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

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Abstract

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.

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About the authors

J. J. D. Korobkina

Center for Theoretical Problems of Physicochemical Pharmacology of the RAS

Author for correspondence.
Email: asve6nikova@yandex.ru
Russian Federation, Moscow

A. A. Mishukov

Center for Theoretical Problems of Physicochemical Pharmacology of the RAS

Email: asve6nikova@yandex.ru
Russian Federation, Moscow

E. O. Osidak

Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology

Email: asve6nikova@yandex.ru
Russian Federation, Moscow

A. N. Sveshnikova

Center for Theoretical Problems of Physicochemical Pharmacology of the RAS; Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology

Email: asve6nikova@yandex.ru
Russian Federation, Moscow; Moscow

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Supplementary files

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2. Fig. 1. Development of a mouse model of thrombopoiesis: (a) - characteristic view of growing thrombus, substrate - collagen, 200 μg/ml; (b) - characteristic view of growing thrombus, substrate - fibronectin, 4 mg/ml; (c) - characteristic view of growing thrombus, substrate - collagen 200 μg/ml + fibronectin 4 mg/ml; (a-c) - 25 μm scale bar, blue - platelet granule DNA/polyphosphates (Hoechst33342), yellow - thrombus (DiOC3(6)); (d) - comparison of thrombus area for different substrates; (e) - comparison of thrombus areas for different substrates: Col + Fbn (collagen 200 μg/ml + fibronectin 4 mg/ml), Col (collagen 200 μg/ml), Fbn (fibronectin 4 mg/ml); (f) - comparison of the number of PMNL that adhered to different substrates for the whole duration of the experiment. Col + Fbn (collagen 200 μg/ml + fibronectin 4 mg/ml), Col (collagen 200 μg/ml), Fbn (fibronectin 4 mg/ml); (f) - number of PMNLs that adhered to collagen-fibronectin substrate at different experiment times. Mann-Whitney test was used to compare samples. * - corresponds to p < 0.05.

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3. Fig. 2. Disruption of thrombosis in mice with breast cancer: (a) - typical field of view after 15 min of experiment in mouse with breast cancer; (b) - typical field of view after 15 min of experiment in healthy mouse; (c) - mean clot size at 5th and 10th min is significantly smaller in mice with breast cancer than in healthy mice. There is no statistically significant difference at 25 min; (d) - typical leukocyte appearance for healthy mice; (e) - typical leukocyte appearance outside the thrombus in mice with tumours. Blue - DNA/polyphosphates of platelet granules (Hoechst33342), yellow - thrombi (DiOC3(6)). Scale bar 25 μm.

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4. Fig. 3. Features of leukocyte-platelet interaction in mice with PMNL: (a) - thrombus areas at the 5th and 10th min are significantly smaller in mice with PMNL than in healthy mice. There is no statistically significant difference at 25th min; (b) - the rate of PMNL movement is not significantly different for healthy controls and mice with tumours; (c) - the ratio of PMNL to thrombus areas is not significantly different for healthy controls and mice with tumours; (d) - the proportion of PMNL outside the thrombus is significantly higher for mice with tumours compared to healthy controls; (e) - the amount of PMNL outside the thrombus correlates with tumour size for mice with RRM. Data for each mouse with tumour are indicated by symbols: square - mouse #1, triangle - mouse #2, star - mouse #3, inverted triangle - mouse #4, circle - mouse #5 and rhombus - mouse #6. The Mann-Whitney test was used for comparison. * - p < 0.05, ** - p < 0.01.

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