Development of Coagulation Factor Probes for the Identification of Procoagulant Circulating Tumor Cells
Authors: Cianchetti Flor A. , Tormoen Garth W. , Bock Paul E. , McCarty Owen J.T. 
Published: October 2019

Abstract
Abstract 634
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. Patients with cancer have a 4–10 fold increased risk of developing thrombosis. Recurrent thrombosis can be clinically managed with anticoagulant therapy; however, the risk of bleeding complications associated with the use of anticoagulants has prevented routine prophylactic anticoagulation for patients with cancer who have not yet developed thrombosis. Therefore, 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. There is currently a lack of laboratory assays capable of identifying which patients with cancer are at risk of developing thrombosis.
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. In this study, we characterized the binding of fluorescently-labeled active site-inhibited factors VIIa, Xa and IIa to the metastatic breast cancer cell line, MDA-MB-231, the non metastatic colorectal cell line, SW480, or the metastatic colorectal cell line, SW620, either in a purified system, in plasma, or in whole blood.
Using flow cytometry, labeling of cancer cells in a purified system showed cell and factor-specific characteristics for labeling efficacy. Our data show that a concentration of 50 nM FVIIa-based probe was sufficient to label both the MDA-MB-231 and SW620 cells, while a concentration of 500 nM of the FXa- or FIIa-based probes was required to label both MDA-MB-231 and SW620 cells. A concentration of 0.5 μM FVIIa, 5 μM FXa and 5 μM FIIa was shown to label MDA-MB-231 and SW620 cells in anticoagulated plasma and in plasma under conditions of coagulation.
We designed a series of experiments to determine whether our labeling strategy was amenable to a cell processing protocol that utilizes cancer cells being plated onto glass slides. We immobilized MDA-MB-231, SW480, and SW620 cells on functionalized glass surfaces and exposed them to fluorescently labeled FVIIa (0.5 μM), FXa (5 μM), FIIa (10 μM). We found that in a purified system, the MDA-MB-231 cells were robustly labeled with the FVIIa and FXa probes. The FVIIa and FXa probes weakly labeled the SW480 cells and SW620 cells. The FIIa probe failed to label any of the three cell lines. Under conditions of coagulation, the FVIIa probe labeled all the adherent MDA-MB-231 cells. Heterogeneous FVIIa-labeling was observed for both the SW480 and SW620 cell lines, with some of the adherent cells labeling brightly, while other cells on the same slide were not labeled by the FVIIa-probe. The FXa probe showed complete labeling of all three cell lines. The FIIa-probe showed complete labeling of the MDA-MB-231 and SW620 cell lines and heterogeneous labeling of the SW480 cells under conditions of coagulation. In whole blood, the FVIIa probe showed heterogeneous labeling of the MDA-MB-231 cells, SW480 and SW620 cells. Heterogeneous labeling of all three cell lines with the FXa and FIIa probes was observed, with very few SW480 or SW620 cells labeled. All three cell lines were labeled with an anti-TF mAb.
In summary, we demonstrated the use of fluorescently-modified, active site-inhibited coagulation factors to label procoagulant cancer cells. We demonstrated that coagulation factors based-probes bound to cancer cell lines in purified systems and in whole blood, yet failed to bind to peripheral blood cells. Labeling of cancer cells was demonstrated via flow cytometry in purified systems, as well as on an immobilized-cell platform similar to what is currently used in some CTC-detection platforms. This work is the first step in the development of a function-based CTC labeling strategy to determine whether CTCs are procoagulant, and whether CTC enumeration and procoagulant characterization strategies are clinically useful in predicting thrombosis in patients with cancer.

Disclosures:
No relevant conflicts of interest to declare.