Phosphatidylserine (PS) exposure occurs during the cell death program and fluorescein-labeled lactadherin permits the detection of PS exposure earlier than annexin V in suspended cell lines. Adherent cell lines were studied for this apoptosisassociated phenomenon to determine if PS probing methods are reliable because specific membrane damage may occur during harvesting. Apoptosis was induced in the human tongue squamous carcinoma cell line (Tca8113) and the adenoid cystic carcinoma cell line (ACC-2) by arsenic trioxide. Cells were harvested with a modified procedure and labeled with lactadherin and/or annexin V. PS exposure was localized by confocal microscopy and apoptosis was quantified by flow cytometry. The detachment procedure without trypsinization did not induce cell damage. In competition binding experiments, phospholipid vesicles competed for more than 95 and 90% of lactadherin but only about 75 and 70% of annexin V binding to Tca8113 and ACC-2 cells. These data indicate that PS exposure occurs in three stages during the cell death program and that fluorescein-labeled lactadherin permitted the detection of early PS exposure. A similar pattern of PS exposure has been observed in two malignant cell lines with different adherence, suggesting that this pattern of PS exposure is common in adherent cells. Both lactadherin and annexin V could be used in adherent Tca8113 and ACC-2 cell lines when an appropriate harvesting procedure was used. Lactadherin is more sensitive than annexin V for the detection of PS exposure as the physical structure of PS in these blebs and condensed apoptotic cell surface may be more conducive to binding lactadherin than annexin V.
Background: Cancer patients are considered to be prothrombotic with major disturbances in hemostasis that are associated with an increased risk of venous thromboembolism, especially in patients with advanced cancer. Recently reports show that the high levels of circulating microparticles (MPs) have procoagulant activity (PCA) in oral squamous cell carcinoma (OSCC). However, this study did not address the question of what specific mechanism might underlie the PCA in OSCC. Neutrophil extracellular traps (NETs) are activated neutrophil-derived web-like structures, which have emerged as important mediators in cancer progression, metastasis and cancer-associated thrombosis. Additionally, the cytokines and neutrophils were known to become aggregated in cancers and are usually present in high numbers in OSCC patients and are associated with poor outcomes. The exact molecular mechanisms responsible for modulation of neutrophils procoagulant functions in OSCC are, however, poorly understood. Thus, we hypothesized that cytokines might activate neutrophils to release NETs, thereby predisposing OSCC patients to a hypercoagulative state. Moreover, we evaluated NETs interaction with human umbilical vein endothelial cells (HUVECs) and their association with pathological lesions in this disease. Methods: OSCC patients (n = 58) were divided into four stages according to the 2009 guidelines of the American Joint Committee on Cancer staging classification, and compared to healthy controls (n = 25). Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. ELISA was used to detect MPO-DNA complexes, TAT (thrombin-antithrombin) complexes, neutrophil elastase, nucleosomes, and cytokines. PCA of NETs was evaluated using coagulation time and purified coagulation complex and fibrin production assays. Phosphatidylserine (PS) exposure, fibrin strands, and FVa/Xa binding on cells were observed using confocal microscopy. Results:Plasma levels of NET markers in patients with stage III/IV OSCC were significantly higher than those in stage I/II patients or controls (all p<0.05), and positively correlated with thrombin-antithrombin (TAT) complex and fibrinogen levels. Interestingly, neutrophils from OSCC patients with stage III/IV were more prone to release NETs compared to those from stage I/II patients and controls. Additionally, we found that plasma from patients with stage III/IV OSCC was able to prime neutrophils to generate higher amounts of NETs than from stage I/II patients and controls. Depleting IL-8, IL-6 and TNF-a reduced plasma-enhance NETs release. In addition, NETs released by stage III/IV OSCC neutrophils significantly increased the potency of control plasma to generate thrombin and fibrin, greatly shortened the coagulation time (all p<0.05). These effects were attenuated by DNase I. Finally, isolated NETs induced ECs to lose normal morphology and retract from their cell-cell junctions, converting them to a pro-coagulant phenotype. DNase I attenuated this cytotoxicity. Conclusion s :These results suggest that OSCC creates a systemic inflammation environment that primes neutrophils to release procoagulant NETs in patients with stage III/IV OSCC. The NETs formation correlated positively with the parameters of disease severity. The information that results from these investigations may serve as a rational basis for the design of future drug intervention trials that target coagulation reactions, mechanisms and/or interactions relevant to OSCC. Disclosures No relevant conflicts of interest to declare.
The specific function of phosphatidylserine (PS) in the context of the development of a hypercoagulable state among individuals with oral squamous cell carcinoma (OSCC) is uncertain. The goal of this study was therefore to assess the exposure of PS on microparticles (MPs) as well as on endothelial and blood cells and to assess procoagulant activity (PCA) as a function of the stage of OSCC progression. We recruited patients with OSCC ( n = 63) as well as healthy controls ( n = 26) to participate in this study. PS exposure was then assessed via confocal microscopy and flow cytometry, revealing that patients with stage III/IV OSCC exhibited higher frequencies of PS-exposing blood cells, MPs, and serum-cultured endothelial cells (ECs) than did patients with stage I/II OSCC or healthy controls. When we conducted functional coagulation assays, we discovered that PS+blood cells, MPs, and serum-cultured ECs from patients with stage III/IV OSCC mediated more rapid coagulation and more substantial production of FXa, thrombin, and fibrin as compared with controls. When samples were treated with the PS antagonist lactadherin, this resulted in an 80% disruption of PCA. Strikingly, when pre- and postoperative samples were compared from patients with stage III/IV OSCC undergoing resective surgery, PCA was significantly reduced in the postoperative samples. After stimulating ECs with inflammatory cytokines, we found by confocal microscopy that they expose PS on their cell membranes, thus generating FVa and FXa binding sites and mediating the formation of fibrin. Together our findings provide evidence that PS+blood cells and MPs are important mediators of the development of a hypercoagulable and prothrombotic state among individuals afflicted by advanced-stage OSCC. As such, a PS blockade may be a viable therapeutic strategy for treating such patients.
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