Genetically Engineered Oncolytic Newcastle Disease Virus Effectively Induces Sustained Remission of Malignant Pleural Mesothelioma Free

Malignant pleural mesothelioma is a highly aggressive tumor that arises from multipotent cells of the pleura. These tumors are related to asbestos exposure, which leads to fiber deposition deep in the lung. Inflammation, free radical production, and direct DNA damage are recognized as the pathogenic features of asbestos exposure (1–3). Tumor formation may also be related to previous SV40 infection and possibly to a genetic predisposition (4, 5). A loss of tumor-suppressor gene activity has been shown to cause development of malignant pleural mesothelioma (6).

Currently, this tumor causes about 20,000 deaths a year in the United States. However, because of a latency period of ∼40 years, it is estimated that the annual incidence will continue to increase through the next decade (7). Unfortunately, this tumor is usually detected at an unresectable stage of disease (8, 9). Because of the very limited effect of chemotherapy and radiation therapy, this disease is almost uniformly fatal. Average survival time after diagnosis varies between 10 and 16 months (4). Alternative therapies, such as immunotherapy, gene therapy (including oncolytic viral therapy), and photodynamic therapy, are currently under clinical investigation but have no accepted clinical role (10–14).

Oncolytic viral therapy uses the natural life cycle of viruses to infect and lyse cancer cells (15). Such oncolytic viruses can also be used as vectors for local delivery of anticancer agents such as cytokines (15). Kelly et al. (16) recently reported encouraging results in an orthotopic mesothelioma animal model treated with vaccinia virus (17). In the current study, the oncolytic efficacy of Newcastle disease virus (NDV) on malignant pleural mesothelioma is tested in vitro and in vivo using an orthotopic pleural mesothelioma tumor model. NDV has already showed tumor cell infection efficacy in several cancer cell lines (18–23).

NDV contains a single-stranded, negative-sense, nonsegmented RNA genome and belongs to the genus Avulavirus in the family Paramyxoviridae (24). The genomic RNA is 15,186 nucleotides in length and contains six genes that encode at least seven proteins (25, 26). The tumor selectivity of NDV has been attributed to the defective activation of type I IFN in tumor cells. In addition, recent studies showed altered response to IFN-β treatment in tumor cells (27).

The aim of the present study was to determine the oncolytic efficacy of NDV against malignant pleural mesothelioma in vitro and in vivo. For this purpose, we established an orthotopic pleural mesothelioma tumor model, which enabled long-term follow-up of NDV treatment in mice using bioluminescence imaging.

Molecular imaging enables the visualization, characterization, and quantification of biological processes at the cellular and subcellular levels within living subjects (28). Bioluminescence imaging uses the light produced by an enzymatic reaction of a luciferase enzyme with its substrate. Firefly (Photinus pyralis) luciferase (FLuc) is the most frequently used enzyme for molecular imaging. This enzyme oxidizes its substrate, luciferin, in a reaction that requires oxygen and ATP, emitting light with a broad emission spectrum and a peak at 560 nm (29). In the present study, we used a mesothelioma cell line that was stably transduced with the gene for FLuc to image mesothelioma tumors in vivo and to investigate the efficacy of the oncolytic NDV in therapy of malignant pleural mesothelioma.

We studied seven human malignant mesothelioma cancer cell lines of various histologic subtypes, including epithelioid (H-2452, HMESO), sarcomatoid (H-2052, H-2373, and VAMT), and biphasic (MSTO-211H and JMN). The MSTO-211H cell line was obtained from American Type Culture Collection. The JMN and VAMT cell lines were a kind gift from F. Sirotnik (Memorial Sloan-Kettering Cancer Center). Cell lines H-2052, H-2452, and H-2373 were a kind donation from H.I. Pass (Karmanos Cancer Institute, Wayne State University). HMESO cell lines were obtained from the National Cancer Institute. All cells were grown in appropriate media and were maintained at 37°C in a humidified incubator supplied with 5% CO₂.

Transduction was done using the vector SFG-tdRFP-cmvFLucSFG-tdRFP-cmvFLuc (tdRFP, tandem repeat red fluorescent protein; ref. 30) and FLuc-encoding cDNAs were placed under constitutive promoters, long terminal repeat and cytomegalovirus (31). RFP-positive cells were sorted by fluorescence-activated cell sorting.

The fusogenic NDV mutants with modified F cleavage site [NDV(F3aa)] were previously described (32). To generate NDV(F3aa) virus expressing green fluorescent protein (GFP), a DNA fragment encoding GFP flanked by the appropriate NDV-specific RNA transcriptional signals was inserted into the XbaI site created between the P and M genes of pT7NDV/F3aa. Viruses were rescued from cDNA using methods described previously (33) and sequenced by reverse transcription-PCR for insert fidelity.

Cells were plated at 4 × 10⁴ per well in 12-well plates in 1 mL of appropriate media per well. After incubation for 6 hours, cells were infected with NDV(F3aa)-GFP at multiplicities of infection (MOI) of 1.00, 0.10, 0.01, and 0 (control wells). Viral cytotoxicity was measured every other day for 7 days. Cells were washed with PBS and lysed in 1.35% Triton X (200 μL per well; Sigma-Aldrich) to release intracellular lactate dehydrogenase, which was quantified using a CytoTox 96 kit (Promega) and a spectrophotometer (EL321e; Bio-Tek Instruments) at 490 nm. Results are expressed as the percentage of surviving cells. This percentage was determined by comparing the measured lactate dehydrogenase of each infected sample with that in uninfected control cells. All samples were analyzed in triplicate.

The status of viral infection in cell culture was monitored by GFP microscopy (Nikon Eclipse TE 2000) every 12 hours during the first 48 hours of virus infection and every 24 hours afterward.

Athymic female mice were purchased from the National Cancer Institute and were provided with food and water ad libitum. All animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996), and animal protocols were approved by the Institutional Animal Care and Use Committee at Memorial Sloan-Kettering Cancer Center. Anesthesia was induced with a mixture of isoflurane (2 L/min) and oxygen (4 L/min) in an induction chamber and was maintained with a nasal cone. Mice were placed in the left lateral position. The right chest was prepared with 10% povidone-iodine solution. A 3- to 5-mm incision was made over the fourth to fifth intercostal space. Sharp dissection was carried out, exposing but not breaching the parietal pleura. The underlying lung was thereby easily visualized through the thin membrane. Slowly, a transduced MSTO-211H malignant mesothelioma cellular suspension (1 × 10⁷ cells in 100 μL PBS) was injected into the pleura with a 27-gauge needle. After injection, the skin was closed with surgical staples. Recovery was observed for 15 minutes before mice were returned to their cages.

Intrapleural treatment with virus was done in a fashion similar to the technique previously described for cancer cell injection. Viral treatment was administered intrapleurally at a dose of 1 × 10⁷ plaque-forming units suspended in 100 μL PBS. After injection, animals were gently rotated from side to side to help distribute the virus throughout the pleural cavity.

The following study groups were established (each group originally consisting of five animals).

• Single treatment group:

  • Group S1 (T1S): single viral dose injection 1 day after tumor implantation

  • Group S10 (T10S): single viral dose injection 10 days after tumor implantation

• Multiple treatment group:

  • Group M1 (T1M): four viral dose injections every other day starting 1 day after tumor implantation

  • Group M10 (T10M): four viral dose injections every other day starting 10 days after tumor implantation

The control group consisted of 10 mice that received an intrapleural injection of 100 μL PBS either 1 day (five animals) or 10 days (five animals) after tumor implantation. Animals were regularly assessed for weight loss and tachypnea throughout the experimental period. Tumor burden was assessed with bioluminescent imaging every other day for the first 20 days after tumor instillation and every 5th day thereafter. Animals suffering from end-stage tumor burden were sacrificed by CO₂ narcosis.

The IVIS Imaging System (Caliper Life Sciences) was used for bioluminescence image acquisition and analysis. Firefly d-luciferin potassium salt was purchased from Xenogen (Caliper Life Sciences), diluted to 30-mg/mL stock in PBS, and filtered through a 0.22-μm filter before use. After initial anesthesia with a mixture of isoflurane (2 L/min) and oxygen (4 L/min) in an induction chamber mice were injected with 100 μL of the d-luciferin solution (150 mg/kg body weight) i.p. Ten minutes after injection, the animals were again anesthetized in an induction chamber and placed in the bioluminescence chamber in a standardized way. Images were acquired for 1 second under general anesthesia maintained over a nasal cone. Each treatment group of five mice was placed in the specimen chamber mounted with the charge-coupled device camera cooled to −120°C, with a field of view set at 25 cm above the sample shelf. Photon emission transmitted from cell samples and mice was measured in prone and supine position. The gray scale photographic images and bioluminescence color images were superimposed using LIVINGIMAGE version 2.11 software overlay (Caliper Life Sciences) and IGOR image analysis software (version 4.02 A; WaveMetrics). A region of interest was manually selected over the signal intensity. Values were expressed in photons per second per square centimeter per steradian and represent the mean values from the prone and supine positions of each animal.

From all animals, representative tissue samples were taken before sacrifice. In tumor-bearing animals, tumor samples from the chest wall were harvested. In healed animals at the final sacrifice, several representative tissue samples from several locations within the chest wall were harvested. Tissue samples were frozen in Tissue Tek embedding medium (Sakura Finetek) and sectioned by cryotome for histologic examination. Slides were fixed with paraformaldehyde and stained with hematoxylin and eosin.

In the animal experiments, tumor signal was represented graphically as the mean value of back and front imaging at each time point for each treatment group. The time-tumor signal curve of each animal was represented using the area under the curve, which is interpreted as the total tumor burden of the animal. A logarithmic transformation to normalize the area under the curve was followed by an ANOVA for group comparisons with an adjustment for multiple comparisons using resampling. All significance testing was done at the P < 0.05 level, protecting the familywise error rate.

We used several existing human malignant pleural mesothelioma cell lines to assess their sensitivity to NDV oncolysis. Several cell lines showed significant sensitivity to NDV(F3aa)-GFP. JMN (Fig. 1A), H-2452 (Fig. 1B), MSTO-211H (not shown), and VAMT (Fig. 1C) showed significant cell kill at MOIs of 0.1 and 1 by day 7 after infection (67/88%; 77/93%; 90/93%; 91/97%, respectively). Significant cytotoxicity was even produced with MOI as low as 0.01 by day 7 after infection (JMN, 27% cell kill; H-2452, 64%; MSTO-211H, 80%; and VAMT, 73%). The transduced MSTO-211H* cell line showed comparable results to MSTO-211H (40%/75%/87% with MOI 0.01/0.1/1, respectively, by day 7; Fig. 1D).

We proceeded to test the oncolytic efficacy of genetically engineered NDV in an orthotopic murine mesothelioma model. Intrapleural treatment with NDV(F3aa)-GFP proved to be a safe procedure, even in animals that received several treatments every other day. Based on a standard animal care facility-defined dietary regimen, it was observed that all animals continued to exhibit normal activity, feeding, and grooming and were able to maintain their body weights within the first 2 weeks after intrapleural virus injection. This was comparable with our previous experience with NDV, in which i.v., i.p., or intratumoral injection of the virus was shown to be safe and did not result in significant change in weight or activity (21, 34). Significant weight loss was observed only in animals with severe tumor burden. All animals with a weight loss >15% of their initial weight were sacrificed within 5 days because of overwhelming tumor burden.

All control animals that received only PBS instead of virus were sacrificed within 24 days after tumor injection because of disease burden. Sixty percent of animals with either single or multiple early treatments (T1S, T1M) survived the whole follow-up period of 4 months. In the T1 treatment groups, there was only a 60% survival rate in animals treated once (T1S) compared with 100% in animals receiving multiple treatments (T1M) 50 days after tumor injection. Even in the late treatment groups (T10S, T10M), 40% of animals survived >80 days after tumor instillation. After 100 days, survival rates of 0% for T10S versus 40% for T10M were recorded. Survival curves for all treatment groups are shown in Fig. 2.

To assess for tumor burden, we used bioluminescent imaging of the transduced MSTO-211H* cells with luciferase substrate. Animals were injected i.p. with luciferin substrate, and the pleural signal was visualized using the IVIS Imaging System as outlined in the Materials and Methods section. In vivo bioluminescence images of a representative animal from the control group at several follow-up time points are shown in Fig. 3A. Images of a representative animal from treatment group TS1 at several follow-up time points are shown in Fig. 3B and for an animal from treatment group T10M in Fig. 3C.

Progression of the bioluminescence signal during follow-up of each treatment group is shown in Fig. 4. Each graph represents the mean bioluminescent values of a single animal calculated using the mean value of prone and supine position. The threshold of signal intensity to provide a visible image by bioluminescence was 7 × 10⁴ p/s/cm²/sr.

All control animals showed a continuous increase in tumor signal until sacrificed. For the rest of the animals, the following results were observed:

• Group TS1 (single treatment day 1 after cancer cells injection; Fig. 4A): Four of 5 animals lost the bioluminescent tumor signal during the first 2 weeks after treatment and showed no bioluminescent signal 13 days after treatment. Three of 5 animals survived the entire 4-month follow-up period without showing a tumor signal with bioluminescence. No histologic signs of tumor were found in these animals at the final follow-up time point, when these animals were sacrificed.

• Group TM1 (several treatments every other day; Fig. 4B): The bioluminescent tumor signal was eradicated in all animals within 12 days, and 80% of these animals survived 90 days of follow-up. All animals surviving the complete follow-up period showed no histologic signs of tumor when sacrificed.

• Group TS10 (single injection day 10; Fig. 4C): Two nonresponders were observed. One animal showed significant tumor signal decrease after 10 days of treatment (reduction of signal by >10²). The other two animals showed no tumor signal 10 days after treatment. Sixty percent of animals survived 60 days after tumor instillation.

• Group M10 (multiple treatments day 10; Fig. 4D): Two animals showed a significant decrease of tumor signal within 10 days (−log 0.5); one of these animals progressed to complete tumor signal disappearance 30 days after the start of treatment. Two other animals showed tumor signal regression within the first 3 weeks after the start of treatment. One animal showed no decrease in the tumor signal. Two animals (40%; both showed complete tumor signal extinction 30 d after treatment) survived the whole follow-up period and did not show any histologic signs of tumor when they were finally sacrificed after 137 days of tumor follow-up.

Animals receiving multiple treatments starting at days 1 and 10 (groups M1 and M10) showed a significantly higher decrease of tumor signal within the first 10 days after the start of treatment (day 1, P < 0.01; day 10, P < 0.01) compared with animals with only single treatment (groups S1 and S10). Absolute log-difference decrease of tumor signal within the first 10 days after viral treatment initiation was 0.3 for single versus multiple treated animals in the early treatment groups (T1S versus T1M) and 0.32 for the late treatment groups (T10S versus T10M).

Overall, multiple-dose treatment with NDV(F3aa)-GFP showed significant survival benefit when compared with single treatment (P = 0.005). Interestingly, comparing the different treatment groups according to treatment start time showed no significant difference in survival (1 versus 10; P = 0.84). Nevertheless, survival was significantly correlated to tumor burden detectable by bioluminescence. Animals with tumor signal either at day 5, 10, 20 or 30 had significantly poorer survival compared with cured animals (all P < 0.005).

Malignant pleural mesothelioma is a highly malignant cancer resulting in poor long-term survival. There is great hope for novel therapeutic options because of the limited effectiveness of currently established chemotherapeutics and radiotherapy (35, 36). Gene therapy strategies have been investigated in several studies with varying benefit (37). Several replication-competent oncolytic viruses have already been designed and tested for targeted cancer therapy. Examples include herpes simplex virus, adenovirus, vesicular stomatitis virus, myxoma virus, lentivirus, reovirus, and vaccinia virus (38–40).

In particular, herpes simplex and vaccinia have already shown promising efficacy in the detection and treatment of malignant pleural mesothelioma (16, 17, 41). NDV is another promising agent that recently re-emerged in the field of oncolytic virotherapy. The oncolytic effects of NDV were first described in the mid-1950s by Sinkovics and Flanagan (42, 43). Numerous characteristics make NDV an attractive oncolytic vector, and several clinical trials recently showed its safety and therapeutic efficacy (44–46). Several cellular mechanisms have been proposed for NDV antineoplastic properties. First of all, oncolytic viral strains may kill tumor cells directly by inducing apoptosis (47, 48). Secondly, replication of NDV occurs in the cytoplasm and is associated with the production of single- and double-stranded viral RNA. NDV infection of tumor cells introduces danger signals that can be recognized by RIG-I and PKR in the cytoplasm and by toll-like receptors in endosomes, leading to induction of an antiviral state and apoptosis (49, 50). Thirdly, application of NDV may stimulate the host to produce cytokines such as IFNs or tumor necrosis factor, which in turn leads to the activation of natural killer cells, monocytes, macrophages, and sensitized T cells, which are supportive in tumor clearance (51, 52).

Additionally, NDV was shown to be an effective vaccine vector capable of eliciting a potent immune response targeted to the encoded vaccine antigens (20). Infection with NDV induces a strong immune response within the tumor, helping the host immune system overcome tumor-induced immunologic barriers. The ability of oncolytic NDV to induce tumor-specific immune responses has been shown in clinical trials (53).

Using NDV as a vector for tumor gene therapy offers several advantages over other viral expression systems. First of all, it is an avian virus and most humans have no pre-existing immunity to the virus. Second, it is an RNA virus replicating in the cytoplasm without a DNA stage, thus limiting the possibility for genetic recombination with host cell DNA (51).

In this study, for the first time, NDV (F3aa)-GFP was successfully used to treat malignant pleural mesothelioma in an orthotopic mouse model. Viral efficacy was monitored with bioluminescence in a long-term follow-up setting for >4 months.

During in vitro testing, mesothelioma cell lines proved to be susceptible to NDV oncolysis and showed encouraging results even with low MOIs. Nevertheless, differences were found in the grade of efficacy to viral treatment in different cell lines. Still, the exact mechanism of susceptibility of cancer cells to NDV is not clearly understood and is part of intensive investigation. Schirrmacher and Fournier (15) described that the induction of an antiviral state depends on the expression of IFN-stimulated factors such as OAS and PKR. In support of the latter, our previous studies showed that type I IFN response plays a strong role in inhibiting NDV replication and spread (21, 54).

On the other hand, Puhlmann et al. (55) showed that H-Ras was essential for viral replication and the GTPase Rac1 for viral susceptibility. We speculate that the factors above may be responsible for the differential sensitivity to the virus; however, further studies need to be done.

To confirm these findings in vivo, a firefly luciferase-transduced MSTO-211H cell line was incorporated into an orthotopic pleural mesothelioma tumor model and treated with locally administered NDV (F3aa)-GFP. Bioluminescence was used as a long-term follow-up investigation tool to detect tumor progression in live animals. In addition, bioluminescence imaging enabled indirect evaluation of the efficiency of tumor lysis in the pleural cavity. Consistent with the in vitro results, tumor signal decrease was noted starting with days 1 to 3 after viral injection and was obvious within the first 10 days after treatment. This suggested that the inoculated viruses continued to replicate in the tumor cells for several cycles. Interestingly, a higher oncolytic potency of the virus was detectable within the early compared with the late treatment groups, which did not reach statistical significance.

Survival rates of ∼40% or higher could be achieved in all treatment groups after 80 days of tumor follow-up compared with sacrifice of all control animals within 24 days. Earlier treatment resulted in higher survival, likely secondary to lower tumor burden during the earlier stages. These data may provide prognostic speculation about response to NDV treatment in more advanced stages of malignant mesothelioma. Animals surviving until day 137 after tumor injection showed no signs of tumor in their pleural cavity after final sacrifice. NDV proved to be safe even after multiple intrapleural virus applications. No signs of viral toxicity were observed, and all animals maintained normal food intake and normal activity.

Virotherapy with either single or multiple treatments showed significant response to oncolytic therapy with NDV(F3aa)-GFP. Nevertheless, overall survival was significantly better in the multiple-treatment groups. Similar results were found in previous studies with human neuroblastoma xenografts and melanoma cell lines (54, 56). Multiple dosing likely allows the delivery of higher virus titers, which may improve viral diffusion to cells deeper within the tumor.

This is especially true in the case of NDV, which has been previously shown to be highly susceptible to the effects of mammalian type I IFN response (54).

In conclusion, NDV showed promising results as a cytotoxic agent against malignant pleural mesothelioma. The virus produced no signs of toxicity and offered prolonged survival in animals. These findings prompt further investigation of NDV as a therapeutic agent against this highly malignant tumor.

No potential conflicts of interest were disclosed.