Chemovirotherapeutic Treatment Using Camptothecin Enhances Oncolytic Measles Virus-Mediated Killing of Breast Cancer Cells

Oncolytic virotherapy represents an emerging development in anticancer therapy. Although it has been tested against a variety of cancers, including breast cancer, the efficacy of oncolytic viral vectors delivered as a monotherapy is limited. Enhancing viral oncolytic therapies through combination treatment with anticancer agents is a feasible strategy. In this study, we considered a chemovirotherapeutic approach for treating breast adenocarcinoma using oncolytic measles virus (MV) and the chemotherapeutic agent camptothecin (CPT). Our results demonstrated that co-treatment of MV with CPT yielded enhanced cytotoxicity against breast cancer cells. Low dosage CPT combined with MV was also found to elicit the same therapeutic effect as high doses of CPT. At the lower dosage used, CPT did not inhibit the early stages of MV entry, nor reduce viral replication. Further studies revealed that co-treatment induced significantly enhanced apoptosis of the breast cancer cells compared to either MV or CPT alone. Overall, our findings demonstrate the potential value of MV plus CPT as a novel chemovirotherapeutic treatment against breast cancer and as a strategy to enhance MV oncolytic activity.


Synergistic effect of MV plus CPT treatment against breast cancer cells. Cells seeded in 96-well
plates (10 4 cells per well) were studied in three different experiments of MV plus CPT administration. The first tested for 'drug sensitization' with the addition of CPT to cells at different concentrations (10,30, and 50 nM) for 2 days, prior to the infection of cells with MV at a MOI 0.1 for 3 days. The second experiment consisted of a 'viral sensitization' treatment, where cells were first infected with MV at MOI 0.1 for 2 days, followed by CPT treatment at varying concentrations (10,30, and 50 nM) for 3 days. Finally, the third experiment consisted of a 'co-treatment' group, where the cells were infected with MV at MOI 0.1 and at the same time, treated with varying concentrations of CPT (10,30, and 50 nM) for a total of 5 days. For all experiments, MV infection was performed for 1.5 h at 37 °C and cells were washed with phosphate buffered saline (PBS) before and after viral challenge. Efficacies of the different modes of treatment were then evaluated by determining cell survival with the MTT assay as described above in the 'Cell viability assay' . Combination index (CI) values were calculated using the Chou-Talalay method 28 to quantitatively deduce synergistic, additive or antagonistic effects of MV plus CPT.
Effect of CPT on free oncolytic MV particles. The assay is performed as reported elsewhere 29 with some modifications. Cell-free virus particles were incubated with CPT at different concentrations at 37 °C in 5% CO 2 for 3 h. The virus-drug mixture was then diluted 20-fold with 2% FBS DMEM to effective concentrations of CPT, after which the inoculum was added to MCF-7 cells seeded in 96-well plates (10 4 cells per well; final MOI 0.1). The cells were incubated at 37 °C in 5% CO 2 for 3 days. After 3 days, the plates were scanned and analyzed for viral www.nature.com/scientificreports www.nature.com/scientificreports/ reporter fluorescence using the Typhoon 9410 variable mode imager (Amersham Biosciences; Baie d'Urfe, QC, Canada) to evaluate MV-EGFP infections. Fluorescence intensity was quantified using Image Quant TL software (Amersham Biosciences).
Effect of CPT on MV attachment. The assay is performed as described before 29 with some modifications. MCF-7 cells were co-treated with MV (MOI 0.1) and varying concentrations of CPT (10,30, and 50 nM) at 4 °C for 1.5 h. Subsequently, the virus-drug inocula were removed, and the cells were washed with PBS. Fresh culture medium was added to the cells, which were then incubated at 37 °C for 3 days. EGFP fluorescence intensities were measured as described above.
Effect of CPT on MV penetration. The experiment was performed as previously reported 29 with some modifications. MCF-7 cells were infected with MV (MOI 0.1) and incubated at 4 °C for 1.5 h. Following which, viral inocula were removed and cells were treated with varying concentrations of CPT (10, 30, and 50 nM) and shifting the incubation to 37 °C for 1.5 h. CPT was subsequently removed and the cells were washed with PBS before adding fresh culture medium. After further incubation at 37 °C for 3 days, viral EGFP signal intensities were detected and analyzed as previously described.
time-of-drug-addition assays. Pre-treatment, co-addition, and post-infection CPT drug treatments were performed as previously described 27 to determine potential antiviral effect of CPT on oncolytic MV infection at different time-points. For the pre-treatment group, MCF-7 cells seeded in 96-well plates (10 4 cells per well) were treated with CPT (10, 30, and 50 nM) for 24 h and washed before infection with MV (MOI 0.1) for 48 h. For the co-addition treatment group, MCF-7 cells seeded in 96-well plates (10 4 cells per well) were treated with virus-drug inocula containing both MV (MOI 0.1) and varying concentrations of CPT (10, 30, and 50 nM) simultaneously for 1.5 h before the wells were washed and refreshed with complete culture medium for 3 days of incubation. For the post-infection treatment group, MCF-7 cells seeded in 96-well plates (10 4 cells per well) were first infected with MV (MOI 0.1) for 1.5 h, washed, and then immediately treated with varying concentrations of CPT (10, 30, and 50 nM) for 3 days. For all three treatment groups at the end-point of the experiments (72 h), the viral reporter fluorescence was measured using the Typhoon 9410 variable mode imager and the data obtained was analyzed using Image Quant TL software. For readouts based on viral titer in the above time-of-drug-addition experiments, MCF-7 cells were seeded in 12-well plates (2 × 10 5 cells per well) and were similarly treated with CPT (10, 30, and 50 nM) as above, either 24 h prior to, concurrently to (0 h), or right after (1.5 h) the MV infection (MOI 0.1); wash steps were included as described earlier. Supernatant was harvested from each well at 3 days post-infection for determination of viral titer by TCID 50 analysis.
Cell cycle analysis. MCF-7 cells seeded in 6-well plates (3 × 10 5 cells per well) were grown overnight, before being subjected to co-treatment with MV (MOI 0.1) and varying concentrations of CPT (10, 30, and 50 nM) at 37 °C for 1.5 h. Subsequently, the virus-drug inocula were discarded and replaced with fresh culture medium containing the respective concentrations of CPT for 5 days incubation at 37 °C. At the end of the experiment, the cells were collected by trypsinization and transferred into 15 ml tubes, where they were washed with ice-cold PBS and then fixed overnight in 70% ethanol at 4 °C. Following fixation, the cells were washed twice with PBS and incubated at 37 °C for 30 min in PBS solution containing 10 mg/ml ribonuclease A from bovine pancreas (RNase A; Sigma-Aldrich). Propidium iodide (PI; Sigma-Aldrich; 40 µg/ml) was then added to the cells before incubation at 37 °C in the dark for 15 min, and cell cycle analysis was performed using the Beckman coulter FC500 flow cytometer (Beckman Coulter Inc.; Brea, CA, USA).
Apoptosis analysis using propidium iodide and Annexin V staining. For apoptosis analysis using PI and Annexin V, MCF-7 cells were seeded in 6-well plates (3 × 10 5 cells per well) and co-treated with MV (MOI 0.1) and CPT (10, 30, and 50 nM). At the end of the 5 days incubation period, the cells were collected by trypsinization, transferred into 15 ml tubes, and washed with ice-cold PBS. After washing, the cells were resuspended in binding buffer containing 1 µl/ml PI and 1 µl/ml Annexin V APC-conjugated (Enzo Life Sciences, Inc; East Farmingdale, NY, USA). Apoptosis analysis, which was based upon the cell surface exposure of phosphatidyl serine that binds to Annexin V, was performed using the Beckman coulter FC500 flow cytometer (Beckman Coulter Inc.).
Western immunoblot analysis. MCF-7 cells seeded in 6-well plates (3 × 10 5 per well) or 6 cm dishes (8 × 10 5 cells) were grown overnight, before being subjected to co-treatment with MV (MOI 0.1) and varying concentrations of CPT (10, 30, and 50 nM) at 37 °C for 1.5 h. Following the treatment, the virus-drug inocula were discarded and replaced with fresh complete culture medium containing the respective concentrations of CPT for 5 days incubation at 37 °C. At the end of the experiment, the MCF-7 cells were harvested and lysed using RIPA buffer containing protease inhibitors (Roche Molecular Biochemicals; Indianapolis, IN, USA). Protein sample concentrations were then determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc.; San Jose, CA, USA). Proteins were resolved by subjecting the lysates to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membrane for western immunoblot analysis. The membrane was first blocked with 5% non-fat milk prepared in Tris-buffered saline with 0.1% Tween ® 20 (TBST), before probing target proteins with primary antibodies at appropriate dilutions: poly (ADP-ribose) polymerase (PARP) antibody (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) and β-actin antibody (1:10000; Cell Signaling Technology, Inc.). Antibody binding was detected by incubating the blot with horseradish peroxidase (HRP)-conjugated secondary antibody (GIBCO-Invitrogen) followed by treatment with the HRP chemiluminescent substrate (Millipore).
www.nature.com/scientificreports www.nature.com/scientificreports/ Chemiluminescence was measured using the UVP BioSpectrum 500 imaging system (UVP; Upland, CA, USA). Protein signals were quantified and compared against the β-actin loading control using densitometry analysis. statistical analysis. All data are expressed as means ± standard error of the means (SEM). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison. P < 0.05 was considered statistically significant. All assays were conducted with at least three independent experiments.

Efficacy of cell killing of MCF-7 breast cancer cells by oncolytic MV and CPT as separate agents.
We first evaluated the ability of CPT and oncolytic MV to kill breast cancer cells as separate agents. For this purpose, we used various concentrations of CPT or different MOIs of a recombinant wild-type-based MV containing and EGFP reporter gene to treat and infect MCF-7 breast adenocarcinoma cells. The MCF-7 cells express Nectin-4/PVRL4 over their cell surfaces and are highly susceptible to MV infection 12 . Both CPT (Fig. 1A) and oncolytic MV (Fig. 1B) decreased cell viability of MCF-7 in a dose-dependent manner after either 3 or 5 days of treatment. At concentrations > 50 nM, CPT could reduce cell viability by over 50% and its CC 50 values were determined to be 381 nM and 58.5 nM for 3 and 5 days of treatment, respectively (Fig. 1A). On the other hand, infection with oncolytic MV at a MOI > 0.1 could reduce MCF-7 cell viability by 50% or more (Fig. 1B). Similar results were obtained using lactate dehydrogenase (LDH) release cytotoxicity detection assay ( Supplementary  Fig. S1). Based on these results, concentrations of CPT (10, 30, and 50 nM) and oncolytic MV (MOI 0.1) were chosen for subsequent experiments designed to determine their synergistic effects in combination treatments.
Combination treatments with oncolytic MV and CPT exert enhanced killing of human MCF-7 breast cancer cells. In order to explore the effects of combination therapy with oncolytic MV and CPT, we examined three different modes of treatment (schematically represented in Fig. 2A). These consisted of 'drug sensitization' (pre-treatment of cells with CPT prior to MV infection), 'viral sensitization' (pre-infection first with MV, followed by CPT treatment), and 'co-treatment' (concurrent addition of oncolytic MV and CPT) to determine the most effective treatment regimen. The data from each combination model were assessed by the www.nature.com/scientificreports www.nature.com/scientificreports/ Chou-Talalay method 28 for level of synergy, where the CI value quantitatively defines synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1). CPT was administered at 10 nM (Fig. 2B), 30 nM (Fig. 2C), or 50 nM (Fig. 2D) in combination with MV (MOI 0.1) and cytotoxicity was measured by MTT assay. Our results showed The CI value of combination models were measured by Chou-Talalay method where CI value quantitatively defines synergism (CI < 1), additive effect (CI = 1) and antagonism (CI > 1). All data shown are means ± SEM (*P < 0.05 compared to MV treatment, and # P < 0.05 compared to CPT treatment) from three independent experiments. www.nature.com/scientificreports www.nature.com/scientificreports/ that the co-treatment model yielded the greatest cytotoxicity against MCF-7 cells, at all three concentrations (10, 30, and 50 nM) of CPT tested. This was evidenced by the reduction in MCF-7 cell viability to 44.45%, 34.87%, and 30.54% by MV plus 10, 30, or 50 nM CPT co-treatment, respectively ( Fig. 2B-D). The percentage of cell death with co-treatment was 20 ~ 30% higher as compared to either MV or CPT treatment alone. Similar results were obtained using the LDH release detection assay ( Supplementary Fig. S2A), and enhanced cytotoxic effect was noted by microscopy ( Supplementary Fig. S2B). Consistently, the lowest CI values (0.11, 0.21, and 0.31) were obtained for 10, 30, and 50 nM CPT co-treatment (Fig. 2E), respectively, suggesting that the MV and CPT co-treatment model exhibits the strongest synergistic oncolytic activity against MCF-7 cells. On the other hand, in both drug sensitization and viral sensitization models, the CI values reflected antagonism, regardless of the CPT concentrations being tested (Fig. 2B-E).

Cpt treatment does not influence the early viral stages of oncolytic MV nor exert antiviral activity against MV infection.
In order to examine potential antagonistic interaction(s) between co-administered CPT and MV, we first investigated whether CPT interferes with the early viral entry steps of MV infection of MCF-7 breast cancer cells. More precisely, we assessed the impact of CPT treatment on free oncolytic MV particles, its attachment to the host cells, and the post-binding viral entry/fusion steps during penetration of the cell (Fig. 3A). CPT was added to the virus and/or cells at specific time-points and viral EGFP reporter fluorescence was measured following incubation with the cells. As shown in Fig. 3B-D, CPT treatments at all doses tested did not produce significant inhibition during the early stages of virus infection. On the other hand, the positive control, punicalagin (PUG), a small molecule known to block MV entry stages 26 , effectively reduced MV infectivity. These results suggest that CPT does not inactivate cell-free MV particles or affect the early entry steps of MV infection in breast cancer cells. To rule out other potential antiviral effects, we used CPT to treat MCF-7 cells prior, during, and after infection with the oncolytic MV (Fig. 4A). Interferon-α (IFN-α) was included as control. As depicted in Fig. 4, pre-treatment of the breast cancer cells with CPT (Fig. 4B), concurrent addition of CPT to the MV inoculation (Fig. 4C), and post-infection CPT treatment (Fig. 4D) had minimal impact on the MV infectivity. Likewise, CPT treatment at any time-point from 24 h prior to and immediately after the MV infection did not cause significant difference on the resulting viral titers at the end-point of the experiment (Fig. 4E).

Oncolytic MV and CPT combinatorial treatment causes sub-G1 cell cycle arrest and induction of apoptosis in human MCF-7 breast cancer cells.
To explore the underlying mechanism of the synergistic oncolytic effect elicited by co-treatment with MV plus CPT, we analyzed how the MCF-7 cell cycle was affected by the 2 agents using flow cytometry. Our results indicated that the oncolytic MV alone increased the sub-G1 cellular population to 25% as compared to untreated cells, while CPT alone dose-dependently induced www.nature.com/scientificreports www.nature.com/scientificreports/ G2/M cell cycle arrest at the three concentrations (10,30, and 50 nM) tested (Fig. 5A). In contrast, as compared to treatment with each agent alone, the combination of MV and CPT treatment substantially increased the sub-G1 population at the three CPT doses used (10, 30, and 50 nM), together with corresponding reductions in G1 and G2 populations (Fig. 5A). The observed increase in sub-G1 population suggested that there was an increase in apoptotic cells. This premise was substantiated by flow cytometry using the Annexin V/PI double staining assay. We observed a shift in the number of apoptotic cells from about 25% with MV alone and 13% with CPT alone to 40% ~ 60% when both agents were used in combination (Fig. 5B). Finally, to further confirm that apoptosis was enhanced by co-treatment of MCF7 cells with MV and CPT, we analyzed PARP cleavage using Western blot analysis. Consistent with the Annexin V/PI analysis, levels of cleaved PARP, which are also indicative of apoptosis, were found to substantially increase in MCF-7 cells that were treated with a combination of MV and CPT (Fig. 6A). A dose-dependent increase in PARP cleavage was observed over the three different CPT concentrations (10,30, and 50 nM) following densitometry analysis of the cleaved products (Fig. 6B). Taken together, our results suggest that the combination treatment consisting of oncolytic MV with CPT produces a synergistic oncolytic effect against human breast cancer cells, and this enhanced efficacy is mediated by increased levels of apoptosis.

Combination of oncolytic MV and CPT exerts similar enhanced killing effect on T-47D breast cancer cells.
Finally, to examine whether the combined treatment of oncolytic MV and CPT was effective against another breast cancer cell line, we also tested their influence as separate agents or in combination on the human breast cancer T-47D cells using the same methods and co-treatment model described in Figs 1 and 2, respectively. The T-47D cells are permissive to MV infection 12 , and were observed to be sensitive to a dose-dependent cytotoxic effect induced by CPT (10-50 nM) and MV (MOI 0.01-10) as separate agents, with 50 nM CPT and MV at MOI of 0.1 being near or below the 50% cell viability threshold (Fig. 7A,B). More importantly, the combined treatment of CPT (10, 30, and 50 nM) and MV (MOI 0.1) using the co-treatment model generated significantly more cell death compared to each agent alone (Fig. 7C). Comparable results were obtained between the MTT analysis (Fig. 7) and the LDH release detection assay (Supplementary Fig. S3).
Altogether, our results demonstrated that CPT and oncolytic MV can be used in combination using a co-treatment approach, and that the combined treatment induces enhanced cell death in the breast cancer cells. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In the current management of breast cancer, chemotherapy plays a major role in treating patients with larger tumor burdens, lymph node invasion, or recurrent/metastatic breast cancer 30 . The most commonly used chemotherapeutics for treating breast cancer include anthracyclines and taxanes, which are characterized by adverse effects such as cardiotoxicity 31 , neurotoxicity, alopecia 32 , and bone marrow suppression 31,32 . Oncolytic virotherapy using MV is potentially an effective strategy specifically targets tumor cells, but can also facilitate the detection and elimination of micro-and macrometastases 3 .
To overcome the limitation of viral oncolytics as a monotherapy, the combination of oncolytic virus and chemotherapeutic agents has been explored. This approach was explored based on the premise that the two treatment modalities may enhance or synergize with each other 33,34 , improving the outcome of cancer treatment. However, compatibility and synergism of the two types of treatment could be context-dependent 35 . To date, very few chemotherapeutics have been investigated in combination with MV therapy besides cyclophosphamide (CPA) 36,37 and doxorubicin 38 . Our finding that co-treatment of CPT can boost MV efficacy adds to the list of therapeutic strategies that enhance MV oncolytics. Some other studies had explored the use of prodrug convertase-encoding MV to catalyze the conversion of chemotherapeutic prodrugs 15 . These include arming MV with purine nucleoside phosphorylase (MV-PNP) [39][40][41][42] , which converts fludarabine and 6-methylpurine-2′-deoxyriboside (MeP-dR) into 2-fluoroadenine and 6-methylpurine, respectively, or super cytosine deaminase (MV-SCD) 43-47 that converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). In the case of CPT, its clinically available analog irinotecan serves as a prodrug and is converted in the liver into the active metabolite and topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin (SN-38), which circulate to kill the cancerous cells 48 . Eventually, the majority of SN-38 is transformed to its inactive metabolite SN-38 glucuronide (SN-38G) and released into the intestinal lumen for elimination, where bacterial beta-glucuronidase could regenerate SN-38 and results in the side effect of diarrhea 49 . Huang et al. has demonstrated that the combination of intratumoral injection of adenovirus expressing beta-glucuronidase and intravenous injection of irinotecan significantly enhanced the in vivo antitumor activity as compared to single-agent treatment 50 . This might indicate the potential use of beta-glucuronidase-expressing MV vector in combination with CPT or its derivatives as future strategies.
Given the limited expression of Nectin-4/PVRL4 in normal tissues including the skin, hair follicles, trachea, and lung 51,52 , but elevated expression in many adenocarcinomas such as breast, lung, bladder, pancreatic, and ovarian cancers 8,10,11,53,54 , Nectin-4/PVRL4 has emerged as an important tumor marker and therapeutic target. In breast cancer, it is a hallmark of advanced stage or highly metastatic cancer 11,55 and promotes cell survival and proliferation by stimulating the c-Src kinase pathway 56 . In addition, a soluble form of Nectin-4/PVRL4 is present in the sera of breast and lung cancer patients 8,55 , which could have diagnostic applications for the www.nature.com/scientificreports www.nature.com/scientificreports/ screening of these cancers. Nectin-4/PVRL4 has also been proposed as a therapeutic target of primary and metastatic triple-negative breast cancers, as well as of lung, bladder, and pancreatic cancers which could potentially be treated with Nectin-4/PVRL4 antibodies conjugated to anti-neoplastic agents 57,58 . Compared to vaccine strain, the wild-type MV is more specific to Nectin-4/PVRL4 since it does not engage CD46 14 . The above reasons along with the results from our study suggest that wild-type MV backbone may serve as a suitable oncolytic vector for treating breast cancer.
In this study, we demonstrated, for the first time, that recombinant wild-type MV combined with low doses of CPT (10, 30, or 50 nM) enhances oncolytic killing of human breast cancer cells. We illustrated a synergistic killing effect during the co-treatment of these cells with both agents. Mechanistically, the synergistic combination treatment increased the accumulation of sub-G1 cell population and led to enhanced apoptosis as evidenced by elevated levels of cleaved PARP (Figs 5 and 6). Given that both MV and CPT treatments can each eventually lead to the induction of cellular apoptosis [21][22][23][24] , this outcome was largely anticipated. Interestingly, MV infection is known to induce autophagy as a pro-viral mechanism, wherein sustained autophagy delays apoptosis and facilitates MV cell-to-cell transmission or syncytia formation before the eventual cell death 59 . On the other hand, CPT has been observed to induce both autophagy and apoptosis 60 , with low-doses (50 nM and less) being capable of triggering premature senescence and autophagy 61 . Since both MV and CPT at the concentrations www.nature.com/scientificreports www.nature.com/scientificreports/ used in this study are known to induce autophagy, co-treatment of CPT and oncolytic MV could potentially amplify the autophagy process, leading to a better viral spread and further sensitizing the breast cancer cells to the eventual apoptotic cell death. Indeed, preliminary experiment indicates induction of the autophagy marker LC3 ('LC3II') with monotreatments using CPT or MV at 24 h and 48 h post-addition, respectively ( Supplementary  Fig. S4A). Interestingly, at 48 h post-treatment, we noted a concomitant decrease in the lipidation of LC3II with increasing CPT concentration in combination with MV ( Supplementary Fig. S4A). This observation was likely not due to inhibition of autophagy but rather its potentiation ("faster turnover"), since treatment with the lysosomal inhibitor bafilomycin to block the autophagic flux caused a substantial change in the accumulation of LC3II in the CPT treatment groups with and without MV combination, as compared to MV infection alone ( Supplementary Fig. S4B). Such amplification by CPT treatment on the inefficient autophagic flux induced by MV could potentially lead to autophagic flux perturbation, an event previously observed to promote apoptotic cell death 62 . Further experiments are required to explore this phenomenon and fully elucidate the nuance of virusand drug-induced autophagy, prior to the cells undergoing apoptosis in the observed synergistic effect from MV and CPT combination.
Altogether, our results suggest that the oncolytic MV plus CPT chemovirotherapy is a potential synergistic combination treatment against breast cancer cells. CPT and MV act together since CPT does not have an antiviral effect when used alone. The synergistic therapeutic effects of the combinatorial treatment also reduce the effective dosages required for each agent. Our findings demonstrated that low doses of CPT (10, 30, and 50 nM) combined with lower MOI of MV (MOI 0.1) gave similar therapeutic effects as high doses of CPT (100 nM) and high MOI of MV (MOI 3) alone in breast cancer cells. This synergistic effect could lower toxicity associated with each reagent 25 , particularly the bone marrow suppression and gastrointestinal toxicity that has been reported for CPT and its derivatives topotecan 63 and irinotecan 64 . In conclusion, the data presented in this paper emphasizes the importance of combination chemovirotherapy and support the future development of co-treatment with oncolytic MV and CPT as a potential therapy for the management of breast cancer.