Most studies performed thus far with in situ zymography using quenched fluorogenic substrates dealt with gelatinolytic activity. However, other quenched fluorogenic substrates are also available. These are DQ-collagen type I, DQ-collagen type IV, DQ-elastin, DQ-bovine serum albumin (BSA), DQ-ovalbumin, and DQ-casein (Jones et al. 1997). These substrates were initially developed to demonstrate activity of proteinases in solution. However, the substrates can in principle be used also to detect proteinase activity in unfixed cryostat sections or cell preparations when LGT-agar or another gelling compound is added to the incubation medium. It must be emphasized that development of other natural substrates of MMPs with quenched fluorescence, such as laminins and fibronectins, should be encouraged.
DQ-collagen type I was applied to fixed cryostat sections of endometrial biopsy specimens in the presence of gelatin (Zhang and Salamonsen 2002). Collagenase activity was observed in small foci within the tissue in all phases of the menstrual cycle, but collagenase activity was most abundant premenstrually and during the menstrual phase. Collagenase activity was localized extracellularly and was abolished when sections were pretreated with phenanthroline. It was concluded that MMP-1 was responsible for the breakdown of collagen type I because the protein showed a similar localization pattern. MMPs appear to play a critical role in matrix degradation during menstruation.
DQ-collagen type IV has been mainly applied to cells cultured on matrices containing the substrate (Horino et al. 2001; Sameni et al. 2001; Elner 2002; Premzl et al. 2003). Invasion of fibrosarcoma cells in gelatin matrix was accompanied by collagenolytic activity due to MMP activity (Horino et al. 2001). Sameni et al. (2000)(2001) investigated the site of proteolysis in a three-dimensional gelatin matrix embedded with DQ-collagen type IV and human cancer cells cultured on top of it. Cells at all levels in the matrix accumulated fluorescent degradation products of DQ-collagen IV intracellularly in vesicles. Based on findings using inhibitors, it was concluded that lysosomal proteinases, such as cathepsin B, are responsible for intracellular degradation of collagen type IV in human breast cancer cells, glioma cells, and colon cancer cells, and are involved in cancer cell invasion.
A similar approach was used by Premzl et al. (2003), who localized the breakdown of DQ-collagen IV by ras-transformed breast cancer cells during invasion in matrigel. Intracellular and extracellular cleavage of collagen type IV was detected that was largely due to intracellular and extracellular cathepsin B activities. Yan and Blomme (2003) demonstrated breakdown of DQ-collagen type IV in cryostat sections of rat tibia. Fluorescence was observed in the distal portion of the hypertrophic zone of growth plates of the tibia. MMP-9 was held responsible for collagenolytic activity because EDTA prevented the production of fluorescence, and protein and mRNA of MMP-9 were similarly localized as the fluorescence due to collagen type IV breakdown.
In situ zymography of uPA was introduced by Sappino et al. (1991) using plasminogen and casein as substrate. Plasminogen was converted by uPA to plasmin, which degraded casein. de Vries et al. (1995) demonstrated with this approach uPA activity in sprouting capillaries in melanomas. The production of fluorescent peptides from DQ-casein was recently used for the precise localization of uPA activity in unfixed cryostat sections of mouse liver with colon cancer metastases (Ackema, unpublished results). Figure 5 shows the localization of fluorescent breakdown products of Bodipy-casein in colon cancer metastases of mouse liver. DQ-BSA has also been used instead of DQ-casein as substrate for the localization of uPA activity in matrices containing fibrosarcoma cells (Horino et al. 2001) and retinal pigment cells (Elner 2002). The specificity of extracellular proteolysis of BSA by uPA was assessed by inhibition with PAI-1. BSA can be cleaved by many proteinases and specificity tests are therefore very important. Koblinski et al. (2000) showed that intracellular cathepsin B is responsible for the intracellular accummulation of degraded DQ-BSA in transfected fibroblasts grown on a gelatin matrix containing DQ-BSA.
Summarizing, dye-quenched fluorogenic natural substrates other than gelatin have mainly been used in gel matrices containing cultured cells and have rarely been applied to cryostat sections. The use of these substrates has potential for the localization of activity of proteinases, but it should be emphasized that proper controls must be performed to establish the proteinases involved, such as the use of inhibitors and combination with detection methods such as IHC and zymography.
In conclusion, precise localization of proteinase activity using natural substrates containing quenched fluorescence is a valuable tool to study its role in (patho)physiological processes. The value of the method may even increase when fluorescence production can be measured locally. Fluorescence should then be measured locally during incubation, and measurements should fulfill a series of criteria as formulated by Stoward (1980), as has been established for many other enzyme histochemical procedures (Van Noorden and Frederiks 1992). Moreover, in situ zymography localization should be combined with IHC, preferably on the same sections or at least on serial sections. Finally, zymography of cell or tissue homogenates should also be performed to assess the nature of the protein and, as a consequence, the type of proteinase that cleaves the substrate.
This is the method of choice to detect gelatinolytic activity with in situ zymography and DQ-gelatin in cells or tissues.
- Dissolve 1 g LGT agarose in 100 ml PBS, pH 7.45, under continuous stirring and heating in a water bath (80C) until a clear solution is obtained.
- Store the clear agarose-containing solution at 4C in air-tight vials.
- Heat the agarose-containing solution to 60C to obtain a clear solution before incubation.
- Cool the desired volume of the solution to 37C before incubation.
- DQ-gelatin (Molecular Probes) is dissolved in a concentration of 1 mg/ml in distilled water.
- The DQ-gelatin solution is diluted 1:10 in the agarose-containing solution.
- Use unfixed cells or unfixed cryostat sections of the tissue to be investigated (8–10 µm thick).
- The DQ-gelatin agarose mixture (40 µl) is put on top of the dried cells or sections and covered with a coverslip of 24 x 40 mm.
- The agar is gelled at 4C.
- Incubation is performed for 1–24 hr at RT dependent on the enzyme activity.
- Fluorescence of FITC is detected with excitation at 460–500 nm and emission at 512–542 nm.
- Control incubations should be carried out on cells or serial cryostat sections by adding 20 mM EDTA or a selective MMP inhibitor to the incubation medium.
- Cells or sections should be preincubated for 1 hr at RT with the different inhibitors dissolved in PBS.
- The presence of autofluorescence in cells or sections should be tested by incubating in the agarose-containing medium that lacks DQ-gelatin.
- Nuclei can be counterstained by adding DAPI (1 µg/ml) or propidium iodide (0.5 µg/ml) to the incubation medium.
- Analyze the cells of sections: compare the fluorescence of FITC formed after incubation in the presence of DQ-gelatin with the fluorescence produced after incubation in the absence of DQ-gelatin or in the presence of DQ-gelatin and MMP inhibitors.
This is the procedure of choice to detect gelatinolytic activity with in situ zymography in cells or tissues in combination with IHC.
- Cells or cryostat sections are air-dried for 1 hr at RT.
- Cells or cryostat sections are fixed in acetone for 10 min at RT. Crosslinking fixatives should be avoided, because they affect enzyme activity.
- The IHC procedure for the desired antigen is performed according to standard procedures using a fluorescently labeled secondary antibody with spectral properties other than FITC.
- The procedure as described in protocol 1 is performed to detect gelatinolytic activity.
- Analyze the cells or sections: compare the localization of the fluorescence of FITC with that of the other fluorophore.
We are grateful to Prof Dr C.J.F. Van Noorden for most valuable comments on the manuscript. We wish to thank Ms H. Vreeling and Ms E. Ackema for providing their micrographs, Mr J. Peeterse for preparation of the micrographs, and Ms T.M.S. Pierik for careful preparation of the manuscript.
Received for publication January 7, 2004; accepted February 27, 2004