Role of proteolytic enzymes in human prostate bone metastasis formation: in vivo and in vitro studies

Abstract

Prostate cancers ability to invade and grow in bone marrow stroma is thought to be due in part to degradative enzymes. The formation of prostate skeletal metastases have been reproduced in vitro by growing co-cultures of prostatic epithelial cells in bone marrow stroma. Expression of urokinase plasminogen activator, matrix metalloproteinase 1 and 7 by prostatic epithelial cells were identified using immunocytochemistry. Also, in vivo tissue sections from human prostatic bone marrow metastases were stained. To establish the role of these enzymes on colony formation, inhibitory antibodies directed against urokinase plasminogen activator, matrix metalloproteinase 1 and matrix metalloproteinase 7 were added into primary prostatic epithelial cells and bone marrow stroma co-cultures. All prostatic epithelial cell cultures stained positively for matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator. Generally prostatic epithelial cells derived from malignant tissues showed increased staining in comparison to epithelia derived from non-malignant tissue. In agreement with in vitro co-cultures, the in vivo tissue sections of prostate bone marrow metastases showed positive staining for all three enzymes. Inhibition studies demonstrated that blocking matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator function reduced the median epithelial colony area significantly in bone marrow stroma co-cultures in vitro. Using a human ex-vivo model we have shown that matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator play an important role in the establishment of prostatic epithelial cells within bone marrow.

Figure 1

Enzyme characterisation of primary prostate epithelial cells and the prostate cell lines grown with BMS. Immunocytochemical staining showing typical colonies of cytokeratin (CK) positive (DAB brown staining) and fluorescent dual staining of epithelial cells grown in BMS co-culture (each picture is unrelated). Enzyme stained photographs are shown as dual fluorescence: red=cytokeratin; green=location of either uPA, MMP-1 or MMP-7; yellow=double staining; blue=DAPI nuclear counterstain (n=5). All negative controls were black. Arrows indicate retracted stromal edge.

Figure 2

Serial sections of a prostatic bone marrow metastasis. Typical examples of the enzyme staining found from seven samples (see Table 2). Each photograph shows the same region of sample immunohistochemically (DAB) labelled for: (A) negative control (haematoxylin), (B) cytokeratin indicating the colonies of epithelial cells within the bone marrow space, (C) MMP-1, (D) MMP-7 and (E) uPA.

Figure 3

Antibody inhibition of epithelial colony area (%). A comparison of the inhibition of prostate epithelial colony area in the presence of enzyme antibodies was made for a variety of primary epithelial samples. Typical immunocytochemical (DAB) cytokeratin staining of a BPH epithelial cell/BMS co-culture in the presence of non-specific mouse IgG1, anti-MMP-1, anti-MMP-7 and anti-uPA antibodies (2 μg day⁻¹) after 7 days in culture is shown in (A). Median area of >100 colonies per sample were calculated and inhibition was calculated as a per cent of the control area. Median percentage inhibition is represented by the bar: (B) CaP, MMP-1 and MMP-7 (n=6), uPA n=4; (C) BPH, MMP-1 and MMP-7 (n=7), uPA (n=3).