Recombinant oncolytic Newcastle disease virus displays antitumor activities in anaplastic thyroid cancer cells

Chicken embryo fibroblast cell line, DF1 (cat no. GNO30), was obtained and authenticated by the Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). THJ-16 T and THJ-29 T cells were kindly provided by the Mayo Foundation for Medical Education and Research to Dr. Quentin Liu [25]. THJ-16 T cells were cultured in RPMI-1640 containing 5% FBS, 10 mM HEPES (Thermo Fisher) and 1 mM sodium pyruvate (Thermo Fisher). THJ-29 T cells were cultured in RPMI-1640 containing 5% FBS and 1 mM sodium pyruvate. A virulent strain of NDV/FMW (GenBank accession number: GU564399) was prepared as reported previously [20]. SB203580, a specific p38 inhibitor, was purchased from Selleckchem which was prepared with dimethyl sulfoxide (DMSO) and stored at − 20°C.

The construction of the recombinant NDV/FMW expressing GFP was performed essentially as described in our previous study for the generation of the recombinant NDV/FMW expressing apoptin [24]. To construct rFMW-GFP, a GFP-labeled fragment flanked by the appropriate NDV-specific RNA transcriptional signals was inserted into the ApaI site created between the P and M genes of pT7NDV/FMW. The resulted plasmid was named as rFMW-GFP and sequencing verified. Viruses were rescued from complementary cDNA using methods described previously [24]. The resultant recombinant virus, rFMW/GFP was prepared, stored and titered as previously described [20‐22, 24].

THJ-16 T and THJ-29 T cells were cultured in 6-well plates and infected with NDV/FMW or rFMW/GFP at a multiplicity of infection (MOI) of 10. Cells were observed using fluorescence microscopy (Olympus IX81). Live cell imaging of bright-field and fluorescence was recorded at 24 h post-infection.

THJ-16 T and THJ-29 T cells for immunofluorescence assay were seeded on coverslips (NEST, 801008) in 24 wells plate and fixed in 4% paraformaldehyde (PFA) for 30 min, then the cells were permeabilized in 0.2% Triton X-100 for 15 min. Non-specific binding sites were blocked by incubation with 3% Bovine Serum Albumin (BSA) for 60 min. Cells were then incubated with primary anti-HN antibody (1:50) overnight at 4 °C. After washed, secondary antibodies (1:1000) were added to appropriate wells. After 60 min, Nuclei were stained with DAPI (5 μg/mL, Sigma) in PBS. Images were acquired using a confocal microscope (Leica TCS SP5 ×) and images were captured with a camera controlled. Images from each experiment were acquired using the same exposure time during the same imaging session.

THJ-16 T and THJ-29 T cells were seeded in 60-mm dishes and infected with vehicle or rFMW/GFP at 10 MOI. Cells were harvested using scraper and lysed in lysis buffer (Roche, USA) at 6, 12 or 24 h. Cell samples were loaded and separated by 10 or 15% SDS-PAGE and subsequently transferred to nitrocellulose membranes (Applygen Technologies Inc. Beijing, China) using a transblot turbo system. Membrane was blocked with 5% milk diluted in TBST buffer (0.05% Tween-20) for 3 h and incubated with primary antibody at 4 °C overnight. The antibodies for GFP-tag (1:10000, Sigma, SAB4301138), HN (1:500, Santa cruz, SC-53562), β-actin (1:10000, Sigma, A1978), caspase-3 (1:1000, Cell signaling technology, 9662S), PARP (1:1000, Cell signaling technology, 9532S), phospho-p38 (1:1000, Cell signaling technology, 9215S), total p38 (1:1000, cell signaling technology, 9212S), phospho-Erk1/2 (1:8000, Promega, V803A), total Erk1/2 (1:8000, Promega, V114A), phospho-JNK (1:2000, Cell signaling technology, 9251S) and total JNK (1:1000, Cell signaling technology, 9253S) were used. After washed three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:10000, Invitrogen, USA) for 1 h at room temperature with continuous rocking. The blots were detected using ECL Western Blot Substrate kit (Thermo Fisher, USA) [20].

rFMW/GFP (1 × 10⁷ TCID50 per dose) was injected intravenously (i.v) into BALB/c mice. To assess GFP expression in organs, mice were euthanized 24 h following virus injection. Heart, liver, spleen, lungs and kidneys were harvested. Cells were lysed and GFP protein was visualized by IB.

DF1 cells were seeded in 96-well plates and then infected with 10-fold serially diluted viruses. Viral titer was measured by end-point dilution assay (50% tissue culture infective dose (TCID50]/ml) and the TCID50 was calculated by the method of Reed and Muench (Reed and Muench, 1938).

Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on the formation of formazan crystals from tetrazolium by living/metabolically active cells. THJ-16 T and THJ-29 T cells were seeded in 96-wells plates (5000 cells/well), and then the cells were vehicle-infected or infected with varying MOI of rFMW/GFP (0.01, 0.1, 1, and 10) for 24, 48, 72 h. Cell growth inhibition was determined as previously described [22].

THJ-16 T and THJ-29 T spheroids were prepared from monolayer cells which were trypsinised and plated in ultra-low attachment 96-well plates (1000 cells/well). The cells were containing in serum-free DMEM/F12 medium supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF) and 1 × B27. After 7 days, the propagated spheroid bodies were observed and counted by light microscope.

Female age-matched (6 weeks old) nude mice were housed in specific pathogen-free (SPF) conditions. THJ-16 T cell suspension (5 × 10⁶ cells in 100 μL PBS/mouse) was injected subcutaneously in the right flank to induce tumor development. When tumors reached an average volume of 200 mm³, the rFMW/GFP treatments were initiated by intratumoral injection. Mice were randomly divided into two groups (eight mice per group): (a) vehicle control, (b) intratumoral administration with rFMW/GFP (1 × 10⁷ TCID50 per dose). Mice were injected three times weekly. After 1 week, four mice (of eight) were euthanized to make into slices. The slices were subjected to either hematoxylin-eosin (H&E) staining or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as previously described [24]. The total proteins of tumor tissue samples were harvested from the other four mice in each group to test GFP and HN expression by immunoblot assay.

For the in vivo oncolysis study of growth curve, ten mice were included in each group ((a) vehicle control, (b) intratumoral administration with rFMW/GFP (1 × 10⁷ TCID50 per dose), (c) intratumoral administration with NDV/FMW (1 × 10⁷ TCID50 per dose), was treated for 3 weeks. Tumor growth was monitored at 5-day intervals for 50 days using and volume was determined with digital caliper according to the formula: volume = (greatest diameter) × (smallest diameter)²/2. Euthanasia: Treat mice in an inhalation anesthesia machine (Shanghai Biowill Co., LTD, Model: BW-AM503) with 5% isoflurane (Sigma, cat no. Y0000858); 2.4 L/min N2O; 1.2 L/min O2. Observe the mice. (The depth of anesthetization is sufficient when the following vital criteria are reached: regular spontaneous breathing. No reflex after setting of pain stimuli between toes, and no response to pain.) Carotid after anesthesia mice was killed off. The animals were tested in a biosafety cabinet of the SPF laboratory animal center of the Dalian Medical University (Dalian, China), complying with the national guidelines for the care and use of laboratory animals and were approved by the experimental animal ethics committee at Dalian Medical University.

We have previously shown that the NDV strain FMW (NDV/FMW) exhibits strong oncolytic activity in vitro and in vivo [20‐23]. To better monitor the NDV/FMW infection process in cancer cells, we generated recombinant NDV carrying the GFP reporter gene (Fig. 1a). The construction of the recombinant NDV/FMW expressing GFP (hereafter as rFMW/GFP) was performed utilizing the reverse genetics rescue system, which was employed in our previous study for the generation of the recombinant NDV/FMW expressing apoptin [24].

GFP expression in rFMW/GFP-infected ATC cells (THJ-16 T and THJ-29 T) was demonstrated by direct fluorescence and immunoblotting (Fig. 1b and c). The expression of HN protein was detected in NDV/FMW-infected cells (Fig. 1c). Importantly, detection of GFP was accompanied by the expression of HN protein in rFMW/GFP-infected cells (Fig. 1c), indicating the replication of rFMW/GFP in infected cells. In addition, the stability of the recombinant virus was evaluated by inoculating rFMW/GFP into SPF embryonated chicken eggs after five serial passages. GFP insertion was subsequently confirmed by RT-PCR assay (data not shown). To determine the in vivo distribution of rFMW/GFP, expression of GFP protein in organs from mice intravenously injected with rFMW/GFP was analyzed by immunoblotting. GFP protein was detected in spleen and lung (Fig. 1d), indicating the tissue tropism of rFMW/GFP and demonstrates the successful construction of a recombinant oncolytic NDV expressing GFP.

To investigate whether GFP insertion caused any replication defect in NDV/FMW, the growth characteristics of rFMW/GFP and its parent virus were evaluated in a single-step growth cycle in DF-1 cell lines. The insertion of GFP into the NDV/FMW genome did not significantly influence viral replication kinetics and total viral yield during the 72 h post-infection (hpi) period compared to the parental NDV/FMW strain (Fig. 2a). However, detection of GFP was accompanied by the expression of HN protein in rFMW/GFP-infected THJ-16 T and THJ-29 T cells by confocal microscopy (Fig. 2b), indicating the successful replication of rFMW/GFP in the ATC cell lines tested.

We proceeded to determine whether rFMW/GFP induced growth inhibition in ATC cell lines. rFMW/GFP infection induced a significant reduction in viability of THJ-16 T and THJ-29 T cell lines in a time and concentration-dependent manner compared to mock infections (Fig. 3a). Similar results were obtained in parent NDV/FMW-infected THJ-16 T and THJ-29 T cells (data not shown). To examine whether the rFMW/GFP-induced growth inhibition of ATC cells was due to apoptosis, caspase-3 and Poly (ADP-ribose) polymerase (PARP) cleavage, two classical markers of apoptosis, was demonstrated in rFMW/GFP-treated THJ-16 T and THJ-29 T cells at 12 hpi as assessed by immunoblotting (Fig. 3b). Together, these data indicated that rFMW/GFP induced apoptosis in ATC cells.

Given that tumor spheroids are considered a useful in vitro model to mimic the biological properties of tumors, we examined whether the effects of rFMW/GFP on spheroids derived from ATC cells in a three-dimensional (3D) culture system. Upon rFMW/GFP infection at an MOI of 10 for 48 h, rFMW/GFP-infected THJ-16 T or THJ-29 T cells did not grow under these conditions compared to mock-infected cells (Fig. 3c).

We sought to explore the potential signaling pathways involved in rFMW/GFP-triggered oncolytic cell death in ATC cells. We examined the role of the MAPK pathway (Erk1/2, JNK and p38) as potential downstream signaling MAPK proteins, as we had previously shown these to be implicated in NDV/FMW-induced cytotoxic effects on lung cancer cells [20, 21]. p38 MAPK signaling was activated in both THJ-16 T and THJ-29 T cells upon rFMW/GFP infection at 12 hpi and in THJ-29 T cells at 24 hpi. While phosphorylation of either Erk1/2 or JNK was not significantly altered in either cell line (Fig. 4a). In addition, we examined other critical pathways such as Akt and p53 signaling which are generally involved in cell survival and apoptosis in THJ-16 T and THJ-29 T cells infected with rFMW/GFP. As shown in the Additional file 1: Figure S1, total AKT level and p53 level were all downregulated after a 24-h infection with rFMW/GFP, suggesting that both Akt and p53 signaling might play a role in the antitumor effects by rFMW/GFP. But change in p-Akt (S473) was not consistent between the two cell lines upon infection with rFMW/GFP (Additional file 1: Figure S1). To investigate the role of p38 MAPK pathway in rFMW/GFP-induced cell death in ATC cells, the specific p38 MAPK inhibitor, SB203580, was added to THJ-16 T and THJ-29 T cells 30 min prior to virus infection. Treatment with SB203580 significantly reduced rFMW/GFP-induced cell death in both cell lines at 24 hpi. Compared to mock-infected controls (virus only) (Fig. 4b). Inhibition of p38 MAPK activity by SB203580 decreased rFMW/GFP-induced cleavage of caspase-3 and PARP in THJ-16 T and THJ-29 T cells (Fig. 4c). These data suggest a role for p38 MAPK in rFMW/GFP-induced oncolysis of ATC cells.

The oncolytic effects of rFMW/GFP and its parent virus NDV/FMW were investigated in mice bearing tumors derived from THJ-16 T cells. The design of these in vivo experiments were based on previous studies from our lab and others [21, 22, 24, 26, 27]. Tumor sections were examined by H&E staining and TUNEL assay. H&E staining of tumors treated with rFMW/GFP showed characteristic apoptotic cells (Fig. 5a). In addition, the TUNEL assay revealed pyknotic chromatin in virus-inoculated tumors (Fig. 5a). In contrast, fewer necrotic and apoptotic cells were detected in PBS-treated controls. Interestingly, immunoblotting analysis of tumor lysates demonstrated GFP and HN expression in rFMW/GFP-inoculated tumors but not in PBS-treated tumors (Fig. 5b). Moreover, viruses isolated from tumors infected with rFMW/GFP, was shown to be infectious (data not shown), indicating that rFMW/GFP replicated in virus-treated tumors.

Consistent with these in vivo data, significant tumor regression was observed in mice inoculated with either rFMW/GFP or parent virus compared to control groups (Fig. 5c). However, there was no significant difference in tumor growth inhibition between rFMW/GFP and parent virus. Non-tumor-bearing mice injected with either rFMW/GFP or parent virus survived and remained healthy during the course of this in vivo study.