Abstract
Abstract 853
The ability of tumor cells to bind plasminogen is highly correlated with their invasive and metastatic potential. Here we evaluate cell surface plasminogen binding in human breast cancer. In a xenograft model for aggressive, triple-negative human breast cancer, tumor cells harvested from orthotopic mammary fat pad (mfp) tumors of the invasive human breast cancer line, MDA-MB-231, exhibit dramatic increases in tumor growth and lung and lymph node metastases, compared to parental cells. The breast cancer cells isolated from the mfp have been designated as 231mfp cells. Although the levels of most secreted proteins are similar, the plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue type plasminogen activator (tPA) are highly upregulated in 231mfp cells, compared with MDA-MB-231 parental cells, suggesting a role for the plasminogen activation system in the differences in growth and metastasis between these two cell lines (Jessani et al., Proc Natl Acad Sci USA 101:13756, 2004). Further evaluation of the plasminogen activation system in this model revealed that plasminogen bound to both 231mfp and MDA-MB-231 cells and plasminogen binding was specifically inhibited by the lysine analog, ε-aminocaproic acid (EACA). 231mfp cells exhibited a 3-fold greater capacity for plasminogen compared to MDA-MB-231 cells: Quantitative FACS analysis yielded a Bmax of 3.8 ± 0.5 × 105 plasminogen binding sites with a Kd of 1.2 μM for the 231 mfp cells and a Bmax of 1.2 ± 0.3 × 105 binding sites with a Kd of 1.5 μM for the parental cells.
We recently discovered a novel cell surface receptor for plasminogen, Plg-RKT (Andronicos et al., Blood 115:1319, 2010). Plg-RKT enhances enzymatic activation of plasminogen to plasmin and localizes and spatially orients plasmin on the cell surface. We compared cell-surface expression of Plg-RKT on 231mfp and MDA-MB-231 cells using a specific monoclonal antibody raised against the C-terminus of human Plg-RKT, termed mAb7H1. In FACS analysis with mAB7H1, expression of Plg-RKT was markedly (5-fold) higher on 231mfp cells, compared to the parental cells. These data were confirmed in Western blotting. To further determine expression of Plg-RKT in breast cancer, human tissues were stained with anti-Plg-RKT antibodies. We found very high expression of Plg-RKT in human invasive ductal carcinoma and ductal carcinoma in situ and minimal expression in normal breast ducts and lobules in control subjects. We considered whether Plg-RKT expressed on breast cancer cells can a therapeutic target and we tested the ability of anti-Plg-RKT mAb7H1 to inhibit lung metastasis of 231mfp cells in immune deficient mice. Mice were injected into the tail vein with cells either mixed with mAb7H1 or with buffer control. The antibody-treated group received a second dose of mAb7H1 24 hr later. Four weeks after tumor cell injection, mice were sacrificed and tumor foci on the lungs were compared between the two groups. Anti-Plg-RKT mAb7H1 markedly reduced the number of 231mfp lung metastases per mouse: mean number of lung foci in the mAb7H1 treated group was 109 (± 47.8) and in the vehicle-treated control group 270.4 (± 61.7), probability of no difference between the groups by t-test P<0.05. The size distribution of the individual foci was not different between the groups. These data demonstrate that anti-Plg-RKT mAb7H1 has activity in the mouse model of metastatic breast cancer and suggest that it interferes with an early step in the establishment of lung metastases. In summary, the highly metastatic 231mfp breast cancer cells have increased plasminogen binding capacity and increased expression of the plasminogen receptor, Plg-RKT, compared to parental MDA-MB-231 cells. Plg-RKT was highly expressed in human breast cancer tissue but not in normal mammary glands. Furthermore, anti-Plg-RKT mAb inhibited establishment of lung metastases in a model of human breast cancer. Therefore, the presence of Plg-RKT on breast cancer cells may represent a new control point in the establishment of breast cancer metastases.
Disclosures:
No relevant conflicts of interest to declare.
Radiographically Occult Carcinomatous Spread of Breast Cancer to the Liver: A Challenging Case
