Isolation and culture of human glioblastoma cells
Human brain tumor samples were obtained from the Neurosurgery Department at Tian Tan Hospital in Beijing, China, after patients with glioma provided informed consent. Specimens were reviewed by a neuropathologist to assess the grade and tumor type before assays were performed. Human glioma samples were transferred to a Petri dish, washed three times in phosphate-buffered saline (PBS, Gibco, USA), and cut into 1-mm3 pieces. Next, 0.05% trypsin (Gibco, USA) was added to the tumor specimens. Then, the single pieces were digested for 10 min, filtered with a 70-µm nylon mesh (Corning, USA) and centrifuged at 1000 rpm for 5 min. Single cells were resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS; Invitrogen, China) and 100 U/ml penicillin/streptomycin (Gibco, USA), seeded into 25-cm2 culture flasks (Corning, USA) and incubated at 37 °C and 5% CO2 in a humidified chamber. The media was changed 2–3 times a week. Primary cells at 70–80% confluence were passaged using 0.05% trypsin (Gibco, USA) and were used for different experiments after purification. This line of primary GBM cells was named BT-01 and identified by short tandem repeat (STR), moreover, the biological characteristics of the primary GBM cells can be found in our previous study [27].
Cell lines
GL261, U251, U87, BV2 and HMC3 cells were purchased from the American Type Culture Collection (ATCC, Gaithersburg, MD, USA), and human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (MD, USA). Cell lines GL261, U251, U87, BV2 and HMC3 were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS; Invitrogen, China) and 100 U/ml penicillin/streptomycin (Gibco, USA). HUVECs were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, China) and 1% penicillin/streptomycin (Gibco, USA). All cells were cultured in a 37 °C humidified incubator with 5% CO2.
Recombinant oncolytic adenovirus
The Ki67 promoter gene was amplified and obtained. The virus skeleton plasmids BamHI/XhoI and pTE-Mel containing the GFP gene were successfully constructed by the laboratory at Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). The plasmids were linked with Ki67 promoter to form pTE-Ki67MEl/pSh5-GFP and were treated with incision enzyme Mfe I. pSh5-GFP was linked to pTE-MEl and pTE-Ki67MEl to construct pSh5-MEl-GFP and pSh5-Ki67-GFP, followed by treatment with the restriction enzyme PME I. Then, plasmids were extracted, and pAd5-GFP and pAd5-Ki67/GFP were obtained. Similarly, in order to construct pAd5-Ki67/IL-15, GFP was replaced with the IL-15 gene. The above constructed plasmids were cotransfected with pAd5-GFP and pAd5-Ki67/GFP into 293T cells using Top10. After viral plaque purification, the identified adenoviruses were termed Ad5-GFP, Ad5-Ki67/GFP and Ad5-Ki67/IL15.
Collection of conditioned media
For all experiments, glioma cells, GL261, U251, and U87, primary cells BT-01, and microglial cells, BV2 and HMC3, were seeded into T25 tissue culture flasks in DMEM with 10% FBS containing penicillin (100 U/ml)/streptomycin (100 mg/ml) (Gibco, Grand Island, USA). After culturing for 24 h, each oncolytic adenovirus (Ad5-GFP, Ad5-Ki67/GFP, Ad5-Ki67/IL-15) at a multiplicity of infection (MOI) of 40 was added separately to the T25 tissue culture flasks, and glioma cells were treated with the viruses for another 24, 48, 72 or 96 h. At various time points, different glioma conditioned media (CM, Ad5-GFP-CM, Ad5-Ki67/GFP-CM and Ad5-Ki67/IL-15-CM) were collected from the flasks and centrifuged at 2000 rpm for 10 min to remove cells and cellular debris. Microglial cells, BV2 and HMC3, were cultured in standard medium, and respectively treated with different viruses (Ad5-GFP, Ad5-Ki67/GFP and Ad5-Ki67/IL-15, MOI = 40) for 12 h, then the BV2 and HMC3 cells were washed three times with PBS to remove the viruses, and continued to be cultured in standard medium for another 72 h, at which time different microglial cells conditioned media (HMC3/BV2-Ad5-GFP-CM, HMC3/BV2-Ad5-Ki67/GFP-CM, and HMC3/BV2-Ad5-Ki67/IL15-CM) were obtained and centrifuged at 2000 rpm for 10 min to remove cells and cellular debris. Afterward, all collected conditioned media were stored at − 20 °C prior to use.
Fluorescence microscopy
Glioma cells, GL261, U251, and U87, primary cells BT-01, and microglial cells, HMC3 and BV2, were treated with Ad5-GFP and Ad5-Ki67/GFP at a multiplicity of infection (MOI) of 40 and were observed under an Olympus microscope. Images were taken 72 h after infection. GFP expression levels were analyzed in three random fields of view per well using ImageJ software (NIH, USA).
Cell proliferation assay
Cell viability was analyzed using the Cell Counting Kit-8 (CCK-8 Kit, Dojindo Laboratories, Japan). Glioma cells, GL261, U251, and U87, primary cells BT-01 and BV2, and HMC3 cells were seeded into 96-well plates (3000 cells/well) and cultured overnight. Then, the oncolytic adenoviruses (Ad5-GFP, Ad5-Ki67/GFP, and Ad5-Ki67/IL-15) were added to the 96-well plates at different MOI values and cultured for 72 h. In addition, Ad5-GFP, Ad5-Ki67/GFP and Ad5-Ki67/IL-15 were added to 96-well plates containing glioma cells at an MOI of 40 and cultured for 1, 2, 3, and 4 days, with standard media as a control. Moreover, the conditioned media of virus-treated microglial cells were added to 96-well plates containing glioma cells and cultured for 1, 2, and 3 days, and untreated conditioned media were used for controls. At various time points, 10 µl of CCK-8 was added to each well and incubated for 2 h at 37 °C and 5% CO2. Finally, 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 condition. Assays were repeated at least three times.
Detection of IL-15 and VEGF by ELISA assay
IL-15 levels in conditioned media collected from Ad5-Ki67/IL-15-treated glioma cells for 24, 48 h, 72, and 96 h were measured using their respective human ELISA kits (Neobioscience, China). VEGF levels in conditioned media collected from virus-treated glioma cells for 72 h were measured using their respective human ELISA kits (Neobioscience, China). All procedures were performed as described in the manufacturer’s instructions, and absorbance was measured at 450 nm. Repeated wells were used for each media sample.
RNA extraction and RT-PCR
GL261, U251, and U87 glioma cells and primary cells BT-01 were treated with Ad5-Ki67/IL-15 at an MOI of 40. Total RNA was isolated 48 h postinfection from all glioma cells using TRIzol reagent (Invitrogen, USA). Then, qPCR assays were performed using SYBR-Green PCR Master Mix (Applied Biosystems) on a QuantStudio 6 Flex system (Applied Biosystems). cDNA synthesis was performed using the Reverse Transcription System Kit (Promega A3500) according to the manufacturer’s instruction. The codon-optimized IL-15 with the following primers: forward primer, 5′-CATGTACGTTGCTATCCAGGC-3′; reverse primer, 5′-GGTCTTCTCCTCC AGCTCCT-3′. Each target was run in triplicate, and GAPDH was used as an internal standard. Relative gene expression was compared to a housekeeping gene. The results were calculated using the 2−ΔΔCt method.
Tube formation assay
Growth factor-reduced Matrigel (BD, USA) was added to flat-bottomed, precooled, 96-well plates. After incubation at 37 °C and 5% CO2 for 40 min, HUVECs pretreated with different viruses (Ad5-GFP, Ad5-Ki67/GFP and Ad5-Ki67/IL-15, MOI = 40) for 72 h and untreated HUVECs were labeled using Calcein AM (Tocris, USA). Treated HUVECs (2 × 104/well) were seeded into wells containing standard medium. The untreated HUVECs were seeded into wells containing different U251 conditioned media (CM, Ad5-CM, Ad5-Ki67-CM and Ad5-Ki67/IL-15-CM). In addition, human recombinant vascular endothelial growth factor (VEGF, 6 ng/ml, Peprotech, USA) was added to the extra wells containing CM, Ad5-CM, Ad5-Ki67-CM and Ad5-Ki67/IL-15-CM. Then, 96-well plates were incubated at 37 °C and 5% CO2. Three wells were used for each media sample. After 6 h, tube formation was imaged with an Olympus microscope. Capillary-like tube formation of HUVECs was analyzed in three random fields of view per well using ImageJ software (NIH, USA).
Statistical analysis
Statistical analyses were performed using GraphPad Prism. Unless specifically noted, all data are representative of at least three separate experiments. Error bars represent the standard deviations (SDs) and were calculated using Prism. The specific statistical tests used were t-test for single comparisons or ANOVA followed by Tukey’s test for multiple comparisons, and all P-values < 0.05 were considered statistically significant.