2003). that TIMP-2 XL647 (Tesevatinib) can differentially affect MMP activity and cellular invasiveness in both an MMP dependent and independent manner. More specifically, MMP activity and invasiveness is increased with the addition of exogenous TIMP-2 in poorly invasive cell lines whereas it is decreased in highly invasive cells lines (MDA-MB-231). Conversely, the addition of ALA + TIMP-2 resulted in decreased invasiveness regardless of cell line. and However, these effects were obtained with TIMP-2 levels ranging from 2.5 KPSH1 antibody to 10?g/ml which are 25 to 100-fold higher than found in normal tissues or biological fluids (10-100?ng/ml) (Larsen et al. 2005). In this study, we treat breast cancer cells with TIMP-2/ALA + TIMP-2 conditioned media containing 22?ng/ml of the respective proteins, a level well within and in the low end of physiological concentrations. Surprisingly, when we assayed for the invasiveness of MCF-7 cells treated with TIMP-2 conditioned media, we saw a significant in the invasive nature of these cells. Accordingly, we decided to look at additional cell lines to see if this increased invasiveness in the presence of an MMP inhibitor was a peculiarity of MCF-7 cells. We examined cells that are both similar to, and different from, MCF-7 cells with respect to characteristics crucial to this study. T47D cells, like MCF-7 cells are regarded as less tumorigenic when compared to MDA-MB-231 cells. Of importance to us is that both MCF-7 and T47D cells show low levels of expression of TIMP-2, MMP2 and many other MMPs (Balduyck et al. 2000; Figueira et al. 2009; Jones et al. 2003). Further, MCF-7 and T47D share similar invasive capabilities, and their invasive capabilities mirror each other even when cells are treated with extracellular reagents, such as the protein tenascin-C (Hancox et al. 2009). These shared attributes of low MMP expression levels and low invasiveness are not shared by MDA-MB-231 cells which express high levels of TIMPs and MMPs, and whose invasive characteristics are also distinct (Balduyck et al. 2000; Hancox et al. 2009; Jones et al. 2003). Here we demonstrate that T47D cells, which are similar to MCF-7 cells, also increased their invasiveness, though not significantly, when treated with TIMP-2 conditioned media. When we isolated media from these treated T47D cells to assay for MMP activity, we saw that it was significantly increased in MMP activity. As MCF-7 and T47D cells both have low endogenous levels of MMPs, we hypothesize that high levels of exogenous TIMP-2 works to activate pro-MMPs when they are expressed at low levels by these cell lines. Conversely, when we treated MDA-MB-231 cells (which express high levels of active MMPs) with TIMP-2, this resulted in a significant decrease in both MMP activity and invasiveness. Here addition of exogenous TIMP-2 inhibited the active MMPs that are already present at high levels. Gelatin zymography was then used to examine the activation of specific MMPs. However, it has been previously reported that gelatin XL647 (Tesevatinib) zymography is not sensitive enough to detect subtle changes in proMMP-2 activation, especially in cell lines that endogenously express low levels of TIMPs and MMPs (Ratnikov et al. 2002). Indeed studies have published that proMMP-2 and -9 activity is not detectable in MCF7 cell media using zymography (Ehrenfeld et al. 2011; Lauber and Gooderham 2011), while others demonstrate the contrary (Abdallah et al. 2007; Bartsch et al. 2003). Similarly, zymography reports using conditioned media from T47D cells have conflicting results. Some report the absence of pro-MMP-2 and -9 activity (Janowska-Wieczorek et al. 2006) while others report the presence (Abdallah et al. 2007). However, despite differences in the absolute levels of pro-MMP-2 or -9 in MCF-7, T47D or MDA-MB-231 media, these reports agree on the relative differences between these cells lines, with MDA-MB-231 showing the highest levels of activity, and MCF-7 the lowest amongst these 3 cell lines (Ehrenfeld et al. 2011; Janowska-Wieczorek et al. 2006; Jones et al. 2003). Here we demonstrate a TIMP-2 dependent increase in global MMP activity that is not detected through zymography. Given that other MMPs in addition to MMPs XL647 (Tesevatinib) 2 and 9 could be activated, and with limitations and inconsistencies of zymography in detecting subtle changes, other approaches need to be utilized to understand how TIMP-2 is facilitating changes XL647 (Tesevatinib) in cell behavior. To further investigate the nature of this phenomenon, we used an ALA + TIMP-2 mutant that cannot inhibit MMP activity (Wingfield et al. 1999). We found that treatment with ALA.