Expression of matrix metalloprotease-9 in vascular pericytes in human breast cancer.
Matrix metalloprotease-9 (MMP-9; 92-kd type IV collagenase, gelatinase B) is regarded as important for degradation of the basement membrane and extracellular matrix during cancer invasion and other tissue-remodeling events. Expression of MMP-9 was analyzed in 22 cases of human ductal breast cancer by immunohistochemistry and in 8 of these cases also by in situ hybridization. For immunohistochemistry we used affinity-purified polyclonal antibodies as well as a MMP-9-specific monoclonal antibody (clone 6-6B). Three different stromal cell types with a positive MMP-9 immunoreaction were identified morphologically: neutrophils and macrophage-like cells in all cases and vascular cells in 16 of 22 cases. Double immunofluorescence with antibodies to CD68 conclusively demonstrated MMP-9 expression in macrophages. To identify the positive vascular cells, we employed antibodies to von Willebrand factor and PAL-E for identification of endothelial cells, high molecular weight melanoma-associated antigen for pericytes, and alpha-smooth muscle actin for vascular smooth muscle cells. Using conventional and confocal double immunofluorescence microscopy, colocalization of MMP-9 was seen with high molecular weight melanoma-associated antigen, the pericyte marker, whereas little or no coexpression was seen with alpha-smooth muscle actin. Virtually no coexpression was seen with the endothelial cell markers PAL-E and von Willebrand factor. In situ hybridization showed that MMP-9 mRNA colocalized with MMP-9 immunoreactivity in macrophages and vascular structures, whereas no MMP-9 mRNA was detected in neutrophils. No MMP-9 immunostaining or in situ hybridization signal was detected in cancer cells in any of the cases. Based on these results, it is concluded that MMP-9 in human breast cancer is located in tumor-infiltrating stromal cells, including neutrophils, macrophages, and vascular pericytes, and that the latter two cell types also produce this metalloprotease. We suggest that the MMP-9 produced in pericytes may play a role in extracellular matrix degradation during tumor angiogenesis.
Nielsen
B S
BS
Department of Pathology, Rigshospitalet, Copenhagen, Denmark.
Sehested
M
M
Kjeldsen
L
L
Borregaard
N
N
Rygaard
J
J
Danø
K
K
D000199:Actins / Q000032:analysis; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D001943:Breast Neoplasms / Q000737:chemistry / Q000378:metabolism*; D018270:Carcinoma, Ductal, Breast / Q000737:chemistry / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism*; D003239:Connective Tissue Cells / Q000737:chemistry / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008264:Macrophages / Q000737:chemistry / Q000378:metabolism; D020780:Matrix Metalloproteinase 9; D008545:Melanoma / Q000276:immunology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D014841:von Willebrand Factor / Q000032:analysis; D000097382:CD68 Molecule
D000199:Actins; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D017364:Collagenases; D020780:Matrix Metalloproteinase 9; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D012333:RNA, Messenger; D014841:von Willebrand Factor; C067980:CD68 antigen, human; D000097382:CD68 Molecule