thromboplastin and Osteosarcoma

Coagulation activation is associated with cancer progression and morbidity. Recently, mechanisms through which coagulation proteases drive the tumor microenvironment (TME) have been elucidated. This review aims to develop a new strategy dependent on the coagulation system for treating osteosarcoma (OS). We focused on tissue factor (TF), the main initiator of the extrinsic coagulant pathway, as a target for OS treatment. It was found that cell surface-TF, TF-positive extracellular vesicles, and TF-positive circulating tumor cells could drive progression, metastasis, and TME in carcinomas, including OS. Thus, targeting tumor-associated coagulation by focusing on TF, the principle catalyst of the extrinsic pathway, TF is a promising target for OS.

Topics: Blood Coagulation; Bone Neoplasms; Humans; Osteosarcoma; Thromboplastin; Tumor Microenvironment

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.

Topics: Carcinogenesis; Cell Line, Tumor; Enhancer Elements, Genetic; Epigenomics; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Lung Neoplasms; Neoplasm Metastasis; Osteosarcoma; Proteins; Selection, Genetic; Thromboplastin; Transcription Factor AP-1; Tumor Microenvironment

Over the last three decades, the prognosis of osteosarcoma has remained unchanged; the prognosis for patients with lung metastasis is still poor, and the development of new treatments is urgently required. We previously showed that aggressive osteosarcoma cells express more tissue factor (TF) and demonstrate enhanced extrinsic pathway capacity. Furthermore, tumor growth can be suppressed with the anticoagulant low molecular weight heparin. However, the molecular mechanisms underlying TF regulation are still unclear. Here, we report that transforming growth factor-β (TGF-β) upregulates TF, which can occur via activated platelets. TF was found to be expressed on osteosarcoma cell surfaces, which mediated the production of Xa and thrombin. TF induction by TGF-β was observed in several osteosarcoma cells, and especially in MG 63 cells. Both TF expression by TGF-β and extrinsic pathway activity through TF were rapidly increased. This reaction was inhibited by a TGF-β type I receptor inhibitor and TGF-β neutralizing antibody. Although TGF-β was found to phosphorylate both Smad2 and Smad3, their roles were markedly disparate. Surprisingly, Smad2 knockdown resulted in no inhibitory effect, whereas Smad3 knockdown completely suppressed TGF-β-induced TF expression. Next, data suggested that platelets were the source of TGF-β. We confirmed that thrombin-activated platelets and osteosarcoma cells could release TGF-β, and that platelet-derived TGF-β could induce TF expression. These processes were also inhibited by a TGF-β type I receptor inhibitor and Smad3 knockdown. Moreover, CD42b, TF, TGF-β, Smad2/3, and p-Smad2/3 were also detected in a biopsy sample from an osteosarcoma patient. Collectively, these finding suggested that the interaction between osteosarcoma cells and platelets, via thrombin and TGF-β, results in a continuous cycle, and that anti-platelet or anti-TGF-β therapy could be a promising tool for disease treatment. © 2018 American Society for Bone and Mineral Research.

Topics: Animals; Blood Platelets; Cell Line, Tumor; Cell Membrane; Humans; Intracellular Space; Mice; Models, Biological; Osteosarcoma; Phosphorylation; Signal Transduction; Smad2 Protein; Smad3 Protein; Thrombin; Thromboplastin; Transforming Growth Factor beta

Osteosarcoma is the most common primary malignant bone tumour. Patients often develop lung metastasis and have a poor prognosis despite extensive chemotherapy and surgical resections. Tissue Factor is associated with poor clinical outcome in a wide range of cancer types, and promotes angiogenesis and metastasis. The role of Tissue Factor in OS tumourigenesis is unknown. Fifty-three osteosarcoma pre-treatment biopsies and four osteosarcoma cell lines were evaluated for Tissue Factor expression, and a possible association with clinical parameters was investigated. Tissue Factor function was inhibited in an osteosarcoma cell line (143B) by shRNA knockdown or specific antibodies, and pro-tumourigenic gene expression, proliferation, matrigel invasion and transwell migration was examined. 143B cells were implanted in mice in the presence of Tissue Factor-blocking antibodies, and tumour volume, micro-vessel density and metastases in the lung were evaluated. Tissue Factor was highly expressed in 73.6 % of osteosarcoma biopsies, and expression associated significantly with disease-free survival. Tissue Factor was expressed in all four investigated cell lines. Tissue Factor was knocked down in 143B cells, which led to reduced expression of IL-8, CXCL-1, SNAIL and MMP2, but not MMP9. Tissue Factor knockdown or inhibition with antibodies reduced matrigel invasion. Tissue Factor antibodies limited 143B tumour growth in vivo, and resulted in decreased intra-tumoural micro-vessel density. Furthermore, lung metastasis from the primary tumour was significantly reduced. Thus, Tissue Factor expression in osteosarcoma reduces metastasis-free survival in patients, and increases pro-tumourigenic behaviour both in vitro and in vivo.

Topics: Adolescent; Animals; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Child; Disease-Free Survival; Female; Gene Expression; Gene Knockdown Techniques; Humans; Male; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Osteosarcoma; RNA, Messenger; RNA, Neoplasm; Thromboplastin; Young Adult

To measure thrombin generation by high and low tissue factor (TF)-expressing canine cancer cell lines.. Canine cell lines CMT25 (high TF-expressing mammary gland tumor cell line) and HMPOS (low TF-expressing osteosarcoma cell line).. Thrombin generation by cancer cells was measured in pooled normal canine plasma by use of calibrated automated thrombography without added trigger reagents. Results were expressed as lag time, time to peak thrombin concentration, peak thrombin concentration, and total thrombin concentration or thrombin generation potential. Corn trypsin inhibitor, hirudin, and annexin V were used to inhibit contact activation, thrombin formation, and phosphatidylserine activity, respectively. Pooled normal human plasma deficient in coagulation factors VII, VIII, IX, X, XI, or XII was used to assess the role of individual coagulation factors on thrombin generation.. CMT25 generated significantly more thrombin than did HMPOS (mean ± SD, 3,555 ± 604 nM thrombin•min and 636 ± 440 nM thrombin•min, respectively). Thrombin generation of CMT25 was dependent on factor VII and phosphatidylserine and was independent of contact activation. In contrast, thrombin generation of HMPOS was attributed to contact activation.. High TF-expressing canine mammary cancer cells generated thrombin in a plasma milieu in vitro in a factor VII- and phosphatidylserine-dependent manner. These findings support a role for TF in hypercoagulability detected in dogs with mammary gland tumors and potentially for other tumors that strongly express TF.

Topics: Animals; Blood Coagulation Factors; Cell Line, Tumor; Dog Diseases; Dogs; Female; Male; Mammary Glands, Animal; Mammary Neoplasms, Animal; Osteosarcoma; Thrombin; Thromboplastin

The cells responsible for bone formation express protease-activated receptors. Although serine protease thrombin has been shown to elicit functional responses in bone cells that impact on cell survival and alkaline phosphatase activity, nothing is known about tissue factor, factor VIIa, and factor Xa, the serine proteases that act upstream of thrombin in the coagulation cascade. This paper demonstrates that tissue factor is expressed in the osteoblast-like cell line SaOS-2 and, that tissue factor in a factor VIIa-bound complex induces a transient intracellular Ca(2+) increase through protease-activated receptor-2. In SaOS-2 cells, factor Xa induced a sustained intracellular Ca(2+) response, as does SLIGRL, a PAR2-activating peptide, and PAR-1-dependent cell viability.

Topics: Alternative Splicing; Calcium; Calcium Signaling; Cell Line; Cell Line, Tumor; Cell Survival; Factor VIIa; Factor Xa; Gene Expression; Humans; Immunohistochemistry; Osteosarcoma; Peptide Fragments; Reverse Transcriptase Polymerase Chain Reaction; Thromboplastin; Time Factors

Tissue factor (TF), a transmembrane glycoprotein, plays a role in the initiation of blood coagulation at sites of vascular injury. Activated products of coagulation may then enhance inflammatory responses. The present investigation assesses the ability of rat osteosarcoma (UMR-106) cells cultured on titanium alloy (Ti6Al4V) to express differential surface TF activity in response to cyclic mechanical strain. Strains ranged from -2000 micro-strain to +2000 micro-strain, and durations from 5, 10, and 20 min per day over 5 days to 24 h continuous stimulation. ROS cells exhibited significant TF activity as demonstrated by the conversion of Factor X to Factor Xa. Strains of +2000 micro-strain with 5-20-min duration exhibited decreased TF activity with duration from 1.4E-04 nM/cell to 8.7E-05 nM/cell. Additionally, ROS cells stimulated with calcium ionophore (A23187) exhibited at least twice the activity of nonstimulated cells. Strains of +1340 micro-strain with 5-20-min duration exhibited an increasing trend with 4.15E-05 nM/cell to 7.38E-05 nM/cell. Strain direction had no significant effect on TF activity. Thus, both mechanical and chemical stimuli induce differential expression of TF activity by ROS cells cultured on Ti6Al4V, a phenomenon that may potentiate or regulate the inflammatory responses associated with the implantation of orthopedic biomaterials.

Topics: Alloys; Animals; Biocompatible Materials; Calcimycin; Cell Culture Techniques; Cell Line, Tumor; Ionophores; Osteosarcoma; Rats; Stress, Mechanical; Surface Properties; Thromboplastin; Titanium

Tissue factor (TF), a transmembrane glycoprotein expressed by numerous cell types, plays a critical role in the initiation of blood coagulation at sites of vascular injury. Activated products of the coagulation cascade may then enhance the inflammatory responses associated with wound healing. In the present investigation the ability of rat osteosarcoma (ROS) cells to express TF activity was examined following their growth on tissue-culture polystyrene (TCPS) and selected orthopedic biomaterials (titanium and zirconium alloys, and stainless steel). ROS cells exhibited significant TF activity as evidenced by the conversion of Factor X to Factor Xa in the presence of TF, Factor VIIa, and Ca2+. Factor Xa concentrations ranged from 1.0 fM per cell at 10 min to 6.0 fM per cell after 60 min. Additionally, ROS cells stimulated with calcium ionophore (A23187) exhibited approximately twice the activity of non-stimulated cells when grown on TCPS but not on the metallic substrates. ROS cells (stimulated or unstimulated) adherent to the zirconium alloy generated lower amounts of Factor Xa compared to those bound to the other alloys and unstimulated cells grown on TCPS. These results indicate that ROS cells cultured on these synthetic surfaces differentially express procoagulant activity and that cells grown on TCPS, but not the metallic alloys, exhibit increased TF activity in response to stimulation by calcium ionophore. This procoagulant activity may potentiate subsequent inflammatory responses associated with the use of orthopedic biomaterials and thereby influence the tissue compatibility of the implant.

Topics: Alloys; Animals; Biocompatible Materials; Calcimycin; Cell Adhesion; Colorimetry; Factor VII; Factor X; Gene Expression Regulation; Ionophores; Orthotic Devices; Osteosarcoma; Polystyrenes; Rats; Stainless Steel; Thromboplastin; Titanium; Tumor Cells, Cultured; Zirconium

Systemic infusion of low concentrations of tumor necrosis factor/cachectin (TNF) into mice that bear TNF-sensitive tumors leads to activation of coagulation, fibrin formation, and occlusive thrombosis exclusively within the tumor vascular bed. To identify mechanisms underlying the localization of this vascular procoagulant response, a tumor-derived polypeptide has been purified to homogeneity from supernatants of murine methylcholanthrene A-induced fibrosarcomas that induces endothelial tissue factor synthesis and expression (half-maximal response at approximately 300 pM), and augments the procoagulant response to TNF in a synergistic fashion. This tumor-derived polypeptide was identified as the murine homologue of vascular permeability factor (VPF) based on similar mobility on SDS-PAGE, an homologous NH2-terminal amino acid sequence, and recognition by a monospecific antibody to guinea pig VPF. In addition, VPF was shown to induce monocyte activation, as evidenced by expression of tissue factor. Finally, VPF was shown to induce monocyte chemotaxis across collagen membranes and endothelial cell monolayers. Taken together, these results indicate that VPF can modulate the coagulant properties of endothelium and monocytes, and can promote monocyte migration into the tumor bed. This suggests one mechanism through which tumor-derived mediators can alter properties of the vessel wall.

Topics: Amino Acid Sequence; Animals; Blood Coagulation Factors; Cell Line; Cells, Cultured; Chemotaxis, Leukocyte; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Culture Media; Endothelial Growth Factors; Endothelium, Vascular; Guinea Pigs; Humans; Immune Sera; Leukocytes, Mononuclear; Lymphokines; Mice; Molecular Sequence Data; Osteosarcoma; Recombinant Proteins; Sequence Homology, Nucleic Acid; Thromboplastin; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Cultured SK-OS-10 cells (human osteogenic sarcoma metastatic to lung) shed microvesicles (dia. 300-1000 nm) that contained procoagulant and proaggregatory activities inhibitable by hirudin, by anti-tissue factor antibody and by phospholipase A2. These results show that SK-OS-10 cells belong to a group including U87MG human glioblastoma and HL-60 promyelocytic leukemia in which these activities are due to a thrombin-dependent mechanism arising from the presence of tissue factor on the surface of the tumor cells and their shed microvesicles.

Topics: Antibodies; Blood Platelets; Cell Line; Creatine Kinase; Hirudins; Humans; Inclusion Bodies; Lung Neoplasms; Osteosarcoma; Phosphocreatine; Phospholipases; Platelet Activating Factor; Platelet Aggregation; Thromboplastin