Application of interferon modulators to overcome partial resistance of human ovarian cancers to VSV-GP oncolytic viral therapy

Abstract

Previously, we described an oncolytic vesicular stomatitis virus variant pseudotyped with the nonneurotropic glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, which was highly effective in glioblastoma. Here, we tested its potency for the treatment of ovarian cancer, a leading cause of death from gynecological malignancies. Effective oncolytic activity of VSV-GP could be demonstrated in ovarian cancer cell lines and xenografts in mice; however, remission was temporary in most mice. Analysis of the innate immune response revealed that ovarian cancer cell lines were able to respond to and produce type I interferon, inducing an antiviral state upon virus infection. This is in stark contrast to published data for other cancer cell lines, which were mostly found to be interferon incompetent. We showed that in vitro this antiviral state could be reverted by combining VSV-GP with the JAK1/2-inhibitor ruxolitinib. In addition, for the first time, we report the in vivo enhancement of oncolytic virus treatment by ruxolitinib, both in subcutaneous as well as in orthotopic xenograft mouse models, without causing significant additional toxicity. In conclusion, VSV-GP has the potential to be a potent and safe oncolytic virus to treat ovarian cancer, especially when combined with an inhibitor of the interferon response.

Figure 1

Ovarian cancer cell lines are efficiently infected and killed by VSV-GP. (a) Tropism of VSV*ΔG-GP and VSV*ΔG-G was determined for different human ovarian cancer cell lines as well as a murine cancer cell line (ID8) and a benign cell line (HOSE). Cells were infected with serial dilutions of VSV*ΔG-GP or VSV*ΔG-G. As a control in each experiment, BHK21 cells were infected in the same way. Sixteen hours post infection the percentage of GFP positive cells was determined by flow cytometry and the titer for both viruses on each cell line was calculated. Titers are given relative to the reference cell line BHK21. Bars represent the mean ± SEM (standard error of mean) of one representative of at least two independent experiments using duplicate or triplicate samples. For the control cell line, BHK21, the mean and SEM of one representative experiment is shown. (b) For replication kinetics, cells were infected with an MOI of 0.1 of either VSV or VSV-GP. One hour after infection, inoculum was removed, cells were washed with PBS and fresh medium was added. Twenty four hours post infection, the supernatant was collected and titrated on BHK21 cells using TCID₅₀ assay. Data represent mean ± SEM of at least n = 2 independent experiments using duplicates. The limit of detection for the TCID₅₀ assay (3.16 TCID₅₀/ml) is shown as dashed line in the graph. For the killing assay, ovarian cancer cell lines were seeded as monolayers and cells were infected with an MOI of 0.1 of VSV or VSV-GP (c). Viability was determined at indicated time points using WST-1 assay. As a reference, PBS treated cells were used. The human benign cell line HOSE was infected with an MOI of 1 and viability was determined after 72 hours. Bars represent mean ± SEM of one representative experiment of at least two independent experiments performed in decaplicates or dodecaplicates. MOI, multiplicity of infection; PBS, phosphate buffered saline; TCID₅₀, 50% tissue culture infective dose; VSV, vesicular stomatitis virus.

Figure 2

VSV-GP shows effective antitumor activity in an ovarian cancer xenograft mouse model but most tumors recur. 5 × 10⁶ A2780 cells were injected subcutaneously (s.c.) into both flanks of NOD/SCID mice. Tumors were treated at a size of 0.1 cm³ with intratumoral (i.t.) injection of either PBS, 2 × 10⁵ PFU VSV or 2 × 10⁵ PFU VSV-GP and treatment was repeated 1 week later. Tumor growth was measured using a caliper. At a tumor size of 0.8 cm³ or when mice showed signs of neurotoxicity mice were sacrificed. (a, b) Graphs show the volume of individual tumors treated with PBS and VSV (a) or PBS and VSV-GP (b), respectively. (c) Kaplan–Meier survival curve for death due to neurotoxicity in VSV treated animals. (d) Kaplan–Meier survival curve for death due to tumor volume in VSV-GP treated animals. To see if recurring tumors are still susceptible to VSV-GP treatment, s.c. tumors were established by s.c. injection of 5 × 10⁶ A2780 cells into one flank of NOD/SCID mice. At a size of 0.1 cm³, tumors were treated i.t. with two doses of 5 × 10⁶ PFU VSV-GP in a 7 day interval. Control animals received PBS injections. (e) Tumor growth was monitored until recurrence (tumor volume > 0.2 cm³) was observed. (f) Recurring tumors (volume > 0.2cm³) from virus treated animals were randomly divided into two groups and treated i.t. either with PBS or two doses of 1 × 10⁷ PFU VSV-GP in a 7-day interval. Animals were sacrificed at a tumor size of 0.8 cm³. Data points represent mean ± SEM. Dashed lines in a–e indicate time point of second treatment, in f time point of first and second treatment. PBS, phosphate buffered saline; PFU, plaque forming unit; SEM, standard error of mean; TCID₅₀, 50% tissue culture infective dose; VSV, vesicular stomatitis virus.

Figure 3

Ovarian cancer cells respond to type I interferon and produce IFN upon viral infection. Ovarian cancer cell lines were seeded at 5 × 10⁴ and pretreated with 0, 20, 100 or 500 Units of universal type I IFN for 24 hours. After 24 hours, cells were infected with VSV-GP at an MOI of 0.1 (a) or 10 (b). 24 hours post infection supernatants were collected and titrated on BHK21 cells using a TCID₅₀ assay. A cell line was defined as responsive (marked with #), when a difference of more than 2-log in viral titer with and without IFN pretreatment was observed. Bars represent the mean ± SEM of at least two independent experiments using duplicates. (c) For an indirect type I IFN secretion analysis, cell lines were infected with an MOI of 3 of the single-cycle infective, attenuated VSV*M_QΔG-GP virus or left uninfected. Supernatants, potentially containing IFN or other antiviral biomolecules, were collected 24 hours after infection. For analysis of biologically active IFN, Vero cells or L929 cells (for analysis of human or murine ovarian cancer cell lines respectively) were incubated either with the supernatant of noninfected cells (SN) or with potentially IFN containing supernatant from virus infected cells (VSV-SN). After 24 hours preincubation, cells were infected with an MOI of 1 of VSV-GP and the supernatant was collected 24 hours post infection. Supernatants were titrated on IFN-nonresponsive G62 cells using a TCID₅₀ assay to determine virus replication. Cells were defined as IFN producing cells when the difference in viral titers was more than 2-logs between SN and VSV-SN treated cells. Vero cells were used as negative control for cells not producing IFN. Bars represent the mean ± SEM of at least two independent experiments using triplicate samples. IFN, interferon; MOI, multiplicity of infection; SEM, standard error of mean; TCID₅₀, 50% tissue culture infective dose; VSV, vesicular stomatitis virus.

Figure 4

Ruxolitinib can inhibit antiviral effects of IFN. (a) A2780 cells were infected with an MOI of three of the single-cycle infective, attenuated VSV*M_QΔG-GP or left noninfected. Supernatants were collected 24 hours post infection. Fresh A2780 cells were treated with either medium, the supernatant of noninfected cells (SN), virus infected cells (VSV-SN), or 500 U/ml recombinant universal type I IFN. Additionally, 1 μmol/l ruxolitinib was added to half of the wells. After 18 hours, cells were infected with an MOI 0.1 of VSV-GP and 24 hours after infection, supernatants were collected and analyzed for viral replication using TCID₅₀ assay on G62 cells. Bars represent mean ± SEM of at least two independent experiments performed in triplicates. ***P < 0.001 (Unpaired, two-tailed t-test). (b) A2780 cells were incubated with different concentrations of ruxolitinib alone (0.1, 1, and 5 µmol/l) or in combination with 1ml IFN-containing supernatant from VSV*M_QΔG-GP infected A2780 cells. After 24 hours lysates were prepared and analyzed for expression of MxA. As loading control blots were probed with an antiactin antibody. (c) A2780, OVCAR3, HTB77 or SKOV6 cells were preincubated with 500 U IFN for 16 hours. Cells were subsequently infected with an MOI of 0.1 with VSV-GP or remained uninfected. Half of the wells were additionally treated with 1 µmol/l ruxolitinib. After 48 hours, viability of cells was analyzed via WST-1 assay. Bars show mean ± SEM of hexaplicates. IFN, interferon; MOI, multiplicity of infection; SEM, standard error of mean; TCID₅₀, 50% tissue culture infective dose; VSV, vesicular stomatitis virus.

Figure 5

Oncolytic effect of VSV-GP is enhanced by combination treatment with ruxolitinib in a subcutaneous xenograft model. Subcutaneous A2780 tumors were established by injection of 5 × 10⁶ A2780 cells into the flank of NOD/SCID mice. Tumors were treated at a size of 0.1 cm³ with (i) 30 μl PBS i.t. at days 0 and 7; (ii) 30 μl PBS i.t. at days 0 and 7; and with 45 mg/kg ruxolitinib diluted in 200 μl methylcellulose i.p. for 11 consecutive days starting day 0; (iii) 5 × 10⁶ PFU VSV-GP i.t. at days 0 and 7; and (iv) 5 × 10⁶ PFU VSV-GP i.t. at days 0 and 7 and with 45 mg/kg ruxolitinib diluted in 200 μl methylcellulose i.p. for 11 consecutive days starting day 0. (a) Tumor growth was monitored until day 100 post-treatment start. Mean ± SEM for each treatment group are shown, n = 5 for PBS group, n = 4 for ruxolitinib alone, n = 10 for VSV-GP alone, and n = 9 for ruxolitinib + VSV-GP combination group. (b) Kaplan–Meier curve showing time till tumor relapse (tumor size > 0.2 cm³) . PBS, phosphate buffered saline; PFU, plaque forming unit; SEM, standard error of mean; TCID₅₀, 50% tissue culture infective dose; VSV, vesicular stomatitis virus.

Figure 6

Oncolytic effect of VSV-GP is enhanced by combination treatment with ruxolitinib in an orthotopic xenograft model. A2780 tumors stably expressing luciferase were established in nude mice by i.p. injection. After 4 days, mice were either left untreated or treated with ruxolitinib alone (for 11 consecutive days at a dose of 45 mg/kg i.p.), with VSV-GP alone (by i.p. injection of 10⁷ PFU at days 0, 3, and 7) or with a combination of ruxolitinib and VSV-GP, n = 8. VSV-GP only and VSV-GP plus ruxolitinib combination treated mice received a second treatment cycle identical to the first one starting at day 38 after tumor implantation. (a) At indicated time points mice were analyzed for luciferase signal using bioluminescence imaging. The graph shows mean ± SEM for the average radiance (p/s/cm2/sr) for each mouse. (b) Kaplan–Meier survival curve of treated animals. (c) Kaplan–Meier curve for response rate. Full response was defined when radiance dropped below 1,000 (background value). (d) At day of sacrifice tumors were weighted. The graph shows mean ± SEM. Significances were determined using a one-way ANOVA test with a Tukey’s post hoc test (ns. non-significant, ***P-value < 0.0001). (e) Exemplary BLI pictures for one mouse from each group are shown, upper panel shows day 4 post-tumor cell implantation, lower panel shows day 35 post-tumor cell implantation. ANOVA, analysis of variance; BLI, bioluminiscence imaging; PFU, plaque forming unit; SEM, standard error of mean; VSV, vesicular stomatitis virus.

Figure 7

Combination of VSV-GP and ruxolitinib enhances virus replication in the tumor and is safe in immunodeficient mice. (a) A2780 tumors stably expressing luciferase were established in nude mice by subcutaneous injection. At a tumor size of 0.1 cm³ mice were either treated with VSV-GP alone or VSV-GP plus ruxolitinib. Ruxolitinib was injected once a day at a dose of 45 mg/kg ruxolitinib diluted in 200 μl methylcellulose i.p. starting 4 hours prior to the virus injection for 11 subsequent days. VSV-GP was injected at a dose of 5 × 10⁶ PFU intratumorally on days 0 and 7. Virus replication was monitored daily via IVIS. Dashed line indicates time point of second treatment. Graph shows mean ± SEM of four (VSV-GP only) or six mice (combination group). (b) Nude mice were injected i.p. with 10⁸ or 10⁹ PFU of VSV-GP containing luciferase. One half of each group additionally received ruxolitinib by i.p. injection once a day (45 mg/kg) for four consecutive days starting 4 hours prior to virus injection, n = 4 for 10⁹ PFU, n = 6 for 10⁸ PFU, and n = 1 for mock. Mice were weighed at indicated time points. The weight of each mouse prior to virus injection was set to 1. The graph shows mean ± SEM. PFU, plaque forming unit; SEM, standard error of mean; VSV, vesicular stomatitis virus.