Cell lines and virus
Human glioblastoma SNB19, U251, and U373 (kindly provided by Dr. H. Okada, University of Pittsburgh), neuroblastoma SK-N-AS (ATCC, Manassas, VA), and osteosarcoma U2OS (ATCC) cell lines were cultured by standard methods. The JD0G mutant HSV-1 virus is deleted for ICP0 and the joint repeat elements, as described elsewhere (Reinhart et al., submitted). JD0G virus stocks were prepared and titered on U2OS cells.
Plasmid construction and transfection
A human MMP-9 cDNA clone in pCMV6-XL4 was purchased from OriGene Technologies (Rockville, MD) and cloned into pIRES1neo (Clontech Laboratories, Palo Alto, CA). Stably transfected SK-N-AS/MMP9 cells were obtained by selection with G418 (Invitrogen Corp, Carlsbad, CA).
Cells were lysed in 1% NP40 buffer, lysates electrophoresed through 10% SDS-polyacrylamide gels, and protein blots reacted with polyclonal anti-MMP-9 antibody (1:1,000 dilution) (Abcam, Cambridge, MA) and HRP-conjugated anti-rabbit secondary antibody (Sigma, St. Louis, MO). Blots were developed with chemiluminescence substrate (Amersham Pharmacia, Piscataway, NJ). The lower portion of each blot was reacted with polyclonal anti-β-actin antibody (Santa Cruz Biotechnology, CA) to detect loading differences.
Conditioned media were separated on a 10% SDS-polyacrylamide gel containing 1mg/mL gelatin. The gel was washed in 10mM Tris (pH 7.5), 2.5% Triton X-100, incubated at 37°C for 16h in 50mM Tris (pH 7.5), 5mM CaCl2, 1μM ZnCl2, stained with Coomassie brilliant blue R-250, and destained.
5×104 cells per well were plated in Matrigel-coated or uncoated Biocoat Invasion Chambers (BD Biosciences, San Jose, CA). At 22h, cells attached to the lower surface of the membrane were stained and counted.
Spheroid culture and imaging
Spheroids were grown to ca. 1 mm in diameter in 0.5% soft agar and individually infected in microfuge tubes with 5×104 pfu of JD0G virus for 2h. At 24hpi, the spheroids were fixed in 4% paraformaldehyde and 3μ Z section images of GFP expression were obtained by two-photon microscopy (Leica Microsystems Inc., Bannockburn, IL). MetaMorph software (Molecular Devices, Downingtown, PA) was used for 3-D image reconstitution analysis. Separately, infected spheroids were trypsinized and GFP expressing cells counted under a fluorescence microscope.
Animals and tumor model
4-5-week-old female BALB/c nude mice (Charles River Laboratories, Wilmington, MA) were anesthetized and stereotactically injected with 5×105
SK-N-AS/MMP9 or control cells into the right frontal lobe as described (4
). Animals were observed daily until death or stereotactically injected at the same coordinates with viral vector (500 pfu/2μl of JD0G) after 3 weeks. Animals were sacrificed 2 days later for analysis of the distribution of virus-infected cells in the tumor mass (IACUC protocol # 07-11-383).
Histology and immunostaining
Brain tissues were fixed in 4% paraformaldehyde for 24h and incubated in 30% sucrose for 48h. Tissues were frozen in 2-methylbutane/dry ice, embedded in OCT, sectioned at 6μm thickness, and imaged for GFP expression under an Olympus Provis fluorescence microscope (Olympus, Center Valley, PA). Alternate sections were stained with Hoechst to visualize tumor boundaries. Selected GFP-imaged sections were stained with anti-HSV antibodies (GeneTex, Irvine, CA) and Cy3-conjugated sheep anti-rabbit IgG (Sigma).