Collagenase-3 (MMP-13) is Expressed by Tumor Cells in Invasive Vulvar Squamous Cell Carcinomas  Nina Johansson, Maarit Vaalamo, Seija Grénman, Sakari Hietanen, Pekka Klemi, Ulpu Saarialho-Kere, Veli-Matti Kähäri  The American Journal of Pathology  Volume 154, Issue 2, Pages 469-480 (February 1999) DOI: 10.1016/S0002-9440(10)65293-5 Copyright © 1999 American Society for Investigative Pathology Terms and Conditions

Figure 1 Expression of MMP-13 and MMP-1 in vulvar SCCs. A and B: Corresponding dark-field and bright-field exposures of in situ hybridization showing MMP-13 mRNA positive tumor cell islands (arrows) in primary vulvar SCC (patient 4). C: Higher magnification of B showing expression of MMP-13 mRNA in tumor cells. D: No signal is detected in a parallel section hybridized with the sense MMP-13 probe. E: Expression of MMP-13 mRNAs by tumor cells at the invasive front of a primary vulvar SCC (patient 3). F: Bright-field exposure of panel E. G: Dark-field exposure of a section parallel to that shown in panel F hybridized with MMP-1 antisense probe, showing MMP-1 mRNA in isolated tumor cells. H: Dark-field exposure of normal vulvar epithelium hybridized with MMP-13 antisense probe shows no signal. t, tumor; s, stroma. Bars: 24 μm (A, B, and D-H) and 6 μm (C). The American Journal of Pathology 1999 154, 469-480DOI: (10.1016/S0002-9440(10)65293-5) Copyright © 1999 American Society for Investigative Pathology Terms and Conditions

Figure 2 Colocalization of MMP-13, MT1-MMP, and MMP-2 expression in vulvar SCCs. A: Dark-field exposure of an in situ hybridization showing MMP-13 mRNA positive tumor cells in a primary vulvar SCC (patient 7). B: Bright-field photomicrograph corresponding to panel A. C: Immunostaining of a section parallel to that shown in panel B with anti-MT1-MMP antibody, showing MT1-MMP-positive tumor cells and stromal fibroblasts. D: Dark-field exposure showing expression of MMP-13 mRNA by tumor cells in a recurrent vulvar SCC (patient 9). E: Bright-field exposure corresponding to panel D. F: Immunostaining of a section parallel to that shown in panel E with anti-MT1-MMP antibody showing MT1-MMP-positive tumor cells and stromal fibroblasts (arrows). G: Immunostaining of a section parallel to that shown in panel E with anti-MMP-2 antibody showing MMP-2 expression in stromal cells and in tumor cells in the periphery of tumor islands. H: Immunostaining of a section parallel to that shown in panel E with anti-MMP-13 antibody showing MMP-13-positive tumor cells and stromal fibroblasts (arrows). I: Control immunostaining of section parallel to that shown in panel E using preimmune mouse ascites fluid in place of primary antibody shows no positive staining. t, tumor; s, stroma. Bars: 24 μm (A-E, I) and 10 μm (F-H). The American Journal of Pathology 1999 154, 469-480DOI: (10.1016/S0002-9440(10)65293-5) Copyright © 1999 American Society for Investigative Pathology Terms and Conditions

Figure 3 Colocalization of MMP-13- and MMP-9-expressing cells in vulvar SCCs. A: Immunostaining of a vulvar SCC (patient 3) with anti-MMP-9 antibody, showing MMP-9-positive tumor cells (thin arrows) and mononuclear inflammatory cells (thick arrows). B: In situ hybridization of a section parallel to that shown in A with MMP-13 antisense probe showing MMP-13 expression by tumor cells. Bars: 6 μm. The American Journal of Pathology 1999 154, 469-480DOI: (10.1016/S0002-9440(10)65293-5) Copyright © 1999 American Society for Investigative Pathology Terms and Conditions

Figure 4 Expression of MMP-13 in cell lines from vulvar SCCs. A: Vulvar SCC cell lines UM-SCV-1B1 and UM-SCV22 were cultured in DMEM supplemented with 6 mmol/L glutamine, non-essential amino acids, and 10% FCS. B: Cell lines UM-SCV-7, UT-SCV-2, and UT-SCV-3 derived from vulvar SCCs were incubated for 24 hours in serum-free medium alone (control) or in the presence of TNF-α (20 ng/ml), TGF-β1 (5 ng/ml), or TGF-α (10 ng/ml), as indicated. A, B: Total RNA was extracted and 20-μg aliquots were analyzed by Northern blot hybridizations for expression of MMP-13, MMP-1, MMP-7, MMP-9, MT1-MMP (MMP-14), and GAPDH mRNAs, as indicated. For details of the cell lines, see Table 2. The American Journal of Pathology 1999 154, 469-480DOI: (10.1016/S0002-9440(10)65293-5) Copyright © 1999 American Society for Investigative Pathology Terms and Conditions

Figure 5 Expression of MMP-13 mRNA in cell lines derived from cervical carcinomas. Cervical SCC cell lines and a glassy cell carcinoma cell line, UM-GCC-1, were cultured in DMEM supplemented with 6 mmol/L glutamine, non-essential amino acids, and 10% FCS. Total RNA was extracted and 15-μg aliquots were analyzed for expression of MMP-13 and GAPDH mRNA by Northern blot hybridizations. The American Journal of Pathology 1999 154, 469-480DOI: (10.1016/S0002-9440(10)65293-5) Copyright © 1999 American Society for Investigative Pathology Terms and Conditions

Figure 6 Expression of MMP-13 mRNA in a cell line derived from vaginal premalignant lesion. UT-DEC-1 cells established from a premalignant vaginal intraepithelial dysplasia were maintained in Keratinocyte Serum Free Medium (passage 21, p21) or serum-free DMEM (passage 89, p89) without or with TNF-α (20 ng/ml) or TGF-β1 (5 ng/ml), as shown, for 24 hours. Total RNA was extracted and 15-μg aliquots were analyzed for expression of MMP-13 and GAPDH mRNA by Northern blot hybridizations. The American Journal of Pathology 1999 154, 469-480DOI: (10.1016/S0002-9440(10)65293-5) Copyright © 1999 American Society for Investigative Pathology Terms and Conditions