelial growth factor (VEGF) expression, microvessel density, and venous thromboembolism in resected pancreatic cancer.. Tissue cores from a tri-institutional retrospective series of patients were used to build tissue microarrays. TF expression was graded semiquantitatively using immunohistochemistry in normal pancreas (n=10), intraductal papillary mucinous neoplasms (n=70), pancreatic intraepithelial neoplasia (n=40), and resected or metastatic pancreatic adenocarcinomas (n=130).. TF expression was observed in a majority of noninvasive and invasive pancreatic neoplasia, including 77% of pancreatic intraepithelial neoplasias, 91% of intraductal papillary mucinous neoplasms, and 89% of pancreatic cancers, but not in normal pancreas. Sixty-six of 122 resected pancreatic cancers (54%) were found to have high TF expression (defined as grade >or=2, the median score). Carcinomas with high TF expression were more likely to also express VEGF (80% versus 27% with low TF expression, P<0.0001) and had a higher median MVD (8 versus 5 per tissue core with low TF expression, P=0.01). Pancreatic cancer patients with high TF expression had a venous thromboembolism rate of 26.3% compared with 4.5% in patients with low TF expression (P=0.04).. TF expression occurs early in pancreatic neoplastic transformation and is associated with VEGF expression, increased microvessel density, and possibly clinical venous thromboembolism in pancreatic cancer. Prospective studies evaluating the role of TF in pancreatic cancer outcomes are warranted.

Topics: Aged; Capillaries; Female; Humans; Male; Middle Aged; Neovascularization, Pathologic; Pancreatic Neoplasms; Survival Analysis; Thromboplastin; Vascular Endothelial Growth Factor A; Venous Thrombosis

Tissue Factor (TF) expression is observed in many types of cancer, associated with more aggressive disease, and thrombosis. Alternatively-spliced human tissue factor (asHTF) has recently been identified in which exon 5 is deleted. asHTF is soluble due to the substitution of the transmembrane and cytoplasmic domains of exon 6 with a unique COOH-terminal domain.. We examine the expression and function of asHTF and full-length Tissue Factor ((FL)TF) in six human pancreatic cancer cells. Further, we transfected asHTF, (FL)TF, and control expression vectors into a non-expressing, human pancreatic cancer line (MiaPaCa-2). We studied the procoagulant activity of asHTF and (FL)TF and the effect on tumor growth in mice.. asHTF is expressed in 5 of 6 human pancreatic cancer cell lines, but not in normal human fibroblasts, nor the MiaPaCa-2 line. (FL)TF conferred procoagulant activity, but asHTF did not. Transfected cells were injected subcutaneously in athymic mice. Interestingly, compared with control transfection, (FL)TF expression was associated with reduced tumor growth (mean 7 mg vs 85 mg), while asHTF-expression was associated with enhanced tumor growth (mean 389 mg vs. 85 mg). asHTF expression resulted in increased mitotic index and microvascular density.. These data suggests that asHTF expression promotes tumor growth, and is associated with increased tumor cell proliferation and angiogenesis in vivo. Our results raise a new perspective on the understanding of the relationship between TF expression and cancer growth, by showing a dissociation of the procoagulant activity of (FL)TF and the cancer-promoting activity of asHTF.

Topics: Alternative Splicing; Animals; Blood Coagulation; Cell Division; Cell Line, Tumor; Clone Cells; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Pancreatic Neoplasms; Thromboplastin; Thrombosis; Transfection

Tissue factor (TF) is overexpressed in many malignant tumours and is linked to the pathogenesis and prognosis of such malignancies. In vitro studies have proved that reduced expression of TF has inhibitory effect on the angiogenesis and cell proliferation of the malignant tumour. Therefore, TF suppression has been raised as a possible treatment for malignant tumours. Here we investigated the effect of celecoxib on TF expression induced by tumour necrosis factor alpha (TNFalpha) in PANC-1 cells and a possible molecular mechanism underlying the celecoxib effect.. Various doses of celecoxib solution were added to standard cell numbers of PANC-1 cells mixed with equal dose of TNFalpha for 6 hours. The expression of tissue factor was detected quantitatively by Western blot, whilst the activation of nuclear factor kappaB was tested by electromobility shift assay.. As the doses of celecoxib increased, the tissue factor expression was decreased in PANC-1 cells and so was the activation of nuclear factor kappaB.. Celecoxib can downregulate the expression of tissue factor induced by TNFalpha in PANC-1 cells. This antitumour effect of celecoxib can be explained indirectly via its suppressive role in activation of nuclear factor kappaB.

Topics: Celecoxib; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Gene Expression Regulation; Humans; NF-kappa B; Pancreatic Neoplasms; Pyrazoles; Sulfonamides; Thromboplastin; Tumor Necrosis Factor-alpha

Tissue factor (TF) is a transmembrane glycoprotein that serves as the prime initiator of blood coagulation and plays a critical role in thrombosis and hemostasis. In addition, a variety of tumor cells overexpress cell-surface TF, which appears to be important for tumor angiogenesis and metastasis. To elucidate the mechanism involved in the upregulation of TF in human tumor cells, a comprehensive analysis of TF mRNA from various normal and tumor cells was performed. The results of these studies indicate that, in addition to possessing a normal full-length TF transcript and minor levels of an alternatively spliced transcript known as alternatively-spliced tissue factor (asTF), human tumor cells express additional full-length TF transcripts that are also generated by alternative splicing. Reverse transcriptase-polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA ends- (5'-RACE) based analyses of cytoplasmic RNA from normal and tumor cells revealed that there is alternative splicing of the first intron between exon I and exon II resulting in 2 additional TF transcripts. One of the transcripts has an extended exon I with inclusion of most of the first TF intron (955 bp), while the second transcript is formed by the insertion of a 495 bp sequence, referred to as exon IA, derived from an internal sequence of the first intron. The full length TF transcript with alternatively spliced novel exon IA, referred to as alternative exon 1A-tissue factor (TF-A), represented approximately 1% of the total TF transcripts in normal cells, but constituted 7-10% of the total TF transcript in tumor cells. Quantitative real-time RT-PCR analysis indicated that cultured human tumor cells contain 10-25-fold more copy numbers of TF-A in comparison to normal, untransformed cells. We propose that high-level expression of the novel TF-A transcript, preferentially in tumor cells, may have utility in the diagnosis and staging of a variety of solid tumors.

Topics: Adenocarcinoma; Alternative Splicing; Base Sequence; Carcinoma, Hepatocellular; Carcinoma, Transitional Cell; Cytoplasm; Exons; Humans; Introns; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Molecular Sequence Data; Neoplasm Staging; Neoplasms; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Thromboplastin; Tumor Cells, Cultured; Up-Regulation

To study expression of tissue factor (TF) in pancreatic cancer and its role in the development of thromboembolism.. TF expression was studied in eight human pancreatic carcinoma cell lines by Northern blot and indirect immunofluorescence. Expression of alternatively spliced TF (asTF) was assessed by RT-PCR. In addition, TF expression was determined by immunofluorescence in pancreatic tissues of 19 patients with pancreatic adenocarcinoma (PCa), 9 patients with chronic pancreatitis (CP) and 20 normal controls. Plasma samples (30 PCa-patients, 13 CP-patients and 20 controls) were investigated for soluble TF levels and coagulation activation markers [thrombin-antithrombin III complex (TAT), prothrombin fragment 1 + 2 (F1 + 2)].. All pancreatic carcinoma cell lines expressed TF (8/8) and most of them expressed asTF (6/8). TF expression at the protein level did not correlate with the differentiation of the carcinoma cell line. All but two pancreatic cancer tissue samples stained positive for TF (17/19). In all samples of CP weak staining was restricted to pancreatic duct cells, whereas only a few subendothelial cells were positive in 9/20 of normal controls. TF and TAT levels in PCa patients were significantly elevated compared to controls whereas elevated F1 + 2 levels did not reach statistical significance compared to controls. In CP patients TAT and F1 + 2 levels proved to be significantly elevated compared to controls, although TAT elevation was less pronounced than in PCa patients.. We conclude that in addition to the upregulated expression of TF on the cell membrane, soluble TF might contribute to activation of the coagulation system in pancreatic cancer.

Topics: Adenocarcinoma; Aged; Blood Coagulation; Cell Line, Tumor; Female; Humans; Male; Middle Aged; Pancreatic Neoplasms; Thromboembolism; Thromboplastin

Tissue factor (TF) is a transmembrane glycoprotein that plays roles in the blood coagulation and intracellular signaling pathways, and has also been suggested to modulate the biological behavior of cancer cells. In order to examine the clinicopathologic significance of TF expression in pancreatic ductal adenocarcinoma, TF expression was determined by immunohistochemistry using a newly raised anti-TF monoclonal antibody in 113 patients who had undergone surgical resection of pancreatic ductal adenocarcinoma. According to the incidence of tumor cell immunopositivity, patients were divided into "negative TF" (0%), "weak TF" (<25%), or "high TF" (25% or more) groups, which accounted for 11.6% (n = 13), 44.2% (n = 50), and 44.2% (n = 50) of the total, respectively. Increased TF expression was correlated with the extent of the primary tumor (P = 0.0043), lymph node metastasis (P = 0.0043), lymphatic distant metastasis (P = 0.0039), advanced tumor-node-metastasis stage (P = 0.0002), and high tumor grade (P = 0.0164). Multivariate analysis using the Cox proportional hazards model showed that high TF expression was an independent negative predictor for survival (hazard ratio, 2.014; P = 0.0076). Moreover, patients with TF-negative tumors had a significantly better prognosis even if lymph node metastasis was present (P < 0.0001). We also showed that TF knockdown by RNA interference suppressed the invasiveness of a pancreatic adenocarcinoma cell line in vitro. These results indicate that TF expression may contribute to the aggressiveness of pancreatic ductal adenocarcinoma by stimulating tumor invasiveness, and that evaluation of the primary tumor for TF expression may identify patients with a poor prognosis.

Topics: Aged; Aged, 80 and over; Animals; Antibodies, Monoclonal; Antibody Specificity; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Middle Aged; Multivariate Analysis; Neoplasm Staging; Pancreatic Neoplasms; Prognosis; RNA Interference; RNA, Small Interfering; Survival Analysis; Thromboplastin

Blood coagulation is activated commonly in pancreatic carcinoma but the role of the tumor cell in this activation is undefined. Immunohistochemical procedures were applied to fixed sections of 22 cases of resected adenocarcinoma of the pancreas to determine the presence of components of coagulation and fibrinolysis pathways in situ. Tumor cell bodies stained for tissue factor: prothrombin: and factors VII, VIIIc, IX, X, XII, and subunit "a" of factor XIII. Fibrinogen existed throughout the tumor stroma, and tumor cells were surrounded by fibrin. Staining for tissue factor pathway inhibitor, and plasminogen activators was minimal and inconsistent. Plasminogen activator inhibitors -1, -2, and -3 were present in the tumor stroma, and on tumor cells and vascular endothelium. Extravascular coagulation activation exists associated with pancreatic carcinoma cells in situ that is apparently unopposed by naturally occurring inhibitors or the plasminogen activator-plasmin system. We postulate that such local coagulation activation may regulate growth of this malignancy. These findings provide a rationale for testing agents that modulate the blood coagulation/fibrinolytic system (that inhibit tumor growth in other settings) in pancreatic carcinoma.

Topics: Adenocarcinoma; Aged; Blood Coagulation Factors; Endothelium, Vascular; Female; Fibrin; Fibrinogen; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; Plasminogen Activator Inhibitor 2; Protein C; Protein S; Prothrombin; Stromal Cells; Thrombophilia; Thromboplastin

A 54-year-old man was diagnosed as having pancreatic cancer and disseminated intravascular coagulation. His plasma tissue factor level on the 11th hospital day was 996 pg/ml (normal range, 120-270 pg/ml). He was treated with gabexate mesilate, antithrombin III, and low-molecular-weight heparin. However, he died of multiple organ failure on the 17th hospital day. The histological finding was poorly differentiated ductal adenocarcinoma of the pancreas, and the production of tissue factor in this lesion was revealed. Tissue factor is a factor that initiates blood coagulation; thus, its expression in pancreatic cancer is one of the causes of coagulation abnormalities in this disease. Although one report has demonstrated immunoreactivity for tissue factor in pancreatic cancer, the patient's detailed clinical course was not mentioned in that report. This is the first report to prove that pancreatic cancer produced tissue factor in a patient with disseminated intravascular coagulation.

Topics: Adenocarcinoma; Disseminated Intravascular Coagulation; Humans; Male; Middle Aged; Pancreatic Neoplasms; Radiography; Thromboplastin

Tissue factor (TF), the physiological procoagulant, is expressed in pancreatic tissue as a result of malignant transformation. The aim of this investigation was to assess its role in pancreatic tumour cell invasion and primary tumour growth.. The full-length TF gene (1360 base pairs) was cloned into the plasmid DNA vector pcDNA3 in sense and antisense orientations, and these vectors were used to transfect the MIA PaCa-2 human pancreatic adenocarcinoma cell line. TF gene expression was characterized by Northern blot analysis, total cellular antigenic content by enzyme-linked immunosorbent assay and cell surface procoagulant activity by enzymatic assay. Invasion of tumour cells in vitro was determined by a standard Matrigel assay, and primary tumour growth was measured in immunodeficient mice.. Overexpression of the TF gene, confirmed by an increased signal on Northern blotting, was associated with increases in both total antigenic content for TF (P = 0.001) and cell surface procoagulant activity (P = 0.008) in sense cells compared with wild-type cells. Likewise, both in vitro tumour cell invasion (P = 0.001) and primary tumour growth (P = 0.007) were increased in sense transfectants.. Expression of TF enhances in vitro invasion and primary tumour growth of MIA PaCa-2 cells, suggesting that this procoagulant molecule might have a role in pancreatic tumour biology. Presented in part to the 83rd meeting of the Surgical Research Society, Oxford, UK, January 1996 and awarded the David Patey Prize, and in part to the 1997 Annual Meeting of the Association of Surgeons of Great Britain and Ireland, Bournemouth, UK, April 1997.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Cell Division; Female; Humans; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Transplantation; Pancreatic Neoplasms; RNA, Messenger; Thromboplastin; Transfection; Tumor Cells, Cultured

Overexpression of tissue factor (TF) is characteristically observed in advanced pancreatic cancer and has been associated with invasion and metastasis. Functional responses of TF activation are here investigated using as a model system the human pancreatic cancer cell lines SW979 (which overexpresses TF) and MIAPaCa2 (which does not express detectable levels). After stimulation of these cell lines with factor VIIa (FVIIa), the only known TF ligand, expression of urokinase receptor (uPAR) gene was up-regulated in SW979 cells in a dose-dependent manner but not in MIAPaCa2 cells. Interestingly, urokinase (uPA) and its specific inhibitor PAI-1 were not up-regulated. Exposure to functionally inactivated FVIIa did not show any effect on uPAR expression on SW979 cells despite binding to TF with higher efficiency. The neutralizing anti-TF antibody 5G9 blocked the FVIIa-induced up-regulation of uPAR completely, whereas hirudin failed to block this up-regulation. Treatment of SW979 cells with Factor Xa did not up-regulate the expression of uPAR gene, whereas treatment with FVII induced the same level of enhanced uPAR gene expression as that with FVIIa. In the matrigel invasion assay, enhanced invasion of SW979 cell line induced by FVIIa was completely inhibited by anti-TF antibody and alpha2-antiplasmin. Moreover, the endogenous levels of uPAR gene expression were significantly correlated with the level of TF gene expression in 19 human cancer cell lines (P < 0.05). These data suggest that up-regulation of uPAR expression by tumor cells leading to tumor invasion is induced through the TF-FVIIa pathway rather than TF-initiated thrombin generation. This is the first report that TF may be one of the key receptors that can up-regulate expression of the plasminogen activator receptor in human cancer cells to enhance tumor invasion and metastasis.

Topics: Breast Neoplasms; Cell Movement; Factor VIIa; Factor Xa; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Thromboplastin; Transcription, Genetic; Tumor Cells, Cultured; Up-Regulation; Urokinase-Type Plasminogen Activator

The transmembrane cellular receptor tissue factor (TF) is the primary initiator of the coagulation cascade in man. Expression of TF was determined in 55 specimens of ductal adenocarcinoma of the pancreas and was found to correlate strongly with the degree of histological differentiation. A significant linear trend was observed with stronger immunoreactivity observed in poorly differentiated tumours (chi 2 = 6.69, P = 0.0098). No TF staining was seen in pancreatic samples from normal controls (n = 18). As expression of TF may be associated with tumour progression, its analysis could provide useful prognostic information in patients with pancreatic malignancy.

Topics: Adult; Aged; Carcinoma, Ductal, Breast; Humans; Immunohistochemistry; Middle Aged; Pancreatic Neoplasms; Thromboplastin

Tissue factor-like activity was measured in the plasma of 30 patients with disseminated intravascular coagulation(DIC) and 22 patients without DIC using a chromogenic substrate. Twenty-three of the 30 patients with DIC (77%) exhibited tissue factor-like activity levels above normal range (greater than 3.0 U/L), and in eleven of these patients, the levels were more than 10 U/L. Of the 22 patients without DIC, seven patients had elevated levels (3-10 U/L), and had a possibility to be developing DIC. So, we considered them to be in a pre-DIC state. No correlation was found between tissue factor-like activity and alpha 2 plasmin inhibitor-plasmin complex or FDP-D dimer. In a patient with acute monocytic leukemia, the elevated tissue factor-like activity (84.4 U/L) rapidly decreased after the initiation of chemotherapy, whereas in a patient with pancreatic cancer, the level remained elevated (67.4-79.2 U/L). These results suggested that the plasma tissue factor-like activity is differ from the other parameters reflecting the process of DIC and is a useful indicator of the presence of an initiating factor of blood coagulation in some selected patients with DIC or pre-DIC.

Topics: Adult; alpha-2-Antiplasmin; Antifibrinolytic Agents; Cerebral Infarction; Disseminated Intravascular Coagulation; Female; Fibrin Fibrinogen Degradation Products; Fibrinolysin; Humans; Leukemia, Monocytic, Acute; Male; Middle Aged; Pancreatic Neoplasms; Thromboplastin

Coagulation system and platelets play an important role in the stage of lodgement of tumor cells. We examined abilities of human and hamster pancreatic cancer cell lines to aggregate platelets in vitro, and investigated the effect of prostaglandin E1, I2, on artificial liver metastases of pancreatic cancer in Syrian golden hamster. Platelet aggregating activities were found in five out of six human pancreatic cancer cell line and thromboplastin likes activity in five cell lines. Diisopropanolnitrosamine induced hamster pancreatic cancer cells (HPK-1) were able to aggregate platelets both in vitro and in vivo and these activities were inhibited by prostaglandin I2. Hamster was inoculated intraportally with 1 X 10(6) HPK-1 cells. After two weeks autopsy of these hamsters revealed multiple metastatic nodules on liver surface. In this model we administered prostaglandin E1, I2 into the portal vein five minutes before cell inoculation. Number of liver surface nodules were significantly decreased to 33.1 + 7.0, 11.0 + 9.6 in hamster given 100g PGE1 PGI2 before cell inoculation, compared with control group of hamsters (62.0 + 6.6 PH9.3, 66.1 + 13.9 PH7.4). But administration of prostaglandin after cell injection was not effective. In all cases none of extrahepatic metastases were noted. Inhibitory action of PGE1 PGI2 on liver metastasis is suspected to be related to inhibition of platelet aggregation.

Topics: Alprostadil; Animals; Carcinogens; Cricetinae; Epoprostenol; Humans; Liver Neoplasms; Male; Mesocricetus; Nitrosamines; Pancreatic Neoplasms; Platelet Aggregation; Platelet Aggregation Inhibitors; Thromboplastin; Tumor Cells, Cultured

We have studied the effects of two human pancreatic cancer and two human small cell lung cancer cell lines on clotting and platelet aggregation. Both pancreatic lines markedly shortened recalcification times and induced platelet aggregation. The lung cancer lines produced little shortening of recalcification times and no platelet aggregation. The clotting and aggregation activities of the pancreatic lines were further characterized. Recalcification times following the addition of cancer cell line material to plasmas deficient in factors VII and X were markedly prolonged, suggesting that the activity is due to tissue factor. Hirudin, an inhibitor of thrombin from the saliva of leeches, and rabbit polyclonal immunoglobulin G anti-bovine brain tissue factor inhibited both procoagulant and aggregation activities. Apyrase (an enzyme degrading ADP), diisopropylfluorophosphate (a serine protease inhibitor) and L-trans-epoxysuccinylleucylamido(4-guanidino)butane (a cysteine protease inhibitor) failed to inhibit these activities. Increasing concentrations of heparin inhibited platelet aggregation. Subcellular fractionation studies showed these activities to be localized to the plasma membrane. The association between mucin and the acceleration of clotting has been well described. The absence of mucin in electron micrographs of these pancreatic whole cells, membrane fractions, and shed microvesicles, as well as the failure of chaotropic agents (i.e., agents stripping material extrinsic to the cell membrane such as mucin) to abrogate this activity support these activities being intrinsic to the plasma membrane. These data strongly suggest that these activities are due to tissue factor which appears to be released as microvesicles in vitro. The release of tissue factor via microvesicles in vivo is one possible mechanism for the coagulopathy sometimes seen in patients with pancreatic carcinoma.

Topics: Adenocarcinoma; Blood Coagulation; Calcium; Carcinoma, Small Cell; Cell Membrane; Humans; Lung Neoplasms; Microscopy, Electron; Pancreatic Ducts; Pancreatic Neoplasms; Platelet Aggregation; Thromboplastin; Tumor Cells, Cultured

We measured in 193 patients, admitted to our wards for symptoms and signs suggestive of pancreatic or digestive malignancy, the serum levels of five tumor-associated antigens (CA 19-9, CA 50, CA 125, TPA, CEA) and we evaluated their diagnostic accuracy both when used alone and in combination. For CA 19-9 and CA 50 a sensitivity for pancreatic cancer as high as 92 and 88%, respectively, and specificity of 91.8% were found. A lower sensitivity vs. pancreatic cancer was found for the other tumor markers, and vs. the other digestive and nondigestive malignancies for all tumor markers (apart for CA 19-9 and CA 50 vs. biliary carcinomas). As for the combined assays, the best figures were found vs. pancreatic cancer for CA 19-9 plus CA 50, CA 50 plus CEA, CA 50 plus CA 125; a sensitivity by far worse vs. the other gastrointestinal cancers was found for all the possible combinations. We conclude that in selected symptomatic patients some tumor-marker determinations can be useful in identifying those with a high probability of harboring a pancreatic cancer, to be further studied or operated upon. The clinical relevance of this in patients already symptomatic is at present unclear.

Topics: Adult; Aged; Antibodies; Antibodies, Monoclonal; Antigens; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Carcinoembryonic Antigen; Female; Humans; Male; Middle Aged; Pancreatic Neoplasms; Thromboplastin

Topics: Blood Cell Count; Blood Coagulation; Blood Platelets; Blood Protein Disorders; Cholestasis; Diagnosis, Differential; Duodenal Neoplasms; Factor VII; Hemorrhage; Heparin; Humans; Liver Neoplasms; Neoplasm Metastasis; Pancreatic Neoplasms; Syndrome; Thromboplastin

Topics: Humans; Kidney Diseases; Pancreatic Neoplasms; Thromboplastin