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. Author manuscript; available in PMC: 2018 May 9.
Published in final edited form as: Thromb Res. 2017 Dec 29;164(Suppl 1):S48–S53. doi: 10.1016/j.thromres.2017.12.018

Mouse models of cancer-associated thrombosis

Yohei Hisada 1,2, Nigel Mackman 1,3
PMCID: PMC5942599  NIHMSID: NIHMS937362  PMID: 29306575

Abstract

Cancer patients have an increased risk of venous thromboembolism (VTE) compared with the general population. Mouse models are used to better understand the mechanisms of cancer-associated thrombosis. Several mouse models of cancer-associated thrombosis have been developed that use different mouse strains, tumors, tumor sites and thrombosis model. In this review, we summarize these different models. These models have been used to determine the role of different pathways in cancer-associated thrombosis. For instance, they have revealed roles for tumor-derived tissue factor-positive microvesicles and neutrophil extracellular traps in thrombosis in tumor-bearing mice. A better understanding of the mechanisms of cancer-associated thrombosis may allow the development of new therapies to reduce thrombosis in cancer patients.

Keywords: cancer, mouse model, neutrophil extracellular traps, tissue factor, venous thrombosis

Introduction

Cancer patients have an increased risk of venous thromboembolism (VTE) compared with the general population. Biomarkers studies have identified a number of distinct pro-coagulant pathways activated in cancer patients that may contribute to VTE [1]. Importantly, the incidence of VTE varies in different types of cancer [2–5]. This suggests that there may be cancer site-specific mechanisms of VTE [6]. However, the underlying mechanisms leading to VTE in each type of cancer have not been elucidated.

Mouse models are used to study mechanisms of cancer-associated thrombosis. A number of different models have been developed that vary by mouse strains, cancer-types and thrombosis models employed. This makes direct comparison difficult. Here, we review the different models and the studies on the role of various pathways in cancer-associated thrombosis.

Choice of mouse strain and cancer cell

The strains of mice used to study cancer-associated thrombosis are largely dictated by the type of cancer-cells used. Immunocompetent mouse strains, most commonly C57BL/6 and BALB/c, are used to generate allograft models with murine cancer cells (Table 1). It is important that the murine cancer cell line is compatible with the strain of mice. The major strength of immunocompetent models is that the host is able to mount a full immune response to the tumor that may contribute to thrombosis. In addition, one can analyze the role of a given host protein using knockout mice. However, there are a limited number of murine cancer cell lines available. The three most commonly used murine cell lines for studies on cancer-associated thrombosis are 4T1, Lewis lung carcinoma (LLC) and Panc02. The 4T1 cell line is a thioguanine-resistant derivative of the 410.4 line that was isolated from a spontaneous mammary tumor in BALB/cfC3H mice [7]. The LLC cell line was isolated from a spontaneous carcinoma in the lung of C57BL/6 mice [8]. The Panc02 cell line was derived from a tumor formed in the pancreas of C57BL/6 mice treated with 3-methyl-chloronthrene [9]. Genetically engineered mice, such as the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) model, develop primary tumors within the pancreas that look and behave like human pancreatic cancer [10]. The KPC model was developed using C57BL/6 mice. Three cell lines (KPC 4684, KPC 4112 and KPC 4580P) have been established from KPC mouse tumors that have been engineered to express luciferase under a Pdx-1-Cre promoter (Dr. J-J Yeh, unpublished data). We are currently comparing these cell lines to Panc02.

Table 1.

Summary of allograft models of cancer-associated thrombosis.

Mouse Cancer cell Tumor site Thrombosis model Blood vessel Observation Ref
C57BL/6 Panc02, LLC1 s.c. FeCl3 mesenteric venules Reduced time to occlusion 47
C57BL/6 Panc02 s.c. Laser-induced injury FeCl3 mesenteric venules Tumor-derived MVs accumulated at the site of injury 47
C57BL/6 Panc02 s.c. Laser-induced injury cremaster muscle microvessels Tumor derived MV accumulation at the site of injury 54
C57BL/6 Panc02 s.c. IVC stenosis IVC Increased incidence and thrombus weight 55
C57BL/6 M27
Eμ-myc		 orthotopic FeCl3 IVC Gas6 deficient mice had reduced thrombus size compared with controls 65
BALB/c 4T1 orthotopic FeCl3 carotid artery Reduced time to occlusion. DNAse I abolished enhanced thrombosis. 63
BALB/c 4T1 orthotopic Rose Bengal jugular vein Reduced time to occlusion. DNAse I reduced thrombus size 63
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IVC: infrarenal vena cava, s.c.: subcutaneous

Immunodeficient mice are used to generate xenograft models (Table 2). Immunodeficient mice include nude mice that lack T-cells, severe combined immunodeficient (SCID) mice that lack T and B cells, and nonobese diabetic (NOD)/SCID mice that lack T and B-cells, complement and have reduced NK cell activity. Nude mice are most often used. In these models, tumors are generated using human cancer cell lines or patient-derived xenografts (PDXs). The strength of using immunodeficient mice is the large variety of human cancer cell lines available. We and others have evaluated a variety of human colorectal and pancreatic cells lines. Interestingly, colorectal cancer cell lines with mutations in both K-ras and p53, such as the HCT116 subline 379.2, express higher levels of tissue factor (TF) than those with mutations in only K-ras, such as HCT116 [11]. There is also a wide range of TF expression in the different human pancreatic cell lines; HPAF-II, HPAC and BxPc-3 express high levels of TF, L3.6pl express medium levels, PANC-1 expresses low levels and MIAPaCa-2 do not express TF [12–14]. The A549 epithelial-like lung cancer cell line was isolated from human cancerous lung tissue [15, 16]. A weakness of using immunodeficient mice is that they are not able to mount a full immune response to the tumor.

Table 2.

Summary of xenograft models of cancer-associated thrombosis.

Mouse Cancer cell Tumor site Thrombosis model Blood vessel Observation Ref
Nude HPAF II orthotopic IVC stenosis IVC No enhancement of thrombosis 13
Nude HPAF II orthotopic FeCl3 saphenous vein Shortened time to occlusion 13
Nude BxPc-3 orthotopic IVC stenosis IVC Increased thrombus area but no increase in incidence of thrombosis 14
Nude BxPc-3 orthotopic IVC stasis IVC Increased thrombus area and weight 28
Nude A549 orthotopic IVC stenosis IVC Increased thrombus weight 64
Nude A549 orthotopic FeCl3 saphenous vein Reduced time to occlusion 64
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IVC: infrarenal vena cava

Cancer cell lines may have a high or low metastatic potential in mice. It is simpler to use cancer cell lines that have a low metastatic potential for cancer-associated thrombosis studies because the tumor-burden is proportional to the size of the primary tumor and is not affected by secondary metastasis. Highly metastatic tumors may also increase the death of mice (Hisada Y unpublished data).

Human and mouse cancer cell lines are easy to grow in culture. However, the gene expression pattern of these lines may be altered by maintaining the cells in culture. PDXs are considered superior to cancer cell lines because they maintain the pathology [17, 18], gene expression pattern [19] and single nucleotide polymorphisms [20] of primary tumors. PDXs are established by transferring them directly from the patient into mice and then maintained in immunodeficient mice [21]. Success rates of PDXs are variable (23–75%) and it can take 2–12 months to establish the different lines depending on the type of tumor [22]. The fundamental genotypic features of PDXs do not change over several passages in mice [23]. However, PDXs lose the gene expression profiles of primary tumors once the cells are cultured in vitro and these changes are irreversible [24]. The disadvantages of PDXs are that they must be maintained in mice, and it is difficult to transfer them between institutions [22].

Tumor site

There are two choices of site: subcutaneous or orthotopic (Tables 1 and 2). Subcutaneous tumors are typically implanted into the back or the right flank of mice between the dermis and underlying muscle. Matrigel is often used to support the establishment of the tumor. The subcutaneous tumor models are widely used because of the ease with which cells can be implanted and tumor size can be measured using calipers. The weakness of this model is that it does not reproduce the tumor microenvironment in patients [25]. In addition, tumors grown subcutaneously rarely metastasize even if the original tumor is metastatic [25].

Orthotopic tumors are more clinically relevant because the tumor microenvironment more closely resembles that found in patients [25]. Importantly, orthotopic tumors retain their capacity to metastasize. A disadvantage of this model is that it is more difficult to monitor tumor growth. This is important when performing thrombosis experiments because a defined size of tumor needs is required to generate consistent results. To monitor tumor growth reporter genes, such as luciferase or green fluorescence protein (GFP), are introduced into the cell lines to allow non-invasive assessment of tumor size using luminescence or fluorescence molecular tomography. However, we have found that mice bearing orthotopic tumors are more prone to spontaneous or surgery-related death compared with mice bearing subcutaneous tumors (Hisada Y and Cooley BC, unpublished data).

As mentioned above, genetically engineered mice form spontaneous orthotopic tumors and are commercially available for most types of cancer. These models are more clinically relevant than xenograft models because they contain oncogenic mutations that occur in tumors in patients. They also mimic many features of tumor progression, such as angiogenesis, acquisition of secondary mutations and metastasis [26]. The disadvantage of genetically engineered mouse models is that tumor development is variable (typically 3 months or more), which means that it not practical to compare the effect of similarly sized tumors on thrombosis.

Thrombosis models

There are several thrombosis models that have been used to study cancer-associated thrombosis in mice (Tables 1 and 2). The most popular are those involving the infrarenal vena cava (IVC). An advantage of the IVC is that high frequency ultrasonography can be used for non-invasive longitudinal monitoring of thrombus formation [14, 27, 28]. Clot formation is typically analyzed between 0–48 hours.

The IVC stasis model involves complete ligation of the IVC and side branches using non-reactive sutures [29]. Dorsal branches can also be cauterized to maximize clot formation [30]. The strengths of this model are that clot formation is close to 100% and large clots are formed (15–25 mg). This permits histological, cellular and molecular analysis of the clots to identify any changes that have been induced by the presence of the tumor. Weaknesses of this thrombosis model are that there is less blood flow to the injury site compared with the stenosis model and damage to the vessel wall exposes TF, which drives early clot formation and induces inflammation.

The IVC stenosis model is a modification of the St. Thomas model without the vessel injury step [31]. Typically, this involves formation of an area of ~90% stenosis by ligating of the IVC over a spacer with subsequent removal [32]. Some groups ligate the side and back branches [33]. Flow restriction appears to activate the endothelium and trigger clot formation, although a low level of vessel injury may also expose vessel wall TF [32]. The strength of this model is that it better mimics deep vein thrombosis in patients, although the clots form upstream of the stenotic site and grow against the direction of blood flow rather than originating in within valve pockets and growing in the direction of blood flow [31]. Weaknesses of the model are that the incidence of clot formation is quite variable and the clots are smaller (5–20 mg) compared to the stasis model and it is likely that complete occlusion will occur after the formation of a clot.

It is important to note that there are significant differences between the stasis and stenosis models in terms of factors that contribute to clot formation. For instance, vessel wall TF drives thrombosis in the IVC stasis model whereas leukocyte TF drives thrombosis in the stenosis model [34, 35]. In contrast, neutrophils and neutrophil extracellular traps (NETs) contribute to thrombosis in the IVC stenosis model but not in the stasis model [36]. Similarly, mice with a platelet-specific deletion of C-type lectin-like receptor 2 have reduced thrombosis in the stenosis but not the stasis model [37]. These differences should be considered when interpreting data from tumor-bearing mice.

Ferric chloride (FeCl3) application is used to initiate thrombosis in large (IVC) and small (saphenous) veins, as well as the carotid artery and mesenteric microvessels [38, 39]. FeCl3 induces oxidative damage to vessels with different concentrations and application times used to alter the severity of the model. Surprisingly, clots formed in the IVC exposed to FeCl3 are quite small (~5 mg). For the saphenous vein and carotid artery models time to occlusion is measured. The strength of the FeCl3 models are that they are both easy to perform and reproducible. However, the non-physiologic nature of the induction represents a significant weakness [31, 40].

The Rose Bengal photochemical model also generates free radicals that injure the inside of the vessel and time to occlusion is measured. The strengths of this model is that it is easy to conduct and reproducible. However, adjustments to the intensity of illumination, duration of illumination, and dose of dye are required depending on the type of vessel used [41, 42]. As with the FeCl3 models the mechanism of vessel injury is not physiologic.

The laser induced-injury model generates clots on the inner surface of microvessels of the cremaster muscle that are analyzed in real-time by intravital microscopy. Clot formation is generally visualized by the accumulation of labeled antibodies against platelets and fibrin [43]. The strength of this model is that it captures real-time images of clot formation; the weakness is that it analyzes clot formation in small vessels that may not be relevant to deep vein thrombosis in cancer patients [44].

TF-positive microvesicles and cancer-associated thrombosis in mouse models

The TF/FVIIa complex is the primary activator of blood coagulation [45]. High levels of TF are expressed by a variety of tumors, particularly pancreatic tumors, as well as human and mouse pancreatic cancer cell lines [12–14, 46, 47]. In pancreatic cancer patients increased levels of microvesicle (MV) TF activity are associated with VTE [48, 49]. However, this association is not observed in patients with other types of cancer, such as gastric, brain, colorectal, ovarian or lung cancer [50–53] (unpublished data). These clinical studies have spurred a number of investigators to analyze the role of tumor-derived, TF+ MVs in mouse models of cancer-associated thrombosis.

The first series of studies reported increased levels of circulating tumor-derived, TF+ MVs and activation of coagulation in mice bearing human tumors. The first study used SCID mice with subcutaneous human colorectal tumors (HCT116) and found a significant correlation between circulating human TF antigen and the tumor volume [11]. Similar results were observed in nude mice bearing orthotopic human pancreatic tumors (L3.6pl) [12]. The authors reported that tumor weight was correlated with human TF antigen and activation of coagulation, measured by plasma levels of thrombin-antithrombin (TAT) complexes [12]. We found that nude mice bearing orthotopic human pancreatic tumors (HPAF-II) had increased human MV TF activity and TAT complex in their circulation [13]. In contrast, no TF MVs or activation of coagulation were observed when the HPAF-II tumors or two other human pancreatic cell lines (BxPc-3 and AsPc-1) were grown subcutaneously [13] (data not shown). In another study, we used the human pancreatic cell line BxPc-3 [14]. Similar to the studies with HPAF-II, we found that nude mice bearing orthotopic BxPc-3 tumors had increased levels of TAT complex [14, 28]. Importantly, administration of an anti-human TF monoclonal antibody abolished the increase in TAT complex [13]. These studies indicate that tumors release TF+ MVs into the circulation, and human TF expressed by the tumors is responsible for the activation of coagulation in the tumor-bearing mice.

The second series of studies measured the effect of different tumors on thrombosis in mice (Tables 1 and 2). One study used two TF expressing cell lines: the murine pancreatic cancer cell line Panc02 and the murine lung cancer cell line LLC [47]. C57BL/6 mice with subcutaneous Panc02 tumors expressing GFP had detectable GFP-positive MVs in their circulation and these tumor-derived MVs accumulated at the site of FeCl3-induced vessel injury [47]. Furthermore, mice bearing subcutaneous Panc02 or LLC tumors had shortened occlusion times after FeCl3 injury of mesenteric vessels compared with controls [47]. Administration of an anti-P-selectin antibody reduced thrombosis in tumor-bearing mice [47]. Another study from the same group found that C57BL/6 mice bearing subcutaneous Panc02 had increased thrombosis in a laser-induced injury model of cremaster muscle microvessels compared to controls [54]. Finally, it was shown that C57BL/6 mice bearing subcutaneous Panc02 tumors had an increase in incidence of thrombosis and clot weight in the IVC stenosis model at 3 hours [55]. However, in contrast to the results with the mesenteric thrombosis model, the enhancement of thrombosis in the IVC stenosis model was not reduced by P-selectin deletion [55]. Further studies are needed to determine the pathways involved in the increased thrombosis in C57BL/6 mice with Panc02 pancreatic tumors.

We used a xenograft model to study the role of tumor-derived TF+ MVs in thrombosis (Table 2). In our initial study with nude mice bearing orthotopic HPAF-II tumors, we observed a shortening of the occlusion time in the FeCl3 saphenous vein model [13]. However, we did not see any difference in clot weight in the IVC stenosis model at 3 hours between mice bearing orthotopic HPAF-II and control mice, although there was a low incidence of thrombosis [13]. In a second study, we found that nude mice bearing orthotopic BxPC-3 tumors had an increased clot area at 3, 6, 9 and 24 hours after IVC stenosis but the incidence of clots in tumor-bearing mice was not significantly increased compared to control mice [14]. Due to the variability of the IVC stenosis model, we changed to the IVC stasis model to study the role of tumor-derived TF+ MV in cancer-associated thrombosis [28]. In addition, we generated a new line of BxPc-3 that expresses luciferase to allow non-invasive monitoring of tumor size. Consistent with our previous results with showing that there was no increase in human TF+ MVs and TAT complex in mice with subcutaneous human pancreatic tumors. In addition, we did not observe an increase in clot weight in nude mice bearing either BxPc-3 or AsPc-1 subcutaneous tumors compared to clots in control mice (Figure 1). In contrast, we observed that nude mice bearing orthotopic BxPc-3 tumors had significantly larger clots at 48 hours compared with clots in control mice [28]. Analysis of the clot area at different time points revealed an increase in the clot area in tumor-bearing mice at 24 and 48 hours but not at 3 and 6 hours compared with mice without tumors [28]. We believe that at 3 and 6 hours clot formation is primarily driven by vessel wall TF exposed during the surgery. At 24 and 48 hours vessel wall TF no longer drives clot formation and this allows us to detect the contribution of circulating tumor-derived TF+ MV to clot formation. We observed human TF activity in clots from tumor-bearing mice indicating the incorporation of tumor-derived MVs into the clot [28]. Importantly, administration of an anti-human TF antibody abolished the enhancement of clot formation in tumor-bearing mice but did not affect the size of the clot in control mice [28]. These data indicate that tumor-derived, TF+ MVs enhance thrombosis in mice bearing orthotopic BxPc-3 tumors.

Figure 1. Clots from subcutaneous tumor-bearing mice.

Figure 1

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BxPc-3 and AsPc-1 subcutaneous tumors were grown to 0.68 g [0.50–1.08] and 1.01 g [0.65–1.29], respectively (mean weight [range]). Clots were harvested and weighed at 48 hours after IVC stasis. Data are shown as mean ± standard deviation. Data were analyzed one way ANOVA with Dunnett’s test.

Neutrophil Extracellular Traps

NETs are released from activated neutrophils and contain granule proteins and chromatin. NETs were first described as a method of killing bacteria [56]. More recently, they were found in both venous and arterial clots, suggesting that they may contribute to thrombosis [57–59]. Indeed, mouse studies demonstrated that NETs contribute to thrombosis in the IVC stenosis model [33, 60]. In terms of cancer-associated thrombosis, plasma citrullinated histone H3, a marker of NETs, is associated with venous thrombosis in cancer patients [61]. Further, mouse studies sought to determine the role of NETs in cancer-associated thrombosis.

The murine mammary cancer cell line 4T1 has been used to study the role of NETs in mouse models of cancer-associated thrombosis. BALB/c mice with orthotopic 4T1 tumors had increased levels of granulocyte colony-stimulating factor (G-CSF), neutrophils, plasma DNA and citrullinated histone H3 compared to controls [62]. The presence of the tumor also induced a pro-thrombotic state in the mice with elevated levels of plasma von Willebrand factor, soluble P-selectin and fibrinogen [62]. Indeed, fibrin-rich thrombi were observed in veins of the lungs of tumor-bearing mice [62]. However, the study did not analyze the effect of tumors on thrombosis models. A second study also observed increased levels of neutrophils as well as plasma myeloperoxidase and DNA in mice with orthotopic 4T1 tumors [63]. We found increased plasma levels of G-CSF and neutrophils in nude mice with orthotopic BxPc-3 tumors [14] (unpublished data). These studies suggest that the presence of 4T1 and BxPc-3 tumors increases neutrophil levels and NET formation.

Leal and colleagues examined the effect of orthotopic 4T1 tumors on both arterial and venous thrombosis [63]. The presence of 4T1 tumors shortened the occlusion time in the FeCl3 carotid artery model and a Rose Bengal/laser-induced jugular vein injury model [63]. Arterial thrombi from tumor-bearing mice contained Ly6G+ neutrophils, and extracellular fiber-like DNA consistent with the presence of NETs. Interestingly, administration of DNAse I abolished the increase in venous thrombosis in tumor-bearing mice without affecting venous thrombosis in control mice [63]. In contrast, DNAse I treatment prolonged the occlusion time in both control and tumor-bearing mice in FeCl3 carotid artery model [63]. These studies suggest that NETs contribute to arterial and venous thrombosis in mice with orthotopic 4T1 tumors.

Other mouse models of cancer-associated thrombosis

One study analyzed thrombosis in BALB/c nude mice bearing subcutaneous human lung adenocarcinoma tumors (A549) [64]. Tumor-bearing mice had increased clot weights in an IVC stenosis model and shortened occlusion times in a FeCl3 saphenous vein model [64]. Furthermore, it was found that the VEGF inhibitor bevacizumab increased the plasma concentration of tumor-derived PAI-1 and clot weight in the IVC stenosis model at 3 hours [64]. Bevacizumab also increased thrombosis in non-tumor bearing wild-type mice but not in PAI-1 deficient mice [64]. These studies indicate that the presence of subcutaneous A549 tumors increase thrombosis in mice.

Another study investigated the role of growth arrest-specific 6 in a mouse model of cancer-associated thrombosis using the mouse lung carcinoma (M27) and Eμ-myc B-cell lymphoma cell lines [65]. C57BL/6N mice bearing M27 lung tumors or Eμ-myc B-cell lymph node tumors had an increase in thrombus size in the FeCl3 IVC model [65]. Thrombus size was measured by high frequency ultrasonography [65]. Interestingly, the clot size was smaller in growth arrest-specific 6 deficient mice and no increase was observed in the presence of tumors [65].

4. Summary

Different mouse models can be used to study cancer-associated thrombosis. The strengths of using immunocompetent mice are that they will have a full immune response to the tumor and gene knock-out mice can be used to study the role of different proteins in cancer-associated thrombosis. However, there are a limited number of mouse cancer lines available. In contrast, there are numerous human cancer cell lines and PDXs that can be used with immunodeficient mice. A weakness of immunodeficient mice is that they have an impaired immune response. Tumor growth can be more easily monitored in subcutaneous tumor compared to orthotopic tumors. However, a strength of using orthotopic tumors is that the tumor microenvironment is more similar to that found in patients. Although a variety of different thrombosis models have been used, the IVC stenosis or stasis are most common. Further studies are needed to determine the role of different pathways in thrombosis in mice bearing different types of tumors.

Acknowledgments

This work was supported by grants from the NIH (Y.H. T32 HL007149-41), the K. G. Jebsen Thrombosis Research and Expertise Center and the John C. Parker Professorship (NM). Brian C. Cooley and The Animal Surgery Core Laboratory of the McAllister Heart Institute at University of North Carolina at Chapel Hill performed the thrombosis experiments. We would like to thank Dr. Steven P. Grover and Reaves Houston for providing comments on the manuscript.

Footnotes

Conflict of interest statement

The authors state that they have no conflict of interest.

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