doi:10.1016/j.jmb.2006.11.053. significantly more effective at protecting HNSCC cells from VSV oncolysis than was IFN-2a. In contrast, normal keratinocytes and endothelial cells were protected equivalently by both IFN subtypes. Differential responsiveness of tumor cells to IFN- and – was further supported by the finding that autocrine IFN- but not IFN- promoted survival of HNSCC cells during persistent VSV infection. Therefore, IFN- and – differentially affect VSV oncolysis, justifying the evaluation and comparison GDC-0068 (Ipatasertib, RG-7440) of IFN subtypes for use in combination with VSV therapy. Pairing VSV with IFN-2a may enhance selectivity of oncolytic VSV therapy for HNSCC by inhibiting VSV replication in normal cells without a corresponding inhibition in cancer cells. IMPORTANCE There has been a great deal of progress in the development of oncolytic viruses. However, a major problem is that individual cancers vary in their sensitivity to oncolytic viruses. In many cases this is due to differences in their production and response to interferons (IFNs). The experiments described here compared the responses of head and neck squamous cell carcinoma cell lines to two IFN subtypes, IFN-2a and IFN-, in protection from oncolytic vesicular stomatitis virus. We found that IFN-2a was significantly less protective for cancer cells than was IFN-, whereas normal cells were equivalently protected by both IFNs. These results suggest that from a therapeutic standpoint, selectivity for cancer versus normal cells may be enhanced by pairing VSV with IFN-2a. INTRODUCTION The use of viruses to selectively kill cancer cells (oncolytic virotherapy) is a promising alternative therapy for cancer (1). The basis for this treatment approach is that cancer cells frequently have defective antiviral responses that develop as a consequence of cellular transformation (2,C5). As a result, they are more susceptible than their normal cellular counterparts to infection and apoptotic death induced by cytopathic viruses (6, 7). Vesicular stomatitis virus (VSV), a negative-strand RNA virus of the family test, and statistical comparisons were considered significant for 0.05. For ELISA data shown in Fig. 2a, which had large variability, log transformations were performed prior to making comparisons between groups. For analysis of variance (ANOVA) among multiple treatment groups, the data were analyzed by one-way ANOVA with Tukey’s method for adjusting for multiple comparisons. For analyses comparing groups over time (see Fig. 6), two-way ANOVA models were fit with group and time and the group-by-time interaction. The group-by-time interaction term was examined in these models to determine whether there were differences in RRAS2 the change in the outcome (slope) over time. Finally, in the models examining differences over time, we performed pairwise comparisons of groups at 4 days using unpaired tests to determine whether the groups differed on the last observed time point. All analyses were performed using SAS, version 9.3 (Cary, NC). Open in a separate window FIG 2 Production and response to type I IFN inhibition by tumor cells infected with M51R VSV. (a) IFN- levels were measured by ELISA using supernatants taken from JSQ-3 or SQ20B cells 24 h after infection with M51R VSV at the indicated MOIs. Results are expressed as picograms/milliliter of IFN- per 1 105 cells. The means SD from 3 individual GDC-0068 (Ipatasertib, RG-7440) experiments is shown. (b) Neutralizing antibodies to IFN-, IFN-, or a combination of the two antibodies were added to JSQ-3 cells 18 h prior to the addition GDC-0068 (Ipatasertib, RG-7440) of M51R VSV (MOI, 0.1) to the cultures. values were determined by unpaired Student’s test. Open in a separate window FIG 6 IFN- maintains the state of persistent infection in tumor cells. (a) GDC-0068 (Ipatasertib, RG-7440) SQ20B cells that had established persistent M51R VSV infections (PI-SQ20B) were reinfected at the indicated passage (p) number with GDC-0068 (Ipatasertib, RG-7440) M51R VSV at an MOI of 0.1. p0 indicates cells that were infected for the first.