The following reagents were used in the study: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Wisent (Montreal, QC, Canada). Puromycin (PM), penicillin, and streptomycin were purchased from Sigma (St. Louis, MO, USA). SDS-polyacrylamide gel electrophoresis and 10% buffered neutral formaldehyde were purchased from Beyotime (Shanghai, China). Nitrocellulose membranes were provided by Millipore (Billerica, MA, USA). FITC-labeled anti-mouse F4/80, PE-conjugated anti-mouse programmed death ligand 1 (PD-L1), and PE-Cy5-labeled anti-mouse CD80 monoclonal antibodies were obtained from eBioscience (San Diego CA, USA) for flow cytometry analysis. Antibodies against MMP-2, MMP-9, and GAPDH used for Western blotting were manufactured by Proteintech (Chicago, IL, USA). Rat anti-mouse F4/80, CD68, IL-10, TGF-β, VEGF, and anti-mouse major histocompatibility complex class II (MHCII) monoclonal antibodies that recognize cells from both BALB/c and C57 BL/6 mice were manufactured by Abcam (Cambridge, MA, USA) for immunohistochemistry assays. The TNF-α enzyme-linked immunosorbent assay (ELISA) kit was provided by CUSABIO (Wuhan, China). The interleukin-6 (IL-6) ELISA kit was obtained from RayBiotech (Norcross GA, USA). The Greiss Reagent System determined for nitrite was purchased from Promega Biotech Company (Madison, WI, USA).

Toxoplasma gondii tachyzoites of PRU (type II) strain were initially obtained by mouse passages with brain homogenate containing cysts. The open reading frame encoding TgGRA15_II (omitted signal peptide of 1,500 bp, http://ToxoDB.org) was amplified through real-time (RT)-PCR from the total tachyzoites RNA, recombinant plasmids pEGFP-gra15_II were constructed, and recombinant lentivirus (Lv) vectors (LV-pEGFP-gra15_II) were obtained (21).

The murine HCC cells, Hepa1-6, were purchased from the Chinese Academy of Sciences Cell Bank in Shanghai. The murine Mφ cell line, RAW264.7, was preserved in the laboratory. RAW264.7 cells transfected by LV-pEGFP-gra15_II were named LV-gra15_II-Mφ. The mock Lv-infected RAW264.7 cells were termed LV-Mφ. The two cell lines remain high in transfection efficiency when screened with PM. All of these cells were cultured in DMEM supplemented with 10–15% FBS and 1% penicillin–streptomycin at 37°C with 5% CO₂.

The transfection and M1 surface marker analysis of macrophages that express GRA15_II were determined by flow cytometry. Briefly, single cell suspensions of the three groups of Mφ (LV-gra15_II-Mφ, LV-Mφ, and Mφ) were washed in PBS containing 1% FBS and adjusted to 1 × 10⁶ cells per 100 µl PBS with 1% FBS, respectively. The cells were subjected to FITC-labeled anti-mouse F4/80, followed by PE-conjugated anti-mouse PD-L1, and PE-Cy5-labeled anti-mouse CD80 for surface antigens staining. All cells were incubated with the antibodies at 4°C for 20 min in order to protect the cell from light and washed in PBS twice. Cells were analyzed with flow cytometry. Results were analyzed using FlowJo software.

The culture supernatants of LV-gra15_II-Mφ, LV-Mφ, and Mφ, were applied to Hepa1-6 cell in vitro, and the Hepa1-6 viability was determined by MTS assay (22). MTS assay CellTiter 96 Aqueous One Solution reagents were manufactured by Promega (Madison, WI, USA). Briefly, LV-gra15_II-Mφ, LV-Mφ, and Mφ cells (2 × 106) were separately cultured for 48 h, and then the supernatants were collected. Hepa1-6 cells were seeded in 96-well plates at a density of 1 × 104 cells per well in 100 μl DMEM supplemented with 10% FBS for 12 h and then exposed to different supernatants for 48 h. At 4 h before culture termination, 20 μl of MTS reagent was added to the wells for 1 h incubation. The absorbance density was read on a 96-well plate reader (BioTek) at wavelength 490 nm.

Transwell devices were used with a 0.4-μm-pore polycarbonate filter membrane, which allow small and soluble molecules but not cells to pass through. Hepa1-6 cells (1.5 × 10⁶) were seeded in lower chamber. LV-gra15_II-Mφ, LV-Mφ, and Mφ (2 × 10⁶) were seeded in the 1% penicillin–streptomycin. After 72 h incubation, the Hepa1-6 cells were harvested for total RNA and protein detections.

The migration of Hepa1-6 cells, cocultured with the three groups of macrophages as described above, were assessed using the wound healing assay. Hepa1-6 cells (1 × 10⁶) were inoculated in the lower chambers in the 6-well plate, and the macrophages (2 × 10⁶) were inoculated in the upper chambers at the same time. The upper and the lower chambers were separated for culturing. After 24 h of the cell culture with Hepa1-6 cells nearly at 80% confluence, a 10-µl pipette tip was used to make a straight scratch to simulate a wound, and then washed twice with PBS. At least three randomly selected fields along the scraped line in each well were photographed. Finally, the medium in both upper and lower chambers was changed with fresh DMEM containing 3% FBS. The upper chamber was inserted into the lower chamber for coculture without cellular contact. Following 12 and 24 h incubation, the selected fields were photographed again. The gap distance was quantitatively evaluated using Image-Pro Plus 6.0.

The invasion capability of Hepa1-6 cells after coculturing with various transfected Mφ was assessed using transwell assay. Also, 8-μm-pore polycarbonate filter membrane was used within side of the transwell apparatus. The upper chambers were pre-covered with Matrigel diluted with culture medium and incubated at 37°C overnight according to the manufacturer’s instructions. Then, Hepa1-6 (1 × 10⁴) cells were suspended in 100 μl serum-free DMEM and deposited into the upper chamber per well. The lower chamber contained various types of Mφ (1 × 10⁶) suspended in 700 µl 10% FBS-supplemented medium. After incubation for 24 h, the cells inside of the upper chamber were removed using cotton swabs. The cells on the surface of the polycarbonate filter membrane were fixed in 10% buffered neutral formaldehyde for 15 min and washed twice in PBS. Then, the cells were stained with crystal violet for 15 min after drying at the room temperature. Before the observation, the cells were washed twice in PBS and dried again. The invasion ability of cells was quantified by counting the number of cells that have invaded the membranes in five randomly selected fields under a microscope.

Female C57BL/6 mice (6 weeks old) were purchased from Changzhou Gavens Laboratory Animal Company, China (production permit No. Scxk2013-003). All animal care and experimental protocols were conducted in strict accordance with the Chinese National Institute of Health Guide for the Care and Use of Laboratory Animals (1998) and approved by the Institutional Review Board of Anhui Medical University Institute of Biomedicine (permit No. AMU26-080610). Every effort was made to minimize animal suffering during the study.

For the subcutaneous tumor model, 100 µl normal saline containing 3 × 10⁶ Hepa1-6 cells were subcutaneously injected into the right lower inguinal of one naïve mouse. After 3 days, a hard mass could be palpated, and the tumor volume reached 50 mm³. Tumors were seen in all inoculated animals. The mice were randomly divided into three groups (10 mice each) as follows: LV-gra15_II-Mφ, LV-Mφ, and normal saline (NS) control. On the third and fifth day of post Hepa1-6 inoculation, the mice of LV-gra15_II-Mφ and LV-Mφ groups received 100 µl NS within 2.5 × 10⁶ corresponding macrophages through tail vein injection, respectively, and the control mice were given the same volume of NS. On days 3, 5, 7, and 10 following Hepa1-6 inoculation, tumor size was measured using vernier caliper and calculated using the formula 0.52 × a × b² (a, long diameter of the tumor; b, short diameter of the tumor). Ten days later, the animals were scarified under euthanasia for further analyses.

Total RNA from the cocultured cells, tumors, and spleens were extracted using Trizol reagent and reversely transcribed to cDNA using Prime Script first Stand cDNA Synthesis Kit according to the manufacturer’s instructions. The qRT-PCR was performed to examine the expression of TNF-α, IL-12, iNOS, MMP-9, MMP-2, IL-10, IL-6, TGF-β, and VEGF using SYBR Premix Ex Taq kit by the ABI Prism 7500 sequence detection system according to the manufacturer’s guidance. GAPDH gene was used as a reference. All the reactions were run in technical triplicates. The relative mRNA expression was calculated with the comparative ΔCt method using the formula 2^−ΔΔCt. The forward and reverse primers are listed in Table 1.

LV-gra15_II-Mφ, LV-Mφ, and Mφ were separately seeded in 6-well plates (2 × 10⁶ cells per well), suspended in 2 ml common culture medium, and cultured at 37°C with 5% CO₂ for 48 h. Cell supernatants were collected and analyzed for cytokine values (TNF-α) and NO concentration. IL-6 in the tumor homogenate supernatants was also determined by ELISA in accordance with the manufacturer’s instructions. The NO content reflected as the nitrite concentration was analyzed using the Griess Reagent System.

Total proteins were extracted from the Hepa1-6 cocultured with the macrophages and separated on 12% SDS-polyacrylamide gels. The proteins were then transferred onto nitrocellulose membranes. Non-specific binding was blocked with 5% skim milk in PBS-Tween-20 (0.1%) for 6 h at room temperature. The membranes were then incubated with primary antibodies to MMP-9 (1:1,000), MMP-2 (1:1,000), and GAPDH (1:2,000) at 4°C overnight and with the horseradish peroxide-conjugated secondary antibodies at room temperature for 2 h. ECL kit was used to detect the specific signals, and the bands intensity was visualized.

The tumor tissues from each animal were removed immediately under anesthesia and placed in 10% buffered neutral formaldehyde. First, the antigen retrieval was executed under high pressure in 10mM EDTA buffer. Second, the samples were incubated with primary antibodies to F4/80, CD68, IL-10, TGF-β, VEGF, or MHCII at 4°C overnight. Finally, the sample was incubated for 45 min with secondary antibodies. Bright-field images were photographed and analyzed. The positive macrophages were defined by examining F4/80 and CD68 and were counted in 10 randomly selected fields at ×400 magnification.

All experiments were replicated three times with similar results. All data are expressed as mean ± SD and evaluated using a two-tailed unpaired Student’s t-test. Statistical significance was determined by using GraphPad Prism Software, and P < 0.05 was taken as significant.

RAW264.7 cells were transfected by LV-gra15_II-Mφ or LV-Mφ as mock control. The results indicated that the Lv-mediated gene transfer system could efficiently transfer GRA15_II gene into RAW264.7 cells. As shown in Figure 1A, we observed numerous green fluorescent cells using the fluorescence microscope. Also, we detected the transfection efficiency using flow cytometry. The efficiency of LV-gra15_II-Mφ, LV-Mφ, and Mφ was 90.02, 97.1, and 0.00%, respectively.

Compared with LV-Mφ, LV-gra15_II-Mφ expressed high level of PD-L1 (P < 0.01) and CD80 (P < 0.01) determined by FCM (Figures 1B,C). The relative TNF-α (P < 0.05), IL-12 (P < 0.05), and iNOS (P < 0.001) mRNA expression levels in LV-gra15_II-Mφ were significantly increased compared with LV-Mφ (Figure 2A). As for the cell supernatants, the inflammatory factors NO (P < 0.001) and TNF-α (P < 0.01) sharply rose in macrophages infected with LV-gra15_II, which is in accordance with the mRNA expression (Figure 2B).

Tumor was palpable, and tumor size was about 50 mm³ 3 days after Hepa1-6 cells subcutaneous injection in the mice (Figure 3A). Compared with the control group (LV-Mφ), on days 7 (P < 0.001) and 10 (P < 0.001) post Hepa1-6 inoculation, the tumor proliferation was markedly inhibited in mice that received twice LV-gra15_II-Mφ treatments (Figures 3B,C). This result was obtained by measuring the size of the tumor (Figure 3C). Examination of frozen sections by fluorescence microscopy revealed that both LV-gra15_II-Mφ and LV-Mφ reached the tumor site and remained in it for at least 72 h after intravenous injection (Figure 3D). The TAMs infiltration of solid tumor was examined by immunohistochemistry. Figure 4 showed that the number of macrophages in tumor microenvironment was decreased in the LV-gra15_II-Mφ treated group, and reduced expression of IL-10, TGF-β, and VEGF in tumor tissues and increased expression of MHCII molecule in the animals that received LV-gra15_II-Mφ inoculations, compared with the group treated with LV-Mφ. The data were further verified by using qRT-PCR. Consistent with the immunohistochemistry assay, relative mRNA expressions of IL-10 (P < 0.01), TGF-β (P < 0.05), and VEGF (P < 0.05) were significantly decreased, and iNOS (P < 0.05) was remarkably elevated (Figure 5A). More significantly, IL-6 production, which has been considered to be closely associated with tumorigenesis in tumor microenvironment, was impaired. The mice with LV-gra15_II-Mφ injection exhibited a notable decrease in levels of mRNA coding for IL-6 (P < 0.05) when compared with the control group (Figure 5B). Parallel IL-6 expression results (P < 0.05) were confirmed by ELISA in the tumor homogenate supernatants (Figure 5B).

Observation of frozen sections of spleen tissue under the fluorescence microscope revealed that both LV-gra15_II-Mφ and LV-Mφ reached spleen and survived in this site after intravenous injection (Figure 6A). Mice injected with LV-gra15_II-Mφ exhibited notable increase in mRNA transcription of TNF-α (P < 0.01) and IL-12 (P < 0.05), and decrease in IL-6 (P < 0.01) and IL-10 (P < 0.01), in comparison to mice injected with LV-Mφ (Figure 6B).

Cocultured migration and invasion of Hepa1-6 cells with LV-gra15_II-Mφ were inhibited. Obviously, in the wound healing assay, the relative Hepa1-6 cell migration was notably decreased in the LV-gra15_II-Mφ coculture group (P < 0.001) compared with the LV-Mφ coculture group (Figures 7A,B). Similar results were obtained in the invasion assay, the migrated numbers of Hepa1-6 cells were declined in the group cocultured with LV-gra15_II-Mφ (P < 0.001) (Figures 7C,D). The MMP-9 (P < 0.05) and MMP-2 (P < 0.05) mRNA expression levels in the Hepa1-6 cells were downregulated in the LV-gra15_II-Mφ coculture group (Figure 7E). To verify the mRNA data, we identified the MMP-9 and MMP-2 expression by Western blotting. The results revealed that the levels of MMP-9 and MMP-2 were lowered in LV-gra15_II-Mφ coculture group (Figure 7F). However, there was no noticeable apoptosis of Hepa1-6 cells that were cocultured with LV-gra15_II-Mφ (Figure 7G).