**2.5 Cancer cells, including breast, synthesize and secrete fibrinogen**

The origin of tumor-associated fibrin(ogen) and fibrin(ogen) degradation products has historically been thought to be from exudation of plasma Fg due to the increased vascular permeability and subsequent procoagulant or fibrinolytic activity at the tumor site (Rybarczyk & Simpson-Haidaris, 2000). However, because Fg deposition in the stroma, but not fibrin formation, is considered a hallmark of breast cancer (Costantini et al., 1991), we hypothesized that breast cancer cells were capable of endogenous synthesis and secretion of Fg. We demonstrated that human MCF-7 cells are capable of synthesizing Fg chains, although assembly of intact Fg is defective due to degradation of the B chain (Rybarczyk & Simpson-Haidaris, 2000). In addition, we have shown that lung, prostate and breast cancer epithelial cells synthesize and secrete Fg that enhances FGF-2-mediated cell proliferation, assembles into the ECM and binds to cancer cell surface receptors (Rybarczyk & Simpson-Haidaris, 2000; Sahni et al., 2008; Simpson-Haidaris, 1997; Simpson-Haidaris & Rybarczyk, 2001). Others have shown Fg production in cervical (Lee et al., 1996) and intestinal (Molmenti et al., 1993) cancer cell lines. Expression array profiling studies confirmed that Fg genes are expressed in breast (Pentecost et al., 2005) and lung carcinomas (Tan et al., 2005) from patients. Thus, Fg synthesized by cancer cells promotes growth of the primary tumor and supports tumor-associated angiogenesis characterized by localized VEGF production and leaky vessels (Dvorak, 2006). The importance of VEGF in promoting tumor vascular permeability, angiogenesis and leakage of plasma Fg into the perivascular space to induce tumor stroma desmoplasia is well known. However, whether *tumor-associated fibrin(ogen)* contributes to permeability of tumor vessels and breast cancer metastasis is unknown*.*

#### **2.6 Fibrinogen is an extracellular matrix protein**

Although Fg is known for its hemostatic role, we showed that Fg, not fibrin, is a component of the insoluble fibrillar ECM of fibroblasts, alveolar epithelial cells, endothelial cells and breast epithelial cells (Guadiz et al., 1997; Pereira et al., 2002; Sahni et al., 2009; Simpson-Haidaris et al., 2010; Simpson-Haidaris & Sahni, 2010). Upon assembly into matrix fibrils, Fg undergoes conformational changes exposing the cryptic 15-42 epitope in the absence of thrombin cleavage or covalent crosslinking (Guadiz et al., 1997; Simpson-Haidaris & Sahni, 2010). When Fg is pre-established in the ECM of adventitial fibroblasts prior to wounding, increased cell proliferation and migration enhance wound closure (Rybarczyk et al., 2003), which is dependent on *de novo* protein synthesis (Pereira & Simpson-Haidaris, 2001) but independent of added growth factors, PDGF and FGF-2 (Rybarczyk et al., 2003). However, assembly of Fg into mature matrix fibrils of breast epithelial cells appears to correlate negatively with the increasing invasive potential of the cell (**Fig. 5**). We also determined whether the cryptic HBD in soluble Fg (Odrljin et al., 1996b) was accessible in matrix Fg using a specific MoAb (T2G1) (Kudryk et al., 1984). Whereas the T2G1 epitope (15-21) within 15-42 is not accessible for antibody binding in soluble Fg or Fg immobilized to a surface, the

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

The origin of tumor-associated fibrin(ogen) and fibrin(ogen) degradation products has historically been thought to be from exudation of plasma Fg due to the increased vascular permeability and subsequent procoagulant or fibrinolytic activity at the tumor site (Rybarczyk & Simpson-Haidaris, 2000). However, because Fg deposition in the stroma, but not fibrin formation, is considered a hallmark of breast cancer (Costantini et al., 1991), we hypothesized that breast cancer cells were capable of endogenous synthesis and secretion of Fg. We demonstrated that human MCF-7 cells are capable of synthesizing Fg chains, although assembly of intact Fg is defective due to degradation of the B chain (Rybarczyk & Simpson-Haidaris, 2000). In addition, we have shown that lung, prostate and breast cancer epithelial cells synthesize and secrete Fg that enhances FGF-2-mediated cell proliferation, assembles into the ECM and binds to cancer cell surface receptors (Rybarczyk & Simpson-Haidaris, 2000; Sahni et al., 2008; Simpson-Haidaris, 1997; Simpson-Haidaris & Rybarczyk, 2001). Others have shown Fg production in cervical (Lee et al., 1996) and intestinal (Molmenti et al., 1993) cancer cell lines. Expression array profiling studies confirmed that Fg genes are expressed in breast (Pentecost et al., 2005) and lung carcinomas (Tan et al., 2005) from patients. Thus, Fg synthesized by cancer cells promotes growth of the primary tumor and supports tumor-associated angiogenesis characterized by localized VEGF production and leaky vessels (Dvorak, 2006). The importance of VEGF in promoting tumor vascular permeability, angiogenesis and leakage of plasma Fg into the perivascular space to induce tumor stroma desmoplasia is well known. However, whether *tumor-associated fibrin(ogen)* contributes to permeability of tumor vessels and breast cancer metastasis is unknown*.*

Although Fg is known for its hemostatic role, we showed that Fg, not fibrin, is a component of the insoluble fibrillar ECM of fibroblasts, alveolar epithelial cells, endothelial cells and breast epithelial cells (Guadiz et al., 1997; Pereira et al., 2002; Sahni et al., 2009; Simpson-Haidaris et al., 2010; Simpson-Haidaris & Sahni, 2010). Upon assembly into matrix fibrils, Fg undergoes conformational changes exposing the cryptic 15-42 epitope in the absence of thrombin cleavage or covalent crosslinking (Guadiz et al., 1997; Simpson-Haidaris & Sahni, 2010). When Fg is pre-established in the ECM of adventitial fibroblasts prior to wounding, increased cell proliferation and migration enhance wound closure (Rybarczyk et al., 2003), which is dependent on *de novo* protein synthesis (Pereira & Simpson-Haidaris, 2001) but independent of added growth factors, PDGF and FGF-2 (Rybarczyk et al., 2003). However, assembly of Fg into mature matrix fibrils of breast epithelial cells appears to correlate negatively with the increasing invasive potential of the cell (**Fig. 5**). We also determined whether the cryptic HBD in soluble Fg (Odrljin et al., 1996b) was accessible in matrix Fg using a specific MoAb (T2G1) (Kudryk et al., 1984). Whereas the T2G1 epitope (15-21) within 15-42 is not accessible for antibody binding in soluble Fg or Fg immobilized to a surface, the

**2.5 Cancer cells, including breast, synthesize and secrete fibrinogen** 

generation (Costantini et al., 1991).

**2.6 Fibrinogen is an extracellular matrix protein** 

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 cellcell or cell-matrix adhesion are not well understood.

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