Synergistic cytotoxicity of oncolytic reovirus in combination with cisplatin–paclitaxel doublet chemotherapy

Abstract

Oncolytic reovirus is currently under active investigation in a range of tumour types. Early phase studies have shown that this agent has modest monotherapy efficacy and its future development is likely to focus on combination regimens with cytotoxic chemotherapy. Indeed, phase I/II clinical trials have confirmed that reovirus can be safely combined with cytotoxic drugs, including a platin—taxane doublet regimen, which is currently being tested in a phase III clinical trial in patients with relapsed/metastatic head and neck cancer. Therefore, we have tested this triple (reovirus, cisplatin, paclitaxel) combination therapy in a panel of four head and neck cancer cell lines. Using the combination index (CI) method, the triple therapy demonstrated synergistic cytotoxicity in vitro in both malignant and non-malignant cell lines. In head and neck cancer cell lines, this was associated with enhanced caspase 3 and 7 cleavage, but no increase in viral replication. In vitro analyses confirmed colocalisation of markers of reovirus infection and caspase 3. Triple therapy was significantly more effective than reovirus or cisplatin—paclitaxel in athymic nude mice. These data suggest that the combination of reovirus plus platin—taxane doublet chemotherapy has significant activity in head and neck cancer and underpin the current phase III study in this indication.

INTRODUCTION

The past decade has witnessed significant developments in our understanding of the therapeutic potential of oncolytic viruses, such that a number of agents have now undergone extensive preclinical and early-stage clinical assessment. Reovirus type 3 Dearing (Reolysin; Oncolytics Biotech Inc., Calgary, AB, Canada) has been at the leading edge of this process and is currently being tested in phase I, II and III clinical protocols in a variety of tumour types.^1–3 Reovirus is a naturally occurring non-pathogenic, double-stranded RNA virus isolated from the human respiratory and gastrointestinal tracts.^4,5 Exposure to reovirus is almost ubiquitous with up to 100% of healthy adults showing seropositivity.⁶ Detailed analysis of the molecular basis of selective cytotoxicity of reovirus in cancer cells has highlighted the central role of Ras pathway activation, either through Ras gene mutation or overexpression/mutational activation of epidermal growth factor receptor signalling.^7–11

Preclinical testing has confirmed that reovirus has impressive single-agent activity against many common epithelial malignancies both in vitro and in vivo in immunodeficient animal models (reviewed in Comins¹ and Yap et al.² Although first-in-man phase I monotherapy studies, involving both intratumoral and intravenous viral administration, have provided reassuring data on the clinical safety of reovirus, the agent has shown only modest anti-tumour activity.^12–14 As a result, further preclinical evaluation has focussed on combining reovirus with standard anticancer modalities, such as radiotherapy¹⁵ and cytotoxic chemotherapy.^16–18 These approaches have also been translated into phase I/II clinical studies.^19–22

One of the clearest signals that emerged from the preclinical studies was the fact that combinations of reovirus with either platinum- or taxane-based chemotherapy appear to be particularly favourable.^16–18 This, in turn, led to a phase I study of reovirus in combination with both carboplatin and paclitaxel in patients with advanced/metastatic cancers that were refractory to conventional treatment. Data from the initial phase I study showed very significant activity of the combination regimen in patients with relapsed head and neck cancers, leading to a subsequent phase II expansion study for this indication.²² A total of 31 patients were included in the phase I/II study and 26 who received at least two cycles of therapy were fully evaluable for efficacy. Of 14 patients with squamous cell cancers of the head and neck that was progressing at the time of recruitment, 10 were fully evaluable, and in this group there were three partial responses, two major clinical responses in cutaneous disease, three patients with stable disease and two patients with disease progression. All 14 of these patients had received prior platin-based chemotherapy. As a result, a double-blind, randomised phase III study of carboplatin and paclitaxel plus reovirus or placebo has recently opened to recruitment for patients with platin-refractory relapsed/metastatic head and neck cancer.

In parallel, we have conducted a detailed analysis of the in vitro and in vivo activity of reovirus in combination with platin- and taxane-based chemotherapy. Specifically, we have shown that the combination of oncolytic reovirus with doublet chemotherapy is potently and synergistically active against head and neck cancer cell lines.

RESULTS

Reovirus is synergistic with cisplatin and paclitaxel in head and neck cancer

The cytotoxicity of reovirus (R), cisplatin (C) and paclitaxel (P), as single agents or in combination, was measured using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays across a range of multiples of the individual half-maximal inhibitory concentration (IC₅₀) doses (Supplementary Figure S1). Data for Cal27, Detroit-562, HN5 and PJ41 cell lines (Figure 1a) show that combinations of reovirus with cytotoxic chemotherapy are more potent than single-agent therapies or cisplatin—paclitaxel doublet chemotherapy. MEF (mouse embryonic fibroblast cells), MCF10A (breast epithelial cells) and NHM (normal human mesothelial cells) were used as non-malignant cell lines and IC₅₀ values of the three single agents were derived (Supplementary Figure S2a). Formal combination indices were calculated for each of the following treatment conditions: cisplatin—paclitaxel doublet chemotherapy (C:P); reovirus plus paclitaxel (R:P); reovirus plus cisplatin (R:C); and reovirus plus cisplatin—paclitaxel chemotherapy (R:C:P) (Figure 1b and Supplementary Figures S2b and c). These analyses reveal that the C:P combination yields slight to moderate synergy/additive effects in three of the head and neck cancer cell lines at IC₅₀ ratios of 0.5 and 1.0, but is most frequently antagonistic. In direct contrast, the R:P and R:C combinations were synergistic in all head and neck cancer cell lines at ratios between 0.5 and 1.0. The R:C:P combination caused the greatest levels of cell death (Figure 1a) and these translated to synergistic activity across IC₅₀ ratios between 0.5 and 2.0 in all cell lines. (Figures 1a and b). Representative plots of the combination indices at different fractional effects are shown for head and neck cells and confirm the impressive synergy that was particularly evident with R:P and R:C:P combinations (Figure 1c).

Figure 1.

Combined treatment of reovirus with cisplatin and/or paclitaxel enhances cell kill in head and neck cell lines. (a) 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays in head and neck cancer cell lines (Cal27, Detroit-562, HN5 and PJ41) treated with reovirus (R), cisplatin (C) or paclitaxel (P), as single agents or in combination as indicated. Cells were treated for 96 h at 0.25 ×, 0.5 ×, 1 ×, 2 × and 4 ×, the IC₅₀ dose for the individual agents or at constant ratios of the IC₅₀ for combination studies. Data are derived from three independent experiments ± standard error of mean (s.e.m.). (b) Interaction between the treatment combinations was assessed using the method of Chou and Talalay.²⁴ CI values were calculated where CI <0.9 is classed as synergy, 0.9–1.1 is additive and >1.1 is antagonistic. For the purposes of R:C:P triple combination analysis, C:P was considered as a single treatment in this case. (c) Representative CI plots of the respective IC₅₀ doses/ratios. Data represent the algebraic estimates of the CI (±1.96s.d.) for reovirus and chemotherapy combinations.

Importantly, the IC₅₀ values for non-malignant cell lines MEF and MCF10A revealed that they were relatively more resistant than the cancer cells to each of the single-agent therapies. However, when experiments were conducted according to the standard CI methodology, multiples of the IC₅₀ values in combination were capable of mediating synergistic cytotoxicity in both MEF and MCF10A cells. NHM cells however yielded mainly antagonistic interactions (Supplementary Figure S2d). Upon comparison of cell survival of NHM cells vs head and neck cancers with treatment of reovirus vs the triple therapy, cancer cell lines showed dramatic loss in survival with the triple therapy, while NHM cells saw no changes in survival (Supplementary Figure S3a). Similar results were observed with comparisons in cell survival between cisplatin—paclitaxel doublet treatment vs the triple therapy (Supplementary Figure S3b).

Combinations of reovirus and cisplatin/paclitaxel disrupt the cell cycle

It has been proposed that taxane-induced microtubular stabilisation may increase viral replication through enhanced formation of viral factories.¹⁸ Platins have also been found to be synergistic with reovirus, leading to increased apoptosis.^16,17 To further investigate the effects of cisplatin and paclitaxel in combination with reovirus, cell cycle distribution was determined by fluorescence-activated cell sorting analysis for all four cell lines for the following conditions: untreated control; reovirus infection (Cal27, PJ41 at a multiplicity of infection (MOI) of 5 and Detroit-562, HN5 at an MOI of 50); cisplatin/paclitaxel doublet treatment (1 × IC₅₀ of each agent); and the triple therapy (same dose levels as above) (Figure 2). For these analyses, we were confident that the agents were being used at biologically active levels by virtue of their effects on cell survival and, in the case of paclitaxel, because of their ability to stabilise microtubules (Supplementary Figure S4). Reovirus or chemotherapy treatment caused an S-phase accumulation in all cell lines, largely at the expense of the G1 population, with the G2/M fraction remaining relatively stable. Highest levels of accumulation of cells in the S phase were seen in treatment with the triple combination for Cal27, Detroit-562, HN5 and PJ41 (by an extra 7%, 13%, 44% and 14%, respectively, compared with untreated controls). However, the most striking feature was the emergence of a prominent sub-G1 population—observed for cisplatin/paclitaxel in combination, both with and without reovirus. The emergence of a sub-G1 population was present in all four cell lines, but was substantially more pronounced for Cal27 and Detroit-562. These data suggest that the triple therapy exerts a potent pro-apoptotic effect in head and neck cancer cells (see analysis on cisplatin/paclitaxel and reovirus triple therapy increases apoptosis).

Figure 2.

Reovirus and chemotherapy individually and in combination cause cell cycle perturbations and accumulation of a sub-G1 cell population. Cell cycle distribution was determined by fluorescence-activated cell sorting analysis for all four cell lines for the following conditions: untreated control; reovirus (R) infection (Cal27, PJ41 at an MOI of 5 and Detroit-562, HN5 at an MOI of 50); cisplatin-paclitaxel (C:P) doublet treatment (1 × IC₅₀ of each agent); and R:C:P triple therapy (same dose levels as above). Cell cycle status was assessed after 48 h by flow cytometry using propidium iodide (PI) staining. Data are average of at least three independent experiments and error bars represent standard error of mean (s.e.m.).

Cytotoxic chemotherapy does not enhance reovirus replication in head and neck cancer cells

Having seen an increase in S-phase population with reovirus alone and in combination with chemotherapy, we explored the possibility that this cell cycle perturbation may have resulted in enhanced reoviral production and, hence, cytotoxicity. One-step growth curves were generated for reovirus following infection of Cal27 and Detroit-562 cells as described previously.¹⁵ For both cell lines, there was no alteration in the kinetics of reoviral replication in the presence or absence of chemotherapy (Figure 3a). In both Cal27 and Detroit-562 cells, triple therapy with R:C:P gave marginally lower levels of viral replication, but the highest levels of cell kill (Figure 1a). Replication with and without chemotherapy was further assessed by measuring the copy number of reoviral RNA by real-time reverse-transcriptase polymerase chain reaction (PCR) using SYBR green assay. These assays confirmed the data from the one-step growth curves in showing no evidence of enhanced replication with either single-agent or doublet chemotherapy (Figure 3b and Supplementary Figure S5). Finally, we also stained cells for reovirus by immunocytochemistry on cell pellets (Figure 3c).

Figure 3.

Combined treatment of reovirus with cisplatin and/or paclitaxel does not enhance reovirus replication. (a) A one-step viral growth assay was used to measure replication of reovirus alone (R), and the effects of cisplatin (R:C), paclitaxel (R:P) or both cisplatin-paclitaxel (R:C:P) on viral growth in Cal27 and Detroit-562 cells. Cells were treated with cisplatin and paclitaxel at the IC₅₀ dose and viral titre was determined by TCID₅₀ assay on L929 cells. Data represent the mean of three repeats ± standard error of mean (s.e.m.). (b) Viral transcript production by quantitative real-time reverse-transcriptase PCR from 24 and 48 h samples were assessed in Cal27 and Detroit-562 cell lines, with/without chemotherapy present. Data are representative of at least two independent experiments. (c) Reovirus immunohistochemical staining in Cal27 and Detroit-562 cell lines. Cells were treated with 0.5 × (Cal27) or 0.25 × (Detroit-562), the IC₅₀ of reovirus (R), with/without cisplatin (C) and/or paclitaxel (P), at the respective IC₅₀ doses, and stained with polyclonal anti-reoviral antibody (red). Images shown are a magnification of × 200 and represent an area of 190 × 250μm².

Cisplatin-paclitaxel and reovirus triple therapy increases apoptosis Given the data on cell cycle redistribution and the observation of a sub-G1 population with triple therapy (Figure 2), we evaluated the occurrence of apoptosis in all four cell lines. Cal27, Detroit-562, HN5 and PJ41 cell lines were treated with reovirus and chemotherapy as single agents or in combinations and were assessed by a luminescence-based reporter assay for activated caspase 3/7. The triple therapy caused an increase in apoptosis over and above that seen with either reovirus alone or cisplatin-paclitaxel chemotherapy (Figure 4a and Supplementary Figure S6). Cal27, Detroit-562, HN5 and PJ41 treated with all three agents had significantly higher levels of cleaved 3/7 caspase compared with reovirus alone (P = 0.0008, 0.0012, 0.0025 and 0.0412, respectively) and, in the case of Cal27 and Detroit-562 cells, those treated with all three agents had significantly higher levels compared with chemotherapy (C:P) alone (P = 0.01 and 0.0001, respectively). We confirmed these observations in Cal27 and Detroit-562 cells by western analysis (Figure 4b), with clear evidence of increased ZVAD-reversible caspase 3 and 7 cleavage with the triple therapy. Densitometric analysis was performed and confirmed increased levels of apoptosis with triple therapy (Figure 4c). In addition, we used immunocytochemical staining to look for reovirus and active caspase 3 co-expression using a Nuance system. Reovirus (fluorescent red) and active caspase 3 (fluorescent green) were overlaid and show co-expression (fluorescent yellow), in Cal27 cells after treatment with reovirus and triple therapeutic combinations (Figure 4d).

Figure 4.

Reovirus- and chemotherapy-induced apoptosis is mediated via caspase 3/7. (a) Head and neck cell lines treated with reovirus (R) at 0.5 × IC₅₀ and cisplatin-paclitaxel (C:P) at 1 × IC₅₀, as single agents or in combinations were assessed for caspase 3/7 activation via caspase-glo assay at 24 h. Data represent the mean and standard deviation (s.d.) of at least two independent experiments. P-values were derived, where *P = <0.05, **P = <0.01 and ***P = <0.001. (b and c) Cleaved caspase 3 and caspase 7 in Cal27- and Detroit-562-treated cells were analysed by western blot and densitometry at 24 h. Cells were treated with 50 μM ZVAD 1 h before treatment with reovirus and/or cisplatin-paclitaxel, each at 0.5 ×, their respective IC₅₀ doses. (d) Colocalisation of reovirus with cleaved caspase 3. Immunohistochemical staining of Cal27 cells treated with reovirus (0.5 × IC₅₀) and cisplatin—paclitaxel (1 × IC₅₀) at 48 h to show colocalisation of reovirus with active caspase 3. RGB images show reovirus (red) and active caspase-3 (brown). These images were converted using a nuance system to show reovirus (fluorescent red) and active caspase 3 (fluorescent green), and were overlaid to show co-expression (fluorescent yellow). Images shown represent area of 500 × 400 μm².

Additional analysis of the time course of cell death included an early assessment at 24 h. These data suggest that the triple combination may be capable of causing more rapid cell death (Supplementary Figure S7). However, the data on viral growth kinetics (Figure 3) exclude the possibility that this is due to more rapid viral replication.

Triple therapy is effective in vivo

The therapeutic efficacy of doublet chemotherapy (cisplatin 8 mg kg⁻¹, paclitaxel 5mg kg⁻¹), single-agent reovirus (single intratumoral injection of 1 × 10⁸ tissue culture infectious dose-50 (TCID₅₀)) or the triple combination was compared in Cal27 xenograft tumours in nude mice. Survival rates for the combination treatment were significantly higher compared with reovirus alone (P = 0.02), and also for cisplatin-paclitaxel therapy (P = 0.002), and were accompanied with a delay in tumour growth (reovirus alone vs the triple therapy, P = 0.01) (Figure 5a). Biodistribution studies were also performed in separate tumour-bearing animals. Organs and tumours were harvested 3 days after a single intratumoral injection of reovirus with/without chemotherapy. In a separate experiment, tumours and organs were harvested 25 days post-treatment with three intratumoral injections (days 1, 5 and 8) of reovirus, with/without chemotherapy. Tumours and organs were analysed for the presence of replication-competent virus by TCID₅₀ assay (Figure 5b and Supplementary Figure S8). Tumour and organ samples collected were incubated on L929 cells at 1:10, and liver at 1:1000 (due to the toxicity of the sample on L929 cells), to allow for replication of any virus present in the samples and harvested 7 days later. The presence of reovirus was confirmed in these samples by reverse transcriptase PCR (Figure 5c). Reovirus was recovered from the tumour, spleen and liver of all animals that had received intratumoral injections of reovirus at day 3. The highest titres were seen in the tumours (up to 1.1 × 10⁸ TCID₅₀ per g). At 25 days, reovirus was still detectable in the tumour (with titres of up to 2.9 × 10⁶ TCID₅₀ per g). Normal organ distribution of reovirus was limited to the spleen and liver for animals treated with reovirus alone at day 3. In animals treated with reovirus combined with either single-agent or doublet chemotherapy, virus was also recovered from the heart and lung (although at very low levels). By day 25, this difference was no longer evident. There was no evidence of increased weight loss in mice treated with triple therapy compared with controls or animals that received either chemotherapy or reovirus alone (Supplementary Figure S9).

Figure 5.

Combined treatment with reovirus and chemotherapy is significantly more effective than either alone in vivo. (a) Cal27 tumours received a single injection of reovirus (R) intratumorally (1 × 10⁸ TCID₅₀), cisplatin (C) and paclitaxel (P) both injected intraperitoneally (8 and 5 mg kg⁻¹, respectively) or the combination treatment on day 1. Data show survival rates and tumour volumes measured twice-weekly throughout treatment. (b) Organs and tumours were harvested at 3 days after treatment for analysis of functional virus by TCID₅₀ assay. The experiment was repeated where mice received three injections of reovirus (days 1, 5 and 8), with chemotherapy given on day 1, and organs and tumours were harvested 25 days after treatment for analysis of functional virus by TCID₅₀ assay. (c) Tumour and organ samples collected were incubated on L929 cells at 1:10, and 1:1000 (liver) to allow for replication of any virus present and harvested 7 days later. These samples were then analysed for reoviral RNA by reverse-transcriptase-PCR. Data represent an animal from each treatment group (showing most bands positive for reoviral RNA). A 1:10 dilution of reovirus or virus-free media were incubated on L929 cells and harvested as a control, as well as a reoviral RNA-positive and H₂O-negative control used for the reverse-transcriptase-PCR reaction. A blood sample was not available for one animal treated with the triple therapy as indicated. (d) To confirm on-target effect of the agents, Cal27 tumours were injected with reovirus, with or without cisplatin-paclitaxel, and were subsequently harvested at 24,48 and 168 h post-treatment for analysis of caspase 3 cleavage by western blot.

For pharmacodynamic studies, Cal27 tumours were harvested from mice at 24 h, 48 h and 1 week post-treatment with a single dose of reovirus and/or chemotherapy on day 1, for western blotting analysis for cleaved caspase 3. Compared with untreated controls and animals that received cytotoxic chemotherapy without reovirus, significant levels of caspase 3 cleavage were seen at up to 7 days in tumours from animals treated with reovirus alone or reovirus plus doublet chemotherapy (Figure 5d). Cal27 tumours 1 week post-treatment were stained for reovirus. Tumours treated with the triple therapy appeared to show increased reoviral intratumoral spread compared with tumours treated with reovirus alone (Supplementary Figure S10).

DISCUSSION

Preclinical studies have confirmed that oncolytic reovirus is active in vitro and in vivo as a highly selective anticancer agent with significant potential for clinical translation in a broad range of tumour types.^1,2 Indeed, this agent has already been assessed in single-agent phase I studies in patients with relapsed or treatment-refractory cancers.^3,12–14 Those studies demonstrated that local and systemic administration of reovirus is safe and tolerable, even in heavily pretreated patients. However, as a single-agent therapy reovirus has shown only modest response rates. In patients with histologically confirmed recurrent malignant gliomas, Forsyth et al.¹² reported that 1 of 12 patients achieved stable disease following intratumoral injections of reovirus. Vidal et al.¹³ treated 33 patients with intravenous reovirus and reported eight patients with minor response or stable disease by the RECIST criteria. Three patients showed evidence of a response in tumour markers.¹³ In a similar study design using intravenous reovirus in 18 patients, Gollamudi et al.¹⁴ reported one radiologically confirmed partial response and seven patients with stable disease.¹⁴ The results of these single-agent clinical studies are reassuring in that none has demonstrated a dose-limiting toxicity at doses up to 3 × 10¹⁰ TCID₅₀ per day for 5 consecutive days every 4 weeks.

In parallel with studies of single-agent reoviral therapy, a number of studies have assessed potentially advantageous interactions between the virus and standard anticancer therapies. In particular, studies have focused on commonly used cytotoxic chemotherapy drugs. Sei et al.¹⁶ evaluated reovirus in combination with cisplatin, gemcitabine, vinblastine or paclitaxel in non-small-cell lung cancer cell lines and demonstrated synergistic interactions between the cytotoxic agents and reovirus. Importantly, the paclitaxel-reovirus combination was synergistic in all cell lines, regardless of their levels of sensitivity to either agent. Subsequent preclinical studies by our group have confirmed synergistic killing of reovirus plus cisplatin in malignant melanoma¹⁷ and reovirus plus docetaxel in prostate cancer.¹⁸

These previous studies provided the rationale for a programme of early phase clinical trials in which systemic administration of reovirus was combined with cytotoxic chemotherapy. In all of these studies, the aim was to administer standard doses of the cytotoxic agent and to escalate the virus dose up to levels that were reached in the phase I single-agent studies of systemic viral administration. Phase I dose escalation studies were successfully completed with reovirus combined with docetaxel,¹⁹ gemcitabine,²⁰ and carboplatin and paclitaxel.²²

Our preclinical analysis provides clear proof of principle for combining reovirus with platin/taxane doublet chemotherapy in head and neck cancer. Reovirus shows synergistic cell killing with cisplatin and paclitaxel either as single agents or in combination as a chemotherapy doublet in head and neck cancer cells (Figures 1a–c). Indeed, these data could be used to support clinical development of reovirus plus single-agent platin or reovirus plus single-agent taxane regimens. However, this would be out of step with normal clinical practice in this disease where a platin—taxane doublet is frequently used in the second-line setting. Importantly, CI analysis also revealed synergistic activity of the triple combination in two non-malignant cell lines, although these data were derived at relatively high virus and cytotoxic drug doses (as dictated by the IC₅₀ values derived in single-agent studies), but not in a normal human mesothelial cell line. Such data demonstrate that clinical use of oncolytic reovirus in association with cytotoxic chemotherapy may be associated with exacerbation of normal tissue toxicities and this consideration will mandate careful phase I dose escalation studies with appropriate toxicity end points for all new combination approaches. Reassuringly, our recently published phase I/II study of reovirus in combination with carboplatin/paclitaxel doublet chemotherapy did not report dose-limiting normal tissue toxicity during the dose-escalation phase,²² suggesting that the apparent synergistic cytotoxicity in MEF and MCF10A cells seen in this study may not translate to the clinic.

Interestingly, the use of platin- or taxane-based chemotherapy neither inhibits nor enhances viral replication in infected tumour cells (Figures 3a–c). Critically, it appears that the dominant process underlying the increased activity of the reovirus/platin/taxane combination is an enhancement of apoptosis of cells exposed to drug—virus combination therapy (Figures 2 and 4a–c. In vivo studies using intratumoral injections of reovirus confirmed that the platin/taxane doublet chemotherapy enhanced the efficacy of the virus, resulting in a significant reduction in tumour growth, a significant increase in survival and in vivo evidence of caspase cleavage (Figures 5a and d). We also analysed the effect of the various treatment combinations on viral biodistribution in the nude mouse model (Figures 5b and c). At an early time point (3 days), reovirus was recovered from a wider range of normal organs in animals that were co-treated with chemotherapy. However, this effect was no longer evident at 25 days. It is important to note that in a clinical setting, the antiviral immune response in off-target organs is likely to be stronger than that observed in the immunocompromised animal model used in these studies. Significantly, there was no evidence of increased toxicity in animals treated with virus and cytotoxic chemotherapy.

The phase I study with reovirus in combination with carboplatin and paclitaxel provided a strong signal of therapeutic efficacy in patients with relapsed/metastatic head and neck cancer, which was confirmed in a subsequent phase II trial.²² As a result, a randomized, double-blind phase III trial of carboplatin/paclitaxel plus either reovirus or placebo in patients with platin-refractory head and neck cancer is now recruiting. The results of this study give some indication of the potential value of this clinical approach in that synergistic interaction was seen between reovirus and the doublet chemotherapy, even in cell lines that were relatively resistant to platinum in vitro.

In summary, we have shown that the combination of oncolytic reovirus with doublet chemotherapy is potently and synergistically active against head and neck cancer cell lines and provide strong supportive data for the further development of this approach in the clinic.

MATERIALS AND METHODS

Cell lines

We have previously characterised the sensitivity of a panel of 15 head and neck cancer cell lines to the effects of reovirus (Twigger et al.,²³ in press). From this panel, we chose representative cell lines with a range of sensitivities to reovirus. Cal27, Detroit-562, HN5, PJ41 (head and neck cancer; from Dr S Eccles, Institute of Cancer Research, London, UK), MEF (from Professor C Marshall, Institute of Cancer Research) and L929 (mouse fibroblast; Oncolytics Biotech Inc., Calgary, AB, Canada) were cultured in Dulbecco’s modified Eagle’s medium. Media were supplemented with 5% (v/v) fetal calf serum, 1% (v/v) glutamine and 0.5% (v/v) penicillin/streptomycin. MCF10A (non-tumorigenic epithelial cell line, from Professor Alan Ashworth, Institute of Cancer Research) were cultured in mammary epithelial basal medium supplemented with insulin, hydrocortisone, recombinant human epidermal growth factor, bovine pituitary extract (as per kit CC-3150; Lonza/Clonetics Corporation, Basel, Switzerland), 100 ng ml⁻¹ cholera toxin (C8052; Sigma-Aldrich, Dorset, UK) with 5% (v/v) horse serum, 1% (v/v) glutamine and 0.5% (v/v) penicillin/streptomycin. NHMs (from Professor Richard Marais, The Institute of Cancer Research) were cultured in medium 254 supplemented with human melanocyte growth supplement (Life Technologies, Paisley, Scotland), 1% (v/v) glutamine and 0.5% (v/v) penicillin/streptomycin.

Reovirus stocks

Reovirus (Dearing type 3) stocks at 3.2 × 10⁹ TCID₅₀ per ml were obtained from Oncolytics Biotech Inc. and stored in the dark at − 80 °C. Neat stocks were in phosphate-buffered saline (PBS) and 1:10 concentrations in Dulbecco’s modified Eagle’s medium containing 2% (v/v) fetal calf serum, 1% (v/v) glutamine and 0.5% (v/v) penicillin/streptomycin (plating media). New stocks of 1:10 concentrations were made and titred by TCID₅₀ assay on reovirus-susceptible L929 cells.

Cell survival experiments

Cells were plated at 5 × 10³ cells per well in 96-well plates and incubated at 37 °C for 24 h before treatment with reovirus, cisplatin (Sigma-Aldrich) and paclitaxel (Sigma-Aldrich). Cell survival was measured 96 h later by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. IC₅₀ values were interpolated from a sigmoidal dose-response curve fit of the log-transformed survival data.

CI analysis

Cells were plated at 5 × 10³ cells per well in 96-well plates and treated the following day at 4 ×, 2 ×, 1 ×, 0.5 × and 0.25 × IC₅₀ doses (Supplementary Figure S1) alone and in combination with the other agents. Cell survival was assessed 96 h later by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The effect of reovirus in combination with chemotherapeutic agents was assessed by the method of Chou and Talalay.²⁴ CI values were generated using the CalcuSyn software (Biosoft, Cambridge, UK).

Fluorescence-activated cell sorting analysis of cell cycle distribution

Cells were plated at 2 × 10⁶ in T75 flasks and treated the following day. After 48 h, cells were washed three times with PBS, resuspended in 200 μl PBS/0.1% fetal calf serum and 5 ml of ice-cold 70% ethanol was added dropwise while vortexing. Cells were stored at 4°C for >1 h and then 1 × 10⁶ cells were pelleted and washed a further three times with PBS, treated with 100 μg RNase A (Sigma-Aldrich), stained with 40 μg propidium iodide (Sigma-Aldrich) and incubated for 30 min before analysis by flow cytometry.

One-step viral growth assays

Cells were plated at 1 × 10⁵ in 24-well plates and treated the following day with reovirus at an MOI of 5 (to ensure infection of all cells), either alone or in combinations with chemotherapy treated at IC₅₀ dose. After 2 h incubation at 37 °C, cells were washed twice with media and plating media were added to each well and chemotherapy was replaced. At 4, 24 and 48 h after infection, cells were scraped into the media and the lysate was subjected to 3 × freeze thawing between − 80 and 37 °C, centrifuged at 13 000 r.p.m. for 5 min and stored at − 80 °C. The supernatant was used to titre for reovirus by TCID₅₀ assay on L929 cells using serial twofold dilutions.

Analysis of reoviral transcripts by real-time reverse-transcriptase-PCR

RNA was extracted from the 24 and 48 h samples from one-step viral growth experiments (QIAmp viral RNA mini kit; Qiagen, West Sussex, UK) and converted to first-strand cDNA (Omniscript Reverse Transcription; Qiagen). Samples were amplified against reoviral transcripts by PCR with SYBR Green (FastStart DNA Master^PLUS SYBR Green I; Roche, West Sussex, UK) using a 2.0 Lightcycler carousel-based system (Roche).

Caspase 3/7 analysis by caspase-glo assay

Cells were plated at 5 × 10³ cells per well in 96-well plates and treated the next day with reovirus and chemotherapy. At 24 h post-treatment, caspase 3/7 activity was measured by the luminescence-based reporter assay Caspase-Glo 3/7 (Promega, Southampton, UK) following the protocol stated by the manufacturer. As a control, Jurkat cells were treated with 10μM camptothecin for 4 h to induce apoptosis.

Western blotting for caspase 3 in vitro

Cells were plated at 1 × 10⁶ in 60 mm dishes. Cells were treated the following day with 50 μM ZVAD, a pan-caspase inhibitor (R&D Systems, Abington, UK), 1 h before treatment with reovirus and chemotherapy. At 24 h post-treatment, cells were harvested for western blotting. Samples were probed with rabbit anti-caspase 3 (Cell Signalling Technology, Denver, MA, USA) and murine anti-g-tubulin (Sigma-Aldrich) for loading controls. Densitometry of western blots were analysed using ImageJ software.

Immunohistochemical staining for reovirus and caspase 3

Cells were seeded at 1 × 10⁶ in 10cm dishes, treated the following day with reovirus and chemotherapy and were fixed with 10% formalin at room temperature for 4–8 h, and thereafter harvested and centrifuged at 1200 r.p.m. for 5 min. The remaining pellet was resuspended in sterile diethylpyrocarbonate water before analysis by immunohistochemistry staining for reovirus (polyclonal anti-reovirus antibody from Matt Coffey, Oncolytics Biotech Inc.) and anti-active caspase 3 (ab2302; Abcam, San Francisco, CA, USA). A nuance system was used (Cambridge Research Institute, Cambridge, UK) for co-expression data.

In vivo studies

Cal27 tumours were established in female CD1 nude mice (Charles Rivers, Kent, UK) by subcutaneous injection of 3 × 10⁶ cells suspended in PBS in the right flank. Once cells had reached ~5 mm in diameter, mice were allocated treatment groups stratified by tumour size. Mice that received reovirus were injected intratumorally with 1 × 10⁸ TCID₅₀ per ml of reovirus suspended in PBS. Mice that were treated with cytotoxic chemotherapy received cisplatin (injected intraperitoneally at 8 mg kg⁻¹) and/or paclitaxel (5 mg kg⁻¹ intraperitoneally) (both from TEVA, Castleford, UK, obtained from the Royal Marsden Hospital Pharmacy, Surrey, UK). In animals, in groups that were not scheduled to receive reovirus and/or chemotherapy, sham injections of PBS were administered. Tumour volumes were measured at least twice-weekly using Vernier calipers and the tumour volume was estimated from the formula: V= 05 × (length × width²). All experiments were carried out in compliance with the NCRI guidelines, with animals judged to have failed treatment if ulceration occurred or if tumour diameter exceeded 15 mm (humane survival end point).

Organs and tumours harvested for biodistribution analysis were collected by dissection, homogenised with 600 μl of serum-free Dulbecco’s modified Eagle’s medium and spun down at 3600 r.p.m. for 5 min. The supernatant was titred for reovirus by TCID₅₀ assay on L929 cells. Supernatants were also incubated on L929 cells for 7 days to allow for replication of any virus present. These samples were harvested and RNA extracted as before and amplified by reverse-transcriptase-PCR (One Step RT-PCR kit; Qiagen). DNA was visualised using DNA gel electrophoresis.

For in vivo analysis of apoptosis, tumours were harvested from mice 24 and 48 h after treatment. Tumours were snap frozen and homogenised for two cycles of 10s in RIPA buffer. Samples were then centrifuged at 13000 r.p.m. for 15 min at 4 °C and the supernatant transferred to a fresh tube for analysis by western blot for cleaved caspase 3.

Tumours harvested for immunohistochemistry staining were fixed in 10% formalin for 24h and then transferred into PBS. Tumours were processed and embedded in wax before staining for reovirus (polyclonal anti-reovirus antibody from Matt Coffey, Oncolytics Biotech Inc.) and counterstained with hematoxylin.

Statistical analysis

T-tests were used to make comparisons between groups. Survival curves were compared using the Kaplan—Meier method and significance was assessed using the χ² test. P-values were derived, where *P = < 0.05, **P = < 0.01, ***P = < 0.001.