Mouse models of cancer-associated thrombosis

Cancer patients have 4-7 fold increased risk of venous thromboembolism (VTE) compared with a general population ^[1]. Clinical studies have identified biomarkers that are associated with VTE in cancer patients. Interestingly, the incidence of VTE is varied with cancer type, suggesting that there may be cancer-type specific mechanisms ^[2]. However, the underlying mechanisms have not been elucidated. Mouse models are used to investigate the mechanisms of cancer-associated thrombosis ^[3]. Many different mouse models have been established that vary by mouse strains, cancer cells, tumor sites and thrombosis models.

There are 2 choices of mouse strain: immunocompetent and immunodeficient. C57BL/6 and BALB/c immunocompetent mice are most commonly used to generate allograft models with murine cancer cells. The strengths of immunocompetent mice are that the mouse has a full immune system and one can determine the role of a given gene using knockout mice. In addition, genetically engineered mice, such as the LSL-Kras^G12D/+; LSL-Trp53^R172H/+; Pdx-1-Cre (KPC) pancreatic cancer model, develop spontaneous tumors that pathophysiologically resemble human tumors ^[4]. The weakness of using immunocompetent mice is that there are limited number of murine cancer cell lines available. There are several types of immunodeficient mice used to generate xenograft models. Nude mice lack T-cells, severe combined immunodeficient (SCID) mice lack both T- and B-cells, and non-obese diabetic (NOD)/SCID mice lack T- and B-cells, complement and have reduced natural killer cell activity. The strength of immunodeficient mice is that one can use the large repertoire of human cancer cell lines and patient-derived xenografts (PDXs). However, the weakness of immunodeficient mice is that they do not have full immune system. Indeed, recent studies have shown a role of immune cells in thrombosis ^[5].

Cancer cell lines are easy to grow in culture. However, the gene expression pattern and morphology may be altered by culturing. PDXs are deemed superior to cell lines because PDXs maintain the pathology ^[6,7], gene expression pattern ^[8] and single nucleotide polymorphisms ^[9] of primary tumors. The limitations of PDXs are that they are difficult and costly to maintain in mice and they change characteristics once they are cultured in vitro and these changes are irreversible ^{[10, 11].}

Both subcutaneous and orthotopic tumor models are used. Subcutaneous tumors are easy to generate and tumor growth can be measured using a caliper. However, they do not reproduce the tumor microenvironment of the original tumor ^[12]. Orthotopic tumors are regarded as superior to subcutaneous tumors because they better mimic tumors in patients ^[12]. However, reporter genes, such as green fluorescent protein or luciferase, are needed to monitor tumor growth.

There are several venous thrombosis models available. The inferior vena cava (IVC) stenosis model is the most popular model that involves the partial ligation (~90%) of the IVC ^[13-16]. The advantage of this model is that it maintains blood flow and resembles the clinical scenario. The disadvantage is that it has a variable incidence of thrombosis and weight of thrombus (5-20 mg). Alternatively, one can use an IVC stasis model that involves complete ligation of the IVC and side branches, and cauterization of back branches ^[17]. The advantages of the IVC stasis model are that the incidence of thrombosis is almost 100% and weight of thrombus are large and consistent (15-25 mg). The disadvantages are that it has limited blood flow and ligation and cauterization points induce inflammation. The ferric chloride (FeCl₃) model and Rose Bengal photochemical model are used to induce thrombosis in different veins. The strength of these models is that they are easy and reproducible. The weakness of the models is that the induction of thrombosis (oxidative damage) is not physiological ^[18,19]. The laser induced-injury model induces thrombosis in the microvessels of the cremaster muscle. The strength of this model is that one can monitor real-time thrombus formation using an intravital microscopy. The weakness of the model is that it analyzes thrombus formation in small vessels that may not reproduce the thrombotic process observed in cancer patients.

There is a need for additional development of mouse models of cancer-associated thrombosis.

References

1. Y. Hisada, J.E. Geddings, C. Ay, N. Mackman, Venous thrombosis and cancer: from mouse models to clinical trials, J Thromb Haemost 13(8) (2015) 1372-1382.
2. Y. Hisada, N. Mackman, Cancer-associated pathways and biomarkers of venous thrombosis, Blood 130(13) (2017) 1499-1506.
3. Y. Hisada, N. Mackman, Mouse models of cancer-associated thrombosis, Thromb Res 164 Suppl 1 (2018) S48-S53.
4. S.R. Hingorani, L. Wang, A.S. Multani, C. Combs, T.B. Deramaudt, R.H. Hruban, A.K. Rustgi, S. Chang, D.A. Tuveson, Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice, Cancer Cell 7(5) (2005) 469-483.
5. B. Engelmann, S. Massberg, Thrombosis as an intravascular effector of innate immunity, Nat Rev Immunol 13(1) (2013) 34-45.
6. P. Loukopoulos, K. Kanetaka, M. Takamura, T. Shibata, M. Sakamoto, S. Hirohashi, Orthotopic transplantation models of pancreatic adenocarcinoma derived from cell lines and primary tumors and displaying varying metastatic activity, Pancreas 29(3) (2004) 193-203.
7. Y.S. DeRose, G. Wang, Y.C. Lin, P.S. Bernard, S.S. Buys, M.T. Ebbert, R. Factor, C. Matsen, B.A. Milash, E. Nelson, L. Neumayer, R.L. Randall, I.J. Stijleman, B.E. Welm, A.L. Welm, Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes, Nat Med 17(11) (2011) 1514-1520.
8. X. Zhao, Z. Liu, L. Yu, Y. Zhang, P. Baxter, H. Voicu, S. Gurusiddappa, J. Luan, J.M. Su, H.C. Leung, X.N. Li, Global gene expression profiling confirms the molecular fidelity of primary tumor-based orthotopic xenograft mouse models of medulloblastoma, Neuro Oncol 14(5) (2012) 574-583.
9. J. McEvoy, A. Ulyanov, R. Brennan, G. Wu, S. Pounds, J. Zhang, M.A. Dyer, Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma, PLoS One 7(8) (2012) e42739.
10. D. Siolas, G.J. Hannon, Patient-derived tumor xenografts: transforming clinical samples into mouse models, Cancer Res 73(17) (2013) 5315-5319.
11. V.C. Daniel, L. Marchionni, J.S. Hierman, J.T. Rhodes, W.L. Devereux, C.M. Rudin, R. Yung, G. Parmigiani, M. Dorsch, C.D. Peacock, D.N. Watkins, A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro, Cancer Res 69(8) (2009) 3364-3373.
12. R.M. Hoffman, Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts, Nat Rev Cancer 15(8) (2015) 451-452.
13. J.G. Wang, J.E. Geddings, M.M. Aleman, J.C. Cardenas, P. Chantrathammachart, J.C. Williams, D. Kirchhofer, V.Y. Bogdanov, R.R. Bach, J. Rak, F.C. Church, A.S. Wolberg, R. Pawlinski, N.S. Key, J.J. Yeh, N. Mackman, Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer, Blood 119(23) (2012) 5543-5552.
14. G.M. Thomas, A. Brill, S. Mezouar, L. Crescence, M. Gallant, C. Dubois, D.D. Wagner, Tissue factor expressed by circulating cancer cell-derived microparticles drastically increases the incidence of deep vein thrombosis in mice, J Thromb Haemost 13(7) (2015) 1310-1319.
15. J.E. Geddings, Y. Hisada, Y. Boulaftali, T.M. Getz, M. Whelihan, R. Fuentes, R. Dee, B.C. Cooley, N.S. Key, A.S. Wolberg, W. Bergmeier, N. Mackman, Tissue factor-positive tumor microvesicles activate platelets and enhance thrombosis in mice, J Thromb Haemost 14(1) (2016) 153-166.
16. K. Stark, I. Schubert, U. Joshi, B. Kilani, P. Hoseinpour, M. Thakur, P. Grunauer, S. Pfeiler, T. Schmidergall, S. Stockhausen, M. Baumer, S. Chandraratne, M.L. von Bruhl, M. Lorenz, R. Coletti, S. Reese, I. Laitinen, S.M. Wormann, H. Algul, C.J. Bruns, J. Ware, N. Mackman, B. Engelmann, S. Massberg, Distinct Pathogenesis of Pancreatic Cancer Microvesicle-Associated Venous Thrombosis Identifies New Antithrombotic Targets In Vivo, Arterioscler Thromb Vasc Biol 38(4) (2018) 772-786.
17. Y. Hisada, C. Ay, A.C. Auriemma, B.C. Cooley, N. Mackman, Human pancreatic tumors grown in mice release tissue factor-positive microvesicles that increase venous clot size, J Thromb Haemost 15(11) (2017) 2208-2217.
18. J.A. Diaz, A.T. Obi, D.D. Myers, Jr., S.K. Wrobleski, P.K. Henke, N. Mackman, T.W. Wakefield, Critical review of mouse models of venous thrombosis, Arterioscler Thromb Vasc Biol 32(3) (2012) 556-562.