Dynamically Optic Imaging MMP Activity Of Living Tumor Cell

Matrix Metaloproteinases (MMPs) are capable of degrading almost all extracellular matrix (ECM) and basement membrane (BM) components. They play an important role in normal issue remodelling and growth, as well as in many destructive pathological conditions such as tumour growth, invasion and metastasis. MMPs inhibitors have good development and application prospect as novel anti-tumor pharmacotherapy. The approach which real-time detecting MMPs activity has an important role in screening MMPs inhibitorsGenetically encoded fluorescence resonant energy transfer (FRET) probes are used to detect proteinase activity, and FRET imaging can monitor proteinase activity in living cells. In this study, we construct two FRET probes to detect MMPs activity in the living and in vivo. The designing principle is to link CFP and YFP or CFP and DsRed with MMPs substract peptide. In this way, the fusion proteins YFP-MSS-CFP or DsRed-MSS-CFP are constructed.The purified probe can be used to analyze MMP activity by spectrum detection in vitro, and this probe is specific for MMP2. And with a display approach, the fusion proteins are displayed on the cells surface to detect secretory MMPs activity. FRET probe will be intact in the absent of extracellular MMPs, the fluorescence resonant energy transfer between CFP and YFP (or CFP and DsRed) will occur. However, when high extracellular MMPs are secreted, FRET probe will be degraded. And this results in YFP or DsRed out of the cells, but CFP was still on the cells. Therefore, MMPs activity can be detected either by FRET imaging or by YFP (or DsRed) imaging. FRET probes were stably expressed in MDA-MB 435s cells, in which high secretory MMPs were expressed and MCF-7 cells that expressed low levels of MMPs. The FRET sensor YFP-MSS-CFP display can sensitively and reliably monitor MMP activation and GM6001 inhibitor effect in living cells in real-time.The DsRed-MSS-CFP was expressed in MDA-MB 435s cells that highly expressing endogenetic secretory MMP, so that the DsRed-MSS-CFP was cleaved by MMP and the fluorescence ratio of DsRed2 to CFP was decreased. When the cells were treated by GM6001, an MMP inhibitor, it could block the cleavage of DsRed-MSS-CFP and caused an increase of fluorescence ratio of DsRed2/CFP. The same result was achieved by using tumor model that DsRed-MSS-CFP expressing MDA-MB-435s cells inoculated onto the chorioallantoic membrane of developing chick embryos form primary tumors on the membrane. Thus, the fluorescent probes are able to sensitively monitor MMP activation and be used to assess MMP inhibitors for anti-cancer research in vivo.