Cells
293T, 293A, and 4 types of glioblastoma (GBM) cell lines, including U87, U251, A172, T98G, were purchased from ATCC. Another GBM cell line H4 is a kind gift from Sheng Zenghua in our laboratory. 293T, 293A, U87, U251, A172, H4 and T98G were maintained in DMEM (Gibco), supplemented with 10% FBS (Gemini), penicillin (100 U/ml) and streptomycin (100 µg/ml). For in vivo imaging, patient-derived glioblastoma tissue were dissolved using 1‰ collagenase (Solarbio), and then cultured in an incubator at 37℃. 6 days later primary glioblastoma cells were modified to express luciferase by lentivirus transduction, and named GBM19-LUCF after puromycin selection. Primary tumor samples obtained from patients with GBM and blood samples from GBM patients and healthy donors were also approved by West China Hospital of Sichuan University Biomedical Ethics Committee (ethical approval document 2018-061). Written informed consent was obtained from patients with GBM and healthy donors.
Adenovirus Construction
The schematic representation of oAD-IL7) and its blank counterpart are shown in Fig. 1. Both oncolytic adenovirus were based on ADMAX system, which consists of an E1, E3-deleted serotype 5 human adenovirus backbone, pBHglox△E1,E3, and a shuttle plasmid, pDC316-EGFP. A cDNA encoding human IL7 purchased from Sinobiological (Beijing, China) was cloned and then inserted the E1 region, and an E1A gene under the control of hTERT, followed by a E1B gene under the control of TATA box was synthesized by General Biosystems (Chuzhou, Anhui province, China) and inserted into the E3 region. The two types of viruses were rescued from 293A cells 7 days after transduction of the modified backbone and shuttle plasmids for further experiment by three consecutive freeze-thaw cycles. The titration was conducted using plaque assay. Briefly, monolayer 293A cells were infected by gradient-diluted viral solutions. 4 hours later, the infection media were replaced by overlay media consisting of DMEM (Gibco), 0.8% agarose (Sangon Biotech), and 2% FBS (Gemini). The GFP-positive plaques were counted using a fluorescence microscope (Zeiss, AXIO observer Z1) 3 days later. Finally, the viruses were purified by cesium chloride gradient centrifugation.
T-cell Generation
Primary lymphocytes were kindly donated by Dr. Zhang in our laboratory, and were isolated by gradient centrifugation at a speed of 800 g for 15 min. Purified T cells were then cultured in 24-well plate in X-vivo media (Lonza) with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin at a density of 1 × 106 cells/ml in a 37 °C incubator with an atmosphere containing 5% CO2. 100 U/ml interlukin 2 (Life Science), 5 ng/ml IL15 (Miltenyi), and CD3/CD28 T cell Transact (Miltenyi) were added for T cell activation and sustaining. 72 hours later, the activated T cells were transduced by lentivirus at an MOI of 10, with the assistance of 10 µg/well coated retronectin (Takara) in the presence of IL-2. After 12 hours incubation, the T cells were collected and maintained in daily refreshed media (X-vivo, Lonza) with 100 U/ml IL-2, until the following experiment 6–8 days later. The titration of the lentivirus is conducted using TCID50 assay.
Immunofluorescent Staining and Western Blot
Immunofluorescent staining and western blot analysis were conducted as previously described[15]. For western blot, 1 × 106/well U87 cells were incubated in 6-well plate for 12 hours. Then 1 × 107 oncolytic adenovirus were added to each well. The cells were collected 24, 48 and 72 hours after viral infection and lysed for protein extraction and further experiment. For immunofluorescent staining, 1 × 105/well GBM cells were incubated in 24-well plate with poly-L-lysine-treated coverlips for 12 hours, and then adenoviral infection was performed at an MOI of 1. The specimens were incubated in 4% paraformaldehyde followed by 0.1% Triton X-100 incubation for 20 min, and then stained with anti-IL7 and DAPI (Beyotime). The images were captured by confocal microscopy (Zeiss, AXIO observer Z1).
Flow Cytometry
For each flow cytometry specimen, 1 × 106 cells were twice washed by PBS and then incubated in 1% BSA solution with Fc blocker (Biolegend) for 30 min. For ki67 staining, the specimens were fixed and perforated for another hour using BD Cytofix/Cytoperm™ kit after surface staining. Fluorochrome-conjugated antibodies were purchase from Biolegend (CD3, CD4, CD8, CD25, CD69, PD1, LAG3), Huabio (IL7), and BD (CD19, B7H3, ki67). T cell activation analysis were performed using FlowJo 9.3.2, while the transduction efficiency of CAR-T, B7H3 expression on GBM cells, and proliferation assay were measured by ACEA NovoCyte (Agilent Biosciences).
T-cell Activation, Cytokine Secretion and Cytotoxicity
2 × 105 U87 cells were seeded in 24-well plate overnight and cocultured with CAR-T cells for 48 hours. For activation analysis, T cells were collected and stained with CD3, CD4, CD8, CD25, and CD69 for flow cytometry. For cytokine secretion analysis, supernatants were harvested for TNF-α and IFN-γ quantitation by Elisa (R༆D). For cytotoxicity assessment, the U87 cells were incubated in culture media with 0.5 mg/ml MTT reagent (Sangon, E606334-0500) for 4 hours at 37℃. Then the media was carefully aspirated and 100 µl Formazan solubilization solution was added. 10 minutes later, the cytotoxicity was measured according to the absorbance at 570 nm.
CytoTel Blue Proliferation Assay
2 × 105 U87 cells were incubated in 24-well plate overnight, and then viral infection was conducted at an MOI of 1. 24 hours later the U87 cells were cocultured with 1 × 106 T cells for 3 days, and then the T cells were collected for another round of proliferation assay until the day of 7. T cells were labeled with CytoTel Blue (AAT Bioquest) according to manufacturer’s instruction before coculturing. At day 3, 5, and 7, the T cells were collected for proliferation analysis by using ACEA NovoCyte (Agilent Biosciences).
Apoptosis Analysis
On the day 7 of coculture, Cells were collected and stained with Annexin V and 7-AAD and analyzed by flow cytometry. Anti-CD3 fluorescent-conjugated antibody (Biolegend) was added to separate the T cells and U87 cells.
In Vivo Experiment
All animal experiments followed a protocol approved by the Institutional Animal Care and Use Committee of Sichuan University. All xenograft models were constructed based on six to eight weeks old NCG mice, which were purchased from Gempharmatech and were bred in the animal vivarium at the State Key Laboratory of Biotherapy, Sichuan University in a pathogen-free condition.
Xenograft Models
Xenograft models were established by intracranial injection of 2 × 105 GBM-LUCF cells suspended in 5 µl PBS solution for each mouse into the right frontal lobe of the brain located in 2 mm lateral and 1 mm anterior to bregma at a depth of 3 mm. 7 days later, xenografts were confirmed by bioluminescent imaging, and treated with an intratumoral injection of 1 × 108 pfu oAD-IL7 or oAD respectively into the same position aforementioned. B7H3-CART cells were intravenously administrated 2 days after viral injection. Tumors were captured twice a week by bioluminescent imaging, and the bioluminescent signals for each group were recorded using the IVIS system (Caliper Life Sciences). Mouse death served as the endpoint of the experiment.
Bioluminescent Imaging
GBM19 cells expressing luciferase were used to detect tumor growth during in vivo experiment. The mice were treated with intraperitoneal injection of 150 mg/kg D-Luciferin (Beyotime) dissolved in PBS. Bioluminescent imaging was performed using the IVIS system (Caliper Life Sciences) 10 minutes after D-Luciferin injection, and the radiance of tumor region was recorded for each mouse.
Ex Vivo Experiment
Mice underwent delayed treatment were killed and the tumors were harvested 7 days after B7H3-CART administration. For H༆E stain, the specimens were fixed in 4% formaldehyde for 24 hours. For cytometry analysis, the tumors were incubated in 1‰ collagenase for 1 hour and washed by PBS twice.
Statistics
During in vitro assay, three wells were set as a group, and the experiments were repeated for twice. Significance was determined by two-sided Student’s unpaired t-tests. The survival curve was obtained by Kaplan-Meier plot and a two-sided log rank test was applied for mouse survival test. P < 0.05 was considered as significant (*p < 0.05, **p < 0.01, ***p < 0.001). All the statistics were performed using GraphPad Prism v6.04.