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

Thromboinflammation is a complex interaction between the hemostasissystem and the immune system associated with the participation ofneutrophils in the process of thrombosis. An imbalance in the mutualactivation of platelets and neutrophils in various pathologies leadsto thrombosis or bleeding. Previously, we developed a techniquefor ex vivo observation of the process of thrombosis and chemotaxisof neutrophils in parallel-plate flow chambers coated with fibrillarcollagen. The aim of this work was to develop a technique for exvivo observation of the thromboinflammation process in mice, whichallows analyzing the interaction of polymorphonuclear leukocytes(PMN) with growing blood clots. To validate the technique, a breastcancer model was used in BALB/c mice (7 days after orthotopic inoculationwith EMT-6 tumor cell culture). In the blood samples of healthymice, blood clots formed on fibrillar collagen, however, nuclearcells were not observed. The use of a combination of fibronectin andcollagen as a substrate made it possible to induce thrombosis andmonitor the movement and behavior of PMNs in the vicinity of bloodclots. Using the developed model, it was shown that in breast cancer,the growth of blood clots is slowed down—the relative size of bloodclots in breast cancer is 21 ± 11% of the field of view comparedwith 39 ± 10% in healthy mice. At the same time, the number of PMNsadhering to blood clots and the speed of their movement on the substratedo not differ in healthy mice and mice with tumors. However, thenumber of PMNs leaving the thrombus and sliding onto the fibronectin-collagenmatrix was significantly increased in mice with a tumor (39 ± 23versus 15 ± 8 in healthy controls). Thus, using the developed thromboinflammationmodel, it is shown that already at the early stages of tumor development,violations of the thromboinflammation process are observed.