Silencing Proteases in Glioma Figure 5. Wound healing, spheroid migration and Matrigel invasion assays. (A) Reduction in wound healing indicative of reduced migration potential was noticed after treatments with MMP-9 (M-sh), uPAR (U-sh), cathepsin B (C-sh), MMP-9/uPAR (MU-sh) and MMP-9/cathepsin B (MC-sh) plasmid shRNAs in both 4910 and 5310 cells. Untreated and SV-sh transfected cells served as controls. (B) Migration of the cells from 4910 and 5310 spheroids transfected with M-sh, U-sh, C-sh, MU-sh and MC-sh was prominently reduced. (C, D) Quantification of wound healing and spheroid migration assays. Percent wound repair was calculated from the mean of the average width of the wound obtained from 3 independent experiments. Similarly, percent migration was calculated from the mean of the average migration obtained from 3 independent experiments. Error bars indicate SEM. *p,0.05 vs. control. (E) Matrigel invasion assay of 4910 and 5310 cells transfected with SV-sh, MU-sh and MC-sh. MU-sh and MC-sh treatments prominently reduced the invasive potential of both 4910 and 5310 cells through Matrigel. (F) Invasion was quantified by counting the average number of invaded cells in five different fields with each treatment. Percent invasion was calculated from the mean of the average number of invaded cells obtained from 3 independent experiments. Error bars indicate SEM. *p,0.05 vs. control. doi:10.1371/journal.pone.0011583.g005 pivotal role in regulating cellular functions during pathological remodeling processes, such as tumor invasion and metastasis. Tumor ECM and basement membranes of tumor blood vessels have shown tenacin, vitronectin, collagens, fibronectin, laminin, hyaluronan, chondroitin sulfate and heparan sulfate. Collagen, laminin, tenacin, fibronectin and vitronectin serve as permissive substrates for glioma cell migration. Cell adhesion plays a major role in regulating cell movement. Adhesion of glioma xenograft cells to various components of ECM such as collagen, vitronectin, fibronectin and laminin was significantly inhibited by MMP-9, uPAR and cathepsin B shRNA plasmid constructs. Simultaneous targeting of multiple molecules has been more synergistic than additive [30]. As expected, inhibition of adhesion of these cells to ECM components is much more significant with the simultaneous downregulation of MMP-9 and uPAR or MMP-9 and cathepsin B. Integrins expressed in tumor cells contribute to tumor progression and metastasis by increasing tumor cell migration, invasion, proliferation and survival [8]. Integrin adhesion to ECM provides the traction required for tumor cell invasion. Interactions and signaling mechanisms among uPAR, MMP-9 and cathepsin B associated with integrin involvement were well studied and reported [31–34]. Integrins are essential uPAR signaling co- receptors and the interactions between uPAR/b1 and uPAR/b3 have an important role in signaling for cell migration and invasion [34]. It was reported that the cleavage of aV integrin subunit precursor by MMP-14 enhanced cancer-cell migration [35]. Further, cooperation between MMP-9 and integrins is known to activate aVb3, which strongly enhanced tumor migration [36]. Cathepsin B is known to be associated with uPAR and its integrin- mediated signaling ability [37]. Moreover, immunoprecipitation of the 4910 and 5310 glioma xenograft cell lysates with b1 integrin followed by immunoblot analysis with cathepsin B indicated a possibility for the direct or indirect interaction between these two molecules (data not shown). Inhibition of adhesion by MMP-9, uPAR and cathepsin B silencing in the present study resulted in a significant reduction in the expressions of several alpha and beta integrins both at mRNA and protein levels. aV, a6, a9, b1 and b3 integrin mRNA and protein expressions were prominently downregulated in both the xenograft cell lines with all the treatments. Upregulation of a2b1, a3b1, a5b1, aVb3 and a6b1 integrins on GBM have already been reported [28,29]. In 2008, deHart et al., identified that a9b1 integrin increase cell migration by modulation of polyamine metabolism and activation of potassium channels [38]. Unlike other integrin heterodimers, a9b1 was able to both increase cell migration and inhibit cell spreading [39,40]. It was recently found that a9b1 integrin played a significant role in the progression of glioblastoma [27]. Hence, we expected the involvement of aVb3, a6b1 and a9b1 heterodimer combinations on the effects mediated by our shRNA treatments in the xenografts studied in our present investigation. FACS analysis confirmed the expression of aVb3, a6b1 and a9b1 integrins on xenograft cells. Bicistronic constructs significantly reduced a9b1 levels in vitro in 4910 cells. These constructs also reduced the proliferation of xenograft cells in vitro. In vivo studies in nude mice injected with 5310 cells revealed a prominent reductions in the expression of integrin heterodimers (aVb3, a6b1 and a9b1) when treated with MMP-9/uPAR or MMP-9/ cathepsin B shRNAs. Furthermore, drastic reductions in tumor growth and extent of blood vessels in tumor mass (neoangiogen- esis) were noticed in vivo after MU-sh and MC-sh treatments in xenograft cells injected nude mice. These results are in agreement with our earlier reports, wherein we have noticed a significant decrease in tumor growth with MU-sh and MC-sh treatments in SNB19 cells injected mouse brains [33,41]. a9b1 has distinguished its functionality from other integrins (aVb3 and a3b1) by facilitating accelerated cell migration [42]. Hence, in the present study, we performed spheroid migration assay and wound healing assay in order to address the effect of silencing proteases on migratory potential of glioma xenografts. All the treatments with single (M-sh, U-sh and C-sh) and bicistronic constructs (MU-sh and MC-sh) significantly reduced the migration of glioma xenografts in all the above-mentioned assays. Our results clearly revealed that the bicistronic constructs were more effective than the single constructs in reducing the migrating potential of glioma xenografts. Further, in concurrence with adhesion assay results, we have noticed a marked reduction in migration in all the assays with MC-sh as compared to MU-sh. Integrin a9b1 is classified within a two member sub-family of integrins highlighted in part by its specialized role in cell migration [43]. Unlike other integrins, a9b1 has been proposed to utilize inducible nitric oxide synthase (iNOS)-nitric oxide (NO) and spermidine/spermine acetyl transferase (SSAT)-inward rectifier potassium channel (Kir) pathways along with common integrin signaling proteins such as Src and FAK to transduce cell migration [43]. Significant inhibition of uPAR, MMP-9 and cathepsin B mediated migration in 5310 xenograft cells by a9b1 integrin blockade, clearly demonstrated its possible role. Gliomas are highly invasive and the invasive nature of gliomas has an important role in the ineffectiveness of current treatment modalities, as the remaining cancer cells inevitably infiltrate the surrounding normal brain tissue and lead to tumor recurrence [37]. Hence, we felt it is crucial to evaluate the effect of shRNA treatments on the invasive potential of xenografts and as expected, the MU-sh and MC-sh treatments significantly inhibited the invasion of both 4910 and 5310 cells through the Matrigel. In this study, downregulation of uPAR and the proteases, MMP-9 and cathepsin B showed a significant reduction in the adhesion, migration and invasive potential of xenografts and also affected the levels of several alpha and beta integrins. Like other integrins a9b1 ligation can activate signaling through Src and FAK mediated tyrosine phosphorylation of multiple proteins including p130Cas and paxillin [40,44]. Hence, we suspected that our treatments played a significant role in regulating the molecules associated with integrin-mediated signaling that leads to migra- tion. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11583