Figure 5.
NSCs Enhance CRAd-s-pk7 Efficacy
(A and B) NSC protects CRAd-S-pk7 from adenovirus neutralization. (A) Blot analysis assessing recognition of adenovirus antigens hexon, penton base, and fiber by antibodies present in ascetic fluid. Dilutions of purified CRAd-S-pk7 virus were subjected to SDS-PAGE. Following semi-dry blotting and blocking in non-fat milk, membranes were incubated with ascites pooled from three patients with ovarian cancer. Bound antibodies were visualized using horseradish-peroxidase-conjugated anti-human IgG secondary antibodies. OVCAR8 human ovarian cancer cells served as a negative control. (B) Neutralization of adenovirus infectivity by ascites fluid. Two thousand OVCAR8.EGFP.ffluc cells/96-well plate were co-cultured with either 2,000 NSC.CRAd-S-pk7 (2.5 × 107 pfu) for 5 days or free CRAd-S-pk7 (2.5 × 107 pfu) for 3 days or NSC-CRAd-S-pk7 in the presence of serial 2-fold dilutions of ascetic fluid obtained from three different ovarian cancer patients. Ascitic fluid was replaced after 24 h with culture media. Data are presented as average raw luminescent signal ± SD. (C and D) NSC improves CRAd-S-pk7 delivery in vivo. (C) qPCR quantification of increased adenoviral E1A gene copy number in mouse tumors treated with NSC.CRAd-S-pk7 (black bars) in comparison with free virus (gray bars) 1 day after administration. (D) Tumor volume was determined by weighing the omentum (primary site of tumor formation) after three treatment rounds; each point indicates an individual mouse. Data for (C) and (D) represent mean ± SEM.