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

封面

如何引用文章

全文:

开放存取 开放存取
受限制的访问 ##reader.subscriptionAccessGranted##
受限制的访问 订阅存取

详细

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.

全文:

受限制的访问

作者简介

J. J. Korobkina

Center for Theoretical Problems of Physicochemical Pharmacology of the RAS

编辑信件的主要联系方式.
Email: asve6nikova@yandex.ru
俄罗斯联邦, Moscow

A. Mishukov

Center for Theoretical Problems of Physicochemical Pharmacology of the RAS

Email: asve6nikova@yandex.ru
俄罗斯联邦, Moscow

E. Osidak

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

Email: asve6nikova@yandex.ru
俄罗斯联邦, Moscow

A. 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
俄罗斯联邦, Moscow; Moscow

参考

  1. Darbousset R, Mezouar S, Dignat-George F, Panicot-Dubois L, Dubois C (2014) Involvement of neutrophils in thrombus formation in living mice. Pathol Biol 62: 1–9. https://doi.org/10.1016/j.patbio.2013.11.002
  2. Italiano Jr JE, Battinelli EM (2009) Selective sorting of alpha-granule proteins. J Thromb Haemost 7: 173–176. https://doi.org/10.1111/j.1538-7836.2009.03387.x
  3. Parsons MEM, Szklanna PB, Guerrero JA, Wynne K, Dervin F, O’Connell K, Allen S, Egan K, Bennett C, McGuigan C, Gheveart C, Ní Áinle F, Maguire PB (2018) Platelet releasate proteome profiling reveals a core set of proteins with low variance between healthy adults. Proteomics 18: 1800219. https://doi.org/10.1002/pmic.201800219
  4. Sveshnikova AN, Adamanskaya EA, Panteleev MA (2024) Conditions for the implementation of the phenomenon of programmed death of neutrophils with the appearance of DNA extracellular traps during thrombus formation. Pediatr Hematol Immunopathol 23: 211–218. https://doi.org/10.24287/1726-1708-2024-23-1-211-218
  5. Schönichen C, Montague SJ, Brouns SLN, Burston JJ, Cosemans JMEM, Jurk K, Kehrel BE, Koenen RR, Ní Áinle F, O’Donnell VB, Soehnlein O, Watson SP, Kuijpers MJE, Heemskerk JWM, Nagy M (2023) Antagonistic roles of human platelet integrin αIIbβ3 and chemokines in regulating neutrophil activation and fate on arterial thrombi under flow. Arterioscler Thromb Vasc Biol 43: 1700–1712. https://doi.org/10.1161/ATVBAHA.122.318767
  6. Morozova DS, Martyanov AA, Obydennyi SI, Korobkin J-JD, Sokolov AV, Shamova EV, Gorudko IV, Khoreva AL, Shcherbina A, Panteleev MA, Sveshnikova AN (2022) Ex vivo observation of granulocyte activity during thrombus formation. BMC Biol 20: 32. https://doi.org/10.1186/s12915-022-01238-x
  7. Nechipurenko DY, Receveur N, Yakimenko AO, Shepelyuk TO, Yakusheva AA, Kerimov RR, Obydennyy SI, Eckly A, Léon C, Gachet C, Grishchuk EL, Ataullakhanov FI, Mangin PH, Panteleev MA (2019) Clot Contraction Drives the Translocation of Procoagulant Platelets to Thrombus Surface. Arterioscler Thromb Vasc Biol 39: 37–47. https://doi.org/10.1161/ATVBAHA.118.311390
  8. Schattner M, Jenne CN, Negrotto S, Ho-Tin-Noe B (2020) editorial: Platelets and immune responses during thromboinflammation. Front Immunol 11: 1079. https://doi.org/10.3389/fimmu.2020.01079
  9. Sveshnikova AN, Tesakov IP, Kuznetsova SA, Shamova ЕМ (2024) Role of platelet activation in the development and metastasis of solid tumors. J Evol Biochem Physiol 60: 211–227. https://doi.org/10.1134/S0022093024010150
  10. Sohal S, Thakur A, Zia A, Sous M, Trelles D (2020) Disseminated intravascular coagulation and malignancy: A case report and literature review. Case Rep Oncol Med 2020: 1–5. https://doi.org/10.1155/2020/9147105
  11. Li B, Lu Z, Yang Z, Zhang X, Wang M, Chu T, Wang P, Qi F, Anderson GJ, Jiang E, Song Z, Nie G, Li S (2023) Monitoring circulating platelet activity to predict cancer-associated thrombosis. Cell Rep Methods 3: 100513. https://doi.org/10.1016/j.crmeth.2023.100513
  12. De Meo ML, Spicer JD (2021) The role of neutrophil extracellular traps in cancer progression and metastasis. Semin Immunol 57: 101595. https://doi.org/10.1016/j.smim.2022.101595
  13. Kakumoto A (2024) Prognostic impact of tumor-associated neutrophils in breast cancer. Int J Clin Exp Pathol 17: 51–62. https://doi.org/10.62347/JQDQ1527
  14. Taucher E, Taucher V, Fink-Neuboeck N, Lindenmann J, Smolle-Juettner F-M (2021) role of tumor-associated neutrophils in the molecular carcinogenesis of the lung. Cancers 13: 5972. https://doi.org/10.3390/cancers13235972
  15. Jin L, Kim HS, Shi J (2021) Neutrophil in the pancreatic tumor microenvironment. Biomolecules 11: 1170. https://doi.org/10.3390/biom11081170
  16. Margaroli C, Cardenas MA, Jansen CS, Moon Reyes A, Hosseinzadeh F, Hong G, Zhang Y, Kissick H, Tirouvanziam R, Master VA (2020) The immunosuppressive phenotype of tumor-infiltrating neutrophils is associated with obesity in kidney cancer patients. OncoImmunology 9: 1747731. https://doi.org/10.1080/2162402X.2020.1747731
  17. Fridlender ZG, Sun J, Kim S, Kapoor V, Cheng G, Ling L, Worthen GS, Albelda SM (2009) Polarization of tumor-associated neutrophil phenotype by TGF-β: «N1» versus «N2» TAN. Cancer Cell 16: 183–194. https://doi.org/10.1016/j.ccr.2009.06.017
  18. Schaider H, Oka M, Bogenrieder T, Nesbit M, Satyamoorthy K, Berking C, Matsushima K, Herlyn M (2003) Differential response of primary and metastatic melanomas to neutrophils attracted by IL‐8. Int J Cancer 103: 335–343. https://doi.org/10.1002/ijc.10775
  19. Musiani P, Allione A, Modica A, Lollini PL, Giovarelli M, Cavallo F, Belardelli F, Forni G, Modesti A (1996) Role of neutrophils and lymphocytes in inhibition of a mouse mammary adenocarcinoma engineered to release IL-2, IL-4, IL-7, IL-10, IFN-alpha, IFN-gamma, and TNF-alpha. Lab Investig J Tech Methods Pathol 74: 146–157
  20. Martyanov AA, Morozova DS, Sorokina MA, Filkova AA, Fedorova DV, Uzueva SS, Suntsova EV, Novichkova GA, Zharkov PA, Panteleev MA, Sveshnikova AN (2020) Heterogeneity of integrin αIIbβ3 function in pediatric immune thrombocytopenia revealed by continuous flow cytometry analysis. Int J Mol Sci 21: 3035. https://doi.org/10.3390/ijms21093035
  21. Martyanov AA, Tesakov IP, Khachatryan LA, An OI, Boldova AE, Ignatova AA, Koltsova EM, Korobkin J-JD, Podoplelova NA, Svidelskaya GS, Yushkova E, Novichkova GA, Eble JA, Panteleev MA, Kalinin DV, Sveshnikova AN (2023) Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon. Blood Adv 7: 4936–4949. https://doi.org/10.1182/bloodadvances.2022009590
  22. Korobkin J-JD, Deordieva EA, Tesakov IP, Adamanskaya E-IA, Boldova AE, Boldyreva AA, Galkina SV, Lazutova DP, Martyanov AA, Pustovalov VA, Novichkova GA, Shcherbina A, Panteleev MA, Sveshnikova AN (2024) Dissecting thrombus-directed chemotaxis and random movement in neutrophil near-thrombus motion in flow chambers. BMC Biol 22: 115. https://doi.org/10.1186/s12915-024-01912-2
  23. Sagiv JY, Michaeli J, Assi S, Mishalian I, Kisos H, Levy L, Damti P, Lumbroso D, Polyansky L, Sionov RV, Ariel A, Hovav A-H, Henke E, Fridlender ZG, Granot Z (2015) Phenotypic diversity and plasticity in circulating neutrophil subpopulations in cancer. Cell Rep 10: 562–573. https://doi.org/10.1016/j.celrep.2014.12.039
  24. Tucker EI, Verbout NG, Leung PY, Hurst S, McCarty OJT, Gailani D, Gruber A (2012) Inhibition of factor XI activation attenuates inflammation and coagulopathy while improving the survival of mouse polymicrobial sepsis. Blood 119: 4762–4768. https://doi.org/10.1182/blood-2011-10-386185
  25. Jackson SP, Darbousset R, Schoenwaelder SM (2019) Thromboinflammation: challenges of therapeutically targeting coagulation and other host defense mechanisms. Blood 133: 906–918. https://doi.org/10.1182/blood-2018-11-882993
  26. Mishukov AA, Gaur S, Adamanskaya E-IA, Panteleev M, Sveshnikova AN (2024) Evaluation of platelet functional activity in healthy BALB/c mice and in EMT-6 breast cancer orthotopic model. J Evol Biochem Physiol 61.
  27. Tavera-Mendoza LE, Brown M (2017) A less invasive method for orthotopic injection of breast cancer cells into the mouse mammary gland. Lab Anim 51: 85–88. https://doi.org/10.1177/0023677216640706
  28. Pulikkot S, Hu L, Chen Y, Sun H, Fan Z (2022) Integrin regulators in neutrophils. Cells 11: 2025. https://doi.org/10.3390/cells11132025
  29. Ingham KC, Brew SA, Isaacs BS (1988) Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen. J Biol Chem 263: 4624–4628. https://doi.org/10.1016/S0021-9258(18)68828-3
  30. Ni H, Yuen PST, Papalia JM, Trevithick JE, Sakai T, Fässler R, Hynes RO, Wagner DD (2003) Plasma fibronectin promotes thrombus growth and stability in injured arterioles. Proc Natl Acad Sci U S A 100: 2415–2419. https://doi.org/10.1073/pnas.2628067100
  31. Everitt EA, Malik AB, Hendey B (1996) Fibronectin enhances the migration rate of human neutrophils in vitro. J Leukoc Biol 60: 199–206. https://doi.org/10.1002/jlb.60.2.199
  32. Boneschansker L, Jorgensen J, Ellett F, Briscoe DM, Irimia D (2018) Convergent and divergent migratory patterns of human neutrophils inside microfluidic mazes. Sci Rep 8: 1887. https://doi.org/10.1038/s41598-018-20060-6
  33. Trivanović D, Mojsilović S, Bogosavljević N, Jurišić V, Jauković A (2024) Revealing profile of cancer-educated platelets and their factors to foster immunotherapy development. Transl Oncol 40: 101871. https://doi.org/10.1016/j.tranon.2023.101871
  34. Kawano T, Hisada Y, Grover SP, Schug WJ, Paul DS, Bergmeier W, Mackman N (2023) Decreased platelet reactivity and function in a mouse model of human pancreatic cancer. Thromb Haemost 123: 501–509. https://doi.org/10.1055/s-0043-1761419
  35. Biermann H, Pietz B, Dreier R, Schmid KW, Sorg C, Sunderkötter C (1999) Murine leukocytes with ring-shaped nuclei include granulocytes, monocytes, and their precursors. J Leukoc Biol 65: 217–231. https://doi.org/10.1002/jlb.65.2.217
  36. Gruijs M, Sewnath CAN, Van Egmond M (2021) Therapeutic exploitation of neutrophils to fight cancer. Semin Immunol 57: 101581. https://doi.org/10.1016/j.smim.2021.101581
  37. Brewer G (2023) Activated neutrophils: the anti-hero. Nat Rev Cancer 23: 190. https://doi.org/10.1038/s41568-023-00555-9

补充文件

附件文件
动作
1. JATS XML
下载
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.

下载 (904KB)
索引源数据
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.

下载 (721KB)
索引源数据
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.

下载 (233KB)
索引源数据

版权所有 © Russian Academy of Sciences, 2025