The electrical resistance breakdown of the Madin-Darby canine kidney (MDCK) cell monolayer provides a continuous assay system for cancer invasion that detects functional changes before morphological alterations. In this study, we address the question of whether physical contact between tumor cell and epithelial monolayer is a prerequisite for tumor cell invasion. When human melanoma cells were seeded directly (i.e., physical contact) on top of an electrically tight epithelial cell layer (5,800 ± 106 Ω · cm2), electrical monolayer leakage led to an 18 ± 3% reduction of transepithelial electrical resistance within 24 h. However, when melanoma cells were seeded close to the basolateral surface of the epithelial cell monolayer but separated by a filter membrane (i.e., no physical contact), electrical leakage occurred even more quickly (42 ± 3% reduction in 24 h). Atomic force microscopy detected discrete structural changes between cells. Electrical leakage was effectively blocked by α2-macroglobulin or ilomastat, inhibitors of matrix metalloproteinases. We conclude that exocytosis of soluble proteases causes electrical breakdown of the MDCK monolayer, independently of physical contact between tumor cells and the monolayer.
- basement membrane
- melanoma cells
- matrix metalloproteinases
metastasis is characterized by spreading of tumor cells through vessels, lymphatic ducts, or cavities to form new colonies away from tumor origin. Therefore, tumor cells must fulfill two prerequisites: the ability 1) to degrade matrix proteins and 2) to migrate actively through physiological barriers formed by cell monolayers and basement membranes (19). Extracellular matrix proteins are cleaved by proteases that are secreted by tumor cells or by surrounding nonneoplastic stromal cells. A clear correlation between protease secretion of cancer cells and their potency of invasion has been documented (9, 19, 35). Four major classes of cellular proteases were identified: the aspartate-, cysteine-, serine-, and metalatom-dependent enzymes. Although representatives of all four classes of proteolytic enzymes have been implicated in tumor invasion and metastasis (1), the matrix metalloproteinases (MMPs) were shown to be especially crucial for invasion (9, 20, 37). Batimastat, ilomastat, and marimastat are broad-spectrum inhibitors of major MMPs and have been shown to prevent or reduce the spreading and growth of a number of different malignant tumors in preclinical and clinical studies (9, 26, 41). Components of the basement membrane, such as laminin, fibronectins, and collagens, are targets of MMPs (9,28). The basement membrane is necessary for the formation of intact epithelial and endothelial cell layers. Basement membranes act as selectively permeable barriers (e.g., renal basement membrane) and serve as a basis for tissue repair. Basement membranes are especially important as a barrier against the spread of tumor cells (33). Although protease secretion is a crucial step in cancer invasion and metastasis, no sensitive method has been available to analyze the impact of these proteases on tumor cell invasion.
Therefore, many studies have been focused on the penetration and destruction of the basement membrane by tumor cells using the Boyden chamber, a histochemical method (2). The Boyden chamber is based on the counting of tumor cells after penetration of a basement membrane-like coated polycarbonate filter. It does not reflect the interaction between cancer cells and normal tissue. Therefore, use of the Boyden chamber prevents the possibility of examining the invasion of cancer cells into an intact cell monolayer. The impact of protease secretion, cell migration, or cellular interaction by physical contact between both cell types cannot be examined.
The electrical resistance breakdown assay, in a study recently published by our laboratory, is a highly sensitive electrophysiological method based on the measurement of the high transepithelial electrical resistance (TEER) of the C7 subclone of MDCK cells (42). TEER measurements allow the quantification of cancer cell invasion into an intact cell monolayer with sufficient time resolution. In this study, we show that this method enables measurement of C7 monolayer violation by cancer cells at the very beginning of invasion, before light microscopy reveals any morphological alterations. However, using high-resolution atomic force microscopy (AFM) (4), discrete structural changes between C7 cells are visible (16). In the present study, we used the invasion assay to examine the impact of protease secretion on cellular monolayer destruction with and without contact between both cell types. We identified secreted MMPs involved in cell monolayer destruction. The data show that cancer cells are able to affect epithelial cell monolayer integrity even without physical contact.
Cells and cell culture.
The electrical resistance breakdown assay as a cell-based system to determine tumor cell invasiveness has been described previously (42). We used the high-resistance MDCK-C7 monolayer that serves as a tight test barrier for the invasion of different human carcinoma cells (12). For a highly malignant cancer cell, we used a human amelanotic melanoma cell line, subclone A7 (kindly provided by Dr. A. Schwab, Institute of Physiology, University of Würzburg). This rare type of cancer cell differs from most other melanoma cells because it does not cloud or change the color of the cell culture medium. Melanoma cells were transfected with actin-binding protein to maximize migration activity (7, 8). Melanoma cells have high migration activity that has been measured in basement membrane matrix-coated dishes (36). MDCK-C7Focus (F) cells were generated by culturing MDCK-C7 cells in alkaline medium (24,38, 40).
Melanoma, C7, and F cells were cultured under standard conditions as previously described (42). To prevent contamination, penicillin (100 U/ml) and streptomycin (100 μg/ml) were present in the media. Cells were seeded in filter membrane cups (growth area, 4.2 cm2; pore diameter, 0.4 μm; thickness, 20 μm; Falcon, Heidelberg, Germany) for TEER measurements at a density of 400,000 cells/cup (see Fig. 1, A andB). The filter membrane was tested for transmigration and found to be impermeable to the C7 and melanoma cells. In a second experimental setup (see Fig. 1, C and D), C7 cells were grown on the reverse side of the filter. C7 cells (106) were dropped onto the reverse side of an upside-down-oriented membrane filter cup. After 4 h, the supernatant was removed, and the filter was placed in a six-well culture plate. Medium exchange and TEER measurement started 48 h after seeding the C7 cells. Control experiments were performed by using noninvasive transformed F cells and C7 cells.
For TEER measurements, as previously described in detail (13, 14,25, 32), we used a commercially available electrode chamber (Endohm-24, WPI, Sarasota, FL) that can house an individual filter cup (Fig. 1). Mounted on a heating plate, the electrode chamber is maintained at 37°C. A set of 6 filter cups (from a 6-well plate) can usually be measured within 3 min. Background electrical resistance, including filter and medium, is constant and extremely low (25 Ω · cm2).
C7 cell monolayers were used in our experiments after exhibiting a resistance of ≥5 kΩ · cm2. A resistance ≥1 kΩ · cm2 already implies a tight C7 cell monolayer (34). In control experiments, TEER increased to a plateau of up to 15 kΩ · cm2 within 14 days. However, after TEER reached a plateau, a small decrease could be measured due to ongoing cell proliferation and increasing cell number. Therefore, we added cancer cells to the C7 monolayer before TEER of C7 cell monolayer reached a plateau. In contrast, the maximal transepithelial resistance of melanoma cells was usually 30 Ω · cm2, a value close to the background resistance of the filter membrane (25 Ω · cm2).
Antibodies and fluorescence microscopy.
For indirect immunofluorescence, C7 cells grown on membrane filter units and cocultured with cancer cells in the inverse orientation (as described in Cells and cell culture) were washed with PBS consisting of (in mM) 137 NaCl, 2.7 KCL, 8.1 Na2HPO4, and 1.5 KH2PO4, pH 7.4, and fixed by the addition of formaldehyde solution (4% paraformaldehyde in PBS). After being washed with PBS, 0.2% Triton X-100, and 3% bovine serum albumin, cells were labeled with monoclonal rat anti-ZO-1 antibodies (MAB1520, Chemicon, Temecula, CA) diluted in PBS. Again, after the cells were washed, IgG Alexa Fluor 546-conjugated goat anti-rat antibodies (Molecular Probes, Eugene OR) were used for secondary labeling. Unbound antibodies were removed by intensive washing with PBS. Membrane filters with labeled cells were cut and mounted on coverslips using Mowiol with freshly added 50 mg/ml 1,4-diazabicyclo-[2.2.2]octane (Sigma, Taufkirchen, Germany). Images were taken using a Zeiss Axiovert 100 microscope and a digital camera system (Photometrics CoolSnap HQ, Universal Imaging MetaMorph, Buckinghamshire, UK).
Surface scanning of C7 monolayer with AFM.
Within 24 h of the coculturing of cancer and C7 cells, TEER decreased. However, even when TEER started to decline, the electrical resistance was still ≥1 kΩ · cm2. To visualize C7 monolayer violation by cancer cells, we used AFM (4). AFM is based on the deflection of a fine silicon-nitride tip scanning the surface of a sample. AFM reconstructs an image of a surface fromx, y, and z data to develop a three-dimensional topography of any surface at a nanometer level (31). The procedure for AFM imaging of different biological samples such as MDCK cells in the contact and tapping modes has been described elsewhere (16, 17, 22, 30). For surface scanning, C7 cells were seeded on the reverse surface of the filter membrane, and melanoma cells were added to the upper medium (as described in Cells and cell culture), separated from C7 cells by the filter membrane (no physical contact). As soon as TEER decreased, we incubated the C7 monolayer, after removal of the supernatant, in 3% isosmolar glutaraldehyde. After 10 min of fixation, cells were intensively rinsed in isosmolar HEPES-buffered Ringer solution. Filter membranes were cut and fixed on glass slides by using double-sided tape. The C7 monolayer was imaged in HEPES-buffered Ringer solution [(in mmol/l): 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES] using the BioScope (Digital Instruments, Santa Barbara, CA) in conjunction with an inverted optical microscope (Zeiss Axiovert). Silicon-nitride tips were used in the contact and tapping modes (resonance frequency between 7 and 9 kHz) with a spring constant of 8 mN/m (Park Scientific, Sunnyvale, CA). We applied a vertical force of ∼0.4–1.0 nN during the contact mode. AFM images were generated at line frequencies of 1–2 Hz, with 512 lines/image. Afterward, cell-imaging data were analyzed with the software accompanying the instrument (Digital Instruments).
Protease and protease inhibitors.
Collagenase A (Boehringer Mannheim, Mannheim, Germany) was dissolved in medium and used at a concentration of 11 μM. Protease inhibitors (Boehringer Mannheim; ilomastat from Chemicon) were dissolved in DMSO (pepstatin A, ilomastat) or cell culture medium (leupeptin, α2-macroglobulin). The inhibitors of aspartatic and cysteine proteases, pepstatin A and leupeptin, were combined in the culture medium (20 and 15 μM, respectively). α2-Macroglobulin was used at a concentration of 40 nM. Ilomastat (GM-6001) is a synthetic specific inhibitor of five MMPs [MMP-1 (K i = 0.4 nM), MMP-2 (K i = 0.5 nM), MMP-3 (K i = 27 nM), MMP-8 (K i = 0.1 nM) and MMP-9 (K i = 0.2 nM), whereK i is the inhibition constant] and was used at a concentration of 25 μM (11). All inhibitors were tested for cell toxicity by the trypan blue method. No significant effect on cell viability was found. Conditioned serum-free medium was obtained by cultivating confluent C7 or melanoma cells for 4 days with serum-free medium and glutamine. The medium was centrifuged to remove detached cells and then stored at −70°C. The conditioned medium was used for Western blot analysis and zymography or was supplemented with 10% FCS to evaluate the effect on TEER of confluent C7 cells.
Zymography and Western blot analysis.
Zymography was done according to a method described by Woessner (39). The standard method of Laemmli was performed (21). For Western blot analysis, TCA-precipitated supernatants of melanoma cells and C7 cells (320,000 cells/ml) were subjected to electrophoresis on 10% SDS-polyacrylamide gels under nonreducing conditions. Western blotting was performed according to standard protocols. The filters were incubated with antibodies specific for MMP-1, MMP-2, or MMP-9, respectively (all provided by Chemicon), and with goat anti-rabbit horseradish peroxidase-conjugated antibodies. The filters were developed with enhanced chemiluminescence substrate (Amersham Pharmacia Biotech, Freiburg, Germany).
Data are given as means ± SE. The statistical significance was tested with the U-test (Mann-Whitney-Wilcoxon), with α = 0.05 regarded as significant.
Apical invasion of the C7 monolayer by melanoma cells.
The cell-based invasion assay quantifies the violation of an intact monolayer during coculture with cancer cells. TEER was measured as a parameter that reflects the integrity of the epithelial C7 monolayer that was used. As shown in Fig. 1, different experimental setups are possible to study cancer cell invasion. Briefly, C7 cells were seeded on top of the filter membrane or were grown on the reverse side of the membrane. These setups enable coculturing of melanoma cells in physical contact with the apical surface of the C7 monolayer (Fig.1 A) or separated by the filter membrane close to the basolateral membrane (Fig. 1 C). TEER of the same C7 monolayer was monitored before and during cancer cell invasion until complete C7 monolayer destruction, indicated by a very low TEER indistinguishable from the electrical background (<50 Ω · cm2).
In the first experiments, C7 cells were grown on the filter membrane. C7 cells developed an electrical resistance of ∼5 kΩ · cm2 within 7 days. Subsequently, cancer cells or C7 cells (noninvasive control cells) were added to the upper medium and thereby were exposed to the apical surface of the C7 monolayer, allowing physical contact between both cell types (Fig. 1,setup A). Already 24 h later, a TEER decrease of ∼1 kΩ · cm2 was measured (Fig.2). Two days of coculture caused a significant breakdown of TEER to a resistance of 1.6 ± 0.1 kΩ · cm2 (n = 6). After 72 h, TEER was close to the electrical resistance of the filter membrane (Fig. 2). However, adding C7 cells to C7 monolayers (control experiment) did not decrease the TEER. Melanoma cells added into the lower medium grew on the bottom of the petri dish about 0.9 mm distant from the C7 cells. In this experimental configuration, cancer cells were not able to decrease TEER (Fig. 2). They were not in physical contact with the C7 monolayer and may not have developed local protease concentrations sufficient to affect TEER. The data show that melanoma cells are potent enough to invade and completely destroy a tight epithelial monolayer in <3 days. However, the experimental configuration used in this study cannot distinguish between the impact of physical contact and soluble proteases.
Basolateral invasion of the C7 monolayer by melanoma cells.
In a second experimental setup, C7 cells were grown on the reverse side of the filter (upside down). The advantage of seeding the C7 monolayer under the filter membrane is the possibility of culturing melanoma cells on the filter membrane next to the basolateral side of the tight C7 monolayer. These culture conditions did not affect C7 cell proliferation and the development of TEER. After 7 days of C7 cell culture, a TEER of 5 kΩ · cm2 was reached. Melanoma cells were added to the filter membrane and thus separated from the epithelial monolayer (filter width: 20 μm; pore size of the filter membrane: 0.4 μm). Surprisingly, melanoma-induced TEER breakdown was enhanced (42 ± 3% reduction in 24 h;n = 10) compared with melanoma cells cocultured on the apical surface of the C7 cell monolayer (18 ± 3% reduction in 24 h; n = 6) (Fig.3). TEER had already decreased from 7.9 ± 0.9 to 5.1 ± 0.4 kΩ · cm2within 24 h (control cells: 8.7 ± 0.6 kΩ · cm2). After 48 h, TEER was 0.18 ± 0.06 (SE) kΩ · cm2 (98 ± 0.7% reduction; control cells: 9.6 ± 0.5 kΩ · cm2;n = 10) and was close to 25 Ω · cm2 (background resistance) after 72 h. However, we could not measure any significant TEER decrease when adding noninvasive cells (C7 or F cells) to the top of the filter membrane or culturing any cell type in the lower medium (data not shown). These data show that cancer cells do not need physical contact to destroy C7 monolayers. Therefore, a mechanism based on secretion of proteases seems crucial for the destruction of cellular monolayers. Moreover, direct access to the basolateral surface of the epithelial monolayer induced an even faster TEER breakdown, indicating the involvement of basement membrane-affecting proteases.
Identification of secreted MMPs.
MMPs are known to play a crucial role in cancer cell spread and invasion. To identify putative secreted proteases of melanoma cells, we analyzed the conditioned medium for MMP activity by zymography. Conditioned media of melanoma cells revealed a strong gelatinolytic band of ∼67 kDa and a weak gelatinolytic band of 80 kDa (Fig.4 A). Gelatinolytic bands of 67 and 80 kDa suggest MMP-2 and MMP-9 activity, respectively. No gelatinolytic bands could be found in conditioned media of F cells and C7 cells.
Immunoblotting of conditioned melanoma media with specific antibodies confirmed the presence of MMP-1 (M r 57 kDa), MMP-2 (M r 72 kDa), and MMP-9 (M r 82 kDa) (Fig. 4B), whereM r is relative mass.
MMP inhibitors delay melanoma-induced TEER breakdown.
In the next experiments, we tested whether the identified proteases secreted by melanoma cells are in fact responsible for the observed effects on TEER. To address this issue, we tested different protease inhibitors in our cell-based invasion assay (Fig.5). C7 cells were seeded on the filter membrane, and cancer cells together with α2-macroglobulin (40 nM) were added (Fig. 5). Within the first 24 h of coculture, α2-macroglobulin completely inhibited TEER breakdown, indicating a lack of destructive activity of melanoma cells. Between 48 and 96 h after coculture, TEER was only reduced by 20% compared with control cells and was at least four times higher compared with the C7 monolayer cocultured with cancer cells lacking α2-macroglobulin (Fig. 5). As soon as α2-macroglobulin concentration decreased due to the change to a medium without α2-macroglobulin (96 h after coculture), we could measure a further decrease in TEER (40 ± 4% reduction, n = 6; data not shown) compared with control cells. The presence of the protease inhibitors pepstatin A and leupeptin in the upper or lower medium together with melanoma cells did not prevent TEER breakdown in any experimental setup (Fig. 5).
Inhibition of TEER breakdown by α2-macroglobulin but not by pepstatin A and leupeptin implies that MMPs are involved in destroying C7 monolayers. Therefore, we incubated melanoma cells with the synthetic MMP inhibitor ilomastat. Ilomastat has a high specificity for the activities of five MMPs (MMP-1, -2, -3, -8, and -9) (11), including those identified in our experiments. C7 cells were cultured on the reverse side of the filter membrane, and cancer cells were added to the upper medium. In all experiments, ilomastat (25 μM) significantly delayed the TEER breakdown induced by melanoma coculture (see Fig. 5). During the first 24 h of coculture, melanoma cell-induced TEER breakdown was completely inhibited. After 48 h, TEER was still elevated compared with ilomastat-free experiments. In summary, the decrease in TEER was inhibited significantly, although ilomastat inhibits only five of the known MMPs, and access of secreted proteases to the basement membrane of C7 monolayer was not hindered in the experimental configuration used (Fig. 1 C).
Collagenases mimic secreted proteases in a melanoma cell-free assay.
The experiments presented so far clearly state that melanoma cells can cause a TEER decrease in our experimental setup. We concluded that secretion of specific proteases by melanoma cells and degradation of the C7 basement membrane are responsible for this effect. First, physical contact with the epithelial monolayer is not necessary for TEER breakdown. Second, resistance decrease is faster and more complete when cancer cells are located close to the basolateral side and slower when cancer cells are positioned on top of the C7 layer (hindering free access of proteases to basement membrane most likely by tight intercellular connections). Third, protease inhibitors or specific inhibitors for some MMPs in the medium delay the effect of melanoma cells on TEER.
To verify our conclusions, we used collagenases in all possible experimental setups to imitate MMP activity under defined conditions. Collagenases (11 μM) were added to the upper or lower medium of a tight C7 monolayer, growing either on top of the filter membrane or upside down on the reverse side. The activity of collagenases led to a significant decrease in TEER in all four setups within 24 h (Fig.6). However, perfectly in line with the data obtained during coculturing of melanoma cells with C7 cells, application of collagenases close to the basolateral membrane exhibited an enhanced TEER reduction compared with the addition of collagenases to the medium in contact with the apical membrane of C7 cells (Fig. 6). Moreover, the kinetics of TEER breakdown were strikingly similar, independently of the orientation of C7 cells on the filter membrane (Fig. 1, A and D, andB and C, respectively). Therefore, it is unlikely that the upside-down orientation of the epithelial monolayer as such biased the results. The results show that basement membrane-degrading proteases are more effective when applied to the basolateral side of the monolayer. They also confirm the view that melanoma cells need to constitute a high local concentration of proteases for efficient basement membrane violation, because melanoma cells do not influence TEER when growing on the bottom of the incubation chamber (Fig. 1,B and D). Consistently, replacement of the upper medium of C7 cells with conditioned medium of melanoma or C7 cells (Fig. 1 A) had no significant effect on the TEER of the C7 layer (data not shown). In conclusion, on the basis of our data we suggest that tight junctions represent an effective barrier that hinders tumor cell invasion by inhibiting diffusion of secreted proteases.
Surface topography of confluent C7 monolayers after coculture.
Our data show that after 24 h of coculture with cancer cells, TEER of the C7 monolayer is >1 kΩ · cm2. As already mentioned, a monolayer with a resistance ≥1 kΩ · cm2 still indicates a virtually intact epithelium. Therefore, conventional microscopy is not sufficient to detect morphological alterations that occur with this initial TEER decrease. To detect C7 cell-cell contact alterations on coculture with cancer cells, we performed immunofluorescence stainings of C7 monolayers using anti-ZO-1 antibodies. The ZO-1 protein, as one component of tight junctional formation, is involved in controlling the paracellular barrier and permeability (3, 15, 23). C7 cells were seeded on the reverse side of the filter, and therefore the cancer cells lacked physical contact with them during coculture. Compared with control cells, 24 and 48 h after coculture TEER decreased (5,200 and 2,400 Ω · cm2, respectively) by ∼40 and ∼70%, respectively. However, we could not detect a significant change in ZO-1 staining (Fig.7), indicating the high sensitivity of the TEER measurements compared with the fluorescence measurements. To further investigate alterations in C7 monolayer morphology during coculture with cancer cells, we used high-resolution AFM. AFM enables the production of three-dimensional data of the apical surface and of cell-cell contacts with nanometer resolution (31). Figure8 shows the apical surface topography of a C7 monolayer during control conditions and after coculture with cancer cells. C7 cells were seeded on the reverse side of the filter, and therefore cancer cells lacked physical contact during coculture. Twenty-four hours after coculture, TEER decreased (4,600 Ω · cm2; see also Fig. 3) by 40% compared with control cells. No intercellular gaps of the C7 monolayer were detectable, consistent with the ZO-1 staining experiments (Fig. 7). However, some other discrete morphological alterations could be seen. C7 cell size over the nucleus increased, whereas cell-cell contacts became less prominent. The three-dimensional AFM images reveal that the heights of cell junctions (Fig. 8, height profile; for statistical analysis, see the table in the legend for Fig.9) were significantly reduced compared with those in control cells. Therefore, the decrease in the height of cell junctions could explain electrical leakage of the monolayer due to the increased MMP activity.
In the present study, we used a novel electrophysiological cell-based invasion assay to study the destruction of an intact cell monolayer during cancer cell invasion This cell-based invasion assay uses a tight MDCK epithelial monolayer cultured on a filter membrane (42). The MDCK monolayer develops a high transepithelial resistance, providing a continuous assay system for cancer invasion with very high sensitivity that demonstrates changes before visible morphometric changes. As soon as the monolayer is disturbed by the cancer cells, transepithelial resistance decreases. This method is a novel, highly sensitive approach that can be applicable to a wide variety of either cells or can be used alternatively as a toxicological assay system. One of the key aspects of this system that has broad-reaching implications is the ease of measurement and the improved sensitivity over conventional assay systems, such as the Boyden chamber. This increased sensitivity is illustrated by the ability of the system to detect resistance change before morphometric changes are visible by light or fluorescence microscopy. Cells of the C7 subclone appear especially useful for our new electrophysiological approach because they develop a high TEER compared with other cell types (6, 12, 40). The invasion assay quantitatively measures the destruction of the C7 monolayer induced by cancer cells (42). Seeding cancer cells on either the apical side or adjacent to the basolateral side of epithelial cells mimics cancer cell extravasation or tissue invasion by cancer cells, respectively. Moreover, this technique can also be used to study exocytosis of proteases by cancer cells. Exocytosis in neuronal and epithelial cells has been studied intensively in the last several years (18, 29,31). Although it is known that proteases are important for cancer cell invasion and angiogenesis (10), little is known about the mechanism of protease release in cancer cells.
Our data show that seeding melanoma cells adjacent to the basolateral membrane of C7 cells led to enhanced TEER breakdown compared with seeding melanoma cells directly on the apical surface of C7 cells, even though physical contact was prevented by the filter membrane under the former conditions. Secreted proteases of tumor cells concentrated at the apical surface of epithelial or microvascular endothelial cells must pass the tight junctional barrier before gaining access to the basement membrane. Therefore, TEER breakdown depends on the polarized secretion of basement membrane-affecting proteases. Physical contact between cancer and epithelial cells alone may not be dynamic enough for invasion. Indeed, melanoma cells cocultured on a C7 monolayer failed to induce a TEER breakdown during incubation with α2-macroglobulin.
In addition, the decrease in TEER was significantly delayed in the presence of the specific MMP inhibitor ilomastat. During the first 24 h of melanoma and C7 cell coculture, ilomastat prevented the melanoma-induced TEER breakdown. In contrast, inhibitors of aspartate, serine, and cysteine proteases did not affect TEER breakdown. Gelatin zymography of conditioned medium showed bands at 67 and 80 kDa, indicating the presence of the active forms of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). No gelatinolytic bands could be found in conditioned media of MDCK-F cells and C7 cells. Gelatinases (MMP-2 and -9) and MMP-1 are the dominating proteases during metastasis of melanomas and can be blocked by ilomastat (5, 11, 27). Western blot analysis confirmed the presence of MMP-1, MMP-2, and MMP-9 in melanoma supernatant medium. Taken together, the results indicate that cancer cell invasiveness depends on secretion of specific proteases, mainly MMPs. Our experiments, of course, cannot exclude other proteases involved in cell monolayer violation. To confirm our conclusions that polarized protease secretion and subsequent destruction of the epithelial basement membrane is critical for cancer invasion, we used collagenases to mimick MMP activity in a cancer cell-free setup. We measured enhanced TEER breakdown when collagenases were applied next to the basolateral membrane of C7 monolayers. Application of collagenases next to the apical membrane of C7 monolayers induced a somewhat slower TEER decrease similar to that with cancer cell coculture on the apical surface of C7 cells. However, after 48 h of melanoma-C7 coculture (physical contact), TEER breakdown was apparently more complete compared with TEER of the corresponding collagenase experiments (TEER/TEER control = 0.22 compared with 0.47). The comparison between these experiments envisions a “two-step” model during cancer invasion. During the first step, cancer cells secrete proteases, mainly MMPs, to attack the basement membrane of the intact monolayer. The “additional step” in TEER breakdown in the melanoma-C7 coculture experiments reflects the impact of physical interaction of both cell types. In our assay, this protease activity was shown to be necessary and a prerequisite for subsequent cancer cell invasion. This early step in cancer cell invasion exhibits only discrete morphological alterations of the C7 monolayer, which are not detectable by conventional light and fluorescence microscopy and can only be visualized by AFM. Although TEER was reduced by ∼40 or ∼70% on coculture, there were no discernible changes in the fluorescence staining of the tight junctional protein ZO-1. From the functional point of view, the electrical resistance breakdown assay is sensitive enough to detect this early step in cancer cell invasion. High-resolution AFM was used in the study to detect discrete nanometer changes in cell-cell contacts and to confirm the sensitivity of the electrical measurements. However, AFM does not remain a practical alternative to the screening of large cell populations due to the complexity of this technique. Moreover, AFM is not able to image structures within or between cells but provides information about surface topography with nanometer resolution. AFM revealed discrete structural changes between cells and an increase in cell size over the nucleus. Data indicate that these morphological alterations mainly represent a change in cell-cell contacts, indicating a change in tight junctional formation. However, we found no significant change in the tight junctional protein ZO-1 by using immunofluorescence microscopy. Therefore, on the basis of our AFM data, the individual tight junctions either shrink or slightly separate but remain virtually intact. A possible scenario could be minimal cell retraction followed by a height decrease of the cell periphery and a height increase of the cell body. In a second step (later), cancer cells could migrate into the monolayers and finally replace the dissolved monolayer visualized by conventional light microscopy (data not shown).
Our data demonstrate that secretion of MMPs is an essential and early step in cancer cell invasion. MMP secretion must occur close to the intact epithelium to attain a high local protease concentration. Physical contact between cancer cells and intact tissue is not sufficient for cancer cell invasion. Therefore, inactivation of proteases or inhibition of their secretion is a potential new target for retardation or prevention of cancer cell invasion.
We thank Profs. J. Geibel and E. Boulpaep for insightful comments regarding this manuscript and B. Schneider for preparing the schematic models.
This study was supported by “Interdisziplinäres Zentrum für Klinische Forschung,” the University of Münster, and the Benningsen-Foerder-Preis (S. W. Schneider).
Address for reprint requests and other correspondence: S. W. Schneider, Institute of Physiology, Univ. of Münster, Robert-Koch-Str. 27a, D-48149 Münster, Germany (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
February 26, 2002;10.1152/ajprenal.00327.2001
- Copyright © 2002 the American Physiological Society