Abstract
Metastatic cancer is associated with a hypercoagulable state, and pathological venous thromboembolic disease is a significant source of morbidity and the second leading cause of death in patients with cancer. Here we aimed to develop a novel labeling strategy to detect and quantify procoagulant circulating tumor cells (CTCs) from patients with metastatic cancer. We hypothesize that the enumeration of procoagulant CTCs may be prognostic for the development of venous thrombosis in patients with cancer. Our approach is based on the observation that cancer cells are capable of initiating and facilitating cell-mediated coagulation in vitro, whereby activated coagulation factor complexes assemble upon cancer cell membrane surfaces. Binding of fluorescently labeled, active site-inhibited coagulation factors VIIa, Xa, and IIa to the metastatic breast cancer cell line, MDA-MB-231, non-metastatic colorectal cell line, SW480, or metastatic colorectal cell line, SW620, was characterized in a purified system, in anticoagulated blood and plasma, and in plasma under conditions of coagulation. We conclude that a CTC labeling strategy that utilizes coagulation factor-based fluorescent probes may provide a functional assessment of the procoagulant potential of CTCs, and that this strategy is amenable to current CTC detection platforms.
Highlights
IntroductionA method to identify which cancer patients are at imminent risk to develop thrombosis would allow for an objective means by which to administer personalized anticoagulant prophylaxis, reducing the morbidity, and mortality for patients with cancer
CLOTTING TIMES To investigate whether the metastatic breast cancer cell line, MDAMB-231, non-metastatic colorectal cell line, SW480, or metastatic colorectal cell line, SW620, were sufficient to initiate and propagate blood coagulation, washed cells were suspended in serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) and added to recalcified human plasma
Our results show that the anti-tissue factor (TF) mAb abrogated the ability for SW480 and SW620 cells to clot plasma, and prolonged the clotting times for MDA-MB-231 cells (288.6 s vs. 38.5 s, respectively)
Summary
A method to identify which cancer patients are at imminent risk to develop thrombosis would allow for an objective means by which to administer personalized anticoagulant prophylaxis, reducing the morbidity, and mortality for patients with cancer. Activation of coagulation is restricted to sites of blood vessel injury through the localized exposure of tissue factor (TF), a transmembrane protein constitutively expressed by extravascular tissue not normally exposed to the circulating blood. The TF·FVIIa complex initiates the extrinsic blood coagulation pathway by activating factor X (FX) and factor IX (FIX). Activated factor X (FXa) and activated factor IX (FIXa) are initially inhibited by TF pathway inhibitor (TFPI) present in blood at a low concentration (∼2.4 nM) by forming a quaternary FXa·TF·FVIIa·TFPI complex (Baugh et al, 1998; Lu et al, 2004). Pathologically excessive coagulation, or the initiation of coagulation at sites other than blood vessel injury, can result in thrombosis
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