Detection of tissue factor-positive extracellular vesicles by laser scanning confocal microscopy

Abstract

Introduction: 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).

Methods: 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.

Results: 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.

Conclusion: LSCM enables visualization of TF+EVs in the supernatant from cultured cells and in clinical samples.

Keywords: Cancer; Extracellular vesicles; Laser scanning confocal microscopy; Tissue factor.