F6 was most reliable at killing GSCs, while MG18L was similar to G47, except in GBM8 (Fig 3A). determined using Chou-Talalay analysis. efficacy studies were performed using a clinically relevant mouse model of GSC-derived glioblastoma. Results MG18L was severely neuroattenuated in mice, replicated well in GSCs, and had anti-glioblastoma activity against a number of cancer cell lines, including glioma U87 and T98G, and against U87 subcutaneous tumors; (2) synergized with PI3K/Akt inhibitors; and (3) was safe after systemic delivery in the periphery (18). BTZ043 (BTZ038, BTZ044) Racemate In order to extend these BTZ043 (BTZ038, BTZ044) Racemate findings to human GSCs and intracerebral glioblastoma tumor models for possible clinical translation, we constructed a new multi-mutated oHSV, MG18L (Us3-deleted and UL39 (ICP6)-negative), which is safe after intracerebral inoculation, replicates well in GSCs, and synergizes with PI3K/Akt pathway inhibitors in killing GSCs and through enhanced apoptosis. Materials and Methods Cell lines and reagents U87 and T98G human glioma and Vero (African green monkey kidney) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and used at low passage number. Human astrocytes were obtained from ScienCell (San Diego, CA). Cells were maintained in Dulbeccos modified Eagles medium supplemented with 10% FCS (DMEM-FCS) at 37C and 5% CO2. Human GSCs were isolated as previously described and cultured in EF20 medium composed of Neurobasal medium (Invitrogen, Carlsbad, CA) supplemented with 3mM L-Glutamine (Mediatech, Manassas, VA), 1 B27 supplement (Invitrogen), 0.5 N2 supplement (Invitrogen), 2 g/ml heparin (Sigma), 20 ng/ml human EGF (R&D systems, Minneapolis, MN), 20 ng/ml human FGF-2 (Peprotech, Rocky Hill, NJ) and 0.5 penicillin G/streptomycin sulfate/amphotericinB complex (Mediatech) (15). The stem-cell features of GBM4, GBM8 and BT74 have been previously described (15). Spheres were dissociated using NeuroCult Chemical Dissociation kit (StemCell Technologies, Vancouver, BC, Canada). Passaged cells were confirmed to be mycoplasma-free. LY294002 (LC Laboratories, Woburn, MA), triciribine (Akt inhibitor V) (Santa Cruz Biotechnology, Santa Cruz, CA), GDC-0941 (Chemdea, Ridgewood, NJ), BEZ235 (Chemdea), and Z-VAD-FMK (Tocris bioscience, Ellisville, MI) were dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO). Viruses All viruses BTZ043 (BTZ038, BTZ044) Racemate were constructed on a HSV-1 strain F background. G207 (34.5, Rabbit Polyclonal to Rho/Rac Guanine Nucleotide Exchange Factor 2 (phospho-Ser885) ICP6?, LacZ), G47 (34.5, ICP6?, ICP47/Us11pro, LacZ), and F6 (ICP6?, LacZ) have been previously described (14, 15, 19). R7041 (Us3-deleted) was provided by Dr. B. Roizman (University of Chicago) (20). Viruses were grown, purified, and titered on Vero cells (19). Construction of MG18L Construction and characterization of MG18L was as described (19). Briefly, the 5.3-kb fragment of pKX2-G3 (from S.K. Weller, University of Connecticut Health Center), containing the E.coli sequence inserted in-frame in UL39, was co-transfected with R7041 viral DNA into Vero cells using Lipofectamine (Invitrogen). Recombinant viruses, isolated by limiting dilution and identified as plaques staining blue after X-gal histochemistry (Fig S2A), were plaque purified three times in Vero cells. The genomic structure of MG18L was confirmed by restriction endonuclease digestion and Southern blot analysis (Fig S1). Viral replication assay Cell were seeded into 24-well plates (2104 cells/well) in 0.5 ml of media and infected at a MOI of 1 1.5 in triplicate. LY294002 was added 6 hours post-infection and titers determined by plaque assay on Vero cells. Cell susceptibility assays and Chou-Talalay analysis Cells were seeded into 96-well plates (5000 cells/well), and 3.5 days after infection or 3 days after drug treatment MTS assays (Promega, Madison, WI) were performed according to manufacturers instructions. For Chou-Talalay analysis (21), experiments were performed as described (22). Dose-response curves and 50% effective dose values (ED50) were obtained, and fixed ratios of drug and virus and mutually exclusive equations used to determine combination indices (CIs). Briefly, combined dose-response curves were fitted to Chou-Talalay lines (21), which are derived from the law of mass action.