Tissue Inhibitor of Metalloproteinase-1 Is Confined to Tumor-Associated Myofibroblasts and Is Increased With Progression in Gastric Adenocarcinoma

Abstract

The tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibits the extracellular matrix-degrading activity of several matrix metalloproteinases, thereby regulating cancer cell invasion and metastasis. Studies describing the expression pattern and cellular localization of TIMP-1 in gastric cancer are, however, highly discordant. We addressed these inconsistencies by performing immunohistochemistry and in situ hybridization analyses in a set of 49 gastric cancer lesions to reexamine the TIMP-1 localization. In addition, we correlated these findings to clinicopathological parameters. We show that strong expression of TIMP-1 protein and mRNA was observed in a subpopulation of stromal fibroblast-like cells at the periphery of the cancer lesions. In a few cases, a small fraction of cancer cells showed weak expression of TIMP-1 protein and mRNA. The stromal TIMP-1-expressing cells were mainly tumor-associated myofibroblasts. In the normal-appearing mucosa, scattered TIMP-1 protein was only found in chromogranin A positive cells. TIMP-1-positive myofibroblasts at the invasive front of the tumors were more frequently seen in intestinal than in diffuse histological subtype cases (p=0.009). A significant trend to a higher number of cases showing TIMP-1 staining in myofibroblasts with increasing tumor, node, metastasis (TNM) stage was also revealed (p=0.041). In conclusion, tumor-associated myofibroblasts are the main source of increased TIMP-1 expression in gastric cancer.

Keywords: TIMP-1; gastric cancer; immunohistochemistry; in situ hybridization; invasion; myofibroblasts; neuroendocrine cells.

Figure 1.

Localization of TIMP-1 protein in gastric cancer tissue. Tissue sections from intestinal (A–C, E–G, J–l) and diffuse (D, H, I) subtypes were processed for immunoperoxidase staining with mAbs against TIMP-1 (A-H, J), chromogranin A (I), anti-TIMP-1 mAb preincubated with TIMP-1 recombinant protein (K), and an mAb against TNP (L). In intestinal subtype gastric cancer, TIMP-1 is focally upregulated in the stromal tissue at the invasive front (A, B, and J; Ca: cancer, St: stroma) and toward the luminal surface (C; Lu: lumen). TIMP-1 expression at the periphery of the tumors is mainly conferred to fibroblast-like cells (arrows in panels B and C). In diffuse subtype, TIMP-1 is also observed in stromal fibroblast-like cells surrounding cancer cell nests (arrows in panel D). Weak TIMP-1 staining is seen in cancer cells in a few cases of intestinal and diffuse subtypes (E–G). In nonneoplastic gastric mucosa, scattered TIMP-1-positive cells are observed (H) in similar location to those positive for chromogranin A staining on adjacent sections (I). TIMP-1 staining (J) is precluded in adjacent sections incubated with anti-TIMP-1 mAb preabsorbed with TIMP-1 recombinant protein (K) or TNP mAb (L). Scale bars: A, C, D, E, F = 100 µm; B, G = 50 µm; H, I = 100 µm; J–L = 200 µm. Abbreviations: TIMP-1, tissue inhibitor of metalloproteinase-1; mAb, monoclonal antibody; TNP, trinitrophenyl.

Figure 2.

Localization of TIMP-1 mRNA in gastric cancer. Tissue sections from intestinal (A, C, D, F–I) and diffuse (B and E) subtypes were processed for in situ hybridization using TIMP-1 mRNA antisense (A–G) and sense (H and I) probes. The TIMP-1 mRNA signal is visualized as white spots in dark-field illumination (A–C, I) and black silver grains in bright-field illumination (D–H). TIMP-1 mRNA is observed in the stromal compartment at the periphery of invasive cancer cells in both intestinal (A, D, G; Ca: cancer, St: stroma) and diffuse (B and E) subtype tumors. TIMP-1 in situ hybridization signal is primarily detected in spindle-shape cells surrounding cancer cells in both histological subtypes (arrows in panels B, E, and G). TIMP-1 mRNA is also detected in a minor fraction of cancer cells (arrows) in a few tumor lesions of both histological subtypes (C and F). No specific TIMP-1 mRNA signal is observed in tissue sections incubated with a TIMP-1 sense probe (H and I), compared with an adjacent section hybridized with TIMP-1 antisense probe (G). Scale bars: A–D = 200 µm; B, C, E–I = 100 µm. Abbreviation: TIMP-1, tissue inhibitor of metalloproteinase-1.

Figure 3.

Identification of TIMP-1-positive cells in gastric cancer. Adjacent paraffin-embedded tissue sections were processed for double immunohistochemistry for TIMP-1 and α-SMA (A, D), TIMP-1 and CK (C), TIMP-1 and CD34 (E), or TIMP-1 and CD68 (B, F) using the EnVision G|2 Double System kit from Dako. The TIMP-1 staining was visualized with DAB (brown) and the stainings for α-SMA, CD68, CK, and CD34 were visualized with Permanent Red (magenta). Numerous TIMP-1-positive cells were found at the periphery of the cancer (indicated with Ca) in all samples tested. The vast majority of the TIMP-1-positive cells (black arrows in panels A, B, D) are also positive for α-SMA (black arrows in A, D) but negative for CD68 (B), indicating that these cells are myofibroblasts (panels A and B represent adjacent sections). TIMP-1 positivity is also seen in a few cancer cells (red arrows in panel C), in CD34-positive endothelial cells (blue arrows in panel E), and in a few CD68-positive macrophages (green arrows in panel F). Scale bars: A, B = 30 µm; C–F = 120 µm. Abbreviations: TIMP-1, tissue inhibitor of metalloproteinase-1; α-SMA, α-smooth muscle actin; CK, cytokeratin; DAB, diaminobenzidine; mAb, monoclonal antibody.