Modulation of monocyte matrix metalloproteinase-2 by breast adenocarcinoma cells

Abstract

Introduction: The presence of monocyte and macrophage cells in growing breast tumors, and the positive relationship between the degree of immune cell infiltration and tumor growth, suggest a possible paracrine growth regulatory function of immune cells in breast cancer.

Method: To better understand the interaction between monocytes and breast cancer cells, in vitro matrix metalloproteinase and tissue inhibitor of metalloproteinase activity was assessed from the THP-1 myeloid cell line in response to conditioned media from two breast cancer cell lines, MCF-7 and MDA-MB-231.

Results: Enzymography and immunoblotting revealed increased MMP-2 as well as increased levels of TIMP-1 and TIMP-2. Furthermore, a significant increase in the invasive potential of MCF-7 and MDA-MB-231 cells was noted in response to THP-1 cell-conditioned media.

Conclusion: These data demonstrate that monocyte cells in the breast tumor microenvironment play an important role in the modulation of MMPs, which may have a significant effect on the control of tumor growth and metastatic spread.

Figure 1

The effects of MCF-7 breast adenocarcinoma CM on MMP and TIMP activity from THP-1 monocytes. (a) Gelatin zymogram and (b) MMP-2 immunoblot showing the upregulation of monocyte MMP-2 after exposure to MCF-7 CM, and (c) a reverse zymogram to show that the activity of monocyte TIMP-1 and -2 remained the same upon treatment. Lanes 1, 4, and 7 contain CM from THP-1 cells in serum-free media grown in the presence of 100 μl, 200 μl and 300 μl concentrated MCF-7 CM, respectively. Lanes 2, 5, and 8 contain CM from THP-1 cells in serum-free media grown in the presence of 100 μl, 200 μl and 300 μl concentrated serum-free media, respectively. Lanes 3, 6, and 9 contain serum-free media with 100 μl, 200 μl and 300 μl, respectively, of MCF-7 CM in the absence of THP-1 cells. These figures are representative of three independent experiments carried out in duplicate, each of which demonstrates similar results.

Figure 2

The effects of MDA-MB-231 breast adenocarcinoma CM on MMP and TIMP activity from THP-1 monocytes. (a) Gelatin zymogram and (b) MMP-2 immunoblot showing the upregulation of monocyte MMP-2 after exposure to MDA-MB-231 CM, and (c) a reverse zymogram to show that the activity of monocyte TIMP-2 was also upregulated upon exposure to MDA-MB-231 CM, although TIMP-1 activity remained the same upon treatment. Lanes 1, 4, and 7 contain CM from THP-1 cells in serum-free media grown in the presence of 100 μl, 200 μl and 300 μl concentrated MDA-MB-231 CM, respectively. Lanes 2, 5, and 8 contain CM from THP-1 cells in serum-free media grown in the presence of 100 μl, 200 μl and 300 μl concentrated serum-free media, respectively. Lanes 3, 6, and 9 contain serum-free media with 100 μl, 200 μl and 300 μl, respectively, of MDA-MB-231 CM in the absence of THP-1 cells. These figures are representative of three independent experiments carried out in duplicate, each of which demonstrates similar results.

Figure 3

MCF-7 and MDA-MB-231 upregulation of TIMP-1 and TIMP-2 protein levels from THP-1 monocytes. Immunoblots representative of three independent experiments carried out in duplicate using monoclonal (a,b) TIMP-1 and (c,d) TIMP-2 antibodies to show an increased production of both proteins by monocyte cells in response to MCF-7 and MDA-MB-231 CM. Lane 1 contains CM from THP-1 cells in serum-free media grown in the presence of 300 μl concentrated (a,c) MCF-7 CM and (b,d) MDA-MB-231 CM. Lane 2 contains CM from THP-1 cells in serum-free media grown in the presence of 300 μl concentrated serum-free media. Lane 3 contains serum-free media with 300 μl of (a,c) MCF-7 CM and (b,d) MDA-MB-231 CM in the absence of THP-1 cells. Lane 4 contains unconcentrated (a) MCF-7 and (b,d) MDA-MB-231 CM.

Figure 4

The effects of monocyte cells and CM on the invasive potential of breast adenocarcinoma cells. Graphs showing an increase in the number of (a) MCF-7 and (b) MDA-MB-231 cells that migrated through matrigel-coated membranes in response to monocyte cell CM. The following conditions were used in the lower chamber: 1, serum-free media; 2, 10% FBS in serum-free media; 3, THP-1 cell-conditioned media; 4, (a) 5 × 10⁴ MCF-7 cells, (b) 5 × 10⁴ MDA-MB-231 cells; 5, 5 × 10⁴ THP-1 cells; 6, (a) 10 × 10⁴ MCF-7 cells, (b) 10 × 10⁴ MDA-MB-231 cells; 7, 10 × 10⁴ THP-1 cells. *p < 0.05 compared to condition 2; **p < 0.05 compared to condition 4; ***p < 0.05 compared to condition 6. The values are the means of three experiments and the error bars are the standard error of the mean.