Matrix metalloproteinases (MMPs) are a family of -dependent endopeptidases targeting extracellular matrix (ECM) compounds. The secreted MMPs anchor to the cellular surface to mediate ECM degradation and release of latent growth and angiogenic factors. Because of the ability of MMPs to degrade ECM proteins, the principal mechanism by MMPs’ promoting tumor development has been thought to be the proteolytic breakdown of tissue barriers to invasion.1 Moreover, MMP2- and MMP9-knockout mice2 or tumor cells with downregulation of MMPs using RNAi3, 4 reduced tumor growth and invasion. MMP2 and MMP9 are particularly important, since they digest collagen, the main component of basement membranes.5, 6 Many MMP inhibitors have been designed to treat malignant tumors and other diseases associated with pathologic angiogenesis. Several inhibitors currently used in clinical development for cancer therapy include BB-2516, AG3340, COL-3, AE941, and so on.7, 8
To study MMP activity for assessing MMP inhibitors in vivo, a near-infrared fluorescence probe has been designed and used to detect the activity of MMP 2.9 This technique of measuring enzymatic activities has a profound impact on a variety of clinical and experimental studies, but the chemical synthesis of those probes is difficult and the cost is high. Compared with the chemical synthesized probes, a genetically encoded probe can easily be obtained by plasmid construction and used to set up a stable tumor model. In this paper, we design a genetically encoded fluorescent sensor named DMC (DsRed2-MSS-CFP expressed from pDisplay vector), that use DsRed2 and cyan fluorescent protein (CFP) linked by MMP substrate site (MSS) to indicate the MMP activity and evaluate the effect of MMP inhibitors in vivo.
MMPs are secreted as the proenzyme and activated outside of the cells. The sensor DMC was designed to detect the secreted MMPs. If the cells expressed high MMPs, the MMPs would act on the MSS site of DMC and result in DsRed2 and CFP separated and DsRed2 free from the cell’s surface. Hence, the fluorescent signal of DsRed2 of tumor cells was weakened, and the fluorescent ratio of DsRed2 to CFP (DsRed2/CFP ratio) was very low. In reverse, if the cells expressed low MMPs or MMP activity was inhibited, the fluorescent sensor DMC would be intact. The fluorescent signals of both DsRed2 and CFP from DMC could be detected, and the DsRed2/CFP ratio was high. In this way, the DsRed2/CFP ratio could be used to reflect the level of MMP activity in DMC-expressing living cells.
In comparison with the fluorescence resonance energy transfer (FRET) sensor YFP-MSS- published in our previous paper,10 the choice of DsRed2 and CFP as the fluorescent pair to construct the genetically encoded fluorescent sensor DMC is due to the bright fluorescent signal of DsRed2 and less cross talk of the fluorescent signal between CFP and DsRed2. FRET imaging is easy for a cultured living cell using a confocal microscope. However, it is difficult to get a FRET signal using an in vivo optical imaging system because of the lower quality of an optical pathway system, such as a low numerical aperture objective and a light source with an LED or a lamp. This is hardly consistent the width imaging field range with high-resolution FRET images. Thus, the genetically encoded fluorescent sensor DMC combined with DsRed2/CFP ratio imaging is more suitable for monitoring the MMP activity in vivo than YFP-MSS- combined with FRET imaging.
Furthermore, we used shell-less chick embryo chorioallantoic membrane (CAM) as a living subject for in vivo tumor imaging. CAM is a good model for in vivo optical imaging experiments because of its advantageous characteristics. CAM is an immunodeficiency animal model, so preparing various kinds of tumor models is easy. Compared with the tumor model prepared using nude mice, CAM is cheap and convenient to image, with a fast tumor growth rate. Therefore, it is very useful in the study of an in vivo antitumor drug screen. In this study, the fluorescent sensor DMC was used to detect MMP activity in living cells and in the shell-less CAM. This is a novel method for detecting extracellular MMP activity and screening the specific inhibitors of MMPs in vivo.
Materials and Methods
Construction of Plasmids
DsRed2 was amplified with the forward and reverse primers -GGGAAGATCTATGGCCTCCTCCGAGAACGTCATCACCG- and -CGGGATCCGCCTCCCTCCAGGAACAGGTGGTGGCGGC- . The amplified fragment was digested with BglII and BamHI and cloned into pGEM-T. The forward and reverse primers of enhanced cyan fluorescent protein (ECFP) were -CGGGATCCGTGCCCCTTAGCCTGTACAGCGGATCTAGAATGGTGAGCAAGGGCGAGGAGC- and -CAAGTCGACCATGCGGGCGGCGGTCACGAACTCCAGCAGG- , respectively. The polymerase chain reaction (PCR) products of ECFP were cloned into pGEM-T (Promega). The cloned cDNA of DsRed2 was selected with a right-inserted direction, and then the cloned cDNA of ECFP was conformed by sequencing. Next, the ECFP PCR product was digested with BamHI and SalI and subcloned at the BamHI/SalI site of pGEM-T-DsRed2. This subclone was digested with BglII and SalI to produce the cDNA fragment of DsRed2-MSS-ECFP. This was subcloned into the pDisplay vector at the BglII and SalI sites, and also into pET-28a at the BamHI and XhoI sites.
Expression and Purification of Recombinant Proteins
The cDNA of DsRed2-MSS-ECFP was subcloned into vector pET-28a and then was transformed into BL21 (DE3) E. coli (Novagen Corp.) to produce the recombinant 6His-tagged DsRed2-MSS-CFP. Bacteria was cultured to and induced with IPTG (isopropyl-beta-D-thiogalactopyranoside) for at . Then cells were disrupted by sonication in lysis buffer ( Tris. Cl, pH 7.9, , PMSF) followed by centrifugation ( , ). The recombinant protein was further purified by Ni-NTA resin (Qiagen). For further analysis, the recombinant protein was dialyzed in deionized water.
Cell Culture and Transfection
Samples of human breast tumor MDA-MB 435s cells were purchased from the Culture Center of Wuhan University (Wuhan, China), and human hepatic cancer HepG2 cells were kindly provided by the Immunology Laboratory of Tongji Medical College of Huazhong University of Science and Technology. The cells were routinely cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). MDA-MB 435s and HepG2 cells were transfected with plasmid DMC by So-fast transfection reagent (Sunma Biotechnology Corp., Xiamen City, China), according to the instructions of the manufacturer. After transfection, DMC-expressing cells were screened by exposure to G418 (Sigma) for MDA-MB 435s cells and for HepG2 cells, and then stable DMC-expressing cells were cloned.
Cellular Fluorescent Imaging
The fluorescent images of DMC-expressing cells were created using an FV1000 Laser Confocal Scanning Microscope (Olympus, Japan) with a Plan Apo oil immersion objective with a 1.42 numerical aperture. Each fluorescence channel was detected with a PMT (photo-multiplier tube), and Fluoview software was applied to this system. The CFP signals of the DMC-expressing cells were observed by using excitation light at and emission at 465 to . The DsRed2 signals of DMC-expressing cells were observed by using excitation light at and emission at 580 to .
The purification recombinant protein was incubated with purified mature MMP2 (with a buffer of pH 7.5, , and ). The reaction mixtures were transferred to a 1-cm cuvette of a spectrofluorometer (LS-50B, Perkin-Elmer, Norwalk, Connecticut) at . Fluorescence emission spectra from 460 to were recorded upon excitation at (5-nm bandwidth).
The purification DsRed2-MSS-CFP was incubated with or without of the mature MMP2 for and then immediately incubated for at . The sample solutions were loaded for sodium dodecylsulfate-polyacrylamide gel electrophoresis. (SDS-PAGE) (10%). Proteins were separated in the SDS-PAGE at for 2 to and subsequently transferred onto a polyvinylidene difluoride membrane (PVDF; Amersham) with a semidry blotting system. Following blotting, the membrane was probed with an anti-GFP (green fluorescent protien) antibody (1:2000; Clontech) in Tris-buffered saline with 0.5% Tween-20 (TBST). The immunoblot was then probed with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG antibody (1:5000; Bio-Rad), and the bands were detected using the ECL Western blotting analysis system (Amersham).
Chick Embryo Chorioallantoic Membrane (CAM) Model and Tumor Fluorescent Imaging
Fertile chick eggs were incubated at saturation humidity for at . The embryos were then explanted into a shell-less culture using a method adapted from our previous report.11 Briefly, a 10-cm-deep concave well was made using plastic food wrap fixed with elastic bands in the mouth of a polystyrene water cup ballasted with of water. After incubation, the shells were then cracked against the edge of a glass vessel and the contents poured into the wells. Nearly always, the contents settled with the blastodisc uppermost and central. Then the cup was sealed with food wrap and placed in an incubator ( , 60% humidity). At day 10, stable DMC-expressing MDA-MB 435s cells were explanted into CAM, and then GM6001 (Calbiochem Corp.), a kind of potent wide MMP inhibitor,12 was add into PBS solution. The solution was then added on the surface of the CAM near the tumor at a concentration of GM6001 per egg. As a negative control, the same number of cells was explanted into CAMs without GM6001. After three days of incubation, the fluorescence signals of the tumors were detected using a Leica MZ FL III fluorescence stereomicroscope with a mercury lamp (Leica, Germany). The DsRed2 (excitation filter ; barrier filter ) and CFP (excitation filter ; barrier filter ) fluorescence-imaging pictures of the tumors were recorded with a color CCD (Evolution VF, Olympus Corp.)
Results and Discussion
In Vitro Monitoring of MMP2 Cleavage of DsRed2-MSS-CFP Fusion Protein with Spectroscopic Measurements and Western Blot Analysis
The cDNA of DsRed2-MSS-ECFP were constructed into pET-28a, which is able to express the recombinant DsRed2-MSS-CFP in E. coli BL21 (DE3) when induced with isopropyl-β-D-thiogalacto-pyranoside (IPTG) [Fig. 1a ]. Then the recombinant protein was purified through an Ni-NTA column after being induced with IPTG. The peptide of VPLSLYSG in MSS was chosen because of its high value for MMP2.13 To determine the cleavage sensitivity of the fusion protein DsRed2-MSS-CFP to MMP2, we first examined it in vitro. As show in [Fig. 1b], fluorescence resonance energy transfer (FRET) from CFP to DsRed2 took place14 when the DsRed2-MSS-CFP was intact. If MMP2 cleaved the linker peptide between CFP and DsRed2, FRET would be abolished. The changes of FRET in response to MMP2 cleavage were measured with a spectrofluorometer at . Figure 1c shows the emission spectra of the purified DsRed2-MSS-CFP fusion protein with an excitation wavelength of . The two emission peaks of DsRed2-MSS-CFP occurred at and , corresponding to peaks of CFP and DsRed2, respectively. This result indicated that FRET from CFP to DsRed2 occurred and that the DsRed2-MSS-CFP fusion protein was intact. After adding MMP2 into the solution of DsRed2-MSS-CFP, the emission peak of DsRed2-MSS-CFP at the wavelength of disappeared later. Furthermore, Western blotting analysis [Fig. 1d] demonstrated that DsRed2-MSS-CFP was cleaved by MMP2. In vitro analysis indicated that DsRed2-MSS-CFP could be used as a fluorescence sensor to detect MMP2 activity. For in vitro monitoring of MMP2 cleavage of the DsRed2-MSS-CFP fusion protein with spectroscopic measurements, we have to use the FRET property of DsRed2-MSS-CFP. Otherwise, the DsRed2/CFP ratio of the integrated or cleaved DsRed2-MSS-CFP fusion protein will be the same in the solution.
Localization of Fluorescence Sensor
MMPs are secreted as proenzymes and can degrade extracellular matrix (ECM) components once activated. To test the level of MMPs in the living cells, we chose a vector pDisplay (Invitrogen Corp.) that is a mammalian expression vector, allowing the protein under investigation to enter the secretory pathway and anchor on the cellular surface [Fig. 2a ]. Proteins expressed from pDisplay are fused at the N-terminus to the murine Ig -chain signal sequence, which directs the protein to the secretory pathway, and at the C-terminus to the transmembrane domain of platelet derived growth factor receptor (PDGFR), which anchors the protein to the plasma membrane, displaying it on the extracellular side. The expressing probes anchor on the cellular surface, where fluorescence probes can be cleaved by secreted MMP [Fig. 2b]. After the DMC was cleavaged by MMPs, DsRed2 was cut off and free from the DMC-expressing cells, while CFP still remained in the cells. Thus, the MMP activity of tumor cells could be traced through monitoring the decrease of the fluorescent signal of DsRed2. To provide further support on the subcellular distribution of expressed fluorescence proteins, the vector of pDisplay-DMC was transfected into HepG2 cells with low expressing MMPs. As shown in Fig. 2c, fluorescent signals in the cellular secretory pathway and on the cellular surface were visualized in an optical section obtained by the confocal microscope.
In Fig. 2c, the confocal fluorescent images of DMC-expressing HepG2 cells showed the most fluorescence signal of CFP and DsRed2 co-localized at the plasma membrane and the secretory pathway in the same single cells. Because HepG2 cells only low-express MMPs and were not a completely negative control, some regions in the plasma membrane at the tail end of the cell appear with only a CFP signal and no DsRed2 signal. Furthermore, DsRed2 required many hours to attain full fluorescence. CFP forms a chromophore rapidly after translation (half ).15 DsRed2 takes longer to develop a coloration (half ).16 If DsRed2 was used to tag the secretory protein, the fusion protein should be secreted prior to maturation for fluorescence.17 Thus, slow maturing of DsRed2 in living cells resulted in the different fluorescent intensity of CFP and DsRed2 inside the cell and at the cell surface in the same single cell, despite the fact that DMC-expressing HepG2 cells should contain equal parts of CFP and DsRed2.
Using the DsRed2/CFP Ratio to Detect MMP Inhibitor Effects in Living Cells
The fluorescence sensor DMC was designed to trace MMP activity in living cells and small animals. When the cells excrete MMP2, the sensor DMC expressed on the surface of the cell plasma member or on the secretory pathway will be cleaved by the MMP2. Thus, DsRed2 is freed from the cells so that it is barely detected by fluorescent imaging. MDA-MB 435s cells are chosen because they highly express MMP2 and also produce MMP1, MMP3, MMP9, MMP14, MMP15, and MMP16.18 Figure 3 shows that the fluorescence signals of DMC-expressing MDA-MB 435s cells were simultaneously detected in the CFP and DsRed2 channels. MMP inhibitor GM6001 could dramatically increase the DsRed2 signal after of treatment. This indicated that GM6001 could efficiently inhibit the MMPs activity so that the new procreant DMC could be kept intact in living MDA-MB 435s cells.
Detecting MMP Inhibitor Effects in CAMs with DsRed2 and CFP Imaging
In vivo imaging of MMPs will be very useful for medical target assessment. In this experiment, we used the chick embryo CAM in a shell-less culture as our experimental model. This model has an advantage of allowing direct observation of fluorescence in the embryo surface without interference by the skin or other tissue. Monitoring the fluorescence signals of CFP and DsRed2 simultaneously reflected the MMP inhibitor effect during tumor growth on CAM. The fluorescent images of CAM were obtained after 3 days of treatment with or without GM6001 (Fig. 4 ). The tumor treated with GM6001 [the bottom row of Fig. 4a] yielded a brighter DsRed2 fluorescence signal than the control tumor [the top row of Fig. 4a]; however, the CFP signals of the two groups had no obvious difference. Thus, we still use the DsRed2/CFP ratio to more exactly reflect the effect of the MMP inhibitor [Fig. 4b], and the results were taken from experiments three times repeated. MMP inhibitor GM601 could obviously increase the DsRed2/CFP ratio because it inhibited the cleavage of the sensor by active MMPs.
In this paper, we detected the MMP activity in vivo using a fluorescence stereomicroscope, a kind of optical imaging system with a larger imaging field and lower resolution than the confocal microscope. Although DMC is a FRET probe, it is difficult to detect a FRET signal in vivo by fluorescence stereomicroscope because of the low numerical aperture objective and because the light source is a mercury lamp. Furthermore, the FRET efficiency of DsRed2/CFP is much lower than YFP/CFP.10 Thus, we used the DsRed2/CFP ratio to reflect the MMP activity and did not detect the FRET efficiency of DMC.
Here, the CFP signal could be used as an internal reference to eliminate the error due to the difference of fluorescent sensor expression in various cells or the difference of imaging focus, and this could tell us where the tumor cells are if the cells highly secreted MMPs. This supposes that if we construct an expressing display vector containing only DsRed2-MSS, it also can be cleaved by MMPs. However, if DsRed2-MSS was transfected into the tumor cells with high secretory MMPs, we cannot detect an obviously fluorescent signal and we do not know whether this is due to the low transfection efficiency or high MMP activity. According to the mechanism of FRET, the fluorescence signal of CFP will increase if DMC was cleaved by MMPs. Because of this, the DsRed2/CFP ratio could generate a high contrast between its intact and cleaved components. If the cells low expressed MMPs, or if MMP activity was inhibited by an MMP inhibitor, DMC would stay intact and the cells could be detected with the fluorescence signals of both DsRed2 and CFP. The DsRed2/CFP ratio would be high. If the cells highly secrete MMPs, DMC would be cleaved, and the cells would appear with obviously decreased fluorescent signal of DsRed2 and lightly increased fluorescent signal of CFP, so the DsRed2/CFP ratio would decrease. We cannot directly compare the fluorescent signal of DsRed2 or CFP alone because the different expressing of the cells and the imaging focus will effect the fluorescent intensity of DsRed2 and CFP. Thus, the DsRed2/CFP ratio is a good approach to reflect the activity of MMPs in vivo.
In this study, we have demonstrated that the fluorescence sensor DMC can be used to monitor MMP cleavage activity and to analyze MMP inhibitor effects in living cells and in CAMs. Highly sensitive and specific fluorogenic substrates were designed as sensors with the ability to detect small-molecule inhibitors. Fluorescence imaging of DMC in the CAM was sensitive to the MMP inhibitor. This method could be very valuable for developing effective medicines for cancer therapy and also for evaluating inhibitors in vivo. Compared with the YFP-MSS- , a genetically encoded surface-displayed FRET sensor to detect MMP activity in living cells,10 DMC is more suitable for in vivo imaging by the fluorescence stereomicroscope because of the infrequent cross talk between the fluorescent signals of DsRed2 and CFP. Despite the lower FRET efficiency of the DsRed2/CFP pair than the YFP/CFP pair, the DsRed2/CFP ratio of DMC-expressing cells still could sensitively reflect the MMP activity in living cells and in the in vivo tumor model.
In the living cells and CAM tumor models, the CFP and DsRed2 signals were simultaneously detected to assess the effect of the MMP inhibitor. According to the CFP signal, we can continuously detect the position and size of tumors even if the DsRed2 signal disappeared due to the cleavage of DMC by the active MMPs. On the other hand, CFP could be used as an internal reference to eliminate the error due to the difference of DMC expression. This genetically encoded probe has a good application perspective for assessing the MMP inhibitor effect in vivo and in vitro. Many MMP inhibitors have undergone clinical trials, such as BB-2516, AG3340, COL-3, and AE941.19 The currently available approaches to monitor MMP inhibition therapies are based on indirect evidence of therapy, such as tumor size, histological changes, and possibly MMP levels in blood or tumor tissue measured, and these take a long time. These approaches do not provide a reliable result for assessing inhibition of MMP activity in vivo. This study provides a direct method of molecular target assessment, and it will benefit MMP inhibitor studies that are under way.
This work was supported by the National Natural Science Foundation of China (Grant Nos. 90508003 and 60440420131), the National Key Technologies Research and Development Program of China (Grant No. 2005BA711A04), Program for Changjiang Scholars and Innovative Research Team in University, and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (Z. Zhang).