Virus and cell lines
The LaSota NDV strain used in this study was prepared from Razi Institute of Serum and Vaccine Research Center. The strain was propagated in the allantoic cavity of 9- to 11-day-old SPF embryonated chicken eggs, and all allantoic fluid samples were harvested and kept at –80°C until use. The titer of the virus was determined using Embryo Infectious Dose 50 (EID50). In order to inactivate NDV, the sample was exposed to the UV radiation [14] and the result was confirmed by Vero cell line [15, 16]. The finding revealed that the UV-inactivated NDV does not create any plaques in Vero cells. EID50 is commonly used as a titration unit of the NDV. For EID50 to PFU conversion, we used EID/50 ∼ 0.7 PFU formula [17].
The murine TC-1 cell line was purchased from the National Cell Bank of Iran (Pasteur Institute of Iran). Briefly, TC-1 cells were cultured in complete RPMI 1640 media (Gibco BRL, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS) (Gibco, Rockville, MD), 100 U/mL of penicillin, 100 𝜇g/mL of streptomycin and 0.4 mg/mL G418 (all from GIBCO, UK), 0.5 mM sodium pyruvate (Sigma Aldrich, Germany), and 2mM L-glutamine. The EL4 cell line (murine T-cell lymphoma of haplotype H-2b derived from C57BL/6 mice) was cultured in RPMI 1640 supplemented with 10% FBS. In addition, MSC cells were flushed from the femurs and tibia of female 6-8-week-old C57BL/6 mice and seeded onto a petri dish containing MSC DMEM F-12 medium (Gibco, UK), 10% FBS, and 100 U/mL of penicillin, 100 𝜇g/mL of streptomycin. The cells were grown for 2–3 weeks until almost confluent. Adherent cells were then detached by 0.25 % trypsin-EDTA and replated using a 1:3 dilution until the third passage. All the cells were incubated at 37∘C in a humidified 5% CO2 incubator.
Mice
Six- to 8-week-old female C57BL/6 (H2b) mice were purchased from the Institute Pasture of Iran (Tehran, Iran). The mice were adapted to the environment for 1 week before the experiment, and had free access to food and water and were kept in a 12-12 light period. All experiments were performed according to the Ethical Committee for the use and care of laboratory animals of Iran University of Medical Sciences (ethics number: IR.IUMS.FMD.REC 1396.9321540001).
Flow cytometry analysis
To verify the stromal nature of cultured cells, the expressions of surface antigens CD44 and CD105 as MSCs markers and antigens CD45 and CD34 as hematopoietic cell markers with proper control isotypes and flow cytometry (BD FACS ARIA II, Becton Dickinson, San Jose, CA, USA) were assessed following fluorochrome-conjugated monoclonal antibodies, including fluorescein isothiocyanate (FITC)-labeled anti-CD44 (Cat. No. 561859) and -CD105 (Cat. No. 565944) (50 μg/ml), and phycoerythrin (PE)-labeled anti-CD45 (Cat. No. 561087) and -CD34 (Cat. No. 551387) (25 μg/ml) (all from BD Biosciences, USA). Briefly, cultured cells were trypsinized with 2.5% trypsin-EDTA, washed twice with PBS, and incubated with 10µl of mentioned antibodies at room temperature for 30 min in the dark. Permeabilization with 0.1% Triton X-100/PBS for 1 min was performed before incubation with the intracellular markers. The cell populations were then characterized according to the surface markers using a FACS Calibur flow cytometer (BD Bioscience, USA). The data were collected and analyzed using Flowjo software (Version 7.6). In addition, nonspecifically labeled proteins were identified by suitable isotype-matched antibodies.
In vitro osteogenic and adipogenic differentiation potential of MSC
Bone marrow MSCs (BM-MSCs) at third passage had been cultured in 12-well cell culture plate (SPL Inc., Korea) until approximately 90% confluence before adipogenic and osteogenic differentiation media were added as previously described [18]. Adipogenic differentiation medium was made with DMEM supplemented with 10 µg/ml insulin and 10−6 M dexamethasone (all supplements from Sigma, St Louis, MO). Adipogenesis was confirmed after 21 days by Oil Red O (Sigma-Aldrich) staining and the accumulation of neutral lipids in fat vacuoles.
Osteogenic differentiation medium was made with DMEM supplemented with 10−8 M dexamethasone, 10 mM β-glycerophosphate, and 50 µg/ml ascorbic acid (all supplements from Sigma, St Louis, MO). The media was changed twice a week for 3 weeks. Matrix mineralization was confirmed by calcium phosphate deposits after staining with 0.2% Alizarin Red S (2% aqueous solution, pH 4.1–4.3, adjusted with ammonium hydroxide) for 20 min.
Viral infection of BM-MSCs with oncolytic NDV
The isolated MSCs were cultured in a six-well plate at a cell density of 5×105 cells/well for 24 hours. Subsequently, the MSCs were infected with the various multiplicity of infection (MOI) (1-40) of NDV for 1 hour in DMEM F-12 medium. Afterward, NDV-encapsulated MSCs were washed with PBS for removal of the NDV from the supernatant medium. Based on the results, the MOI of 20 was chosen as an optimum dose for BM-MSCs infection.
In vivo tumor treatment experiment
In vivo tumor induction was conducted through subcutaneous (s.c.) injection of 7×105 TC-1 tumor cells/mouse into the right flank area of the mice on day 0, then they randomly divided into six different groups (10 mice/group). Ten days after tumor cells injection, C57BL/6 mice were treated peritumorally (p.t.) with MSC (105 cell/100 μl), MSC/iNDV (105 cell/100 μl containing inactivated NDV), MSC/NDV (105 cell/100 μl containing activated NDV), iNDV (108 PFU/100 μl inactivated NDV), NDV (108 PFU/100 μl activated NDV) and PBS (100 μl) twice at one week intervals. Tumor growth and survival were monitored two to three times a week. Thereafter, mice were monitored twice a week by inspection and palpation. Tumor size was monitored by measuring the length (i.e., the longest dimension) and width (i.e., the shortest dimension) using electronic calipers. Tumor volume was calculated by the following formula: tumor volume = 0.5 × (length + width2).
BM-MSC transduction and GFP reporter gene detection
BM-MSCs were transduced with a lentiviral vector expressing the enhanced green fluorescent protein (eGFP) gene (a gift from Stem Cell Technology Research Center, Tehran, Iran) at a multiplicity of infection of 10 (MOI=10) and the transduction efficiency was evaluated directly in cell culture using fluorescence microscopy (Olympus, Tokyo, Japan) after 24h (Fig 2).
In order to track the migration and distribution of injected BM-MSCs transduced with eGFP (MSC-eGFP) in the tumor microenvironment, 1.5×106 MSCs at the third passage in 100 μL of PBS were injected into tumor-bearing mice through the peritumoral administration procedure. Mice (n=3/group) were sacrificed under deep anesthesia after the injection, and the intensity of fluorescent signal was evaluated in tumor sections.
Lymphocyte proliferation assay (LPA)
In order to investigate whether treatment with the mesenchymal stem cells infected with oncolytic NDV could induce antigen-specific cell-mediated immunity, lymphocyte proliferation was performed in vitro. In this assay, the capability of re-stimulated splenocytes in converting tetrazolium to insoluble purple formazan was evaluated. One week after last treatment, splenocyte culture at 2×105 cells/well was established in 96-well round-bottom plates containing RPMI-1640 supplemented with 10% FBS, 1% L-glutamine, 1% HEPES, and 0.1% penicillin/streptomycin (in triplicate), followed by 72 h incubation at 37°C in a 5% CO2 incubator in the presence of 1 μg/ml E7-specific H-2Db CTL epitope (1 μg/ml, Biomatik, Ontario, Canada, >99% purity), PHA (positive control), and medium alone as negative control. Afterward, the supernatants were removed, and the pellets were solubilized in 100 μl dimethyl sulfoxide attempting to eliminate the possibly produced crystals of formazan. Plates were read at a wavelength of 540 nm and stimulation index was used for expressing the results. This index was obtained as follows:
See Formula 1 in the Supplemental Files
In vitro cytotoxic activity
To confirm whether mesenchymal stem cells infected with oncolytic NDV could induce cytotoxic immune responses by activating antigen-specific cytotoxic T lymphocytes, in vivo cytotoxic T lymphocyte (CTL) assay was performed by the measurement of lactate dehydrogenase (LDH) release. One week after the last treatment, a single-cell suspension of splenocytes was prepared and applied as effector cells. For the preparation of the target cells, EL4 cells were pulsed with 1 μg/ml E7-specific H-2Db CTL epitope. An exact viable number of 4×104 EL4 cells in a volume of 100 μl (as target cells) were co-cultured with effector cells (100 μl) at 50:1 effector-to-target cell (E/T) ratios, in which a maximal release of LDH was observed. After centrifugation, the supernatants (50 μl/well) were transferred to 96-well plates, and CTL activity was measured [19].
Cytokine ELISA assay
Seven days after the second treatment, the spleen of the mice (n=3) were isolated and mononuclear cells from spleen of immunized mice were seeded at a concentration of 2×105 cells/well in 24-well plates (TPP, Switzerland) for three days in RPMI1640 supplemented with 10% FBS, 1% L-glutamine, 1% HEPES, 2.5mM 2-mercaptoethanol, and stimulated with E7-specific H-2Db CTL epitope at a concentration of 1 μg/ml (Biomatik, Ontario, Canada, >99% purity) at 37°C in 5% CO2. The cell supernatants were collected after 48 h and the secretion of IL-4, IFN-γ and IL-12 in the supernatant were evaluated by commercially available ELISA kits (R&D Systems Inc., Minneapolis, Minn, USA) following the manufacturer’s instructions. All samples were performed in triplicate and the plates were read at optical density (OD) 450 nm.
Intratumoral Activity Assay of Caspase 3 and Caspase 9
Intrinsic apoptosis is one of the pathways that may be induced by oncolytic NDV. Caspase-3 and -9 activities in the tumor microenvironment were measured by caspase ELISA kit (Abcam, Cambridge, MA, USA). Briefly, the tumor tissue was extracted from each group (n=3) and 100 mg of discarded tissue homogenized in 0.5 ml lysis buffer (0.1 M Tris-HCl (pH 7.6) and 0.1 M fresh dithiothreitol). After centrifugation at 10,000× g (1min), equal amount of supernatant was added to the substrate‐containing reaction buffer (0.1 M dithiothreitol and 5 μl of 4 mM DEVD-p-NA) and incubated for 120 min at 37 °C. Finally, the caspase-9 and -3 activities were assessed by the microplate reader (BioTek, 800TS, USA) at an absorbance of 405 nm. Each experiment was repeated in triplicate.
Histology and immunohistochemistry (IHC)
For histological analysis, harvested tumor tissues were collected and immersed in 10% buffered formalin and then embedded in paraffin. Specimens were sectioned at 5 µm thickness and stained with hematoxylin/eosin (H&E). Finally, all specimens were observed under microscope (Nikon) and images were captured with digital camera (RT color SPOT). Subsequently, mitotic cells and histological structure between different groups were compared.
To evaluate the level of myeloid and myeloid-derived suppressor cells (MDSCs) in tumor tissue, the tumor sections were also analyzed immunohistochemically using anti CD11b (BioLegend Cat. No. 101207) and anti‐Gr-1 (BioLegend Cat. No. 108407) antibodies, respectively.
Briefly, Tumor tissue sections were deparaffinized and rehydrated in alcohol gradients and then washed and boiled for antigen retrieval (10 min at 95 °C). In the next step, sections were blocked using bovine serum albumin (BSA) and incubated with biotinylated goat anti‐rat secondary antibody (1:500, Sigma Aldrich) for 1 h at room temperature. After washing and incubation with horseradish peroxidase (HRP)-conjugated streptavidin (Sigma-Aldrich, Pro. No. 18-152), the reaction was revealed with DAB (Sigma-Aldrich). Cell counting was performed on randomly taken photographs of IHC-stained sections from four independent samples, using an oil-immersion 100x objective. Finally, image J software (NIH, Bethesda, USA) was used to quantify stained regions.
Statistical analysis
All statistical analysis was performed using the SPSS 16.0 software through one-way ANOVA technique. A value of *P<0.05, **P<0.01 and ***P<0.001 were considered to demonstrate statistical significance.