**3.4 VE-cadherin binding domain of Fg (15-42) enhances transendothelial migration of malignant breast epithelial cells**

Because plasma Fg promotes metastasis of some types of cancer and Fg 15-42 sequences promote endothelial cell permeability, we hypothesized Fg 15-42 sequences would play a role in promoting TEM of breast cancer cells. To test this hypothesis, breast cancer cells were labeled with a fluorescence cell-tracking dye (DiI) before they were mixed with increasing concentrations Fg. Breast cancer cells and Fg were allowed to pre-incubate for 15 minutes prior to addition to the upper chamber of a barrier monolayer of endothelial cells. After 45 minutes incubation, the relative number of breast cancer cells migrating to the underside of the transwell insert membrane were quantified by relative fluorescence and

Fig. 8. Fg enhances TEM of malignant breast epithelial cells (Panel A), induces gap formation between adjacent endothelial cells (Panel B, asterisks), promotes intracellular relocalization (Panel B, arrowheads) of VE-cadherin at membrane cell-cell junctions (Panel B, Control, arrow), assembles into ECM (Panel C, arrowhead), and shows punctate, cell surface receptor-like binding between adjacent endothelial cells (Panel C, arrows). Cells in Panels A and B were treated as described in Section 3.3. In Panel C, endothelial cells were treated for 24 hours with purified plasma Fg conjugated to Oregon Green. Cells were fixed, permeabilized and stained with anti-FGF-2 (red fluoresence). After staining, the coverslip was mounted upside down on a microscope slide so that the basolateral aspect (bottom of cells) and the subendothelial ECM appear as the "top" of the cells. Matrix Fg and receptor bound Fg are shown in green fluorescence. Cover Figure ref (Sahni et al., 2009).

nonimmune IgG1 was used for the control. Monoclonal antibodies were used at 3 nM in the absence of Fg, or with 0.3 nM or 30 nM Fg for 45 min. The data were plotted as the mean ± SEM of relative FITC-Dextran Flux and were obtained from three independent experiments with a total sample size of 6-9 per condition. (Reprinted from (Sahni et al., 2009) with

**3.4 VE-cadherin binding domain of Fg (15-42) enhances transendothelial migration of** 

Because plasma Fg promotes metastasis of some types of cancer and Fg 15-42 sequences promote endothelial cell permeability, we hypothesized Fg 15-42 sequences would play a role in promoting TEM of breast cancer cells. To test this hypothesis, breast cancer cells were labeled with a fluorescence cell-tracking dye (DiI) before they were mixed with increasing concentrations Fg. Breast cancer cells and Fg were allowed to pre-incubate for 15 minutes prior to addition to the upper chamber of a barrier monolayer of endothelial cells. After 45 minutes incubation, the relative number of breast cancer cells migrating to the underside of the transwell insert membrane were quantified by relative fluorescence and

Fig. 8. Fg enhances TEM of malignant breast epithelial cells (Panel A), induces gap formation between adjacent endothelial cells (Panel B, asterisks), promotes intracellular relocalization (Panel B, arrowheads) of VE-cadherin at membrane cell-cell junctions (Panel B, Control, arrow), assembles into ECM (Panel C, arrowhead), and shows punctate, cell surface receptor-like binding between adjacent endothelial cells (Panel C, arrows). Cells in Panels A and B were treated as described in Section 3.3. In Panel C, endothelial cells were treated for 24 hours with purified plasma Fg conjugated to Oregon Green. Cells were fixed, permeabilized and stained with anti-FGF-2 (red fluoresence). After staining, the coverslip was mounted upside down on a microscope slide so that the basolateral aspect (bottom of cells) and the subendothelial ECM appear as the "top" of the cells. Matrix Fg and receptor

bound Fg are shown in green fluorescence. Cover Figure ref (Sahni et al., 2009).

permission). P-values can be found in ref (Sahni et al., 2009).

**malignant breast epithelial cells** 

visualized by microscopy. VEGF was used as a positive control to induce endothelial cell permeability and TEM of breast cancer cells. The results indicated that TEM of both MCF-7 and MDA-MB-231 cells was increased in a Fg-concentration-dependent manner (see Fig. 3a of ref (Sahni et al., 2009)) and as visualized by immunofluorescence microscopy showing MDA-MB-231 cells adhered to the bottom side of the transwell filter (**Fig. 8A**).

To determine whether VE-cadherin and/or Fg 15-42 were involved in Fg-enhanced TEM of MDA-MB-231 cells, the assay was repeated in the presence of the neutralizing and control antibodies (as shown in Fig. 3c of ref (Sahni et al., 2009)). To determine whether Fg promoted gap formation between cells, confluent HUVEC were treated with 150 or 480 nM Fg or 100 Units/ml TNF-, a known inducer of endothelial permeability and gap formation, for 30 minutes then cells were fixed, permeabilized and immunostained with an anti-VEcadherin. Fg treatment induced gap formation between adjacent endothelial cells, and such treatment promoted the subcellular relocalization of VE-cadherin from the cell periphery as in control cells into the cytoplasm in Fg- and TNF--treated cells (**Fig. 8B**). Indirect evidence for Fg binding at endothelial cell-cell junctions was obtained by fluorescence microscopy. The data reveal that Fg binds to endothelial cell-cell junctions in a punctate pattern, consistent with cell surface receptor binding to the cell-cell adhesion receptor, VE-cadherin (**Fig. 8C, arrows**). Fg also assembles as part of the fibrillar subendothelial ECM (**Fig. 8C, arrowhead**). Taken together, the data in **Fig. 6-8** demonstrate that the VE-cadherin binding domain defined by residues 15-42 on the -chain of human Fg induces permeability of endothelial but not epithelial cell barriers and enhances TEM of malignant breast cancer cells by a VE-cadherin-dependent mechanism. In contrast, the basal level of TEM of nonmalignant breast epithelial cells was not enhanced by Fg treatment (Sahni et al., 2009).

#### **3.5 Fibrinogen potentiates endothelial cell permeability at low doses of VEGF**

Both FGF-2 and VEGF bind to fibrin(ogen) at distinct sites with high affinity (Sahni & Francis, 2000; Sahni et al., 1998). Fg bound-FGF-2 potentiates endothelial cell proliferation over FGF-2 alone (Sahni et al., 2003; Sahni & Francis, 2004; Sahni et al., 2006; Sahni et al., 1999). Although Fg-bound VEGF remains active, it does not potentiate endothelial cell proliferation over VEGF alone (Sahni & Francis, 2000). Because Fg induces endothelial cell permeability through VE-cadherin binding interactions (Sahni et al., 2009) and VEGF binds to Fg (Sahni & Francis, 2000), we tested the hypothesis that Fg would potentiate VEGFinduced EC permeability (**Fig. 9**).

Fig. 9. Fg enhances permeability induced by low concentrations of VEGF.

The Role of Fibrin(ogen) in Transendothelial Cell Migration During Breast Cancer Metastasis 197

cell proliferation and angiogenesis (*Step 10*) in lung results in metastatic disease (*Step 11*). We *hypothesize* that free peptide 15-42 will bind to VE-cadherin between endothelial cells to block endothelial cell binding to 15-42 on intact fibrin(ogen) found in the tumor stroma or tumor vessels, thereby inhibiting tumor-associated angiogenesis (*Step 2*), intravasation (*Step 3*), extravasation (*Step 8*), and angiogenesis at metastatic tumor sites (*Step 10*) (**as denoted by** 

Fig. 10. Schematic summarizing role of fibrin(ogen) 15-42 in breast cancer metastasis and hypothesis development for employing free peptide 15-42 as a therapeutic strategy to treat

Successful demonstration of peptide 15-42 as an inhibitor of breast cancer metastasis and tumor-associated inflammation and angiogenesis *in vivo* would significantly impact breast cancer treatment in a timely manner. Peptide 15-42, an endogenous fragment of fibrin, is already shown to be well tolerated in humans and effective in reducing damage to heart muscle after a heart attack in preclinical models of IR injury. However, until now, no one has proposed the use of peptide 15-42 as an inhibitor of breast cancer metastasis. A precedent and pipeline for production of viable therapeutics based on peptide 15-42 exists for treatment of damaged heart tissue, and Phase I and Phase II clinical trials are ongoing to test the safety and efficacy, respectively, of free 15-42 peptide for IR injury (Hallen et al., 2010; Petzelbauer et al., 2005a; Petzelbauer et al., 2005b; Roesner et al., 2007; Roesner et al., 2009; Wiedemann et al., 2010; Zacharowski et al., 2006). Therefore, the timeline for

**the lightening bolts at these steps in Fig. 10**).

metastatic breast cancers*.*

The data indicate that 10 g/ml (30 nM) Fg enhanced the flux of FITC-dextran to the bottom chamber of the transwell plate at low doses of VEGF (0.05 and 0.1 ng/ml); however, the additive effect on induction of endothelial cell permeability was lost at 0.5 ng/ml and higher concentrations of VEGF (**Fig. 9**). Fg-enhancement of VEGF-induced permeability is rapid and saturated within 5 min, whereas 5 ng/ml of VEGF is required to induce a similar amount of FITC-dextran flux as 30 nM Fg + 0.05 ng/ml, *i.e.*, 100-fold less VEGF. Studies by others suggest that low-dose VEGF mediates inflammation to promote cell survival of vascular and nonvascular cells such as those of the CNS, prior to induction of angiogenesis (Abumiya et al., 2005; Croll et al., 2004). Furthermore, VEGF colocalizes with exuded Fg at sites of edema in renal cell carcinoma (Verheul et al., 2010). Together with the aforementioned published data, our results suggest that Fg may regulate vascular permeability induced by low doses of VEGF without inducing EC proliferation—such a response would be conducive to fibrinogen induction of breast cancer cell TEM*.*
