**1. Introduction**

182 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

Yu KD, Di GH, Fan L, Chen AX, Yang C, Shao ZM (2009b) Lack of an association between a

functional polymorphism in the interleukin-6 gene promoter and breast cancer risk: a meta-analysis involving 25,703 subjects. *Breast Cancer Res Treat* 122:483-488. Zhang GJ, Adachi I (1999) Serum interleukin-6 levels correlate to tumor progression and prognosis in metastatic breast carcinoma. *Anticancer Res* 19:1427-1432.

> Despite all the modern advances in treatment for breast cancer, metastatic disease remains the hurdle to surmount in curing breast cancer or, at least, in significantly reducing morbidity and mortality to improve long-term survival and quality of life. For over a century, inflammation and thrombosis have been linked to metastatic cancer (Boccaccio & Medico, 2006). In addition to being known for describing the factors leading to venous thromboembolism (alterations in blood flow, vascular endothelial injury, and hypercoagulability) as Virchow's triad, in 1863 Virchow noted a connection between chronic inflammation and cancer based on the recruitment of leukocytes to cancerous lesions (reviewed in (Balkwill & Mantovani, 2001)) (**Fig. 1**).

Fig. 1. The three faces of cancer metastasis. (Portraits obtained from public domain).

The Role of Fibrin(ogen) in Transendothelial Cell Migration During Breast Cancer Metastasis 185

transforming growth factor (TGF)-; adhesive glycoproteins, including fibrinogen (Fg), fibronectin and von Willebrand factor; and lipid mediators such as lysophosphatidic acid, platelet-activating factor, leukotriene B4, and thromboxanes. During secondary hemostasis, coagulation is activated either through the extrinsic pathway via tissue factor (TF)-Factor VII (FVII)/activated FVII (FVIIa) or the intrinsic pathway through Factor XII/FXIIa (**Fig. 2**). These pathways converge at the formation of the tenase complex that activates FX to FXa leading to thrombin activation. Thrombin cleaves soluble plasma Fg into fibrin monomers that form the insoluble fibrin clot after fibrin monomer polymerization and covalent crosslinking and stabilization by activated FXIII (FXIIIa). The fibrin clot provides a provisional matrix upon which injured endothelial cells adhere, proliferate and migrate to restore an intact endothelium lining blood vessels. Furthermore, fibrin and Fg provide a reservoir for sequestration of growth factors including FGF-2 (Sahni et al., 1998; Sahni et al., 1999), VEGF (Sahni & Francis, 2000), and TGF- (Schachtrup et al., 2010), as well as an adhesive substrate for recruitment of leukocytes and stromal fibroblasts to aid in wound repair (Rybarczyk et al., 2003; Ugarova & Yakubenko, 2001). Normal wound repair is selflimiting as the provisional fibrin matrix is dissolved by various proteases, *e.g.*, plasmin, upon resolution of the vascular injury, reduction of inflammation and restoration of normal

In the 1980s, however, Dvorak likened cancer progression to "wounds that never heal" in which Fg and fibrin also play prominent roles (Dvorak, 1986), and as reviewed by Coussens and Werb (Coussens & Werb, 2002). Several key steps in normal wound repair are also manifested during cancer progression (**Fig. 3**). As discussed above, a heighten state of coagulation occurs immediately after wound injury, and the release of chemokines and cytokines from activated platelets to recruit and activate proinflammatory cell types to the

Fig. 3. Normal wound repair is depicted in panel A and mechanisms of wound repair left unchecked in cancer are depicted in Panel B**.** (Figure reprinted from (Coussens & Werb,

wound site amplify the inflammatory response system wide.

2002) with permission from Nature Publishing Group).

function (**Fig. 2**).

Rudolf Virchow, Armand Trousseau and Stephen Paget each provided valuable insight into the pathophysiology of invasive carcinomas—these theories still hold today to explain molecular mechanisms of cancer metastasis. Hypercoagulability is often diagnosed before identification of a coexisting malignancy, and is associated with increased thromboembolic risk (Sorensen et al., 2000). Armand Trousseau (Trousseau, 1865) (**Fig. 1**) identified and described the association between cancer and clot formation in 1865 and, shortly thereafter, self-identified these findings as a consequence of gastric cancer from which he later succumbed (Varki, 2007). Trousseau's Syndrome is associated with hypercoagulability and thromboembolic events in adenocarcinomas (Starakis et al., 2010). Another important contribution that has lead to better understanding of the mechanisms of cancer metastasis was provided by Stephen Paget in 1889 (Paget, 1889) when he propose the seed and soil concept of cancer metastasis (**Fig. 1**). By examining countless autopsy specimen from breast cancer patients, Paget determined that cancer cells, the "seed", had a preference to metastasize to distinct organs of the body based on favorable interactions with the stromal microenvironment, the "soil". As reviewed by Langley and Fiddler (Langley & Fidler, 2011), it is clear that cancer therapy is targeted to either the "seed" through chemotherapy with cytotoxic drugs or the "soil" by manipulating stromal contributions favorable to metastatic growth such as inhibiting angiogenesis.

Fig. 2. Schematic view of intrinsic and extrinsic coagulation pathways.

Red lines denote pathway inhibitors of coagulation and green lines denote thrombin activation of hemostatic factors. (Reproduced from public domain image).

Appropriate activation of the clotting cascade is fundamental to arrest bleeding in response to vascular injury. The immediate response, known as primary hemostasis, involves vasoconstriction of blood vessels and activation and aggregation of platelets to form a plug at the site of vascular injury. Activated platelets release a panoply of stored constituents including: chemokines (IL-8) and growth factors such as platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2 and

Rudolf Virchow, Armand Trousseau and Stephen Paget each provided valuable insight into the pathophysiology of invasive carcinomas—these theories still hold today to explain molecular mechanisms of cancer metastasis. Hypercoagulability is often diagnosed before identification of a coexisting malignancy, and is associated with increased thromboembolic risk (Sorensen et al., 2000). Armand Trousseau (Trousseau, 1865) (**Fig. 1**) identified and described the association between cancer and clot formation in 1865 and, shortly thereafter, self-identified these findings as a consequence of gastric cancer from which he later succumbed (Varki, 2007). Trousseau's Syndrome is associated with hypercoagulability and thromboembolic events in adenocarcinomas (Starakis et al., 2010). Another important contribution that has lead to better understanding of the mechanisms of cancer metastasis was provided by Stephen Paget in 1889 (Paget, 1889) when he propose the seed and soil concept of cancer metastasis (**Fig. 1**). By examining countless autopsy specimen from breast cancer patients, Paget determined that cancer cells, the "seed", had a preference to metastasize to distinct organs of the body based on favorable interactions with the stromal microenvironment, the "soil". As reviewed by Langley and Fiddler (Langley & Fidler, 2011), it is clear that cancer therapy is targeted to either the "seed" through chemotherapy with cytotoxic drugs or the "soil" by manipulating stromal contributions favorable to metastatic

growth such as inhibiting angiogenesis.

Fig. 2. Schematic view of intrinsic and extrinsic coagulation pathways.

activation of hemostatic factors. (Reproduced from public domain image).

Red lines denote pathway inhibitors of coagulation and green lines denote thrombin

Appropriate activation of the clotting cascade is fundamental to arrest bleeding in response to vascular injury. The immediate response, known as primary hemostasis, involves vasoconstriction of blood vessels and activation and aggregation of platelets to form a plug at the site of vascular injury. Activated platelets release a panoply of stored constituents including: chemokines (IL-8) and growth factors such as platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-; adhesive glycoproteins, including fibrinogen (Fg), fibronectin and von Willebrand factor; and lipid mediators such as lysophosphatidic acid, platelet-activating factor, leukotriene B4, and thromboxanes. During secondary hemostasis, coagulation is activated either through the extrinsic pathway via tissue factor (TF)-Factor VII (FVII)/activated FVII (FVIIa) or the intrinsic pathway through Factor XII/FXIIa (**Fig. 2**). These pathways converge at the formation of the tenase complex that activates FX to FXa leading to thrombin activation. Thrombin cleaves soluble plasma Fg into fibrin monomers that form the insoluble fibrin clot after fibrin monomer polymerization and covalent crosslinking and stabilization by activated FXIII (FXIIIa). The fibrin clot provides a provisional matrix upon which injured endothelial cells adhere, proliferate and migrate to restore an intact endothelium lining blood vessels. Furthermore, fibrin and Fg provide a reservoir for sequestration of growth factors including FGF-2 (Sahni et al., 1998; Sahni et al., 1999), VEGF (Sahni & Francis, 2000), and TGF- (Schachtrup et al., 2010), as well as an adhesive substrate for recruitment of leukocytes and stromal fibroblasts to aid in wound repair (Rybarczyk et al., 2003; Ugarova & Yakubenko, 2001). Normal wound repair is selflimiting as the provisional fibrin matrix is dissolved by various proteases, *e.g.*, plasmin, upon resolution of the vascular injury, reduction of inflammation and restoration of normal function (**Fig. 2**).

In the 1980s, however, Dvorak likened cancer progression to "wounds that never heal" in which Fg and fibrin also play prominent roles (Dvorak, 1986), and as reviewed by Coussens and Werb (Coussens & Werb, 2002). Several key steps in normal wound repair are also manifested during cancer progression (**Fig. 3**). As discussed above, a heighten state of coagulation occurs immediately after wound injury, and the release of chemokines and cytokines from activated platelets to recruit and activate proinflammatory cell types to the wound site amplify the inflammatory response system wide.

Fig. 3. Normal wound repair is depicted in panel A and mechanisms of wound repair left unchecked in cancer are depicted in Panel B**.** (Figure reprinted from (Coussens & Werb, 2002) with permission from Nature Publishing Group).

The Role of Fibrin(ogen) in Transendothelial Cell Migration During Breast Cancer Metastasis 187

Cell-associated TF expression by cancer cells correlates with disease severity and poor prognosis {reviewed in (Palumbo & Degen, 2007)}. Although tumor cell-associated TF expression is not required for the growth of primary tumors, it is necessary for their metastatic spread (Palumbo et al., 2007). Similarly, FXIII and Fg are important for the metastatic spread of tumor cells through both the circulation and lymphatic systems but not primary tumor growth (Palumbo et al., 2008; Palumbo & Degen, 2001; Palumbo & Degen, 2007; Palumbo et al., 2000; Palumbo et al., 2002; Palumbo et al., 2007; Palumbo et al., 2005). Moreover, FXIII, Fg and platelets are important substrates or cell targets for thrombin action demonstrating the critical role played by the hemostatic system in promoting cancer metastasis. Degen and colleagues suggest that tumor cell-associated TF mediates thrombin generation to support the early survival of micrometastases by at least two mechanisms: 1) the formation of platelet-fibrin microthrombi to protect newly formed micrometastases from natural killer (NK) cell-mediated cytotoxicity, and 2) by promoting mechanical stability of tumor cell emboli within vascular beds at distant metastatic sites (Palumbo et al., 2008; Palumbo & Degen, 2001; Palumbo & Degen, 2007; Palumbo et al., 2000; Palumbo et al., 2002;

**2.2 Chronic inflammation is associated with cancer initiation and progression** 

Systemic inflammation is clearly linked with adverse prognosis in patients with cancer, and is characterized by elevated expression of pro-inflammatory mediators including interleukin (IL)-6 (Gao et al., 2007; Knupfer & Preiss, 2007). IL-6 is the major cytokine responsible for upregulation of specific plasma proteins in the liver during an acute phase response (Baumann & Gauldie, 1994), and also in chronic inflammation (Barton, 2001; Lin & Karin, 2007; Neurath & Finotto, 2011). IL-6 induces expression of target genes, including Fg, by activation of Stat3 (Duan & Simpson-Haidaris, 2003); Stat3 is often constitutively active in breast cancer, and tumor growth can become dependent on Stat3 signaling (Pensa et al., 2009). Both IL-6 and Fg levels are elevated in patients with advanced lung cancer (Yamaguchi et al., 1998). In breast cancer patients, serum IL-6 correlates with increasing numbers of involved sites, liver metastasis, and disease progression (Knupfer & Preiss, 2007; Salgado et al., 2003). In 2002, Drix et al demonstrated that IL-6, VEGF and D-dimer levels are elevated in patients with progressive breast cancer; these markers correlate positively with disease severity, and serum IL-6 is an independent prognostic factor in patients with metastatic disease (Dirix et al., 2002). Elevated levels of Fg, D-dimers, IL-6, VEGF and soluble P-selectin, an indicator of platelet activation, were also found in the plasma of breast cancer patients by Caine et al, who furthered demonstrated that IL-6 induces dosedependent release of VEGF from platelets in vitro (Caine et al., 2004). Steinbrecher et al demonstrated a direct link between fibrin(ogen), elevated IL-6 levels and the development of inflammation-driven cancer using a mouse model of colitis-associated cancer (Steinbrecher et al., 2010). IL-6 serves as a marker to predict which patients will respond poorly to anti-endocrine chemotherapy (Zhang & Adachi, 1999), as a marker of tumor staging and a predictor of micrometastases (Ravishankaran & Karunanithi, 2011). IL-6 also induces VEGF expression (Cohen et al., 1996) and invasion and migration of breast cancer cells (Walter et al., 2009). Furthermore, overexpression of Her2 in breast cancer cells upregulates IL-6 leading to Stat3 activation and altered gene expression resulting in an autocrine feedback loop promoting cell survival (Hartman et al., 2011). Together, these reports substantiate the importance of fibrin(ogen) and inflammation in cancer metastasis.

Palumbo et al., 2007; Palumbo et al., 2005).

Systemic inflammation is best characterized by the innate acute phase response to injury or infection whereby the synthesis of a host of plasma proteins by the liver is altered to immediately respond to disruptions of homeostasis (Baumann & Gauldie, 1994). Of note, Creactive protein and Fg are two positive (upregulated) acute phase proteins whose expression is also elevated in malignancies (Jones et al., 2006; Yamaguchi et al., 1998; Yigit et al., 2008). Coagulation and deposition of a provisional fibrin matrix occurs within minutes of vascular injury, and changes in expression of adhesion molecules on the surface of activated endothelium leads to the rolling and slowing of circulating leukocytes, firm attachment and the processes of diapedesis, *i.e.*, transmigration across the endothelial cell barrier into interstitial spaces. Neutrophils are the first proinflammatory cells to appear in the wound space where they release molecules to kill invading microorganisms and promote recruitment of stromal cells such as fibroblasts and endothelial cells to the wound space. Locally deposited growth factors promote cell proliferation and migration leading to the formation of granulation tissue over several days to a few weeks, which is the result of fibroblasts/myofibroblasts depositing extracellular matrix constituents (*e.g.*, collagens) and endothelial cells forming new blood vessels to facilitate would closure. In the case of cutaneous wounds, re-epithelialization begins to close the wound, the provisional fibrin matrix is dissolved, and infiltrating monocytes/macrophages clean up wound debris in preparation for matrix remodelling, deposition of a complete basement membrane (*e.g*., laminin) and, over weeks to months, gradual restoration of the tensile strength of the tissue (Coussens & Werb, 2002). In contrast, the orderly array of signaling components that turn on and off cell migration, cell proliferation, and angiogenesis during wound repair goes array during cancer such that cell growth is unchecked, mechanisms of apoptosis are overridden and the stromal compartment is dramatically altered to perpetuate angiogenesis, tumor growth and cell migration to promote metastasis (**Fig. 3**).

Metastatic disease remains the prevailing reason for treatment failure and death from solid tumors including breast cancers. Only recently have three major areas of research outside the realm of the primary tumor cells themselves been considered viable for development of new therapeutic strategies to prevent the initiation, progression and metastasis of tumors. These include hemostatic factors, the tumor stromal microenvironment, and chronic inflammation. The blood coagulation protein Fg and its insoluble counterpart, fibrin, play central roles in inflammation, venous thromboembolism, and as components of the extracellular matrix. The goals of this chapter are three-fold: first, to review the current understanding of the roles of Fg and/or fibrin {commonly referred to as fibrin(ogen)} in cancer progression in general; second, to provide evidence that fibrin(ogen) likely plays a critical role in the metastatic spread of breast cancer; and third, to propose new therapies for treatment and future avenues of research to elucidate the molecular mechanisms that promote the phenotypic switch of breast epithelial cells to a metastatic cell phenotype.

#### **2. Fibrin(ogen) in cancer progression**

#### **2.1 Hemostatic factors and vascular cells promote tumor metastasis**

Molecules and cells linked to the prothrombotic state of Trousseau's syndrome that also facilitate cancer metastasis including thrombin, TF, selectins, platelets, endothelial cells and fibrin (Varki, 2007). It is well known that thrombin contributes to the severity of cancer progression by promoting tumor angiogenesis, cancer cell proliferation and metastasis by mechanisms other than just thrombin generation of fibrin (Nierodzik & Karpatkin, 2006).

Systemic inflammation is best characterized by the innate acute phase response to injury or infection whereby the synthesis of a host of plasma proteins by the liver is altered to immediately respond to disruptions of homeostasis (Baumann & Gauldie, 1994). Of note, Creactive protein and Fg are two positive (upregulated) acute phase proteins whose expression is also elevated in malignancies (Jones et al., 2006; Yamaguchi et al., 1998; Yigit et al., 2008). Coagulation and deposition of a provisional fibrin matrix occurs within minutes of vascular injury, and changes in expression of adhesion molecules on the surface of activated endothelium leads to the rolling and slowing of circulating leukocytes, firm attachment and the processes of diapedesis, *i.e.*, transmigration across the endothelial cell barrier into interstitial spaces. Neutrophils are the first proinflammatory cells to appear in the wound space where they release molecules to kill invading microorganisms and promote recruitment of stromal cells such as fibroblasts and endothelial cells to the wound space. Locally deposited growth factors promote cell proliferation and migration leading to the formation of granulation tissue over several days to a few weeks, which is the result of fibroblasts/myofibroblasts depositing extracellular matrix constituents (*e.g.*, collagens) and endothelial cells forming new blood vessels to facilitate would closure. In the case of cutaneous wounds, re-epithelialization begins to close the wound, the provisional fibrin matrix is dissolved, and infiltrating monocytes/macrophages clean up wound debris in preparation for matrix remodelling, deposition of a complete basement membrane (*e.g*., laminin) and, over weeks to months, gradual restoration of the tensile strength of the tissue (Coussens & Werb, 2002). In contrast, the orderly array of signaling components that turn on and off cell migration, cell proliferation, and angiogenesis during wound repair goes array during cancer such that cell growth is unchecked, mechanisms of apoptosis are overridden and the stromal compartment is dramatically altered to perpetuate angiogenesis, tumor

Metastatic disease remains the prevailing reason for treatment failure and death from solid tumors including breast cancers. Only recently have three major areas of research outside the realm of the primary tumor cells themselves been considered viable for development of new therapeutic strategies to prevent the initiation, progression and metastasis of tumors. These include hemostatic factors, the tumor stromal microenvironment, and chronic inflammation. The blood coagulation protein Fg and its insoluble counterpart, fibrin, play central roles in inflammation, venous thromboembolism, and as components of the extracellular matrix. The goals of this chapter are three-fold: first, to review the current understanding of the roles of Fg and/or fibrin {commonly referred to as fibrin(ogen)} in cancer progression in general; second, to provide evidence that fibrin(ogen) likely plays a critical role in the metastatic spread of breast cancer; and third, to propose new therapies for treatment and future avenues of research to elucidate the molecular mechanisms that promote the phenotypic switch of breast epithelial cells to a metastatic cell phenotype.

Molecules and cells linked to the prothrombotic state of Trousseau's syndrome that also facilitate cancer metastasis including thrombin, TF, selectins, platelets, endothelial cells and fibrin (Varki, 2007). It is well known that thrombin contributes to the severity of cancer progression by promoting tumor angiogenesis, cancer cell proliferation and metastasis by mechanisms other than just thrombin generation of fibrin (Nierodzik & Karpatkin, 2006).

growth and cell migration to promote metastasis (**Fig. 3**).

**2. Fibrin(ogen) in cancer progression** 

**2.1 Hemostatic factors and vascular cells promote tumor metastasis** 

Cell-associated TF expression by cancer cells correlates with disease severity and poor prognosis {reviewed in (Palumbo & Degen, 2007)}. Although tumor cell-associated TF expression is not required for the growth of primary tumors, it is necessary for their metastatic spread (Palumbo et al., 2007). Similarly, FXIII and Fg are important for the metastatic spread of tumor cells through both the circulation and lymphatic systems but not primary tumor growth (Palumbo et al., 2008; Palumbo & Degen, 2001; Palumbo & Degen, 2007; Palumbo et al., 2000; Palumbo et al., 2002; Palumbo et al., 2007; Palumbo et al., 2005). Moreover, FXIII, Fg and platelets are important substrates or cell targets for thrombin action demonstrating the critical role played by the hemostatic system in promoting cancer metastasis. Degen and colleagues suggest that tumor cell-associated TF mediates thrombin generation to support the early survival of micrometastases by at least two mechanisms: 1) the formation of platelet-fibrin microthrombi to protect newly formed micrometastases from natural killer (NK) cell-mediated cytotoxicity, and 2) by promoting mechanical stability of tumor cell emboli within vascular beds at distant metastatic sites (Palumbo et al., 2008; Palumbo & Degen, 2001; Palumbo & Degen, 2007; Palumbo et al., 2000; Palumbo et al., 2002; Palumbo et al., 2007; Palumbo et al., 2005).

#### **2.2 Chronic inflammation is associated with cancer initiation and progression**

Systemic inflammation is clearly linked with adverse prognosis in patients with cancer, and is characterized by elevated expression of pro-inflammatory mediators including interleukin (IL)-6 (Gao et al., 2007; Knupfer & Preiss, 2007). IL-6 is the major cytokine responsible for upregulation of specific plasma proteins in the liver during an acute phase response (Baumann & Gauldie, 1994), and also in chronic inflammation (Barton, 2001; Lin & Karin, 2007; Neurath & Finotto, 2011). IL-6 induces expression of target genes, including Fg, by activation of Stat3 (Duan & Simpson-Haidaris, 2003); Stat3 is often constitutively active in breast cancer, and tumor growth can become dependent on Stat3 signaling (Pensa et al., 2009). Both IL-6 and Fg levels are elevated in patients with advanced lung cancer (Yamaguchi et al., 1998). In breast cancer patients, serum IL-6 correlates with increasing numbers of involved sites, liver metastasis, and disease progression (Knupfer & Preiss, 2007; Salgado et al., 2003). In 2002, Drix et al demonstrated that IL-6, VEGF and D-dimer levels are elevated in patients with progressive breast cancer; these markers correlate positively with disease severity, and serum IL-6 is an independent prognostic factor in patients with metastatic disease (Dirix et al., 2002). Elevated levels of Fg, D-dimers, IL-6, VEGF and soluble P-selectin, an indicator of platelet activation, were also found in the plasma of breast cancer patients by Caine et al, who furthered demonstrated that IL-6 induces dosedependent release of VEGF from platelets in vitro (Caine et al., 2004). Steinbrecher et al demonstrated a direct link between fibrin(ogen), elevated IL-6 levels and the development of inflammation-driven cancer using a mouse model of colitis-associated cancer (Steinbrecher et al., 2010). IL-6 serves as a marker to predict which patients will respond poorly to anti-endocrine chemotherapy (Zhang & Adachi, 1999), as a marker of tumor staging and a predictor of micrometastases (Ravishankaran & Karunanithi, 2011). IL-6 also induces VEGF expression (Cohen et al., 1996) and invasion and migration of breast cancer cells (Walter et al., 2009). Furthermore, overexpression of Her2 in breast cancer cells upregulates IL-6 leading to Stat3 activation and altered gene expression resulting in an autocrine feedback loop promoting cell survival (Hartman et al., 2011). Together, these reports substantiate the importance of fibrin(ogen) and inflammation in cancer metastasis.

The Role of Fibrin(ogen) in Transendothelial Cell Migration During Breast Cancer Metastasis 189

plasmin cleavage fragment E and C-terminal fragments D. N-terminal disulfide knot (NDSK) (dashed line) is the minimal sequence of the central domain after CNBr cleavage and is structurally similar to plasmin E fragment. Residues on Fg for receptor-cell binding domains are: CD11c/CD18, A17-19; integrin RGDF, A95-98 and RGDS, A572-575; ICAM-1,117-133; CD11b/CD18, 190-202, 228-253 and 390-396; platelet (PT) binding, 400-411. The heparin binding domain (HBD) at 15-42 overlaps the VE-cadherin binding site. The first fibrin degradation products (FDPs) released by plasmin cleavage are the 15-42 domain and the Cterminal 2/3rd of the A chain, termed C, which contain several cell binding domains.

The tumor microenvironment is a complex entity composed not only of extracellular matrix (ECM) constituents including: i) growth factors; ii) cytokines and chemokines; iii) proteases; and iv) matrix glycoproteins, glycosaminoglycans and proteoglycans—but also diverse cell populations that influence the behavior of cancer cells including: v) immune cells such as lymphocytes, NK cells, dendritic cells, macrophages and neutrophils; vi) stromal fibroblasts/myofibroblasts, adipocytes and stem cells; and vii) cells of the vasculature including endothelial cells, pericytes and smooth muscle cells (reviewed in (Andre et al., 2010; Anton & Glod, 2009; De Wever et al., 2008; Deryugina & Quigley, 2006; Tlsty & Coussens, 2006; Ulisse et al., 2009)). Although activated inflammatory cells in the tumor microenvironment play important roles in cancer initiation, progression, angiogenesis and metastasis, they are not the most numerous. Cancer-associated fibroblasts, similar to myofibroblasts of healing wounds, are the most abundant stromal cells in the tumor microenvironment (Tlsty & Coussens, 2006), and contribute significantly to chronic inflammation by production of chemokines, cytokines, and pro-angiogenic factors and deposition of matrix constituents that support new blood vessel formation required for tumor growth, cell migration and metastasis (De Wever et al., 2008). Solid tumors need to develop their own blood supply for nutrient delivery and removal of toxic waste. Angiogenesis, the formation of new blood vessels from existing vasculature, requires activation of proteases leading to degradation of the basement membrane, endothelial cell sprouting and pericyte attachment for vessel stabilization. Cancer-associated fibroblasts play an important role in synchronizing these events (De Wever et al., 2008). Furthermore, the topography of the ECM mediates vascular development and regulates the speed at which cells migrate during angiogenesis (Bauer et al., 2009). Vascular endothelial cells play a pivotal role in regulating leukocyte recruitment during inflammation (McGettrick et al., 2007). In most cases, cancers exploit pro-inflammatory mediators and recruited inflammatory cells to benefit their own survival (Lorusso & Ruegg, 2008) (also as reviewed

Fg and fibrin deposition is found within the stroma of most solid tumors (Simpson-Haidaris & Rybarczyk, 2001), and elevated levels of plasma Fg and fibrin degradation products (FDPs) correlate positively with lymph node involvement and metastatic spread of colorectal, ovarian, lung and breast cancers (Sahni et al., 2009; Varki, 2007). Fibrin deposition at the tumor-normal host cell interface as well as in the stroma of primary tumors is well documented, and is thought to protect tumors from infiltrating inflammatory cells by acting as a barrier thereby preventing inflammatory reactions directed towards the tumor cells (reviewed in (Simpson-Haidaris & Rybarczyk, 2001)). The presence of D-dimer, a fibrin degradation product indicative of pathological fibrin formation and dissolution, correlates

**2.4 Fibrin(ogen) in the stromal microenvironment in breast cancer** 

in (Simpson-Haidaris et al., 2010)).

#### **2.3 Fibrin(ogen) functions as a bridging molecule in cell-cell interactions during coagulation and inflammatory cell trafficking**

Excessive fibrin deposition is accompanied by local expression of proinflammatory mediators, vascular leakage, and inflammatory cell recruitment and activation, leading to amplification of the inflammatory response (Clark, 1996; Simpson-Haidaris & Rybarczyk, 2001; van Hinsbergh et al., 2001). Specific structural features of fibrin(ogen) modulate the functions of a variety of different cell types including endothelial, epithelial, leukocytes, platelets and fibroblasts (**Fig. 4**). Cell receptors that bind to fibrin(ogen) include: 3 integrins (IIb3 and v3) (Bennett et al., 2009); 2 integrins (CD11a/CD18 and CD11b/CD18) (Altieri et al., 1993; Flick et al., 2004; Lishko et al., 2004; Loike et al., 1991; Ugarova et al., 2003; Yakovlev et al., 2005); and 1 integrin, 51 (Asakura et al., 1997; Suehiro et al., 1997). Nonintegrin adhesion molecules that bind to fibrin(ogen) include intercellular adhesion molecule (ICAM)-1 (Languino et al., 1993; Pluskota & D'Souza, 2000), vascular endothelial (VE)-cadherin (Bach et al., 1998b) and heparan sulfate proteoglycans (HSPG) (Odrljin et al., 1996a; Odrljin et al., 1996b). Fibrin(ogen) also modulates a number of signaling molecules important in innate immunity. Fg-bound FGF-2 induces expression of uPA, uPA receptor and PAI-1, and fibrin(ogen) induce IL-8, MCP-1 or IL-1 expression in endothelial cells (Guo et al., 2004; Harley & Powell, 1999; Kuhns et al., 2001; Lee et al., 2001; Qi & Kreutzer, 1995; Ramsby & Kreutzer, 1994; Sahni et al., 2004). Fg and fibrin activate NF-B and AP-1 (Guo et al., 2004; Sitrin et al., 1998), transcription factors critical for propagation of inflammation.

Fig. 4. Fibrin(ogen) enzyme and CNBr cleavage fragments and cell recognition domains*.* 

Fg AB and chains are held together by 29 pairs of disulfide bonds (approximated by the vertical lines) with the N-termini of all six chains held together in the central domain. Electron microscopy studies indicate that the dimeric Fg molecule appears as a trinodular structure as depicted by the red ball and stick cartoon. Thrombin release of fibrinopeptides, FPA and FPB, from A and B N-termini, respectively, produces soluble fibrin leading to fibrin polymerization into an insoluble gel stabilized by FXIIIA-mediated crosslinks between - and - chains. Lines below the ball and stick cartoon denote N-terminal

Excessive fibrin deposition is accompanied by local expression of proinflammatory mediators, vascular leakage, and inflammatory cell recruitment and activation, leading to amplification of the inflammatory response (Clark, 1996; Simpson-Haidaris & Rybarczyk, 2001; van Hinsbergh et al., 2001). Specific structural features of fibrin(ogen) modulate the functions of a variety of different cell types including endothelial, epithelial, leukocytes, platelets and fibroblasts (**Fig. 4**). Cell receptors that bind to fibrin(ogen) include: 3 integrins (IIb3 and v3) (Bennett et al., 2009); 2 integrins (CD11a/CD18 and CD11b/CD18) (Altieri et al., 1993; Flick et al., 2004; Lishko et al., 2004; Loike et al., 1991; Ugarova et al., 2003; Yakovlev et al., 2005); and 1 integrin, 51 (Asakura et al., 1997; Suehiro et al., 1997). Nonintegrin adhesion molecules that bind to fibrin(ogen) include intercellular adhesion molecule (ICAM)-1 (Languino et al., 1993; Pluskota & D'Souza, 2000), vascular endothelial (VE)-cadherin (Bach et al., 1998b) and heparan sulfate proteoglycans (HSPG) (Odrljin et al., 1996a; Odrljin et al., 1996b). Fibrin(ogen) also modulates a number of signaling molecules important in innate immunity. Fg-bound FGF-2 induces expression of uPA, uPA receptor and PAI-1, and fibrin(ogen) induce IL-8, MCP-1 or IL-1 expression in endothelial cells (Guo et al., 2004; Harley & Powell, 1999; Kuhns et al., 2001; Lee et al., 2001; Qi & Kreutzer, 1995; Ramsby & Kreutzer, 1994; Sahni et al., 2004). Fg and fibrin activate NF-B and AP-1 (Guo et al., 2004; Sitrin et al., 1998), transcription factors critical for propagation of inflammation.

Fig. 4. Fibrin(ogen) enzyme and CNBr cleavage fragments and cell recognition domains*.* 

Fg AB and chains are held together by 29 pairs of disulfide bonds (approximated by the vertical lines) with the N-termini of all six chains held together in the central domain. Electron microscopy studies indicate that the dimeric Fg molecule appears as a trinodular structure as depicted by the red ball and stick cartoon. Thrombin release of fibrinopeptides, FPA and FPB, from A and B N-termini, respectively, produces soluble fibrin leading to fibrin polymerization into an insoluble gel stabilized by FXIIIA-mediated crosslinks between - and - chains. Lines below the ball and stick cartoon denote N-terminal

**2.3 Fibrin(ogen) functions as a bridging molecule in cell-cell interactions during** 

**coagulation and inflammatory cell trafficking** 

plasmin cleavage fragment E and C-terminal fragments D. N-terminal disulfide knot (NDSK) (dashed line) is the minimal sequence of the central domain after CNBr cleavage and is structurally similar to plasmin E fragment. Residues on Fg for receptor-cell binding domains are: CD11c/CD18, A17-19; integrin RGDF, A95-98 and RGDS, A572-575; ICAM-1,117-133; CD11b/CD18, 190-202, 228-253 and 390-396; platelet (PT) binding, 400-411. The heparin binding domain (HBD) at 15-42 overlaps the VE-cadherin binding site. The first fibrin degradation products (FDPs) released by plasmin cleavage are the 15-42 domain and the Cterminal 2/3rd of the A chain, termed C, which contain several cell binding domains.

#### **2.4 Fibrin(ogen) in the stromal microenvironment in breast cancer**

The tumor microenvironment is a complex entity composed not only of extracellular matrix (ECM) constituents including: i) growth factors; ii) cytokines and chemokines; iii) proteases; and iv) matrix glycoproteins, glycosaminoglycans and proteoglycans—but also diverse cell populations that influence the behavior of cancer cells including: v) immune cells such as lymphocytes, NK cells, dendritic cells, macrophages and neutrophils; vi) stromal fibroblasts/myofibroblasts, adipocytes and stem cells; and vii) cells of the vasculature including endothelial cells, pericytes and smooth muscle cells (reviewed in (Andre et al., 2010; Anton & Glod, 2009; De Wever et al., 2008; Deryugina & Quigley, 2006; Tlsty & Coussens, 2006; Ulisse et al., 2009)). Although activated inflammatory cells in the tumor microenvironment play important roles in cancer initiation, progression, angiogenesis and metastasis, they are not the most numerous. Cancer-associated fibroblasts, similar to myofibroblasts of healing wounds, are the most abundant stromal cells in the tumor microenvironment (Tlsty & Coussens, 2006), and contribute significantly to chronic inflammation by production of chemokines, cytokines, and pro-angiogenic factors and deposition of matrix constituents that support new blood vessel formation required for tumor growth, cell migration and metastasis (De Wever et al., 2008). Solid tumors need to develop their own blood supply for nutrient delivery and removal of toxic waste. Angiogenesis, the formation of new blood vessels from existing vasculature, requires activation of proteases leading to degradation of the basement membrane, endothelial cell sprouting and pericyte attachment for vessel stabilization. Cancer-associated fibroblasts play an important role in synchronizing these events (De Wever et al., 2008). Furthermore, the topography of the ECM mediates vascular development and regulates the speed at which cells migrate during angiogenesis (Bauer et al., 2009). Vascular endothelial cells play a pivotal role in regulating leukocyte recruitment during inflammation (McGettrick et al., 2007). In most cases, cancers exploit pro-inflammatory mediators and recruited inflammatory cells to benefit their own survival (Lorusso & Ruegg, 2008) (also as reviewed in (Simpson-Haidaris et al., 2010)).

Fg and fibrin deposition is found within the stroma of most solid tumors (Simpson-Haidaris & Rybarczyk, 2001), and elevated levels of plasma Fg and fibrin degradation products (FDPs) correlate positively with lymph node involvement and metastatic spread of colorectal, ovarian, lung and breast cancers (Sahni et al., 2009; Varki, 2007). Fibrin deposition at the tumor-normal host cell interface as well as in the stroma of primary tumors is well documented, and is thought to protect tumors from infiltrating inflammatory cells by acting as a barrier thereby preventing inflammatory reactions directed towards the tumor cells (reviewed in (Simpson-Haidaris & Rybarczyk, 2001)). The presence of D-dimer, a fibrin degradation product indicative of pathological fibrin formation and dissolution, correlates

The Role of Fibrin(ogen) in Transendothelial Cell Migration During Breast Cancer Metastasis 191

results indicated that 15-21 is exposed on Fg assembled into matrix fibrils (Guadiz et al., 1997; Rybarczyk et al., 2003). Together these data suggest that matrix Fg possesses "fibrinlike" properties in the absence of fibrin polymerization and that Fg deposition rapidly changes the topology of the ECM to provide a surface for cell migration and matrix remodeling during wound repair. However, the mechanisms by which 15-42 modulates cell-

Fig. 5. Plasma fibrinogen assembles into mature matrix fibrils of nonmalinant cells (HFF and HBL-100) but poorly assembles in the matrix of malignant breast cancer cells (MCF-7 and MDA-MB-231). Primary human fibroblasts (HFF), a nonmalignant human breast cancer cell line (HBL-100) and two human breast cancer cell lines (MCF-7 and MDA-MB-231) were grown on gelatin-coated glass coverslips and treated with Fg conjugated to Oregon Green (30 g/ml) for 24 hr. The cells were washed, fixed, stained with anti-fibronectin (FN) polyclonal antibodies followed by rhodamine-goat anti-rabbit secondary antibodies, and visualized by epifluorescence microscopy. Green fluorescence is Fg-specific and red fluorescence denotes FN staining. Colocalization of Fg and FN results in yellow

fluorescence. The loss of FN in the more invasive cell lines (MCF-7 and MDA-MB-231) is likely an explanation for purified plasma Fg binding to the surface of cells but failure to assembly into mature matrix fibrils, as we have shown that assembly of Fg into an elaborate

Fibrin(ogen) 15-42 sequences support a diverse array of biological functions mediated by fibrin(ogen). Although the primary structure of fibrinopeptide B (FPB) is poorly conserved across species, the fibrin 15-42 domain is highly conserved, implying evolutionary conservation of function (Courtney et al., 1994). The 15-42 region constitutes a cryptic domain in soluble Fg that is exposed in fibrin after thrombin cleavage (Odrljin et al., 1996b). Both the HBD and overlapping binding site for VE-cadherin are localized to 15-42. VE-

fibrillar ECM depends on the assembly of FN fibrils as well (Pereira et al., 2002).

**3.1 Importance of Fg peptide 15-42 in Fg-endothelial cell interactions** 

**3. Role of Fibrin(ogen) in breast cancer metastasis** 

cell or cell-matrix adhesion are not well understood.

with poor prognosis in most solid tumors including colon, prostate, lung and breast (Batschauer et al., 2010; Kilic et al., 2008; Knowlson et al., 2010). However, in some malignancies, including breast, evidence demonstrating deposition of fibrin within the primary tumor is lacking (reviewed in (Simpson-Haidaris & Rybarczyk, 2001)). Instead, abundant Fg deposition occurs in breast tumor stroma in the absence of thrombin generation (Costantini et al., 1991).
