Isolation and culture of GA-MSCs
Isolation of human GA-MSCs was performed as described previously [10]. Briefly, the GA-MSCs were separated from the fresh glioma specimens; the tissue was cut into pieces after washed twice in PBS. Afterward, the trypsin was added to the pieces for digestion. Then, the cell suspension was transferred to a 70-μm filter and washed twice again with PBS. GA-MSCs were cultured in DMEM containing 10% fetal bovine serum (FBS; Gibco, USA) and 100 U/ml penicillin/streptomycin (Gibco, USA) in a humidified atmosphere at 37 °C with 5% CO2. All experiments were performed according to the flow chart (Additional file 1).
Mouse lymphocytes
Isolation of mouse spleen lymphocytes was performed according to the instructions. Briefly, fresh spleen was obtained and transferred to a 70-μm filter (Corning, USA) on a 50-ml centrifuge tube, then gently ground, washed continuously with precooled PBS and centrifuged at 1800 rpm for 5 min. The cell pellets were re-suspended in washing solution and separated with lymphocyte fluid at a 1:1 ratio density gradient centrifugation at 2000 rpm for 20 min. The collected cells were washed using washing buffer and cleaning buffer before use in experiments.
Construction of Ad5-Ki67/IL-15
The recombinant oncolytic adenovirus (OAd) has been previously described [25]. Briefly, an intrinsic promoter that controls the type 5 adenovirus E1A gene was replaced by the Ki67 promoter sequence, and GFP gene was inserted into E3 region, thus forming Ad5-Ki67/GFP. Subsequently, GFP gene was replaced with human IL-15 gene to produce Ad5-Ki67/IL-15. Therefore, the targeted OAd Ad5-Ki67/IL-15 was constructed.
Glioma cells
GL261, U87, U251 and BT-01 were cultured in DMEM (Gibco, USA) containing 10% FBS (Invitrogen, China) and 100 U/ml penicillin/streptomycin (Gibco, USA) in a humidified atmosphere at 37 °C with 5% CO2.
Collection of conditioned media
GA-MSCs, U251 and BT-01 cells were seeded in six-well plates containing the viruses at a multiplicity of infection (MOI) of 40 for 72 h. The supernatants were collected and centrifuged at 2000 rpm for 10 min to remove cells and cellular debris.
Immunofluorescence
GA-MSCs were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton, and blocked with 3% bovine serum albumin for 1 h at room temperature. The cells were incubated with primary antibodies against CD44 and CD105 (1:100, Proteintech, China), then overnight at 4 °C. The secondary antibodies used were as follows: Cy3-conjugated antibodies (1:100, Boster, WuHan, China). Nuclei were stained for visualization using DAPI. Immunofluorescence microscopy was performed with an Olympus microscope.
Fluorescence microscopy
GA-MSCs were treated with Ad5-Ki67/GFP at an MOI of 40 and were observed under an Olympus microscope. Images were taken 48 h after infection.
Cell cycle
The GA-MSCs (1 × 105 cells/well) were seeded into 6-well plate with 10% FBS (Invitrogen, China) and 100 U/ml penicillin/streptomycin (Gibco, USA) in a humidified atmosphere at 37 °C with 5% CO2. After the cell adherence, the culture condition was added with the virus Ad5-Ki67/IL-15 (MOI = 40), and incubated for 72 h. The untreated cell was as a control. The cells were fixed in 95% ethanol (4 °C) overnight. Then, the fixed cells were washed twice with precooled PBS and incubated in 2.5 mg/ml DNase-free RNase A (Gene-Protein Link) and PI (1×, Gene-Protein Link) for 30 min at 37 °C. The distribution of the cell cycle was detected using a flow cytometry.
Cell viability
Cell viability was analyzed using the Cell Counting Kit-8 (CCK-8 Kit, Dojindo Laboratories, Japan).
The cells (3000 cells/well) were seeded into a 96-well plate and cultured overnight. Then, the medium was replaced with 100 µl of different media and cultured for 1, 2, 3 days. At every time point, 10 µl of CCK-8 was added to each well and incubated for 2 h at 37 °C with 5% CO2. Then, the absorbance of each well was measured at 450 nm using a microplate reader (PerkinElmer, USA). At least three wells were used for each sample in different media.
Tube formation assay
Tube formation assays were performed according to previous descriptions [25]. Briefly, growth-factor-reduced Matrigel (BD, USA) was pre-added to 96-well plates. Cells (2 × 104 cells/well) were seeded into wells and incubated at 37 °C and 5% CO2. After 6 h, the cells were labeled using Calcein AM (Tocris, USA), and tube formation was imaged with an Olympus microscope. Capillary-like tube formation of GA-MSCs was analyzed in three random fields of view per well using ImageJ software (NIH, USA). The tube segment lengths and number of tubes of GA-MSCs cultured with different conditions were also quantified.
Coculture assay
For the coculture experiments, glioma cells GL261, U251, U87 and BT-01 (1 × 105 cells/well) and lymphocytes (1 × 106 cells/well) were, respectively, placed into the bottom wells of transwell permeable 6-well plate supports (Corning, Corning, NY), then the GA-MSCs (1 × 105 cells/well) were plated in the apical chamber, and the virus Ad5-Ki67/IL-15 (4 × 106 VP) was added into the upper chamber, which was co-cultured for 72 h. Inserts had a pore size of 0.4 μm, permitting the free exchange of molecules but preventing cell migration or contact. The experiments included different groups: cultures of glioma cells with addition of GA-MSCs, cultures of glioma cells with GA-MSCs plus virus, glioma cells with single virus and only glioma cells. All cells were cultured in a supplemented MSCs basal medium. After 3 days in coculture, the cells were collected to perform flow cytometry analysis.
Flow cytometry
Flow cytometry analysis was performed using fluorochrome-conjugated antibodies. Cultured glioma cells were detached with 0.05% trypsin–EDTA (ATCC), and enzymatic action was stopped by adding 10% FBS in PBS (Gibco, USA). The cells were washed in PBS, and then, the pellets were re-suspended in fluorescent-activated cell sorting (FACS) buffer. The glioma cell suspensions were stained with APC-conjugated antibodies against human or mouse PD-L1 (1:20, Proteintech, USA), and the lymphocytes were, respectively, stained with mouse PD-1 (1:100, Proteintech, USA), CD3, CD4 and CD8 (1:100, BioLegend, USA). All cells were incubated in the dark at 4 °C for 20 min. Then, the cells were centrifuged and re-suspended in PBS. Data were acquired within 2 h after staining on a BD Accuri C6 Plus (BD Biosciences), and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
Animal experiments
All animal studies were approved by Experimental Animal Welfare and Ethics Committee of Beijing Tiantan Hospital affiliated with Capital Medical University and approved all the animal experiments. Male BALB/nu-c (6 weeks old; Beijing Vital River Laboratory Animal) were kept in the animal facilities at Beijing Neurosurgical Institute and maintained under specific-pathogen-free conditions. Inhalation of isoflurane was used to anesthetize the animals in all experiments. BALB/nu-c (n = 5/group) was subcutaneously inoculated with a PBS suspension containing 1 × 106 U87-Luc cells (100 ul) in the right underarm using 1 ml syringe. The GA-MSCs suspension (100 ul, 1 × 105 cells) or Ad5-Ki67/IL-15 (3 × 1011 vp) plus GA-MSCs (1 × 105 cells) were injected into the tumor sites 14 days after U87-Luc cell implantation. Tumor volumes were calculated according to the following formula: width2 × length × 0.5.
IVIS imaging
The IVIS spectrum was utilized to obtain luciferase images. In vivo images were obtained on day 0 (prior to virus injection) and on days 3 and 6 after virus and GA-MSCs injection. Each mouse was treated with 100 μl 15 mg/ml d-luciferin (PerkinElmer, Waltham, MA, USA) i.p. in PBS.
Western blotting
The animals were anesthetized and sacrificed after treatment for 10 days. The tumor specimens were harvested from the mice, then washed two times and cut into pieces. The total protein was extracted with RIPA lysis buffers were treated by ultrasound and supplemented with mammalian protease inhibitor (Sigma-Aldrich, St Louis, MO, USA). Protein samples (20 μg per lane) were loaded into gels for separation via sodium dodecyl sulfate polyacrylamide gel electrophoresis. After separation, the proteins were transferred to nitrocellulose membranes (Thermo, Waltham, MA, USA), which were then blocked for 60 min at room temperature in TBST (Tris-buffered saline, 0.1% Tween 20) containing 5% nonfat milk. Membranes were washed with TBST and probed with primary antibodies against PD-L1 (1:800, all from Santa Cruz Biotechnology, USA) at 4 °C overnight. Membranes were washed with TBST and incubated with secondary antibodies for 1 h at room temperature. Signals were detected with an ECL detection system.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 6.0. Sample sizes for each experiment are indicated in the Results or the Material and Methods section. The specific statistical tests used were t test for single comparisons and analysis of variance followed by Tukey’s test for multiple comparisons, and P < 0.05 was considered to indicate statistical significance. Numerical values are reported as mean ± standard deviation.