Baby hamster kidney (BHK-21) fibroblasts were obtained from American Type Culture Collection (Cat. No. CCL-10™; ATCC/LGC Prochem, Boras, Sweden) and cultured in Glasgow’s MEM (Cat. No. 11710; Thermo Fisher Scientific, Boston, MA, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. F7524; Sigma-Aldrich, St. Louis, MO, USA), 10% Tryptose Phosphate Broth solution (Cat. No. T4049; Sigma-Aldrich), 20 mM HEPES (Cat. No. H0887; Sigma-Aldrich), 2 mM l-Glutamine (Cat. No. 25030; Thermo Fisher Scientific), 100 U/mL penicillin, and 100 mg/mL streptomycin (Cat. No. P0781, Sigma-Aldrich). The LLC cell line (also called LLC1; Cat. No. CLS 400263) and mouse macrophage cell line J774A.1 (Cat. No. CLS 400220) were obtained from Cell Lines Service (CLS GmbH, Eppelheim, Germany). The LLC cells were grown in RPMI-1640 medium (Cat. No. 61870; Thermo Fisher Scientific) supplemented with 10% FBS (Sigma-Aldrich). The J774A.1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Cat. No. S0415; Biochrom GmbH, Berlin, Germany). The human lung carcinoma cell line A549 (Cat. No. CCL-185™) and murine fibrosarcoma cell line L929 (Cat. No. CCL-1™) were obtained from American Type Culture Collection (ATCC/LGC Standards GmbH, Wesel, Germany). The A549 cells were propagated in Advanced Dulbecco’s MEM (Cat. No. 12491; Thermo Fisher Scientific) supplemented with 5% FBS (Sigma-Aldrich) and 2 mM L-glutamine. The conditioned medium (CM) of L929 cells was used as a source of macrophage colony-stimulating factor (M-CSF) (58) for the maintenance and differentiation of bone marrow-derived progenitors into macrophages. Briefly, L929-CM was produced by cultivating L929 cells in RPMI-1640 with 10% FBS (Sigma-Aldrich), 100 U/mL penicillin and 100 mg/mL streptomycin in T75 flasks until confluency. The medium was then removed, 30 mL of RPMI-1640 supplemented with 10% FBS (Biochrom) without antibiotics was added, and cells were incubated for 10 days. The L929-CM was harvested and spun at 300 g for 10 min. The collected supernatant was filtered through a 0.22-µm strainer and stored at −20°C until used. All cells were cultured at 37°C in a humidified incubator in an atmosphere containing 5% CO₂ and 95% air.

C57BL/6NRj mice (Janvier Labs, Le Genest-Saint-Isle, France) were bred at the Department of Comparative Medicine, Oslo University Hospital, Rikshospitalet (Oslo, Norway). All animal experiments were approved by and performed in accordance with the regulations and guidelines of the Norwegian Food Safety Authority.

Murine BMDMs were differentiated from bone marrow progenitors obtained from C57BL/6NRj mice as previously described (59, 60) with a few modifications. Femur and tibia were aseptically dissected from 8- to 10-week-old C57BL/6NRj mice, and bone marrow cells were collected by flushing the femurs and tibias with RPMI-1640 supplemented with 10% FBS (Biochrom) using a 25 G needle. After the cells were centrifuged for 5 min at 400 g, the erythrocytes were lysed in lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA-Na₂, pH 7.2–7.4, filtered through a 0.2-µm strainer), and the remaining cells were then filtered through a 70-µm cell strainer (Cat. No. CLS431751; Sigma-Aldrich) and centrifuged. The cells were seeded in 90-mm untreated cell culture dishes (Cat. No. 734-2359; VWR, Radnor, PA, USA) at a concentration of 8 × 10⁶/dish and then differentiated via cultivation for 7 days in medium referred to hereafter as complete BMDM differentiation medium (consisting of RPMI-1640 with 10% FBS and 30% L929-CM containing M-CSF). The adherent cells were considered CD11b+F4/80+ macrophages since flow cytometry revealed that these cells were more than 99% pure (data not shown). After 7 days, the cells were detached by incubating them in cold Dulbecco’s phosphate-buffered saline without Mg²⁺/Ca²⁺ (referred to as PBS−/−; Cat. No. D8537; Sigma-Aldrich) for 15–20 min at 4°C. The harvested cells were centrifuged and frozen in FBS containing 10% DMSO (Cat. No. 0231; VWR). The BMDMs were cultivated in RPMI-1640 supplemented with 10% FBS and 10% L929-CM.

Peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coats via centrifugation in Lymphoprep™ density gradient medium (Cat. No. 1114547; Alere Technologies AS, Oslo, Norway) according to the manufacturer’s protocol. Buffy coats were obtained from the blood bank of St. Olav’s Hospital (Trondheim, Norway). Monocytes were enriched from total PBMCs via plastic adherence in 8-well TC Lab-Tek Chamberslides (Cat. No. 80826; ibidi GmbH, Martinsried, Germany) and maintained in RPMI 1640 medium supplemented with 10% human serum (obtained from the blood bank of St. Olav’s Hospital, Trondheim, Norway). Monocyte-derived macrophages were differentiated from monocytes by incubating the cells in RPMI 1640 containing 10% human serum and 10 ng/mL human M-CSF (Cat. No. SRP6165; Sigma-Aldrich) for 7 days.

The pSFV1-DsRed vector, which carries a gene for Discosoma sp. red fluorescent protein (DsRed), was generated in our lab as previously described (61) using the pSFV1 vector (20). The pSFV1 and pSFV-Helper1 plasmids (20) were generously provided by Garoff (Karolinska Institute, Stockholm, Sweden).

To generate the pSFV1-Tnfa-Flag plasmid, we used the pSFV1-NruI vector, which is a derivate of pSFV1 that was generated in our lab by deleting a 527 bp StuI-HindIII fragment from pSFV1 (bp 7603–8130) and changing a unique SpeI site to a NruI site (62). The murine TNF-α-coding gene (Tnfa) (GenBank No. NM_013693.2) fused to a Flag-tag sequence was inserted into the pSFV1-NruI vector under the control of the SFV 26S subgenomic promoter. The Tnfa-Flag fragment was amplified from the template plasmid pCMV3-mTNF-Flag (Cat. No. MG50349-CF; Sino Biological Inc., Beijing, China) using PCR with the following primers (synthesized by Microsynth AG, Balgach, Switzerland): 5′-GCGGATCCATGAGCACAGAAAGCATGATC-3′ (forward) and 5′-TGCCCGGGTTTACTTATCGTCGTCATCCTTG-3′ (reverse). BamHI and SmaI restriction sites (shown here in bold) were introduced to facilitate the ligation of the PCR fragment into the pSFV1-NruI vector.

The pSFV1/Enh-Luc plasmid was generously provided by A. Merits (Institute of Technology, University of Tartu, Estonia) and has been previously described (63). The pSFV1/Enh-Ifng vector was generated by replacing the XmaI-ApaI fragment (bp 7572-9268), which contained the firefly luciferase gene (Luc) of pSFV1/Enh-Luc, with the murine IFN-γ-coding gene (Ifng) (GenBank No. NM_008337.3). The Ifng gene was amplified from the template plasmid pMD19-mIFNG (Cat. No. MG50709-M; Sino Biological Inc.) using PCR with the following primers (synthesized by Microsynth AG): 5′-TCCGCCCGGGATGAACGCTACACACTGC-3′ (forward) and 5′-TCCGGGCCCTCAGCAGCGACTCCTTTTCC-3′ (reverse). XmaI and ApaI (shown here in bold) restriction sites were incorporated to facilitate cloning into the pSFV1/Enh-Luc vector. The sequences Tnfa-Flag and Enh-2A-Ifng of the constructs were verified by GATC Biotech AG (Constance, Germany) and found to share 100% identity with the reference sequences Tnfa (GenBank No. NM_013693.2), Flag (shown in the product description of pCMV3-mTNF-Flag (Cat. No. MG50349-CF; Sino Biological)), Enh-2A [shown in Ref. (7)] and Ifng (NM_008337.3).

RNAs were synthesized and transfected into BHK-21 cells using electroporation as previously described (63). The plasmids pSFV/Enh-Ifng, pSFV-DsRed, and pSFV-Helper1 were linearized using the restriction enzyme SpeI (Cat. No. ER1251; Thermo Fisher Scientific), and pSFV/Tnfa-Flag was linearized using NruI (Cat. No. ER0111; Thermo Fisher Scientific). The linearized plasmids (1 µg of each) were used as templates for the in vitro transcription (SP6 RNA polymerase; Cat. No. AM2071; Thermo Fisher Scientific) of SFV-Helper1 RNA and recombinant RNAs (rRNAs) carrying Ifng, Tnfa, or DsRed. The transcribed rRNAs were capped by adding 1 mM 3′-O-Me-m⁷G(5′)ppp(5′)G cap-structure analog (Cat. No. S1411S; New England Biolabs, Hitchin, UK) during the transcription reaction. To package the rRNAs into viral particles, each of the rRNAs was co-electroporated with the SFV-Helper1 RNA in 1 × 10⁷ BHK-21 cells by pulsing the mixture twice at 850 V, 25 mF using a Gene Pulser-II apparatus (Bio-Rad, Hercules, CA, USA). The Helper1 RNA provided in trans the SFV structural proteins (20) for encapsidation of the rRNA. The electroporated BHK-21 cells were resuspended in 15 mL of BHK-21 cultivation medium containing 1% FBS (Sigma-Aldrich) and then incubated at 33°C for 48 h. The cell growth medium containing the infectious rSFV particles was then harvested, rapidly frozen in liquid nitrogen, and subsequently used as a source of rSFV particles. The titers of the rSFV particles were determined based on the “one virus particle—one infected cell” hypothesis regarding the replication-deficient virus particles. BHK-21 cells were infected with serial dilutions of the rSFV particles. Infected cells expressing DsRed were counted in 10 viewfields using fluorescence microscopy at 24 h post-infection. The cells that were infected with either SFV-Tnfa or SFV-Ifng were stained with rabbit polyclonal antibodies that were specific for the nsp1 subunit of SFV replicase (generously provided by A. Merits, Institute of Technology, University of Tartu, Estonia) and with a fluorochrome-conjugated secondary antibody (Cat. No. A11034; Thermo Fisher Scientific). The stained cell monolayers were observed using EVOSfl digital inverted fluorescence microscope (Thermo Fisher Scientific), and infection-positive cells were counted in 10 viewfields using fluorescence microscopy. The rSFV particle titers were expressed as infectious units (IFU)/mL relative to virus infectivity in BHK-21 cells and were used later in this study to calculate multiplicity of infection (MOI), which indicated how many infectious viral particles were added per target cell during infection. Cells infected with the rSFV stocks did not secrete infectious virus particles, as shown in cell reinfection experiments, confirming that the virus stocks contained replication-deficient SFV.

Baby hamster kidney, LLC, A549, and J774A.1 cells and murine BMDMs were seeded in 12-well plates (Cat. No. 150628; Thermo Fisher Scientific) or 24-well plates (Cat. No. 142475; Thermo Fisher Scientific) and cultivated until 50–80% confluency. The cells were washed once with PBS containing Mg²⁺ and Ca²⁺ (referred to as PBS+/+; Cat. No. 14040; Thermo Fisher Scientific), and 140—600 µL of an infectious suspension containing SFV-DsRed at a MOI = 10 or MOI = 15 (defined as 10 or 15 IFU in BHK-21 cells) in PBS+/+ was added to duplicate sets of wells. PBS+/+ was used as a negative control. After the cells were incubated at 37°C for 80 min, the infectious suspension was replaced with cell-specific growth medium, and the expression of DsRed in the infected cells was observed 24–48 h later using an EVOSfl digital inverted fluorescence microscope (AMG). The LLC and J774A.1 cells were detached via scraping, the BHK-21 cells were detached using a 0.25% trypsin-EDTA solution (Cat. No. T4049; Sigma-Aldrich), and the BMDMs and A549 cells were detached by treatment with accutase (Cat. No. A6964; Sigma-Aldrich). The cells were then centrifuged and DsRed-positive cells were quantified 24 h post-infection using BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). To evaluate cell viability BHK-21 and LLC cells were labeled 48 h post-infection with 1 µg/mL of fluorescein isothiocyanate (FITC)-conjugated annexin V (annexin V-FITC from Apoptosis Detection Kit; Cat. No. 556547; BD Biosciences) for 15 min followed by staining with 3 µM of DAPI (Cat. No. 422801; BioLegend, San Diego, CA, USA) for 5 min. Cells were immediately quantified using BD LSRFortessa flow cytometer (BD Biosciences). The data were analyzed using FlowJo V10 software (FlowJo LLC., Ashland, OR, USA).

After differentiation in 8-well TC Lab-Tek Chamberslides (Cat. No. 80826; ibidi GmbH), HMDMs were washed once with PBS+/+ and incubated for 1 h with 200 µL of an infectious suspension containing SFV-DsRed at a MOI = 15 (defined as 15 IFU in BHK-21 cells) diluted in PBS+/+. A recombinant paramyxoviral vector Sendai virus (SeV)-GFP that was based on a wild-type infectious SeV clone that carried a supplemental GFP transgene (64) was generously provided by D. Kolakofsky (Department of Microbiology and Molecular Medicine, University of Geneva School of Medicine, Geneva, Switzerland) and used at a MOI = 15 as a positive control for HMDM infection. The infectious suspension was replaced with normal cell growth medium, and the cells were observed at 24 h post-infection using phase-contrast and confocal fluorescence microscopy with a Zeiss Axiovert 100-M inverted microscope equipped with a LSM 510 laser-scanning unit and a 1.4 NA × 63 Plan-Apochromat oil-immersion objective. To calculate the percentage of fluorescent protein-expressing cells, the total number of cells was manually counted in at least 10 viewfields using phase-contrast microscopy. Fluorescent protein-expressing cells were simultaneously counted within the same viewfields using fluorescence microscopy.

Baby hamster kidney cells (1.97 × 10⁴ cells/well) and LLC cells (7.03 × 10⁴ cells/well) were plated in 96-well plates (Cat. No. 3595; Corning, Corning, NY, USA) and incubated for 22 h. The cells were then washed once with PBS+/+ and incubated at 37°C for 80 min with 50 µL of an infectious suspension containing rSFV particles at MOI = 15 (defined as 15 infectous units in BHK-21 cells) diluted in PBS+/+. The infectious suspension was then replaced with normal BHK-21 or LLC growth medium. Cells incubated in PBS+/+ without viral particles were used as a negative control. Cell morphology was observed 47 h after infection using bright-field microscopy, and 1 μCi of [methyl-³H]-thymidine (³H-TdR; Cat. No. MT6032; Hartmann Analytic GmbH, Braunschweig, Germany) per 1 mL of cell growth medium was then added. After a further 24 h of incubation, the cells were subjected to two freeze/thaw cycles and harvested using a Tomtec-96 cell harvester (Tomtec, Hamden, CT, USA) onto fiberglass filters (Cat. No. 1450-421; PerkinElmer Inc., Waltham, MA, USA). Cell growth was determined by measuring the incorporation of ³H-TdR into proliferating cells as counts per minute (cpm) using a microplate scintillation counter (MicroBeta Trilux 1450, PerkinElmer Inc.).

To produce rSFV-encoded TNF-α and IFN-γ, BHK-21 cells were cultivated in 75 cm² cell culture flasks (Cat. No. CLS430641; Sigma-Aldrich) until 80% confluency, washed once with PBS+/+ and then incubated at 37°C for 80 min with 5 mL of a suspension containing 1.92 × 10⁸ IFU of SFV-Tnfa or 1.21 × 10⁸ IFU of SFV-Ifng in PBS+/+. The virus-containing suspensions were then replaced with BHK-21 cultivation medium containing 1% FBS. After 36 h, the cell growth medium was harvested and centrifuged at 300 g for 5 min to remove the cells before the supernatants were transferred to a new tube, which was then centrifuged at 11,000 g for 8 min. The remaining supernatants were stored at −80°C and used as a source of vector-derived (vd) TNF-α or vdIFN-γ. The concentrations within the supernatants of the cytokines vdTNF-α or vdIFN-γ were 332.28 and 795.92 ng/mL, respectively. The levels of cytokines within the supernatants were quantified using Luminex bead-based assay with Bio-Plex Mouse Cytokine Group I IFN-γ or TNF-α sets (Cat. No. 171G5017M and 171G5023M; Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.

Baby hamster kidney and LLC cells were plated in 24-well plates (Cat. No. 142475; Thermo Fisher Scientific) and cultivated until the next day when the cells had reached 100 and 50% confluency, respectively. The cells were washed once with PBS+/+, and 600 µL of infectious suspension containing either SFV-Tnfa or SFV-Ifng particles at a MOI = 1, MOI = 10 or MOI = 40 (calculated according to the titration in BHK-21 cells) in PBS+/+ was added per well. After cells were incubated with the infectious suspension for 80 min at 37°C, the infectious suspension was discarded, and 500 µL of fresh cell growth medium was added per well. The cells were cultured for 24 h before the growth medium was collected and centrifuged for 6 min at 300 g. The supernatant was then transferred to a new tube, which was centrifuged at 11,000 g for 10 min. The supernatants were stored at −80°C, and the levels of secreted vdTNF-α and vdIFN-γ were determined using Luminex bead-based assay with Bio-Plex Mouse Cytokine Group I IFN-γ and TNF-α sets (Cat. No. 171G5017M and 171G5023M; Bio-Rad) according to manufacturer’s recommended protocols.

L929 fibroblasts or LLC cells were plated in 24-well plates (Cat. No. 142475; Thermo Fisher Scientific) and cultivated in RPMI 1640 containing 10% FBS (Sigma-Aldrich) for approximately 24 h until the cells reached 50–90% confluency. To verify the functional activity of vdTNF-α, it was added to L929 cells in parallel with recombinant (r)TNF-α (Cat. No. 315-01A; PeproTech, Rocky Hill, NJ, USA) to reach a final concentration of 6.6, 20, or 60 ng/mL, and the cells were then cultivated for 24 h. L929 cells that were treated with 1 µM staurosporine (Cat. No. S4400; Sigma-Aldrich) for 24 h were used as the positive control for apoptosis induction (65), and untreated cells were used as the negative control. LLC cells were treated with either 50 ng/mL rTNF-α or 100 ng/mL rIFN-γ for 48 h to test cytotoxicity of cytokines to LLC cancer cells. LLC cells were also treated with 50 ng/mL rTNF-α in combination with 100 ng/mL rIFN-γ for 24 h or 48 h. LLC cells were pretreated with 100 ng/mL rIFN-γ for 24 h followed by 24 h treatment with 50 ng/mL rTNF-α. Untreated cells were used as the negative control, whereas L929 cells that were treated with 50 ng/mL of rTNF-α were used as the positive control for apoptosis induction. After treatments L929 or LLC cells were detached using 0.25% trypsin-EDTA and centrifuged before they were labeled with annexin V-FITC and PI using an Apoptosis Detection Kit (Cat. No. 556547; BD Biosciences) according to the manufacturer’s protocol. Briefly, the cell pellet was resuspended in 100 µL 1 x annexin V binding buffer and stained using 1 µg/mL annexin V-FITC and 5 µg/mL PI by incubating the cells at RT for 15–20 min. Then, 400 µL of additional annexin V binding buffer was added. The cell suspension was immediately analyzed using flow cytometry in a BD LSRFortessa instrument (BD Biosciences). The data were analyzed using FlowJo V10 software.

Murine BMDMs (4 × 10⁶ cells/dish) were seeded in 90-mm nontreated cell culture dishes (Cat. No. 734-2359; VWR) and cultivated in complete BMDM growth medium (RPMI-1640 with 10% FBS (Biochrom) and 10% L929-CM) for 2–3 days. The BMDMs were detached by incubating the cells in cold PBS−/− at +4°C for 15–20 min and then harvested via centrifugation for 6 min at 300 g. To block cell proliferation, the BMDMs were incubated with 10 µg/mL mitomycin C (Cat. No. M4287; Sigma-Aldrich) at 37°C for 2 h. The cells were then washed twice with PBS−/− and plated in triplicate at one of three different densities (i.e., 6 × 10⁴, 3 × 10⁴, and 3 × 10³ cells/well) in 96-well plates in 200 µL of macrophage growth medium. The cells were then cultivated for 24 h. To stimulate the macrophages, we treated them with 100 ng/mL of the toll-like receptor (TLR) 2/1 heterodimer agonist Pam3CSK4 (referred to as Pam3; Cat. No. tlrl-pms; InvivoGen, San Diego, CA, USA), 100 ng/mL of recombinant (r)IFN-γ (Cat. No. 315-05; PeproTech) or 100 ng/mL of vdIFN-γ for 24 h. Untreated BMDMs served as the negative control, and BMDMs that were treated with 100 ng/mL of Pam3 in combination with 100 ng/mL of rIFN-γ were used as the positive control. Some BMDMs were treated for 24 h at 37°C with 100 ng/mL of Pam3 in combination with fourfold dilutions of rIFN-γ at concentrations ranging from 0.0015 to 100 ng/mL. Alternatively, other BMDMs were treated with 100 ng/mL of Pam3 in combination with 16-fold dilutions of vdIFN-γ at concentrations ranging from 0.0015 to 100 ng/mL. After 24 h of treatment, 100 µL of the cell culturing medium was replaced with 100 µL of a LLC cell suspension containing 3 × 10⁴ cells/mL to form co-cultures of macrophages and cancer target cells. The resulting macrophage:cancer cell ratios were 20:1, 10:1, and 1:1. BMDMs that were incubated alone served as the negative control for macrophage growth, and LLC cells that were incubated alone served as the positive control for normal LLC cell growth. After 20 h, ³H-TdR was added to all the wells at a concentration of 1 μCi per 1 mL of growth medium, and the cultures were incubated for an additional 24 h. Next, the plates were submitted to two freeze/thaw cycles and then harvested using a Tomtec-96 cell harvester onto fiberglass filters. Cell growth was analyzed by measuring the incorporation of ³H-TdR into proliferating cells as cpm using a microplate scintillation counter (MicroBeta Trilux 1450).

Nitrite (NO2−) levels were measured using the Griess test in cultivation medium harvested from BMDMs at 24 h post-treatment. Cultivation medium (100 µL) was obtained from the wells with the highest densities of BMDMs and transferred to new 96-well round-bottom plates (Cat. No. 3799; Sigma-Aldrich). These were centrifuged for 6 min at 400 g. Then, 50 µL of the supernatant was pipetted into new 96-well flat-bottom plates (Cat. No. 3595; Corning), and an equal volume of a solution containing 1% sulfanilamide (Cat. No. S9251; Sigma-Aldrich) and 5% phosphoric acid (Cat. No. 345245; Sigma-Aldrich) in H₂O was added. The plates were incubated in the dark for 10 min at RT to allow the sulfanilic acid to convert to diazonium salt. After the incubation period, 50 µL of 0.1% N-(1-naphthyl)ethylenediamine (NED; Cat. No. N9125; Sigma-Aldrich) in H₂O was added to the samples, standards, and blank to induce the immediate formation of azo dye. Solutions of sodium nitrite (NaNO₂) in twofold dilutions at concentrations ranging from 3.13 to 100 µM were used as the standard and set up in triplicate. Water was used as the blank. Absorbance was measured at 540 nm using a microplate spectrophotometer (Epoch; BioTek Instruments, Winooski, VT, USA).

We constructed two new replication-deficient SFV-based vectors that encoded either mTNF-α or mIFN-γ (Figure 1A). To express mTNF-α in the SFV vector, a sequence containing the Tnfa gene followed by a Flag-tag-encoding sequence at the 3-prime end was subcloned into the pSFV1-NruI vector (Figure 1A). The pSFV-Ifng vector was made by inserting the mIFN-γ gene (Ifng) into the pSFV1/Enh vector downstream and in-frame with the SFV capsid translation enhancer sequence (Enh) that was fused to the 2A auto-protease sequence of foot and mouth disease virus (FMDV) (Figure 1A). The 2A-encoding sequence was included to induce the cleavage of the vdIFN-γ protein from the 34 amino acid-long peptide that was translated from the Enh sequence. It has been shown that by incorporating an enhancer element within SFV vector, the expression of the heterologous protein can be increased by up to 10-fold due to enhanced translation (7, 66). In contrast to the SFV-Ifng construct, we did not incorporate the enhancer element into the SFV-Tnfa vector aiming to retain low levels of translated TNF-α if necessary since lower levels of TNF-α might be important for normalization of tumor vasculature (46). The previously constructed vector pSFV-DsRed, which encode a fluorescent protein (Figure 1B), was used as a control to detect cell infection. All vectors comprised the prokaryotic SP6 RNA polymerase promoter for in vitro transcription of recombinant SFV replicons, as well as the vectors encoded SFV replicase complex, which consists of non-structural proteins (nsP) 1–4 (Figures 1A–C). The SFV structural proteins C, p62, 6 K, and E1, which are necessary for the formation of infectious virus particles, were encoded by the SFV-Helper1 vector (Figures 1C,D). The heterologous genes (HGs) and the genes encoding the structural SFV proteins were inserted downstream of the SFV 26S promoter (Figures 1A–C). In this study, replication-deficient rSFV particles were produced by BHK-21 cells that were co-transfected with RNA transcribed from the SFV-Helper1 vector and RNA transcribed from a vector encoding a HG (Tnfa, Ifng, or DsRed) (Figure 1D). The rSFV viral particles were quantified in BHK-21 cells and revealed titers ranging from 10⁷ to 10⁸ IFU/mL. More specifically, the mean viral titers were 1.15 × 10⁸ IFU/mL of SFV-Tnfa, 7.23 × 10⁷ IFU/mL of SFV-Ifng, and 1.46 × 10⁸ IFU/mL of SFV-DsRed. The SE of the mean viral titers between decuplicates did not exceed 10%.

We wanted to use rSFV to deliver cytokine genes into cancer cells for subsequent production of the tumor-suppressive factors. Cell susceptibility to viral infection is a prerequisite for successful gene delivery into cells using a viral vector. We investigated the ability of rSFV particles to infect both cancer cells and macrophages using the reporter vector SFV-DsRed. We infected cells with SFV-DsRed at a concentration 15 virus particles per cell (i.e., MOI = 15). We observed the expression of the DsRed fluorescent protein using fluorescence microscopy and quantified the DsRed-expressing cells using flow cytometry at 24-h post-infection (Figures 2A–E). In all the tested cell types, non-infected cells were used as the negative control for DsRed expression. The BHK-21 cell line was used as a positive control due to being highly susceptible to SFV infection, as demonstrated by flow cytometry analysis that showed that 88.2% of the BKH-21 cells were infected under the experimental conditions used in this study (Figure 2A). We verified that both the murine (LLC) and the human (A549) lung carcinoma cell lines were infected by the rSFV particles because 41% of both cell types were DsRed-positive (Figures 2B,C). However, we also found that neither the mouse BMDMs nor the murine macrophage cell line J774A.1 was susceptible to SFV because less than 1.5% of the cells were DsRed-positive (Figures 2D,E). Macrophages remained resistant to SFV infection also when we increased the concentration of SFV particles during infection (data not shown).

To confirm these findings in human macrophages, we differentiated macrophages from blood-derived monocytes and infected the cells with SFV-DsRed virus particles at a MOI = 15 (calculated according to the titer that was determined in BHK-21 cells). The SeV is known to infect human macrophages (67), and we therefore included a SeV that encoded GFP (SeV-Gfp) at a MOI = 15 as a positive control. In parallel, we infected the human lung carcinoma cell line A549 with both types of recombinant viruses at a MOI = 15, and used the infected A549 cells as a positive control for SFV infection. To calculate the percentages of infected cells, we counted the total number of cells as well as the number of fluorescent protein-expressing cells within the same viewfields (multiple viewfields per sample) using phase-contrast and fluorescence microscopy at 24 h post-infection. In these experiments, 30% of the positive control sample (the human cancer cell line A549) was infected with SFV-DsRed, whereas the HMDMs remained uninfected (Figure 3, middle panel). Contrary to our findings for SFV, SeV-Gfp infected both HMDMs and A549 very efficiently, with 82 and 95% of the HMDMs and A549 cells, respectively, being positive for GFP (Figure 3, lower panel). Taken together, these data show that lung carcinoma cells are efficiently infected by rSFV, whereas primary mouse and human macrophages are resistant to SFV infection, suggesting that rSFV may be used to deliver genes for cancer therapy without killing tumor-associated macrophages.

We wanted to test macrophage viability after challenge with rSFV particles. Both J774A.1 macrophages and LLC cancer cells were subjected to rSFV-DsRed particles at MOI = 10 during 80-min infection protocol. Cell infection rate was determined 48 h post-infection by quantifying DsRed-expressing cells using flow cytometry. Cells were also stained with annexin V-FITC and DAPI followed by flow cytometry analysis to determine cell viability.

As expected, we observed that 98% of non-infected macrophages stayed viable after 48 h as determined using flow cytometry and retained round morphology, which is characteristic to J774A.1 cells in normal culture conditions (Figure 4A). Also 48 h after challenge with rSFV-DsRed particles 90% of macrophages remained viable, whereas 6% of macrophages were single-positive for Annexin V (Figure 4B). Interestingly, light microscopy revealed that macrophages changed morphology 48 h after challenge with rSFV by gaining spindle-like or polygonal shape (Figure 4B). LLC cells were used in parallel as a positive control for cell infection. As expected, the non-infected LLC cells showed 92% viability and were characterized by mixed population of adherent and floating cells with round or spindle-like morphology (Figure 4C). However, the infected LLC cells underwent cell death 48 h after infection with rSFV, and only 35% of LLC cells were viable (Figure 4D). We observed that 33% of rSFV-challenged LLC cells were double-positive for Annexin V-FITC and DAPI suggesting apoptotic/necrotic cell death. A LLC population of 30% was single-positive for DAPI 48 h after infection suggesting affected cell membrane permeability (Figure 4D). Light microscopy revealed that LLC cells possess apoptotic morphology characterized by round swollen cells with translucent cytoplasm after infection with rSFV (Figure 4D). We observed microscopically that both DsRed-positive (infected) and DsRed-negative (non-infected) cells undergo apoptosis after challenge with rSFV particles (Figure 4D, red and white arrows), whereas part of infected DsRed-expressing cells remain viable 48 h post-infection (Figure 4D, yellow arrows). Flow cytometry analysis revealed that 69% of all DsRed-expressing cells undergo cell death characterized mostly by being double-positive for Annexin V-FITC and DAPI (Figure 4E). Our data show that macrophages are not only resistant to SFV infection but also remain viable after challenge with rSFV particles.

It has been shown that SFV infection induces cellular death via p53-independent apoptosis (21). To determine whether rSFV has a direct effect on cancer cell growth, we used in vitro assay to evaluate proliferation of infected LLC cells. The cells were cultivated until approximately 70–80% confluency, and they were then infected with SFV-Ifng or SFV-Tnfa viral particles at MOI = 15. To determine cell growth, a radioactive thymidine, which incorporates into new DNA strands during cell division, was added to the cell cultures at 47 h post-infection. The experimental setup is illustrated in Figure 5A.

BHK-21 cells were used in parallel as a positive control for efficient cell infection by SFV and showed nearly complete growth inhibition (Figure 5B). Growth inhibition was similar independent on whether SFV carried a gene encoding TNF-α or IFN-γ (Figure 5B). It was observed using light microscopy that BHK-21 cells lost confluency and gained apoptotic cell morphology characterized by translucent cytoplasm, granularity, or round shape 47 h post-infection (Figure 5C). LLC cell growth was inhibited by half 47 h after infection with SFV-Ifng and by 80% after infection with SFV-Tnfa (Figure 5D). Light microscopy revealed that LLC cells acquired apoptotic cell morphology characterized by round shape, granularity, and translucent cytoplasm 47 h after infection with either SFV-Ifng or SFV-Tnfa viral particles (Figure 5E). The data show that rSFV particles encoding murine IFN-γ or TNF-α efficiently inhibited LLC cell growth at 47 h post-infection.

In the next set of experiments, we sought to determine whether LLC cells secrete cytokines after infection with SFV-Tnfa and SFV-Ifng virus particles. In this experiment, BHK-21 cells were used as a positive control. The LLC and BHK-21 cells were cultivated until 50 and 100% confluency, respectively, before they were infected with SFV-Tnfa or SFV-Ifng at a MOI = 1, MOI = 10, or MOI = 40 (calculated according to the titration in BHK-21 cells). After 24 h, the levels of secreted vdIFN-γ and vdTNF-α in the cell cultivation medium were analyzed using Luminex bead-based assay. LLC cells infected with SFV-Tnfa secreted vdTNF-α (range, 10³–10⁴ pg/mL for all tested MOIs) (Figure 6A). There was no clear correlation between the MOI of the virus particles and the level of vdTNF-α (Figure 6A). The vdTNF-α was not detected in the medium of non-infected cells or in the medium of cells infected with SFV-Ifng, confirming that the secreted vdTNF-α was derived from the Tnfa gene that was delivered using SFV (Figure 6A).

In LLC cells, infection with SFV-Ifng resulted in the secretion of vdIFN-γ into the growth medium at a concentration ranging from 2.9 to 9.9 × 10² ng/mL at 24 h after infection (Figure 6B). The concentration of vdIFN-γ appeared to correlate with the concentration of viral particles, and the highest tested MOI resulted in the highest level of vdIFN-γ (Figure 6B). The vdIFN-γ was not detected in media obtained from non-infected cells or from cells infected with SFV-Tnfa, indicating that the vdIFN-γ originated from the SFV-delivered cytokine gene (Figure 6B). In conclusion, these results show that both BHK-21 and LLC cells produce and secrete vdTNF-α and vdIFN-γ when infected with the rSFV vectors described in this study.

To determine whether the vdTNF-α is biologically active, we utilized a known property of TNF-α which is to induce necrotic cell death in the murine fibrosarcoma cell line L929 by binding to TNF receptor-1 (68). In these experiments, L929 cells were cultivated until 90% confluency; we then added rTNF-α or vdTNF-α at concentrations varying from 2.2 to 60 ng/mL to the cells. The culture medium of BHK-21 cells infected with SFV-Tnfa particles was used as the source of vdTNF-α. At 24 h after treatment, the morphology of the L929 cells was evaluated using bright-field microscopy. Then cells were stained with annexin V-FITC to determine phosphatidylserine exposure, whereas staining with PI was used to determine loss of membrane integrity in order to quantify the numbers of viable (Annexin V−PI−), apoptotic (Annexin V+PI−), and necrotic (Annexin V+PI+) cells using flow cytometry (Figures 7A–J). Untreated L929 cells were used as a control, and viable cells were defined as those that were annexin V-negative/PI-negative (Figure 7A). Treatment with staurosporine was used as the control for apoptotic cell death (65), where annexin V-positive/PI-negative cells were regarded as early apoptotic, whereas annexin V-positive/PI-positive cells were regarded as late apoptotic undergoing secondary necrosis (Figure 7B).

In the absence of any treatment, the L929 cells reached 100% confluency after 24 h and had a normal fibroblast-like, slightly polygonal cell morphology (Figure 7A). The untreated L929 cells were also analyzed using flow cytometry and used to set the gates for the viable (Annexin V−PI−) cell population, which accounted for 99% of all events (Figure 7A). After cells were treated with 1 µM staurosporine, cell confluency was lost, and an apoptotic cell morphology characterized by cell shrinkage, a round shape and granularity was observed (Figure 7B). Only 11% of the staurosporine-treated cells were viable. The remaining cells were undergoing apoptosis and secondary necrosis (Figure 7B). In cells that were incubate with increasing concentrations of rTNF-α or vdTNF-α (2.2, 6.7, 20, or 60 ng/mL), we observed a concentration-dependent increase in the proportion of apoptotic (Annexin V+PI−) and necrotic (Annexin V+PI+) cells (Figures 7C–J) in a similar manner between rTNF-α and vdTNF-α. Treatment with vdTNF-α and rTNF-α at the lowest concentration (2.2 ng/mL) induced apoptosis in 9–13% and necrosis in 19–20% of the cells (Figures 7C,D), whereas the highest concentrations of rTNF-α and vdTNF-α (60 ng/mL) induced apoptosis in 18–23% and necrosis in 46–62% of the cells (Figures 7I,J). Moreover, after treatment with rTNF-α or vdTNF-α, we observed necrotic cell morphology characterized by round, swollen cells with translucent cytoplasm. These data verify the functionality of the vdTNF-α protein because it was able to induce apoptosis and necrosis in L929 cells in a concentration-dependent manner that was similar to that observed for rTNF-α.

It has been previously shown that LLC cells are resistant to cell death induced by TNF-α (69). Based on previous studies, where IFN-γ was shown to enhance TNF-α receptor expression in cancer cells (51, 52), we wanted to test whether IFN-γ may sensitize LLC cancer cells to TNF-α treatment. Therefore, we tested induction of cell death in LLC cells after combined treatment of recombinant cytokines TNF-α and IFN-γ. LLC cells were single-treated with either TNF-α or IFN-γ for 48 h. LLC cells were treated with TNF-α in combination with IFN-γ for 24 h or 48 h. LLC cells were also pretreated with IFN-γ for 24 h followed by 24 h treatment with TNF-α. L929 fibroblasts, which were treated with TNF-α, were used as a positive control. Untreated cells were used as a negative control. Cell morphology after treatments was evaluated using bright-field microscopy. Cells were stained with annexin V-FITC to determine phosphatidylserine exposure, whereas staining with PI was used to determine loss of membrane integrity in order to quantify the numbers of viable (Annexin V−PI−), apoptotic (Annexin V+PI−), and necrotic (Annexin V+PI+) cells using flow cytometry (Figures 8A–H).

In the absence of any treatment for 48 h, 99% of L929 control cells and 94% of LLC cancer cells remained viable and showed normal cell morphology (Figures 8A,B). The positive control of L929 cells, which were treated with TNF-α for 48 h, lost their confluency and showed apoptotic morphology characterized by round swollen cells with translucent cytoplasm (Figure 8C). Only 26% of the TNF-α-treated L929 cells were viable (Figure 8C). By contrast, LLC cells were resistant to treatment with TNF-α for 48 h, showed 86% viability and retained normal cell morphology (Figure 8D). Treatment with IFN-γ for 48 h was not cytotoxic to LLC cells, and 90% of the treated LLC cells remained viable (Figure 8E). Interestingly, combined treatment of LLC cells with both cytokines for 24 h also did not induce cytotoxicity in LLC cells (Figure 8F). However, a prolonged incubation of LLC cells with both cytokines for 48 h induced cytotoxicity in LLC cells resulting in 38% cells being double-positive for Annexin and PI (Figure 8G). Also pretreatment of LLC cells with IFN-γ for 24 h followed by treatment with TNF-α for 24 h was cytotoxic to LLC cells and induced apoptosis in 10% and necrosis in 41% of the cells (Figure 8H). Cell morphology of LLC cells changed after the combined treatments of LLC with both cytokines simultaneously for 48 h or with IFN-γ pretreatment followed by TNF-α (Figures 8G,H). After combined treatments with both cytokines, we observed cells with apoptotic cell morphology as well as live cells that had spread in an elongated shapes possibly due to access of free space resulting from cells dying in proximity (Figures 8G,H). All in all, combined treatment with IFN-γ and TNF-α was effective in inducing cell death in LLC cancer cells, which were resistant to single-treatments with these cytokines.

To verify that vdIFN-γ is a functional protein, we used an in vitro assay based on cancer cell growth inhibition mediated by activated macrophages. The macrophages were treated with mitomycin C to block their proliferation, and they were activated by applying either vdIFN-γ or rIFN-γ in combination with a second stimulus. Based on previous experiments performed in our lab, we chose to stimulate the BMDMs with IFN-γ and TLR2/1 agonist Pam3 because this combination leads to efficient inhibition of cancer cell growth (70). After the BMDMs were stimulated for 24 h, LLC cells were added, and the co-cultures were cultivated for an additional 20 h. Finally, to enable the detection of proliferating cancer cells, we added radiolabeled thymidine to the cultures at 24 h before they were harvested (Figure 9A).

In the first experiment, we treated cells with different concentrations of rIFN-γ in combination with 100 ng/mL Pam3 to identify the lowest effective dose of IFN-γ that is necessary to activate BMDMs toward a cancer-suppressive phenotype (Figure 9B). The results revealed that adding rIFN-γ within the range 100–1.56 ng/mL effectively inhibited cancer cell growth (cpm below 750 when the ratio of BMDMs:LLC cells was 20:1) in a concentration-dependent manner. Some growth inhibition was also observed at 0.098–0.39 ng/mL rIFN-γ, but minor or no effect was observed at concentrations ≤0.024 ng/mL (Figure 9B).

All controls included in this assay produced the expected results (Figures 9B,C). Treating the BMDMs with mitomycin C resulted in counts lower than 650 cpm, confirming that the treatment hindered proliferation (Figures 9B,C; first group of bars from the left). Unstimulated macrophages did not inhibit growth in LLC cells since more than 15,000 cpm were measured in the wells (Figures 9B,C; second group of bars from the left). These results were comparable to the average cpm in LLC cells that were cultivated alone (Figures 9B,C; orange line). Treating cells with either Pam3, rIFN-γ, or vdIFN-γ alone resulted in growth rates similar to those observed in LLC cells cultivated alone (Figures 9B,C).

To analyze the functionality of vdIFN-γ, we treated BMDMs with the same concentrations that were used to test rIFN-γ. The vdIFN-γ was produced by infecting BHK-21 cells with SFV-Ifng viral particles and harvesting the cell culture medium to use as a source of vdIFN-γ. The results were similar to what was observed for rIFN-γ in that incubating macrophages with vdIFN-γ in combination with Pam3 resulted in efficient cancer cell growth inhibition in a concentration-dependent manner. The lowest effective concentration of vdIFN-γ was 6.25 ng/mL, which produced less than 1,050 cpm (Figure 9C). In all the experiments, the most efficient inhibition of cancer cell growth was observed when the ratio of BMDMs to LLC cells was 20:1 or 10:1 (Figures 9B,C). Finally, the pattern of effective IFN-γ doses and the extent of growth inhibition they produced was similar between rIFN-γ and vdIFN-γ (Figures 9B,C). Based on these data, we conclude that vdIFN-γ has biological activity and can substitute for rIFN-γ.

Using TLR ligands in combination with IFN-γ provides a classical set of stimuli that activates macrophages toward an M1-phenotype, which can produce NO through inducible NO synthase (70). We, therefore, determined NO production as a readout to verify that vdIFN-γ triggers macrophage activation potentially toward an M1-phenotype. We measured NO2−, which is the end product of the rapid oxidation of NO, in cell culture media using the Griess test. First, we verified that the BMDMs produced NO in a concentration-dependant manner at 24 h after stimulation with both rIFN-γ and Pam3 (Figure 10A). We then stimulated BMDMs with both vdIFN-γ and Pam3, and the results were similar to those observed for rIFN-γ in that the levels of NO2− varied according to the concentration of vdIFN-γ (Figure 10B). The highest levels of NO2− were observed when rIFN-γ and vdIFN-γ were used in a range from 0.39 to 100 ng/mL. At IFN-γ concentrations ≤0.098 ng/mL, the levels of NO2− were lower (Figures 10A,B). Stimulation with rIFN-γ alone resulted in the production of only small amounts of NO2− in the cell culture medium (Figure 10A). The levels of NO2− were also low in cultures stimulated with vdIFN-γ alone (Figure 10B) or Pam3 alone (Figures 10A,B). Thus, IFN-γ and Pam3 had a synergistic effect on the ability of macrophages to produce NO. The higher levels of NO2− that were observed following stimulation with higher doses of IFN-γ (Figures 10A,B) were well-correlated with the enhanced cancer cell growth inhibition that was observed in the growth inhibition experiments (Figures 9B,C). Taken together, data from the growth inhibition assay demonstrated that vdIFN-γ is a potent agent at very low concentrations that when combined with Pam3 induces an antitumor phenotype in macrophages that suppresses growth in LLC cells.