Synergistic antitumor activity of oncolytic reovirus and chemotherapeutic agents in non-small cell lung cancer cells

Background Reovirus type 3 Dearing strain (ReoT3D) has an inherent propensity to preferentially infect and destroy cancer cells. The oncolytic activity of ReoT3D as a single agent has been demonstrated in vitro and in vivo against various cancers, including colon, pancreatic, ovarian and breast cancers. Its human safety and potential efficacy are currently being investigated in early clinical trials. In this study, we investigated the in vitro combination effects of ReoT3D and chemotherapeutic agents against human non-small cell lung cancer (NSCLC). Results ReoT3D alone exerted significant cytolytic activity in 7 of 9 NSCLC cell lines examined, with the 50% effective dose, defined as the initial virus dose to achieve 50% cell killing after 48 hours of infection, ranging from 1.46 ± 0.12 ~2.68 ± 0.25 (mean ± SD) log10 pfu/cell. Chou-Talalay analysis of the combination of ReoT3D with cisplatin, gemcitabine, or vinblastine demonstrated strong synergistic effects on cell killing, but only in cell lines that were sensitive to these compounds. In contrast, the combination of ReoT3D and paclitaxel was invariably synergistic in all cell lines tested, regardless of their levels of sensitivity to either agent. Treatment of NSCLC cell lines with the ReoT3D-paclitaxel combination resulted in increased poly (ADP-ribose) polymerase cleavage and caspase activity compared to single therapy, indicating enhanced apoptosis induction in dually treated NSCLC cells. NSCLC cells treated with the ReoT3D-paclitaxel combination showed increased proportions of mitotic and apoptotic cells, and a more pronounced level of caspase-3 activation was demonstrated in mitotically arrested cells. Conclusion These data suggest that the oncolytic activity of ReoT3D can be potentiated by taxanes and other chemotherapeutic agents, and that the ReoT3D-taxane combination most effectively achieves synergy through accelerated apoptosis triggered by prolonged mitotic arrest.


Background
The use of viral vectors for cancer gene therapy has been vigorously explored for the last two decades. The overall goal of the strategy is to promote cancer cell death through various means, such as tumor suppressor gene replacement, oncogene inactivation, suicide gene delivery, drug sensitization or enhancement of anticancer immunity. The extensive research efforts to develop tumor cell death-inducing viral vectors have reignited the interest in oncolytic viruses in recent years as a promising group of viral therapeutics that can directly induce tumor cell lysis through viral replication. The latest multidisciplinary research in cancer genomics and proteomics further provides an opportunity to discern various molecular pathways specifically upregulated (or dysregulated) in cancers that can be exploited as part of viral replication and destruction machinery. Indeed, many replication-competent oncolytic viruses currently in development are recombinant viruses engineered to become reliant on such cancer-specific molecules and signaling pathways for viral entry and replication, thus rendering cancer cells more selectively susceptible to virus-mediated oncolysis [1]. Unlike chemical entity-based anticancer agents, these viruses can propagate in susceptible tumor cells, re-target, infect, and destroy remaining cancer cells within the primary tumor or in the metastases, repeating the cycle until viral spread is halted by the host antiviral response or by mechanical barriers such as loss of vasculature and necrotic tissues.
Mammalian reoviruses are ubiquitous, non-enveloped dsRNA viruses, normally associated with relatively benign pathology in humans. The Dearing strain of reovirus serotype 3 (ReoT3D) is a non-engineered wild type reoviral strain and belongs to a growing number of the new generation of oncolytic viruses because of its innate ability to preferentially kill transformed cells [2,3]. The oncolytic potency of ReoT3D has been extensively demonstrated against various cancers in vitro and in vivo, including colon, pancreatic, ovarian and breast cancers, as well as malignant gliomas and lymphoid malignancies [4][5][6][7][8][9]. The safety, feasibility and potential efficacy of ReoT3D cancer therapy are currently being investigated in phase I/II clinical trials [10]. As with other emerging therapeutics for cancer, the combined regimen of ReoT3D and conventional chemotherapeutic agents is expected to play a significant role in future clinical applications. However, it is currently unknown whether conventional chemotherapeutic agents can augment or interfere with the oncolytic effect of ReoT3D. In this study, we evaluated the oncolytic activity of ReoT3D in non-small cell lung cancer (NSCLC), and explored the therapeutic feasibility of ReoT3D-chemotherapeutic combination regimens against NSCLC.

Oncolytic activity of ReoT3D and progeny virion production in NSCLC cell lines
We first examined the in vitro susceptibility of human NSCLC cell lines to ReoT3D, as reoviral oncolytic activity had not been extensively studied in human lung cancer cells. Nine NSCLC cell lines (NCI-H460, A549/ATCC, HOP-62, NCIH322M, NCI-H226, EKVX, NCI-H23, NCI-H522, and HOP-92) included in the NCI-60 tumor cell line panel (Developmental Therapeutics Program, DTP, NCI-Frederick, Frederick, MD) were incubated with serially diluted ReoT3D for cytopathic effect (CPE) determination. Within 48 hours post-infection, ReoT3D induced significant cell death in seven of nine NSCLC cell lines in a dose-dependent manner ( Figure 1). ReoT3D 50 percent effective dose (ED50), defined as the initial virus dose (multiplicity of infection, MOI, expressed in plaque forming units per cell, pfu/cell) that resulted in 50% cell viability at 48 hours post-inoculation as compared to untreated controls, ranged from 1.46 ± 0.12 to 2.68 ± 0.25 (mean ± SD from 3 separate experiments) log 10 pfu/cell in the sensitive cell lines (Table 1). In contrast, NCI-H226 and NCI-H322M were relatively resistant to ReoT3D in this shortterm incubation assay, as indicated by the significantly lower levels of cell death even at the highest inoculum dose compared to those seen in the sensitive cell lines (P < 0.0001) ( Figure 1). While ReoT3D has been shown to induce CPE in murine cells with an activated Ras pathway [11,12], the presence of Ras-activating gene mutations [13] or activated Ras was not necessarily associated with lower ReoT3D ED50 in the NSCLC cell lines tested in our study (Table 1 and Figure 2).

In vitro combination effects of ReoT3D and chemotherapeutic agents against NSCLC cells
Next, we investigated the combination effects of ReoT3D and chemotherapeutic agents in four ReoT3D-susceptible (NCI-H460, HOP-92, NCIH23, and EKVX) and two relatively resistant (NCI-H226 and NCI-H322M) cell lines using the Chou-Talalay method. Growth inhibitory effects of chemotherapeutic agents, paclitaxel, cisplatin, gemcitabine, and vinblastine, were first determined in each cell line by the XTT cytotoxicity assay and expressed as the drug concentration that inhibited cell growth by 50% compared to untreated control (IC50) ( Table 2). Of the 6 cell lines, NCI-H322M and EKVX showed high levels of resistance to multiple compounds, consistent with the previous report that characterized them as multi-drug resistant against a variety of anticancer agents [14] (Table  2). When NCI-H460 cells were incubated with increasing doses of ReoT3D in the absence or presence of paclitaxel, whose concentration was serially increased to maintain a constant ratio to the ReoT3D dosage (the constant ratio combination design) [15], the synergistic anticancer effect of the two agents was clearly demonstrated by a leftward shift of the dose response curve (Figure 3a) as well as isobologram and combination index (CI) analyses [16,17] ( Figure 3b and 3c, respectively). The combination effects of ReoT3D and other chemotherapeutic agents were similarly examined in all 6 cell lines using at least two different dosage ratios, and CI values were obtained for each combination regimen (Table 2). In many instances, the combination of ReoT3D with different chemotherapeutic agents was found to exert moderate to strong synergistic effects at both combination ratios as shown by the CI that were consistently smaller than 1.0 at ED50, ED75 and ED90 (Table 2). Interestingly, with the exception of ReoT3D-paclitaxel combination, antagonistic effects (CI > 1.0) of ReoT3D-chemotherapeutic combinations were typically observed in NSCLC cell lines that exhibited highlevel resistance to the test compounds with IC50 often exceeding the highest test concentration, 100 μM (Table  2). In contrast, the combination of ReoT3D and paclitaxel was consistently synergistic in all the NSCLC cell lines tested, regardless of the level of paclitaxel sensitivity ( Table 2). The relative resistance of NSCLC cell lines to ReoT3D did not appear to negatively influence the outcomes, as the synergistic effect was clearly seen in NCI-H226 for all the combination regimens examined ( Table  2).
These data demonstrated that while the oncolytic activity of ReoT3D could be generally potentiated by the chemotherapeutic agents that alone were cytotoxic to the tested cell lines, paclitaxel appeared to exert unique effects on the cell-death process induced by ReoT3D, even in the cells with reduced sensitivity to the compound.

Progeny virion production from NSCLC cells treated with the combination of ReoT3D and chemotherapeutic agents
Next, we asked whether the synergistic oncolytic effects of ReoT3D-chemotherapeutic combination regimens reflected increases in virally induced lytic cell death, driven by a burst of progeny virion production from the cells. To capture early changes in viral production levels, the amounts of infectious virions released into the culture supernatants in the first 24 hours were compared among Oncolytic effect of reovirus type 3 Dearing strain (ReoT3D) in non-small cell lung cancer (NSCLC) cells NSCLC cell lines infected with ReoT3D in the absence or presence of different chemotherapeutic agents. Notably, the addition of paclitaxel to ReoT3D (MOI = 20) was found to increase the level of progeny virion production as compared to virus alone in all 4 cell lines tested, NCI-H460, NCI-H23, EKVX and HOP-92 (P = 0.001, 0.03, 0.003 and 0.0001, respectively) ( Figure 4a). However, such augmentation of virion production was not unique to paclitaxel nor associated with synergy, as ReoT3D-vinblastine combination also significantly increased the level of progeny virions released from EKVX and NCI-H322M (P < 0.01) (Figure 4b), where the combination was not associated with a synergistic effect (mean CI > 1.0). In contrast to paclitaxel and vinblastine, the addition of gemcitabine was not associated with an increased reoviral progeny virion production in NSCLC cells (Figure 4b). These data suggested that the synergistic effects of ReoT3D-chemotherapeutic combinations did not directly result from lytic necrosis induced by rapid increases in virion release, but probably stemmed from certain changes in programmed cell death pathways.

PARP cleavage in NSCLC cells treated with ReoT3Dchemotherapeutic combination regimens
Reovirus type 3 Abney (ReoT3A) has been shown to induce apoptotic cell death in various cancer cell lines, including in 2 NSCLC cell lines, NCI-H157 and A549 [18]. To further investigate the mechanistic basis for the synergistic activity of ReoT3D-chemotherapeutic combinations in NSCLC cells, we examined the proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) in NCI-H460 treated with 20 MOI of ReoT3D alone or in combination with gemcitabine, paclitaxel or vinblastine for 24 to 30 hours. Western blot analysis of cell lysates demonstrated the cleaved 89-kDa PARP fragment in ReoT3Dtreated NCI-H460, whereas treatment with gemicitabine (1 μM), paclitaxel (1 μM) or vinblastine (0.1 μM) alone resulted in virtually undetectable PARP cleavage in this cell line ( Figure 5). Cells treated with the combination of ReoT3D and paclitaxel showed significantly higher levels of PARP cleavage than ReoT3D alone at 24 and 30 hours post-treatment, accompanied by a gradual decrease in the amount of full-length PARP ( Figure 5). Increased PARP cleavage was also observed with the combination of ReoT3D and other chemotherapeutic agents at 24 or 30 hours post-treatment, although to a lesser extent than ReoT3D-paclitaxel combination ( Figure 5). These data suggested that the synergistic cell death induced by the ReoT3D-paclitaxel combination was mediated at least in part by the enhanced apoptotic process in NCI-H460.

Activation of caspases in NSCLC cells treated with ReoT3D alone or in combination with paclitaxel
To explore whether the enhancement of apoptosis played a role in the synergistic activity of the ReoT3D-paclitaxel combination in other NSCLC cell lines, we examined the level of caspase activity in NCI-H460, NCI-H23, EKVX and NCI-H322M treated with increasing doses of either ReoT3D or paclitaxel alone or both in combination, using the constant ratio combination design [15], the same dosing scheme adopted for CI analyses of Chou-Talalay (see above). In each of 4 cell lines, treatment with ReoT3D alone led to a significant increase in caspase activity in a dose-dependent manner, although the response appeared less robust in NCI-H322M, where higher ReoT3D MOIs were required to achieve similar magnitudes of caspase activation above baseline than in 3 other cell lines ( Figure  6a). The increases in caspase activity were associated with Levels of GTP-bound activated Ras protein in 9 NSCLC cell lines analyzed by Western blot, using anti-pan-Ras antibody Figure 2 Levels of GTP-bound activated Ras protein in 9 NSCLC cell lines analyzed by Western blot, using anti-pan-Ras antibody. Note, high levels of activated Ras were observed with A549/ATCC (K-ras G12S ) [13], HOP-62 (K-ras G12C ) [13], and NCI-H23 (K-ras G12C ) [13], followed by NCI-H460 (K-ras Q61H ) [13], EKVX and HOP-92. The levels of activated Ras were significantly lower not only with ReoT3D-resistant NCI-H322M and NCI-H226, but also with ReoT3D sensitive cell line, NCI-H522. Also see table 1 for comparison of Ras-activating gene mutation status and ReoT3D sensitivity of each cell line. dose-dependent decreases in cell viability as assessed by intracellular ATP content in ReoT3D-treated cells ( Figure  6a). Paclitaxel alone had virtually no effects on caspase activation or ATP content during the 24-hr exposure, except in NCI-H23 that showed moderate increases in caspase activity upon treatment with the compound at ≥ 10 nM ( Figure 6a). When ReoT3D was combined with paclitaxel, the levels of caspase activation appeared more pronounced as compared to ReoT3D single therapy, as seen by a leftward shift of the dose response curves (Figure 6a). Similar results were obtained from 3 separate experiments for all 4 cell lines.
In order to evaluate whether the extent of caspase activation induced by the ReoT3D-paclitaxel combination was significantly different from ReoT3D single treatment in these cell lines, the caspase activity dose-response curves were fitted by non-linear regression using GraphPad Prism (GraphPad Software Inc., San Diego, CA) ( Figure  6b). The best-fit values of a variable, LogEC50, obtained by the Prism analysis from 3 independent experiments were then compared between the two treatment regimens, ReoT3D alone vs. ReoT3D-paclitaxel combination, using a paired t-test. The analysis demonstrated that the activation of caspases was significantly enhanced with the ReoT3D-paclitaxel combination therapy as compared to ReoT3D alone in NCI-H460, NCI-H23, EKVX and NCIH322M (P = 0.004, 0.03, 0.04, and 0.02, respectively). These data suggested that enhanced apoptotic cell death most likely constituted the synergistic cell killing induced by the combination of ReoT3D and paclitaxel in NSCLC cells.
Synergistic activity of ReoT3D and paclitaxel combination in NCI-H460 cells

Dynamic effects of ReoT3D and paclitaxel on cell cycle progression and caspase activation
ReoT3D infection has been shown to induce cell cycle arrest at G1/S and G2/M [19,20]. Antimicrotubule agents, including taxanes and vinca alkaloids, activate the spindle-assembly checkpoint and induce mitotic arrest at the metaphase and anaphase transition, which ultimately leads to cell death by apoptosis [21]. A certain proportion of cells exposed to these antimitotic agents may undergo an aberrant mitotic exit without cytokinesis, forming multinucleated cells in interphase [21]. To gain mechanistic insight into the enhanced apoptotic cell death induced by the ReoT3D-paclitaxel combination, we investigated the effects of ReoT3D and paclitaxel on cell cycle and caspase-3 activation in NSCLC cells using flow cytometry.    (Figure 7b). Paclitaxel treatment invariably increased the proportion of post-G1 cells in these cell lines, although the percentages of cells positive for activated caspase-3 were significantly smaller than in NCI-H23. The ReoT3D-paclitaxel combination consistently led to substantial increases in cells expressing active caspase-3, including in paclitaxel-resistant EKVX and NCI-H322M cells, and these increases appeared more prominent in post-G1 phase (Figure 7b). Indeed, statistical analysis demonstrated that the proportions of active caspase-3positive cells were significantly different among the treatment groups in the post-G1 cell population (P < 0.005, one-way ANOVA), but not in the population with DNA content of 2N or less (sub-G1 and G1) (P = 0.05). In this subpopulation of post-G1 cells, the proportions of active caspase-3-expressing cells were significantly increased with the ReoT3D-paclitaxel combination treatment as compared to single therapy (P = 0.02 for both betweengroup comparisons, ReoT3D alone vs. ReoT3D+paclitaxel 0.1 μM, and paclitaxel 0.1 μM alone vs. ReoT3D+paclitaxel 0.1 μM; P = 0.02 and < 0.04 for between ReoT3D alone vs. ReoT3D+paclitaxel 1 μM, and paclitaxel 1 μM alone vs. ReoT3D+paclitaxel 1 μM, respectively). These data suggested that the combination of ReoT3D and paclitaxel enhanced apoptotic cell death, which was linked to cell cycle perturbation in NSCLC cells.

Ultrastructural analysis of NSCLC cells treated with ReoT3D and paclitaxel
To further examine the relationship between cell cycle perturbation and apoptotic cell death induced by ReoT3D and paclitaxel, we examined the morphological changes of NCI-H23 cells treated with either ReoT3D or paclitaxel alone or in combination, using electron microscopy (EM). After overnight (~20 hours) treatment, the cells were directly fixed in situ and processed for the analysis. The EM study did not include dead cells that had detached before fixation. One hundred cells were surveyed within one 60-nm thin section for each treatment group to document significant changes as compared to untreated control (Table 3). When NCI-H23 cells were exposed to 1 μM paclitaxel overnight, an increased number of cells were (a) Fold increases (mean ± SD) in infectious progeny virion production found to be enlarged and multinucleated (Figure 8b), as verified by the survey (Table 3). These multinucleated cells, detected as post-G1 cells with ≥ 4N DNA content by flow cytometry (Figure 7a and 7b), represented the cells that had exited mitosis without cytokinesis. The cells infected with ReoT3D (MOI = 20) contained numerous viral particles, mostly in viral inclusion bodies that appeared globular in shape, and an increased number of mitochondria (Figure 8c and 8d). The combination of ReoT3D and paclitaxel resulted in notable increases in mitotic cells (Figure 8e), and apoptotic cells characterized by condensed and fragmented chromatin as well as cytoplasmic shrinkage and vacuolation (Figure 8f) (Table 3). Of note, although the number of viral particle-harboring cells that could be detected in the EM specimen was decreased with the ReoT3D-paclitaxel combination compared to ReoT3D alone (Table 3), the level of infectious progeny virion in the corresponding supernatant was higher than that of cells treated with ReoT3D alone (data not shown), indicating that the addition of paclitaxel enhanced reoviral production as discussed earlier.
These data from flow cytometric and electron microscopic analyses strongly suggested that treatment with the combination of ReoT3D and paclitaxel caused prolonged mitotic arrest, which triggered accelerated apoptosis, resulting in synergistic cell killing in dually treated NSCLC cells.

Discussion
Lung cancer is the leading cause of cancer mortality in both men and women in the United States [23] and all cancer deaths worldwide [24]. The most common form of lung cancer is NSCLC that includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Despite the tremendous efforts and progress in lung cancer research, treatment outcomes for non-localized NSCLC remain poor [25]. New treatment strategies are urgently needed to improve survival for advanced NSCLC patients. In the current study, we uncovered a potent oncolytic activity of ReoT3D against a panel of human NSCLC cell lines, in particular, NSCLC cell lines of adenocarcinoma or large cell carcinoma origin. The susceptibility of cancer cells to ReoT3D-mediated cytolysis has been attributed to increased Ras activity [11,12]. However, we did not observe any significant association between the ReoT3Dpermissibility and the presence of Ras-activating gene mutations or activated Ras in human NSCLC cells. The lack of association has also been reported by others in human colon cancers [26]. It is possible that in addition to the activation status of Ras-associated pathways [11,12], there are other molecular determinants of ReoT3D-sensitivity, such as the cell surface density of putative ReoT3D receptors/coreceptors [27][28][29] and intracellular virion uncoating processes [30,31], all of which can affect ReoT3D infection efficiency.
The combination effects of herpesvirus or adenovirusbased oncolytic viral vectors and chemotherapeutic agents have previously been evaluated against different human cancers [32][33][34][35][36][37]. Synergistic activity was reported in the majority of these studies. However, combination regimens selected for the previous studies were mostly limited in scope in terms of dose range and the number of chemotherapeutic agents investigated. In the current study, we demonstrated that the oncolytic activity of ReoT3D against NSCLC cells could be significantly potentiated by a number of chemotherapeutic agents used in the treatment of NSCLC, including paclitaxel, cisplatin, gemcitabine and vinblastine. Combination analysis based on the Chou-Talalay's method [16,17] clearly showed significant levels of synergy between ReoT3D and each chemotherapeutic agent tested. Interestingly, we found that the drug sensitivity of each NSCLC cell line was an important determinant for the in vitro synergistic effect of ReoT3Dchemotherapeutic combination regimens, with the exception of ReoT3D-paclitaxel combination. It is conceivable that certain molecular changes conferring drug resistance can antagonize the process of ReoT3D-mediated cell killing. Our data, therefore, caution against the use of chemotherapeutic agents combined with ReoT3D for the Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage treatment of NSCLC that have developed resistance to the agents. In contrast, the level of ReoT3D sensitivity did not appear to compromise the combination effects in NSCLC cells. Rather, the addition of chemotherapeutic agents may help accelerate ReoT3D-induced cell death process, which is otherwise slowed in NSCLC cells with low susceptibility to ReoT3D infection.
The most intriguing finding from our study was the synergistic effect of ReoT3D-paclitaxel combination consistently observed in all the NSCLC cell lines examined, regardless of the level of sensitivity to the compound. Because previous studies of oncolytic virus-chemotherapeutic combinations, in particular with paclitaxel, did not address the impact of drug resistance on the combination effects, we cannot ascertain whether our finding is unique to reovirus-containing combination therapy. Mammalian reoviruses are known to exploit microtubules for the formation of viral replication complexes (inclusion bodies) [38]. Based on our initial findings that the addition of paclitaxel to ReoT3D significantly increased the level of progeny virion production from all the NSCLC cell lines (a) Levels of caspase activity and ATP content in NCI-H460, NCI-H23, EKVX and NCI-H322M cells treated with increasing doses of either ReoT3D or paclitaxel alone, or both in combination for 24 hours, using the constant ratio combination design [15]. This design format was employed to evaluate the synergistic activity of ReoT3D-chemotherapeutic combination regimens (see text). Shown are the mean ± SD in relative fluorescence units (RFU) and relative luminescence units (RLU) for caspase activity (closed triangle) and ATP content (open circle), respectively, for ReoT3D alone (green), ReoT3D-paclitaxel combination (red) and paclitaxel alone (blue). The data shown are representative of 3 separate experiments. (b) Doseresponse curves of caspase activation induced by treatment with ReoT3D alone or ReoT3D-paclitaxel combination for 24 hours fitted by non-linear regression using GraphPad Prism (GraphPad Software, Inc.). Shown as an example are non-linear regression analyses for NCI-H460 and EKVX. See text for statistical analysis on the best-fit values.
tested, we speculated that microtubule-stabilizing paclitaxel might have enhanced reoviral replication, resulting in a more efficient and synergistic oncolytic effect. However, the increased progeny virion production was not necessarily a unique outcome of ReoT3D-paclitaxel combination, but was also observed with ReoT3D-vinblastine combination in vinblastine-resistant NCI-H322M cells, where the combination of ReoT3D and vinblastine was found to be strongly antagonistic. Moreover, the addition of gemcitabine to ReoT3D treatment was not associated with an increased progeny virion production, regardless of the combination effects (synergy or antagonism) attained. These data suggested that the synergistic effect of ReoT3D and chemotherapeutic agents was not the direct result of enhanced lytic cell death, but more likely the manifestation of accelerated programmed cell death, which was triggered before virion assembly and release.
Reovirus has been shown to induce apoptotic cell death in a variety of cell types, including cancer cells [18,39]. Indeed, increased caspase activity and apoptotic cleavage of PARP were readily detectable in ReoT3D-treated NSCLC cells within 24 hours in the current study, as has been shown in ReoT3A-exposed cancer cells [18]. The combination treatment with ReoT3D and paclitaxel led to more robust caspase activation than ReoT3D alone with a significant leftward shift of the dose response curve in both paclitaxel-sensitive and -resistant NSCLC cell lines, suggesting that the enhanced apoptosis most likely constituted the synergistic cell killing by the combination, regardless of the level of paclitaxel sensitivity. To gain more insight into the mechanistic basis of accelerated apoptosis associated with the ReoT3D-paclitaxel combination, we examined the effects of ReoT3D and paclitaxel on caspase-3 activation in relation to cell cycle progres-  Compared to untreated control (a), the increased number of paclitaxeltreated NCI-H23 cells were found to be enlarged and multinucleated (b), whereas the cells infected with ReoT3D (c and d) contained numerous viral particles, mostly in viral inclusion bodies, which appeared globular in shape (black arrows). In addition, ReoT3D-infected cells appeared to contain an increased number of mitochondria (white arrows). When the cells were exposed to the combination of ReoT3D and paclitaxel, there were increased numbers of mitotic cells (e) as well as apoptotic cells characterized by condensed chromatin, cytoplasmic shrinkage and vacuolation (f). The results from survey of 100 cells for each treatment group are summarized in Table 3. Scale bars: 2 μm for (a), (b), (c), (e) and (f), and 500 nm for (d).
sion. While taxanes and other antimicrotubule agents are known to activate the spindle checkpoint and induce mitotic arrest [21], ReoT3D infection has been shown to effect cell cycle arrest at G1/S and G2/M [19,20]. Because arrests in cell cycle progression induced by anticancer agents are commonly followed by apoptosis [40], we hypothesized that these two agents with differing effects on cell cycle progression may have synergistically activated apoptotic pathways in dually treated NSCLC cells.
We found that the proportion of cells expressing activated caspase-3 was significantly increased by the ReoT3D-paclitaxel combination as compared to either ReoT3D or paclitaxel single treatment in each NSCLC cell line tested. Interestingly, the activation of caspase-3 was found more prominent in post-G1 cell population with ≥ 4N DNA content. Escape from mitotic arrest induced by spindle poisons such as taxanes and other antimicrotubule agents is commonly observed in cancer cells with impaired (weakened) mitotic checkpoint [21,41]. These cells that prematurely exit mitosis without proper cell division form large multinucleated cells with DNA content of 4N or greater, as demonstrated by EM in our study. After such mitotic slippage, some cells may undergo p53-dependent apoptosis, while others survive through senescence or continuing cell cycle (endoreduplication) [42], depending on the functions of p53, MAP kinase pathways, and p21-activated kinase [43,44]. In the current study, we found that NSCLC cells could efficiently escape from mitotic arrest induced by paclitaxel at 0.1 ~1 μM and survive at least for the first 24 hours of exposure, with the exception of NCI-H23 cells that were more prone to apoptosis upon paclitaxel exposure than 3 other NSCLC cell lines examined. While paclitaxel-treated NCIH460 cells ultimately underwent dramatic cell death after 48 hours, EKVX and NCI-H322M demonstrated considerable levels of resistance to paclitaxel-induced CPE. Nonetheless, the combination of ReoT3D and paclitaxel consistently accelerated the apoptotic process in post-G1 cells, including in paclitaxelresistant EKVX and NCI-H322M cells. This enhanced apoptosis appeared to have resulted from prolonged mitotic arrest, as corroborated by the EM data demonstrating that treatment with the ReoT3D-paclitaxel combination resulted in increased numbers of both mitotically arrested and apoptotic cells while decreasing the number of multinucleated cells as compared to paclitaxel alone.
The molecular mechanism of prolonged mitotic arrest induced by the ReoT3D-paclitaxel combination has yet to be elucidated. It is possible that ReoT3D infection may enhance the mitotic checkpoint activity in cancer cells with weakened mitotic checkpoint, for example, by upregulating the expression of mitotic checkpoint proteins (such as Mad1, Mad2, BubR1/Mad3, Bub1 and Bub3) [45], Cdk1 and/or cyclin B [45], or suppressing the anaphase-promoting complex/cyclosome activity [45]. Such ReoT3D-induced alterations in the mitotic regulatory network may reinforce the mitosis-arresting signal of taxanes, leading to prolonged mitotic arrest and apoptosis. Better understanding of the molecular consequences of ReoT3D infection on cell cycle checkpoint function and apoptotic signaling pathways in cancer cells will greatly enhance our ability to design rational combination therapies with proapoptotic oncolytic agent, ReoT3D, and various classes of anticancer agents. Concurrently, it is also of high importance to investigate potential consequences of ReoT3D-chemotherapeutic combinations on normal tissues in order to identify undesirable combination regimens that are associated not only with synergistic oncolytic activity, but also with enhanced toxicity in humans.

Conclusion
The current study showed that the oncolytic activity of ReoT3D could be most effectively potentiated by taxanes through accelerated apoptosis, regardless of the level of taxane-sensitivity. Recently, preliminary findings from ongoing phase I clinical trials evaluating the safety of ReoT3D-taxane combination in patients with chemotherapy-refractory advanced tumors have demonstrated objective anticancer response in some patients without serious side effects [46,47], corroborating that ReoT3D-taxane combination regimens can achieve a durable oncolytic effect in clinical settings and thus should be further explored as a novel treatment modality for NSCLC.

Cell lines, virus and chemotherapeutic agents
Human NSCLC cell lines included in the NCI-60 cell line panel [48]:

Determination of virus-and drug-induced cell death
ReoT3D-or drug-induced CPE was assessed by the XTT assay as described previously [49]. ED50 for ReoT3D was defined as the initial virus dose (MOI expressed in pfu/ cell) that resulted in 50% cell viability at 48 hours postinoculation as compared to untreated controls. Sensitivity of NSCLC cell lines to chemotherapeutic agents was expressed as the drug concentration that inhibited cell growth by 50% compared to untreated controls (IC50). Both ED50 and IC50 were calculated by using the software GraphPad Prism (GraphPad Software, Inc.). Cell viability was also assessed by intracellular ATP content (ATPLite™, PerkinElmer, Waltham, MA), when the level of caspases activity was evaluated in selected NSCLC cell lines treated with ReoT3D, paclitaxel or both agents in combination (see below).

Evaluation of in vitro combination effects by the Chou-Talalay method
The combined effects of ReoT3D and each chemotherapeutic agent on cell survival were analyzed using the software CalcuSyn (Biosoft, Ferguson, MO), which applies the median-effect equation of Chou [16] and the CI equation of Chou and Talalay [17]. NCI-H460, HOP-92, NCI-H23, EKVX, NCI-H226 and NCI-H322M plated in 96-well microplates as above were exposed in triplicate to a serial dilution of each agent or both in combination using the constant ratio combination design [15] for 48 hours, followed by the XTT assay for cell viability determination. Calculated CIs were used to ascertain the presence of strong synergism (CI < 0.3), moderate synergism (0.3 < CI < 0.9), additive effect (CI = 1), antagonism (CI > 1) and strong antagonism (CI > 3.3) [15] between ReoT3D and chemotherapeutic agents.

Reovirus plaque assay
Levels of infectious progeny virions in culture supernatants produced from ReoT3D-infected NSCLC cells were evaluated by plaque assay as previously described [50]. Infectious reovirus titers were expressed as pfu/mL of the original sample.

Determination of activated Ras levels in NSCLC cells
Basal levels of activated Ras in 9 NSCLC cell lines were determined by Ras activation assay Biochem Kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer's instructions. Briefly, cell lysates were prepared from NSCLC cells using the kit lysis buffer, and aliquots of 2 mg protein were incubated with 20 μL of Raf1-RBD beads at 4°C for 1 hr, followed by centrifugation to pellet the Raf1-RBD beads. The pelleted beads were washed with wash buffer twice, and resuspended in 10 μL Laemmli sample buffer. The samples were separated by 12% Trisglycine SDS-PAGE, and subjected to Western blot analysis with anti-Ras antibody (Cell Signaling Technology, Inc., Danvers, MA).

Determination of caspase activity and ATP content by microplate-based assays
The levels of caspase activity and ATP content in NSCLC cells treated with ReoT3D alone or in combination with paclitaxel for 24 hours were evaluated by microplatebased assays, using a fluorometric pan-caspase assay (Homogeneous Caspases Assay; Roche Applied Science, Indianapolis, IN) and luminescence ATP detection assay (ATPLite™; PerkinElmer), respectively.

Flow cytometric analysis of DNA content and caspase-3 activation
Adherent and non-adherent NSCLC cells harvested as above (see Western blot analysis) were fixed and permeabilized with BD Cytofix/Cytoperm™ solution (BD Bio-sciences, San Jose, CA) and stained with FITC-conjugated anti-active caspase-3 antibody (BD Biosciences), followed by incubation with PI/RNase staining buffer (BD Biosciences) for DNA content determination. The proportion of cells expressing active caspase-3 and DNA content were analyzed using a FACScan™ flow cytometer (BD Biosciences) as previously described [51].
Transmission electron microscopy NSCLC cells cultured in 6-well plates were processed in situ for electron microscopic analysis as previously described [52]. Thin sections (60 nm) were examined with a Hitachi H7000 transmission electron microscope.

Statistical analysis
Results are reported as mean ± SD unless otherwise indicated. Statistical significance of differences was determined by one-way ANOVA or Student's t-test as appropriate. Differences were considered statistically significant when P < 0.05 (two-tailed).