Interferon Beta and Interferon Alpha 2a Differentially Protect Head and Neck Cancer Cells from Vesicular Stomatitis Virus-Induced Oncolysis

Abstract

Oncolytic viruses (OV) preferentially kill cancer cells due in part to defects in their antiviral responses upon exposure to type I interferons (IFNs). However, IFN responsiveness of some tumor cells confers resistance to OV treatment. The human type I IFNs include one IFN-β and multiple IFN-α subtypes that share the same receptor but are capable of differentially inducing biological responses. The role of individual IFN subtypes in promoting tumor cell resistance to OV is addressed here. Two human IFNs which have been produced for clinical use, IFN-α2a and IFN-β, were compared for activity in protecting human head and neck squamous cell carcinoma (HNSCC) lines from oncolysis by vesicular stomatitis virus (VSV). Susceptibility of HNSCC lines to killing by VSV varied. VSV infection induced increased production of IFN-β in resistant HNSCC cells. When added exogenously, IFN-β was significantly more effective at protecting HNSCC cells from VSV oncolysis than was IFN-α2a. In contrast, normal keratinocytes and endothelial cells were protected equivalently by both IFN subtypes. Differential responsiveness of tumor cells to IFN-α and -β was further supported by the finding that autocrine IFN-β but not IFN-α promoted survival of HNSCC cells during persistent VSV infection. Therefore, IFN-α and -β differentially affect VSV oncolysis, justifying the evaluation and comparison of IFN subtypes for use in combination with VSV therapy. Pairing VSV with IFN-α2a 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-α2a and IFN-β, in protection from oncolytic vesicular stomatitis virus. We found that IFN-α2a was significantly less protective for cancer cells than was IFN-β, 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-α2a.

FIG 1

Sensitivity of HNSCC lines to M51R VSV and rwt VSV. HNSCC cell lines (JSQ-3, SCC61, and SQ20B) or normal endothelial cells or keratinocytes (HMVEC and NHEK cells, respectively) were infected with eGFP-expressing M51R VSV (a and c) or rwt VSV (b and d) at the indicated MOIs. RKO, a colorectal line that is highly sensitive to VSV, served as a control. The percentage of eGFP-expressing cells, quantified by fluorescence microscopy at 24 h postinfection, is shown in panels a and b. Cell viability as measured by MTT assay at 48 h postinfection is shown in panels c and d. Results are expressed as percentages of cells relative to the number of mock-infected cells that survived infection. The means ± SD from at least 3 individual experiments is shown. *, P < 0.05 for three HNSCC lines, all comparisons; **, P < 0.05 for two normal cell lines versus three HNSCC lines, all comparisons at each MOI. P values were determined by one-way ANOVA with Tukey's posttest.

FIG 2

Production and response to type I IFN inhibition by tumor cells infected with M51R VSV. (a) IFN-β 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-β per 1 × 10⁵ cells. The means ± SD from 3 individual experiments is shown. (b) Neutralizing antibodies to IFN-β, IFN-α, or a combination of the two antibodies were added to JSQ-3 cells 18 h prior to the addition of M51R VSV (MOI, 0.1) to the cultures. P values were determined by unpaired Student's t test.

FIG 3

Protective effect of exogenous IFNs on tumor and normal cells infected with rwt VSV. (a) JSQ-3 cells, (b) NHEK cells, and (c) HMVEC were preincubated for 18 h with recombinant human IFN-α2a or IFN-β at either 100 or 1,000 U/ml and then infected with rwt VSV at an MOI of 10. Viability was measured at 48 h by MTT assay. Results shown are the means ± SD from at least 3 experiments per treatment. *, P < 0.05 for 100 U/ml IFN-β versus 100 U/ml IFN-α; *, P < 0.05 1,000 U/ml IFN-β versus 1,000 U/ml IFN-α, determined by one-way ANOVA with Tukey's posttest. (d) Viral titers in supernatants from JSQ-3 cells and HMVEC cells were measured at 24 h by plaque assay. Two independent experiments for each cell type and virus are indicated by open and closed symbols.

FIG 4

Protective effect of exogenous IFNs on SCC-61, SQ-20B, and RKO cells infected with VSV. SCC61 (a), SQ20B (b), or RKO (c) cells were preincubated for 18 h with recombinant human IFN-α2a or IFN-β (100 U/ml or 1,000 U/ml) and then infected with M51R VSV (left) or rwt VSV (middle) at the indicated MOIs. Viability was measured at 48 h. Results shown are the means ± SD from 4 experiments per treatment. *, P < 0.05 for 100 U/ml IFN-β versus 100 U/ml IFN-α and P < 0.05 for 1,000 U/ml IFN-β versus 1,000 U/ml IFN-α, each determined by one-way ANOVA with Tukey's posttest. Viral titers in supernatants (right) were measured at 24 h by plaque assay. Two independent experiments for SCC61 and SQ20B and three independent experiments for RKO infected with M51R VSV (circles) or rwt VSV (squares) are shown.

FIG 5

Tumor cells persistently infected with M51R VSV resist killing upon superinfection with M51R or rwt VSV. Tumor cells that had established persistent M51R VSV infections, generated as described in Materials and Methods, were reinfected at the indicated passage (p) number with M51R or rwt VSV at the indicated MOIs. p0 indicates cells that were infected with VSV for the first time (not persistently infected). At 48 h after reinfection, viability was measured by MTT assay. Results are expressed as the percentage of cells, relative to the number of mock-infected cells, that survived reinfection. Data from four (SQ20B) or three (JSQ-3 and SCC61) separate passages are shown.

FIG 6

IFN-β maintains the state of persistent infection in tumor cells. (a) SQ20B cells that had established persistent M51R VSV infections (PI-SQ20B) were reinfected at the indicated passage (p) number with M51R VSV at an MOI of 0.1. p0 indicates cells that were infected for the first time (not persistently infected). Neutralizing antibodies to IFN-α, IFN-β, or a combination of the two antibodies were added to some cultures 18 h prior to the addition of virus. At 48 h after reinfection with M51R VSV at the indicated MOI, viability was measured by MTT assay. Results are expressed as the percentage of cells, relative to the number of mock-infected cells, that survived reinfection. Three passages are shown. The results from all three passages were pooled for analysis of variance. The following survival differences (P < 0.05) were found for the treatment groups: (i) anti-IFN-β and anti-IFN-α plus anti-IFN-β groups had decreased survival relative to the no-antibody groups, and (ii) anti-IFN-β groups had decreased survival relative to the anti-IFN-α groups. (b) Persistently infected JSQ-3 cells were maintained continuously in the presence of exogenous IFN-β. When virus was no longer detectable in the cultures, the cells were reinfected with M51R VSV and viability was measured 48 h later. Persistently infected SQ20B (PI-SQ20B) (c) or JSQ-3 (PI-JSQ-3) (d) cells were treated with neutralizing anti-IFN-β or anti-IFN-α antibodies, and viability was measured over 4 days. The number of viable cells per well was determined by light microscopy using the criteria of trypan blue exclusion. The means ± SD from 3 determinations per time point are shown. The following survival differences (P < 0.05) at day 4 were found: (i) the no-antibody group survived in greater numbers than the anti-IFN-β group, and the anti-IFN-α group survived in greater numbers than the anti-IFN-β group for both cell lines. (c and d) Differences in group-by-time interaction (slope) (P < 0.05) were the following: the no-antibody group survived in greater numbers than the anti-IFN-α group, which survived in greater numbers than the anti-IFN-β group (c); the no-antibody group survived in greater numbers than the anti-IFN-β group, and the anti-IFN-α group survived in greater numbers than the anti-IFN-β group (d).