Platelets and fibrinogen facilitate each other in protecting tumor cells from natural killer cytotoxicity

Abstract

The functions of platelets and fibrinogen in protecting tumor cells from natural killer cytotoxicity have been discussed for more than 20 years. However, their exact roles and relationships in the process are still not clear. In this study, we show that tumor cells prefer to adhere to fibrinogen than to platelets, and fibrinogen can enhance the adhesion of tumor cells to platelets. Beta3 integrin plays an important role in the adhesion of B16F10 to platelets enhanced by fibrinogen. In the presence of thrombin, fibrinogen forms dense fibrin(ogen) layers around tumor cells. Tumor cells can induce platelets to aggregate and form thrombin. Platelets, as well as thrombin, can help fibrinogen protect tumor cells from lethal contact with natural killer cells and natural killer cytotoxicity. Hirudin, a specific inhibitor of thrombin, can reverse the effect of platelets on fibrinogen in blocking natural killer cytotoxicity. Our results suggest that fibrinogen helps platelets to adhere to tumor cells, and platelets in turn promote more fibrinogen to aggregate around tumor cells by forming thrombin. They facilitate each other in protecting tumor cells from natural killer cytotoxicity.

Figure 1

Tumor cells prefer to adhere to fibrinogen than to platelets. (A) One hundred microliters of 2 mg/mL fibrinogen (Alexa Fluor488 conjugated, green) or 2 × 10⁸/mL platelets (PKH26 dyed, red) were incubated with 100 µL of 2 × 10⁵ YAC‐1 or B16F10 cells in phosphate‐buffered saline (PBS) containing Ca²⁺ (sample) or ethylenediaminetetraacetic acid (control) for 30 min. Tumor cells adhered to fibrinogen or platelets were counted by fluorescence‐activated cell sorting before and after washing three times with PBS. FS, Forward Scatter; SS, Side Scatter. (B) Statistical results (control and washed samples) from flow cytometry. (C) Fibrinogen or platelet monolayers on glass slides were washed with PBS/0.1% bovine serum albumin for 2 min, tumor cells (1 × 10⁶/mL) were perfused over the layers at the shear stress of 1.2 dyn/cm², and the number of tumor cells adhered to fibrinogen or platelets were counted in 10 randomly selected fields after 3 min. All of the experiments were repeated more than three times. **P < 0.01; ***P < 0.001.

Figure 2

Fibrinogen enhances the adhesion between tumor cells and platelets. (A,B) Platelet monolayers on glass slides were washed with phosphate‐buffered saline (PBS)/0.1% bovine serum albumin for 2 min. YAC‐1 (A) and B16F10 (B) cells were preincubated with or without fibrinogen (2 mg/mL) for 30 min before being used, then the treated cells were perfused over the layers under a series of shear stress from 0.3 to 1.2 dyn/cm². The adhered tumor cells on platelet monolayers were counted in 10 randomly selected fields after 3 min. (C) B16F10 cells were preincubated with the function blocking antibodies (10 µg/mL) against mice β₃ or β₁ integrin before being incubated with fibrinogen (Fib). After incubation, B16F10 cells were allowed to flow over the platelet monolayers under 0.3 dyn/cm² shear stress, and the adhered cells were counted as above. All of the experiments were repeated more than three times. ***P < 0.001.

Figure 3

Thrombin aggregates fibrinogen around tumor cells. One hundred microliters of fibrinogen (2 mg/mL, conjugated with Alexa Fluor488, green) was mixed with 100 µL B16F10 cells (1 × 10⁶/mL). After the addition of 1 U/mL thrombin (A(a,b)), or phosphate‐buffered saline (PBS) (A(c,d)), the mixture was incubated at 37°C for 30 min. After washing three times with PBS, the tumor cells were checked under a fluorescence microscope. (B) Thirty randomly selected photographs were counted for the tumor cells coated with dense fibrin(ogen) layers. (C) PKH26‐dyed B16F10 cells (red; 2 × 10⁵/mouse) were injected into the mice after the injection of fibrinogen conjugated with Alexa Fluor488 (green; 1 mg/mouse), and the fibrin layers formed around tumor cells in the capillary were observed. PBS (a,b) or hirudin (c,d) (10 mg/kg) were injected with tumor cells. (D) B16F10 cells coated with fibrin layers were numbered from 30 randomly selected digital microscope photographs of the frozen sections of mouse lung. ***P < 0.001.

Figure 4

Tumor cells can induce platelets to aggregate and form thrombin. (A) Platelet (2.5 × 10⁸/mL) aggregation induced by B16F10 cells at different densities was monitored by a light aggregometer. (B) B16F10 cells were checked by a chromogenic assay for their ability to induce platelets to form thrombin. The absorbance at 405 nm reflected the level of thrombin formed in the wells. P, wells containing platelets and tumor cells but without platelet‐poor plasma; PPP, wells containing tumor cells and PPP but without platelets; P+PPP, wells containing tumor cells, platelets and platelet‐poor plasma.

Figure 5

Fibrinogen blocks the effector–target cell interaction in a thrombin‐ or platelet‐dependent manner. 100 µL of B16F10 cells (2 × 10⁵/mL) was seeded in each well of the 96‐well culture plate and cultured overnight. After that, 50 µL of platelets (2 × 10⁸/mL), fibrinogen (4 mg/mL), and calcein acetyoxymethyl‐dyed natural killer (NK) cells (2 × 10⁷/mL) were added to the wells, the culture plate was incubated at 37˚C for 1 h. After washing three times with phosphate‐buffered saline (PBS), the fluorescence at 515 nm was checked by a plate reader, which could reflect the quantity of NK cells adhered to B16F10 cells. Thrombin (1 U/mL) was present in (B) but not in (A). All of the wells contained a trace of platelet‐poor plasma (1/100). PBS, wells added with PBS for control; Platelets, wells containing platelets; Fibrinogen, wells containing fibrinogen; PF, wells containing platelets and fibrinogen; PFH, wells containing platelets and fibrinogen after hirudin treatment (0.1 mg/mL). **P < 0.01.

Figure 6

Thrombin or platelets are required for the role of fibrinogen in blocking natural killer (NK) cytotoxicity. Fifty microliters of calcein acetyoxymethyl‐dyed YAC‐1 cells (1 × 10⁵/mL) was mixed with 50 µL NK cells (1 × 10⁷/mL) for NK cytotoxic assay. To check the effect of platelets and fibrinogen on NK cytotoxicity, 50 µL of different concentrations of platelets (A) or fibrinogen (B) was added into the systems. (C) To determine the role of platelets on the blocking effect of fibrinogen on NK cytotoxicity, 50 µL platelets (2 × 10⁸/µL) or fibrinogen (2 µg/µL) was added to wells containing 1 U/µL thrombin, or without thrombin (PF). All of the wells contained a trace of platelet‐poor plasma (1/100). PFH, wells containing fibrinogen and platelets after hirudin treatment (10 mg/kg). (D) Hirudin (10 mg/kg) or phosphate‐buffered saline (PBS) was injected with B16F10 cells (2 × 10⁵/mouse) into the lateral tail vein. After 14 days the animals were killed and the lungs removed. The metastatic nodes on the surface of lung were numbered under an anatomical lens. *P < 0.05; **P < 0.01; ***P < 0.001.