Ex vivo Murine Thromboinflammation Model with Validation on EMT-6 Breast Cancer

Thromboinflammation is a complex interaction between the hemostasis system and the immune system associated with the participation of neutrophils in the process of thrombosis. An imbalance in the mutual activation of platelets and neutrophils in various pathologies leads to thrombosis or bleeding. Previously, we developed a technique for ex vivo observation of the process of thrombosis and chemotaxis of neutrophils in parallel-plate flow chambers coated with fibrillar collagen. The aim of this work was to develop a technique for ex vivo observation of the thromboinflammation process in mice, which allows analyzing the interaction of polymorphonuclear leukocytes (PMN) with growing blood clots. To validate the technique, a breast cancer model was used in BALB/c mice (7 days after orthotopic inoculation with EMT-6 tumor cell culture). In the blood samples of healthy mice, blood clots formed on fibrillar collagen, however, nuclear cells were not observed. The use of a combination of fibronectin and collagen as a substrate made it possible to induce thrombosis and monitor the movement and behavior of PMNs in the vicinity of blood clots. Using the developed model, it was shown that in breast cancer, the growth of blood clots is slowed down—the relative size of blood clots in breast cancer is 21 ± 11% of the field of view compared with 39 ± 10% in healthy mice. At the same time, the number of PMNs adhering to blood clots and the speed of their movement on the substrate do not differ in healthy mice and mice with tumors. However, the number of PMNs leaving the thrombus and sliding onto the fibronectin-collagen matrix was significantly increased in mice with a tumor (39 ± 23 versus 15 ± 8 in healthy controls). Thus, using the developed thromboinflammation model, it is shown that already at the early stages of tumor development, violations of the thromboinflammation process are observed.