Silencing Proteases in Glioma Figure 7. Effect of treatment with bicistronic constructs on the brain tumors in nude mice pre-injected with xenograft cells. (A) shRNA mediated regression of pre-established tumor growth. Hematoxylin and eosin staining performed on the brain sections obtained from various groups of animals revealed a prominent tumor reduction after MMP-9/uPAR (MU-sh) or MMP-9/cathepsin B (MC-sh) treatments. Each group consisted of 6 animals. Yellow curved line in 4910 and 5310 control brain sections indicate the tumor area. (B) Normal nude mice brain section stained with hematoxylin and eosin. (C) Immunohistochemical comparison of control, MU-sh and MC-sh-treated nude mice which are pre-injected with 5310 cells (and sacrificed 2–3 weeks prior to the end of the treatment), to analyze the expression of aVb3, a6b1 and a9b1 integrin heterodimers. Brown staining indicative of aVb3, a6b1 or a9b1 integrin expression was reduced in MU-sh and MC-sh treated sections compared to controls. n = 6. Scale bar = 50 mm. (D) Untreated 4910 and 5310 in vivo tumors were prominently stained for platelet endothelial cell adhesion molecule (PECAM-1) whereas the intensity of brown staining was reduced in 5310 in vivo tumors after MU-sh and MC-sh treatments. doi:10.1371/journal.pone.0011583.g007 the first time, we have shown the involvement of a9b1 integrin in the effects mediated by MMP-9, uPAR and cathepsin B silencing in glioma xenografts. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach for inhibiting integrins while downregulating MMP-9, uPAR and cathepsin B in treating glioma. Materials and Methods Ethics Statement The Institutional Animal Care and Use Committee of the University Of Illinois College Of Medicine at Peoria, Peoria, IL, USA approved all surgical interventions and post-operative animal care. The consent was written and approved. The approved protocol number is 851, dated November 20, 2009. Cell culture and transfection conditions The xenograft cell lines (4910 and 5310) were kindly provided by Dr David James at University of California, San Francisco. These xenografts were generated and maintained in mice and are highly invasive in mouse brain [49]. 4910 and 5310 xenografts were maintained in RPMI 1640, supplemented with 10% fetal bovine serum, 50 mg/ml streptomycin and 50 U/ml penicillin in a humidified atmosphere containing 5% CO2 at 37uC. Xenografts were transfected with SV-sh (scrambled vector), M-sh (MMP-9), U-sh (uPAR), C-sh (cathepsin B), MU-sh (MMP-9 and uPAR) or MC-sh (MMP-9 and cathepsin B) shRNA expressing plasmids using FugeneH HD reagent (Roche Diagnostics, Indianapolis, IN) as per the manufacturer’s instructions. After transfection, cells were incubated in serum-containing medium for a minimum of 60 h. Construction of shRNA and gene expressing plasmids Plasmid shRNAs for MMP9, uPAR, cathespin B, MMP-9/ uPAR and MMP-9/CathespinB designed in our laboratory [31,41,50] were used to transfect the xenograft cells. Briefly, a pCDNA-3 plasmid with a human cytomegalovirus (CMV) PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11583