br refine imaging techniques to confirm
refine imaging techniques, to confirm deep tissue imaging in ortho-topic and metastatic models, and to delineate comparisons of SPECT with PET. It will be interesting to investigate in future experiments whether radioiodine uptake correlates to viral tumor titer throughout all time points and to further refine imaging techniques and timing, using immunocompetent and orthotopic or metastatic models.
In summary, our novel chimeric orthopoxvirus CF33-hNIS encodes a functional sodium iodide symporter, induces caspase-independent ICD, allows real-time noninvasive imaging of viral infection, and most importantly, kills colorectal cancer in vitro and in vivo. Our pre-sent study showed that CF33-hNIS synergizes with I-131. Most notably, this vector can be tracked in real time and used in concert
with radioisotope therapy in order to maximize the reach of CF33-hNIS or similar vectors while minimizing dose. We anticipate that this would decrease toxicity and cost while maximizing tumoricidal activity and allowing for tracking of viral replication and efficacy.
MATERIALS AND METHODS
Cell Lines and Maintenance
For standard viral plaque assays, African green monkey kidney fibro-blasts (CV-1), purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured at 37 C with 5% CO2 in DMEM (Corning, Corning, NY, USA). Human colorectal cell lines were all purchased from ATCC. HCT-116 and HT-29s were main-tained in McCoy’s 5A medium (ATCC, Manassas, VA, USA) per ATCC instructions. All Pyocyanin were kept at 37 C and 5% CO2. Unless
stated otherwise, cells were supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution, both purchased from Corning (Corning, NY, USA). Cell lines were expanded and frozen down after a maximum of three passages. Efforts were made to ensure all experiments took place after less than 10 passages.
CF33 Chimerization and hNIS Cloning
The chimerization process of the CF33 backbone virus has been described in previous publications.14,15 In brief, nine strains of ortho-poxvirus were used to create CF33 by co-infecting CV-1 cells and fostering chimerization. These included cowpox, raccoonpox, rabbit-pox, and multiple vaccinia virus strains, all purchased from ATCC and grown and titrated in CV-1 cells. Following the co-infection, 100 individual plaques were chosen and then thrice purified in CV-1 cells to obtain 100 clonally purified chimeric orthopoxviruses. High-throughput screening was used to compare the cytotoxic effi-cacy of chimeric clones with each other and with parental strains versus the NCI-60 panel of human cancer cell lines. CF33 was selected as the chimeric isolate that demonstrated superior cell killing in the NCI-60 panel compared with all parental viruses and other plaque-purified isolates.
To generate CF33 expressing hNIS, the hNIS expression cassette un-der control of the vaccinia virus H5 synthetic early (SE) promoter was cloned using Sac I and BamHI restriction sites and was inserted at the thymidine kinase locus of CF3316 with transient dominant selection as previously described using the guanine phosphoribosyltransferase marker, as has been similarly described with insertion of firefly luciferase.15,37
In order to examine vaccinia and hNIS co-expression, HT29 and HCT116 cells infected with CF33-hNIS at MOI 0.01 were compared with non-infected controls. Cells were fixed with 4% paraformalde-hyde at room temperature for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min on ice, and blocked for 30 min at 37 C in Tris-NaCl-blocking buffer (0.1 M Tris-HCl, pH 7.5, 0.15 NaCl, and 0.5% Blocking Reagent; category number FP1020; PerkinElmer). After blocking, cells were incubated with anti-sodium iodide symporter antibody (ab17795; Abcam) at 1:50 dilution in Tris-NaCl-blocking buffer and incubated with a secondary Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (IgG) H&L (ab150113; Abcam) at 1:100 dilution. Cells were re-blocked in Tris-NaCl-blocking buffer and stained with anti-vaccinia virus antibody (ab35219; Abcam) at 1:200 dilution and a secondary Alexa Fluor 594-conjugated goat anti-Rabbit IgG H&L (ab150080; Abcam) at 1:100 dilution to document surface expression of NIS on virally infected cells.