Learn More. These cells metastasized into chick embryonic liver more than control cells. Takaoka, Japan. Nagano, Japan. Scrambled oligo was used as a negative control. The assay was done as originally described by Endo et al. Ten eggs were used for the injection of each type of cell. Gels were processed and monitored by an infrared imaging system with low top and high sensitivity bottom.
Cells in collagen culture were photographed under a phase contrast microscope. The expression of each protein was analyzed by Western blotting. Toth et al. This work was supported by the grant from the Japan Society for the Promotion of Science Kakenhi National Center for Biotechnology Information , U.
Journal List Cancer Sci v. Cancer Sci. Published online Mar Author information Article notes Copyright and License information Disclaimer. Hiroshi Sato, Email: pj. Corresponding author. This article has been cited by other articles in PMC. Chick embryo assay The assay was done as originally described by Endo et al.
Open in a separate window. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Disclosure Statement The authors have no conflict of interest. Acknowledgments This work was supported by the grant from the Japan Society for the Promotion of Science Kakenhi References 1. The role of gelatinases in colorectal cancer progression and metastasis. Biochim Biophys Acta ; : 69— Biochim Biophys Acta ; : 37— Nature ; : 61—5.
Here, we demonstrate that endogenous up-regulation of TIMP-2 in glioblastoma cells can lead to increased MMP-2 activation and subsequent invasion. For media conditioning experiments, cells were seeded in the above medium and allowed to adhere overnight, at which point the medium was changed to serum-free MEM. Six- or well Transwell polycarbonate membrane inserts with 8. Filters were then washed, and cells on the upper surface were removed with cotton swabs.
Eight random fields were counted to determine the number of cells invaded. To obtain URes cells, noninvading cells on the upper surfaces of the filters were harvested in a similar fashion and cultured. As described previously, 18 confluent monolayers were scratched with a plastic pipet tip to create a uniform, cell-free wound area that was then inspected at regular time intervals.
At each time point eight photographs of each wound were taken and the distance between the opposing edges was measured at two points on each photograph. The distance migrated in micrometers was calculated as the difference of the scratch width at the beginning of the assay and that at each indicated time point. Photographs of the spheroids were taken, and after overnight incubation the radial migration distance of each sample was examined and compared.
Images were acquired with an AxioCam digital camera Zeiss, Germany mounted on an Axioskop 2 plus microscope Zeiss and analyzed using AxioVision software.
Gelatinolytic activity of conditioned media was detected by gelatin zymography. Gelatinolytic activity was detected as transparent bands on a blue background. TIMP activity was detected as positive staining bands over a clear background. Protein concentrations of the resulting supernatants were determined using a BCA protein assay Pierce. All densitometric analysis was carried out using AlphaEase software version 5. Significance of difference was determined using the independent t -test.
Individual colonies of cells resistant to 0. The TIMP-2 expression level of each clone was determined by reverse zymography of conditioned media. We chose to use collagen type I as a matrix barrier because it has been shown to be ectopically upregulated in glioblastoma tissue. The selection process was performed under serum-free conditions in the absence of chemoattractants so that only differences in cell behavior inherent to the tumor cells would be isolated, and not those due to external cues.
Because cells that had invaded through the Transwell membrane adhered to the underside of the filters and did not progress to attach to the wells of the lower chamber compartments, the invasive cells were harvested by brief and gentle trypsinization from the bottoms of the filters and cultured in new dishes.
The selected cells were subsequently expanded and designated as UC1. Residual cells that had not invaded and remained on the upper surface of the filters were harvested for comparison and designated as URes.
The cellular and molecular phenotypes of the selected cells, described below, were stable at least up to 15 passages in our experiments.
Differences in invasive and motile behaviors associated with selected UC1 cells compared to parental cells were characterized using three independent methods. Interestingly, a single round of selection at this level of stringency sufficiently isolated a stable subpopulation of glioblastoma cells with enhanced invasion. Transwell chambers coated with collagen type I clearly showed that UC1 cells exhibited a marked increase in invasion over parental cells Figure 1.
The invasion rate of residual URes cells was nearly identical to that of parental cells. To confirm this phenotype independently, differences in motility were assessed using a wound assay in which cells are grown to confluence in monolayers, and their ability to migrate into and across a linear cell-free area of the monolayer is followed over time.
No differences in cell proliferation between U87MG and UC1 cells were detected using BrdU incorporation and MTS assays data not shown , ruling out the possibility that the selected cells appeared more motile due to increased cell division. As an additional independent confirmation, differences in motility were also analyzed using a spheroid radial migration assay. Consistent with the Transwell and wound-closing assay results, UC1 cells migrated distances two-fold greater than parental cells away from the initial spheroids in a radial pattern Figure 2b.
The migration of cells from parental spheroids was more compact, the resultant spheroids being slightly smaller in diameter but their shape clearly intact. Assays performed on collagen substrates slightly enhanced the radial motility in both cell lines, but selected cells still demonstrated markedly more diffuse migration.
These results demonstrate that selected UC1 cells display enhanced invasiveness and motility in multiple independent assays. Cells that had penetrated onto the lower surface of the filter were fixed in paraformaldehyde and stained with crystal violet. Eight random fields from each Transwell were photographed and stained cells were visually counted. Error bars represent s. Significance of difference between U87MG and UC1 invasion was estimated using the independent t -test.
UC1 cells are more motile than parental U87MG cells in wound healing and spheroid radial migration assays. Error bars in the chart represent s. Representative photographs of the spheroids before and after migration are shown. To determine whether the differences in invasion and motility correlated with cytoskeletal alterations associated with tumor cell invasion, we performed immunofluorescence staining of the actin cytoskeleton using FITC-phalloidin.
UC1 cells exhibited extensive lamellipodia Figure 3 , arrows in addition to the presence of well-defined stress fibers arrowheads. On the other hand, parental cells presented fewer stress fibers and lacked the lamellipodia indicative of cell motility found in the selected cells. Plated on collagen-coated surfaces, both cell types demonstrated a slight increase in actin-based membrane specializations, but again the differences between U87MG and UC1 cells remained.
These results indicate that UC1 cells feature cytoskeletal rearrangements that correlate with their enhanced invasiveness. Staining of the actin cytoskeleton with FITC-phalloidin revealed extensive lamellipodia arrows as well as formation of organized actin stress fibers arrowhead in UC1 cells. This increase in MMP-2 activation in UC1 cells was observed whether the cells were grown in media with or without serum supplementation Figure 4a , indicating that the effect was not a result of differential responsiveness to serum-borne factors.
Overall pro-MMP-2 expression, as detected by zymography and immunoblot, did not differ between parental and selected cells, indicating a difference specifically in the amount of MMP-2 activation in UC1 cells. In contrast, MMP-9 expression and activation were identical in both parental and selected lines data not shown. Activation level is presented as the mean densitometric ratio compared to U87MG over 15 independent samples; error bars represent s.
The amounts of MT1-MMP expressed on the cell surface corresponded with the immunoblot data, with identical staining patterns and intensities for both cell lines Figure 5b. We next studied expression of soluble TIMP-2 in the conditioned media of the cells by immunoblotting and reverse zymography. Reverse zymography of conditioned media revealed that the level of soluble TIMP-2 protein from UC1 cells was increased two-fold over that of parental and URes cells Figure 6a , as quantitated by densitometry.
Likewise, immunoblots on 10x concentrated conditioned media using a monoclonal antibody against TIMP-2 indicated two-fold higher levels of secreted TIMP Gelatin zymography of the same conditioned media samples confirmed that MMP-2 activation in UC1 cells was increased in the presence of higher levels of secreted TIMP Densitometric ratios of band intensities are indicated.
Exogenous addition of TIMP-2 to UC1 cells likewise stimulated proMMP-2 activation in a dose-dependent manner, except that slightly higher levels of the activated intermediate and fully processed forms were achieved with lower TIMP-2 doses. As expected, reverse zymography of serum-free conditioned media from the control clone showed the same levels of TIMP-2 as untransfected U87MG cells Figure 9. The same results were observed when immunoblots detecting TIMP-2 were performed.
Conditioned media from cells were analyzed for TIMP-2 expression by reverse zymography and immunoblot, while MMP-2 activation was observed with gelatin zymography. One of the critical challenges in glioblastoma research is the identification of mechanisms used by glioblastoma cells to invade the brain.
With a variety of new pathway inhibitors and MMP inhibitors being recently introduced into clinical trials, the importance of discovering mechanisms of invasion that can be pharmacologically targeted is apparent.
In this study, we have successfully used an in vitro selection method to generate an interesting and useful experimental model for identifying candidate mechanisms involved in glioblastoma invasion without having to genetically modify cells. Relative to parental U87MG cells and U87MG-Res cells, UC1 cells derived from this selection process demonstrated a three-fold increase in invasion, displayed cytoskeletal features characteristic of cell motility, and exhibited biochemical alterations that were stable over at least 15 passages.
Although previous studies have indicated that TIMP-2 can inhibit MMP-2 activation and that it is also required in this process, this is, to the best of our knowledge, the first demonstration that endogenous physiological upregulation of TIMP-2 expression can promote MMP-2 activation and subsequent glioblastoma cell invasion. Furthermore, because higher expression of TIMP-2 and MMP-2 in glioblastoma tissue has been previously documented, 23 these data have direct clinical relevance.
Elevated MMP-2 expression and activation are strongly correlated with astrocytic tumor grade and malignancy. Although TIMP-2 was originally characterized as a suppressor of tumor invasion due to its ability to bind and inhibit MMP-2, 11 and overexpression of TIMP-2 was previously shown to reduce invasion and metastasis in a number of tumor cell models, 25 , 26 , 27 it is well known that TIMP-2 is also necessary for the efficient activation of proMMP Points A, B, and C are representative positions along this plot for the scenarios depicted in Figures 10 a — c.
Because TIMP-2 has been shown to promote cell proliferation, 28 , 29 an increase in cell growth resulting from TIMP-2 upregulation could potentially be misinterpreted as increased migratory and invasive phenotypes in our assays. Under our culture conditions, however, this was not the case as UC1 cells exhibited no growth advantage over U87MG cells as determined by cell growth and DNA synthesis assays. Although MMPs and their inhibitors play key roles in the dynamic processes of glioblastoma cell invasion and motility, there are likely to be other mechanisms promoting these phenotypes including those not involving proteases.
Activation of the small GTPase Rho induces stress fiber formation while Rac contributes to the formation of lamellipodia. It is likely that other pathways or intrinsic adaptations exist in UC1 cells to trigger an invasive phenotype.
In glioblastoma, it is probably a combination of various mechanisms in addition to interactions with the surrounding stromal environment that contribute to this pattern of growth and dissemination within the brain.
The development of such therapies would require a careful understanding of the TIMP-MMP balance in each individual patient to better predict their suitability and efficacy. This is made more complicated by the fact that tumor cells may also utilize MMPs and TIMPs produced by stromal cells in the local tumor microenvironment. In conclusion, we have selected a subpopulation of glioblastoma cells that exhibit greater invasive and motile characteristics as well as increased MMP-2 activation mediated by an increase in TIMP-2 expression.
Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu Rev Cell Biol ; 9 — Stetler-Stevenson WG. Dynamics of matrix turnover during pathologic remodeling of the extracellular matrix. Am J Pathol ; — DeClerck YA. Interactions between tumour cells and stromal cells and proteolytic modification of the extracellular matrix by metalloproteinases in cancer. Eur J Cancer ; 36 — Nagase H. Activation mechanisms of matrix metalloproteinases.
Biol Chem ; — Br J Cancer ; 79 — Expression of matrix metalloproteinases and their inhibitors in human brain tumors. Ann N Y Acad Sci ; — Roles of membrane type 1 matrix metalloproteinase and tissue inhibitor of metalloproteinases 2 in invasion and dissemination of human malignant glioma. J Neurosurg ; 94 — Matrix metalloproteinase-2 activation modulates glioma cell migration.
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