The myofibroblast is the predominant plasminogen activator inhibitor-1-expressing cell type in human breast carcinomas - PubMed (original) (raw)
The myofibroblast is the predominant plasminogen activator inhibitor-1-expressing cell type in human breast carcinomas
Birgitte Vrou Offersen et al. Am J Pathol. 2003 Nov.
Abstract
The tumor level of plasminogen activator inhibitor-1 (PAI-1) is an informative biochemical marker of a poor prognosis in several cancer types. However, the tumor biological functions of PAI-1 and the identity of PAI-1-expressing cells are controversial. With the aim of immunohistochemically localizing PAI-1 in formalin-fixed, paraffin-embedded invasive ductal breast carcinoma samples, we raised new polyclonal antibodies against PAI-1 from different expression systems. The antibodies were affinity purified by absorption on immobilized preparations of PAI-1 different from those used for immunization. The specificity of the antibodies was ensured by immunoblotting analysis. In immunohistochemistry, the staining pattern obtained with the antibodies showed a good correlation with the PAI-1 mRNA expression pattern. In all 25 cases analyzed, PAI-1 immunoreactivity was predominantly localized in fibroblast-like cells. Double-immunofluorescence analyses showed co-expression of PAI-1 and alpha-smooth muscle actin in these cells, suggesting that they are myofibroblasts. PAI-1 was also seen in some myoepithelial cells surrounding occasional foci of ductal carcinoma in situ (9 of 25), some endothelial cells (8 of 25), some cancer cells (3 of 25), and some mast cells (6 of 25). In conclusion, we have provided a robust immunohistochemical procedure for detection of PAI-1 and shown that the majority of the PAI-1-expressing cells in invasive ductal breast carcinomas are myofibroblasts.
Figures
Figure 1.
Immunoblotting analysis of the reactivity of anti-PAI-1 antibodies with tumor extracts. The following samples were subjected to SDS-PAGE: a portion of a pool of tumor extracts from eight tumors, corresponding to ∼200 μg of total protein without additions (lane 1); tumor extract supplemented with 25 ng of purified HT 1080 PAI-1 (lane 2); tumor extract supplemented with 50 ng of purified uPA (lane 3); 25 ng of purified PAI-1 (lane 4); 100 ng of purified uPA-PAI-1 complex (lane 5); or 100 ng of purified tPA-PAI-1 complex (lane 6). Blots of the gel lanes on polyvinylidene difluoride filters were prepared and analyzed with AB-1A, AB-1D, AB-2A, or AB-2D, as indicated. The migration of PAI-1, uPA-PAI-1 complex, and tPA-PAI-1 complex are indicated to the left. Two additional bands, appearing after supplementing tumor extracts with purified PAI-1 (lane 2), are labeled PAI-1d and PAI-1h. Please note that the preparations of uPA-PAI-1 complex and tPA-PAI-1 complex contain some free PAI-1, which in the case of the uPA-PAI-1 complex is in the form of reactive center-cleaved PAI-1, migrating slightly faster than native PAI-1. The slowly migrating band in the PAI-1 preparation in lane 4 is aggregated PAI-1.
Figure 2.
Immunoperoxidase staining with affinity-purified anti-PAI-1 polyclonal antibodies in human breast cancer tissue. Four adjacent paraffin sections were analyzed immunohistochemically for PAI-1 with affinity-purified anti-PAI-1 IgG, AB-1A (a), and AB-2A (c), and anti-PAI-1-depleted IgG, AB-1D (b), and AB-2D (d). PAI-1 immunoreactivity obtained with AB-1A and AB-2A was seen in the very same (stromal) cells (arrows), whereas no staining was seen with AB-1D and AB-2D (b, d). The weak background staining observed with AB-1A (a) is related to the difficulties with this antibody preparation generally staining intensely toward the border of the sections. A cryostat section (e) and a paraffin section (f) from the same tumor were incubated with AB-2A. PAI-1 immunoreactivity is seen in stromal cells in both sections (arrows) and no staining is seen in cancer cells. Scale bars: ∼80 μm (a–d); ∼20 (e, f).
Figure 3.
Expression of PAI-1 protein and mRNA in various cell types in human breast cancer. Two adjacent sections were processed for PAI-1 immunoperoxidase staining using rabbit polyclonal affinity purified anti-PAI-1 IgG, AB-2A (a–e) and in situ hybridization with a 35S-labeled PAI-1 anti-sense probe (a′–e′, a″–e″) shown in dark-field (a′–e′) and bright-field (a″–e″) illumination. PAI-1 immunoreactivity and mRNA are seen in the very same cells, as indicated by arrows in fibroblast-like cells surrounding cancer cell (Ca) nodules (arrows in a), vascular endothelial cells (arrows in b), cancer cells (arrows in c), and myoepithelial cells (arrows in d). No PAI-1 mRNA signal (e′–e″) is detected in the PAI-1 immunoreactive stromal mononuclear cells (e) identified as mast cells. The cancer cell compartment is indicated by Ca and the stromal compartment by St. Scale bars: ∼75 μm (a–d); ∼150 μm (e).
Figure 4.
Double immunofluorescence of PAI-1 with α-SMA, CD34 and mast cell tryptase immunoreactivities in human breast cancer. Paraffin sections were incubated with polyclonal anti-PAI-1 antibodies (AB-2A) together with a monoclonal antibody to α-SMA (a–f), monoclonal antibody to CD34 (g–l), or monoclonal antibody against mast cell tryptase (m–o). The polyclonal antibodies were recognized with Cy3-conjugated goat anti-rabbit (a, d, g, j, m) and the monoclonal antibodies with FITC-conjugated goat anti-mouse (b, e, h, k, n). Image overlays of Cy3 and FITC signals were processed (c, f, i, l, o). PAI-1 immunoreactivity is co-expressed with α-SMA immunoreactivity in myofibroblasts surrounding cancer cell (Ca) nodules (arrows in a–c, yellow color in c), and in myofibroblasts located in stromal tissue with multiple myofibroblast (arrows in d–f, yellow color in f). Only a few PAI-1-positive cells were CD34-positive (arrows in g–i, yellow color in i), but most CD34-positive capillary endothelial cells were negative (arrows in j–l). Strong PAI-1 immunoreactivity is seen in two mast cell tryptase-positive cells (white arrows in m–o) close to a PAI-1-positive myofibroblast (blue arrow in m and o). Scale bar, ∼30 μm.
Figure 5.
Double labeling for PAI-1 mRNA and CD68 or α-SMA immunoreactivities in human breast cancer. Sections were first processed for immunohistochemical staining of CD68 (a, b) or α-SMA (c, d; brown color, left), and then for PAI-1 mRNA in situ hybridization [black silver grains in bright-field illumination (a, c) and light grains in dark-field illumination (b, d)]. No PAI-1 mRNA signal (black arrows in a, white arrows in b) is detected in CD68-positive macrophages cells (blue arrows in a, yellow arrows in b), but is clearly seen in the α-SMA-positive myofibroblasts (arrows in c). Scale bar, ∼20 μm.
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