Breast Cancer Research and Treatment 63: 105–115, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. Report Induction of matrix metalloproteinases MMP-1 and MMP-2 by co-culture of breast cancer cells and bone marrow fibroblasts Sonia Saad, Linda J Bendall, Alexander James, David J Gottlieb, and Kenneth F Bradstock Institute for Cancer Research, Westmead Millennium Institute, Westmead Hospital and Faculty of Medicine, University of Sydney, Sydney, Australia Key words: bone marrow fibroblasts, breast cancer, migration, matrix metalloproteinases, MMP-1, MMP-2 Summary Two invasive breast cancer cell lines (MDA-MB-231 and BT-549) were found to be more adherent and have greater migratory capacity on bone marrow fibroblasts than three non-invasive cell lines (MCF-7, T47D and BT- 483). Antibodies to the adhesion molecules CD44, E-cadherin, ICAM-1, and integrin chains α2, α3, α4, α5, α6, αv, β1, β3 and β7 failed to inhibit breast cancer cell migration through bone marrow fibroblasts. Inhibitors of matrix metalloproteases, 1, 10-phenanthroline, Ro-9790, TIMP-1 and TIMP-2 were able to attenuate the migration of MDA-MB-231 cells through bone marrow fibroblast monolayers suggesting a role for these enzymes in the migration of breast cancer cells through bone marrow adherent layers. Co-culture of MDA-MB-231 cells and bone marrow fibroblasts resulted in augmentation of the levels of the matrix metalloproteases MMP-1 and MMP-2 in culture supernatants. Soluble factors produced by bone marrow fibroblasts were responsible for the increase in MMP-1 levels. However, maximal MMP-2 production was dependent on direct contract between the breast cancer cells and the bone marrow fibroblasts. Modulation of MMP production by cell–cell contact or soluble factors suggests a mechanism by which breast cancer cells can enhance their ability to invade the bone marrow microenvironment. Introduction Breast cancer is one of the most common forms of cancer in women but despite being responsive to hor- monal manipulation and chemotherapy, relapse fol- lowing treatment is common, particularly in patients with metastatic disease [1]. Encouraging results have been obtained using autologous hematopoietic stem cell transplantation to facilitate chemotherapy dose in- tensification for the treatment of metastatic and node- positive breast cancers [2]. Breast cancers frequently metastasize to the bone marrow, suggesting that the marrow may provide a favorable microenvironment for the growth of tumor cells and act as a reservoir of disease allowing further hematogenous spread. The presence of breast cancer cells in the bone marrow has raised concerns about malignant cell contamina- tion of reinfused marrow and peripheral blood stem cell preparations [3]. Matrix metalloproteases (MMPs) are a family of zinc dependent enzymes with the capacity to degrade extracellular matrix proteins [4]. MMP expression has a strong correlation with cancer cell invasiveness and metastasis [5–7]. Although some MMPs such as MMP-2 are elevated in breast cancer epithelial cells, most tumor associated metalloprotease activity occurs in the adjacent fibroblasts [8]. This suggests that the MMP activity of non-malignant tissue is modulated by the adjacent malignant epithelial cells. Similarly, some MMPs, including MMP-1 and MMP-9 can be induced in malignant cells by cytokines [9–14]. Crosslinking of antibodies to the β1 integrins VLAs-2 and -3 can induce increased production of pro- and active forms of MMP-2 [15]. We have examined the adhesive interactions between breast cancer cells and bone marrow derived fibroblasts and the ability of breast cancer cells to mi- grate through bone marrow stromal layers. Although the adhesive mechanisms used by breast cancer cells to bind to bone marrow fibroblast monolayers are not clear, it is apparent that MMPs play a role in the migra- tion of invasive breast cancer cells through monolayers